FACULTATIVE PROTEIN SELENYLATION FOR MODULATING

METABOLISM

Cross-Reference to Related Applications

This application claims the benefit of priority to U.S. Provisional Application Serial No. 62/969,877, filed on 04 February 2020; the entire contents of said application is incorporated herein in its entirety by this reference.

Background of the Invention

Oxidation of cysteine thiols by physiological reactive oxygen species (ROS) initiates thermogenesis in brown and beige adipose tissues. Cellular selenols, where sulfur is replaced with selenium, exhibit enhanced reactivity with ROS. Despite their critical roles in physiology, methods for broad and direct detection of cellular selenols are lacking.

In particular, Sec incorporation into proteins is thought to be limited to the so-called selenoproteins, approximately 25 proteins in which Sec is exclusively encoded at a particular site by co-translational cis- and /ra//.s-acting elements (Labunskyy etal. (2014) Physiol Rev 94: 739-777). In this small number of mammalian selenoproteins, Sec typically exists in enzymatic domains to facilitate catalytic redox reactions. The physiological action of these selenoproteins depends strictly on the presence of Sec, while mutagenesis to Cys leads to decreased or abrogated functionality (Labunskyy etal. (2014) Physiol Rev 94: 739-777). Importantly, the breadth of established selenoproteins has been determined strictly on the basis of specific nucleotide sequences in co-translational cis- elements, so-called SECIS nucleotide sequences.

Because the mechanisms that involve the utilization of selenium in the body have not yet been fully elucidated, there is a need for a better understanding of how selenium is used for various processes, especially given that selenols play critical roles in physiology.

Summary of the Invention

The present invention is based, at least in part, on the discovery that selenium is incorporated facultatively, without reliance on a selenocysteine insertion sequence (SECIS), into certain locations in certain proteins, and that such an incorporation can be utilized to modulate energy expenditure in a subject ( e.g ., to induce weight loss).

In some aspects, methods of identifying a selenium-containing amino acid of a selenylated peptide include detecting presence of a selenium isotopic signature in a mass spectrometry (MS) mass spectrum from the selenylated peptide or from a derivatized form of the selenylated peptide having the same selenium; detecting a derivative-mass shift in an MS mass spectrum from a selenium-related derivative of the selenylated peptide; and localizing, with reference to a non-selenylated peptide that corresponds with respect to its amino acid sequence to the selenylated peptide, a selenium-mass shift in a fragment MS mass spectrum from the selenylated peptide to a position that includes a cysteine or a methionine in the non-selenylated peptide.

In some embodiments, the selenium-containing amino acid includes a selenocysteine or a selenomethionine ( e.g ., selenocysteine). In certain embodiments, the selenium isotopic signature includes at least one peak for each of the following isotopes: ⁷⁶ Se, ⁷⁷ Se, ⁷⁸ Se, ⁸⁰ Se, and ⁸² Se. In some embodiments, each said peak has a relative abundance, and in which the numerical order of each said relative abundance among the relative abundances of said isotopes is the same as the numerical order of its corresponding natural abundance among said isotopes. In certain embodiments, the selenium-related derivative is obtained by reacting the selenylated peptide with a thiol-derivatizing agent. In some embodiments, the thiol-derivatizing agent includes N-ethylmaleimide (NEM) or iodoacetamide (IAM). In certain embodiments, the derivative-mass shift is +125.13 Da per unit charge per one derivatization for NEM, and in which the derivative-mass shift is +57.02 Da per unit charge per one derivatization for IAM. In some embodiments, said non- selenylated peptide corresponds with respect to its amino acid sequence to said selenylated peptide if the amino acid sequence of the selenylated peptide is 100% identical with the amino acid sequence of the non-selenylated peptide when selenium is considered as identical with sulfur. In some embodiments, the selenium-mass shift is +47.94 Da per unit charge per selenium. In certain embodiments, said selenocysteine has been incorporated into said selenylated peptide via an in vivo process that does not rely on a selenocysteine insertion sequence (SECIS).

In certain aspects, methods of selecting a subject for modulation of energy expenditure in the subject’s brown adipose tissue via selenium supplementation are provided comprising detecting from the subject one or more levels for one or more biomarkers listed in Table 1; and selecting the subject for modulation of energy expenditure in the subject’s brown adipose tissue via selenium supplementation based on the one or more levels. In some aspects, methods of determining suitability of a subject for weight- loss treatment via selenium supplementation are provided comprising detecting from the subject one or more levels for one or more biomarkers listed in Table 1; and determining that the subject is suitable for said treatment if the one or more levels are lower than a control threshold, and determining that the subject is unsuitable for said treatment if the one or more levels are equal to or greater than the control threshold.

Numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in some embodiments, the one or more biomarkers comprise one or more biomarkers listed in Table 2. In some embodiments, said one or more biomarkers comprise mitochondrial brown fat uncoupling protein 1 (UCP1) containing a selenocysteine instead of a cysteine at position 254 with respect to SEQ ID NO: 1. In some embodiments, the one or more levels comprise, for each biomarker, the ratio of the biomarker amount to its selenylation-agnostic amount. In certain embodiments, each said ratio is less than 40%. In some embodiments, the methods further include stratifying the subject. In some embodiments, said stratifying includes classifying the subject as low obligate, low facultative if the one or more levels are below a first control threshold; high obligate, low facultative if the one or more levels are neither below a first control threshold nor above a second control threshold; or high obligate, high facultative if the one or more levels are above a second control threshold. In some embodiments, said stratifying includes classifying the subject either as low obligate, low facultative or as high obligate, low facultative. In certain embodiments, said modulation of energy expenditure includes increasing the energy expenditure. In some embodiments, said selenium supplementation includes sodium selenite, selenocysteine, or selenomethionine. In certain embodiments, within a cell of the subject, said selenomethionine is used to generate said one or more biomarkers. In some embodiments, said selenium supplementation includes selenium at an amount greater than a basal threshold. In some embodiments, said selenium supplementation includes selenium at a dose of least 50 pg per day and at most 4 mg per day. In some embodiments, said selenium supplementation includes selenium at a dose of least 1.0 mg per day and at most 1.5 mg per day. In some embodiments, said subject is selected based on the one or more levels being lower than a control threshold. In some embodiments, the one or more levels comprise, for each biomarker, the ratio of the biomarker amount to its selenylation-agnostic amount, and in which said control threshold is 2% for each of the one or more biomarkers. In some aspects, compositions include at least one other biomarker listed in Table 2 at an amount that is at least 20% w/w of the total protein content of the composition. In some embodiments, the biomarker is mitochondrial brown fat uncoupling protein 1 (UCP1) containing a selenocysteine instead of a cysteine at position 254 with respect to SEQ ID NO: 1.

In certain aspects, agents that modulate selenium in a subject, for use in modulating a metabolic response in the subject, optionally in which the agent is formulated with a pharmaceutically acceptable carrier, are disclosed. In some aspects, methods for modulating a metabolic response include contacting a cell with an agent that modulates selenium, to thereby modulate the metabolic response.

Numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in some embodiments, selenium is upregulated. In some embodiments, the selenium is upregulated using an agent that can be selected from among sodium selenite, selenocysteine, and selenomethionine. In some embodiments, the agent is conjointly used an additional agent that increases the metabolic response. In some embodiments, the selenium is downregulated. In some embodiments, the selenium is downregulated using an agent that can be selected from among a metabolizer of selenocysteine and a metabolizer of selenomethionine. In some embodiments, the agent is conjointly used with an additional agent that decreases the metabolic response.

In some embodiments, the metabolic response that can be selected from among: a) modified level of a biomarker that can be selected from among the biomarkers listed in Table 1; b) modified thermogenesis in adipose cells; c) modified basal respiration or uncoupled respiration; d) modified whole body oxygen consumption; e) modified obesity; f) modified diabetes; and g) modified activity of UCP1 protein. In some embodiments, the metabolic response is upregulated. In some embodiments, the metabolic response is downregulated. In some embodiments, the metabolic response includes a modified level of a biomarker that can be selected from among the biomarkers listed in Table 2. In some embodiments, the subject is under a high-fat diet.

For embodiments that include contacting a cell with an agent that modulates selenium, the step of contacting occurs in vivo in some embodiments, and occurs in vitro in other embodiments. The cell, in some embodiments, includes a brown adipocyte or a beige adipocyte. In some aspects, methods of preventing or treating a metabolic disorder in a subject include administering to the subject an agent that promotes selenium, in the subject, thereby preventing or treating the metabolic disorder in the subject.

In some embodiments, the agent is administered orally or systemically, optionally in which the administration is in a solution including selenium at a dose of least 50 pg per day and at most 4 mg per day. In certain embodiments, the agent is administered in a pharmaceutically acceptable formulation. In certain embodiments, the metabolic disorder that can be selected from among type II diabetes and obesity. In certain embodiments, the subject is a non-human animal or a human, optionally in which the non-human animal is an animal model of the metabolic disorder.

In certain aspects, methods for preventing or treating a metabolic disorder in a subject include administering to the subject an agent that inhibits or reduces selenium, in the subject, thereby preventing or treating the metabolic disorder in the subject.

In some embodiments, the agent that can be selected from among a metabolizer of selenocysteine and a metabolizer of selenomethionine. In some embodiments, the agent is administered systemically. In some embodiments, the agent is administered in a pharmaceutically acceptable formulation. In some embodiments, the metabolic disorder that can be selected from among obesity-associated cancer, anorexia, and cachexia. In some embodiments, the subject is a non-human animal or a human, optionally in which the non-human animal is an animal model of the metabolic disorder.

In certain aspects, cell-based assays for screening for agents that modulate a metabolic response in a cell by modulating selenium, include contacting the cell in the presence of selenium, with a test agent that modulates selenium, and determining the ability of the test agent to modulate a metabolic response in the cell. In additional aspects, methods for assessing the efficacy of an agent that modulates selenium uptake, for modulating a metabolic response in a subject, include a) detecting in a subject sample at a first point in time, the amount of selenium; b) repeating step a) during at least one subsequent point in time after administration of the agent; and c) comparing the amount detected in steps a) and b), in which a significantly lower amount of selenium in the first subject sample relative to at least one subsequent subject sample indicates that the test agent increases the uptake of selenium in the subject and/or in which a significantly higher amount of selenium in the first subject sample relative to at least one subsequent subject sample indicates that the test agent decreases the uptake of selenium in the subject; optionally, in which a level of a marker listed in Table 1 is further detected in steps a) and b), and comparing the expression and/or activity of the marker, in which a significantly lower level of a marker listed in Table 1 in the first subject sample relative to at least one subsequent subject sample indicates that the test agent increases the metabolic response in the subject and/or in which a significantly higher level of a marker listed in Table 1 in the first subject sample relative to at least one subsequent subject sample indicates that the test agent decreases the metabolic response in the subject.

In various embodiments, the selenium uptake is upregulated. In some embodiments, selenium uptake is downregulated. In some embodiments, the agent that can be selected from among sodium selenite, selenocysteine, selenomethionine, a metabolizer of selenocysteine, and a metabolizer of selenomethionine. In some embodiments, in which between the first point in time and the subsequent point in time, the subject has undergone treatment for the metabolic disorder, has completed treatment for the metabolic disorder, and/or is in remission from the metabolic disorder. In some embodiments, the first and/or at least one subsequent sample that can be selected from among ex vivo and in vivo samples. In some embodiments, the first and/or at least one subsequent sample is obtained from an animal model of a metabolic disorder. In some embodiments, the first and/or at least one subsequent sample that can be selected from among tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow.

In some embodiments, the tissue includes brown adipocytes and/or beige adipocytes. In some embodiments, the first and/or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject. In some embodiments, modulation includes upregulation by at least 25% relative to the second sample. In some embodiments, modulation includes downregulation by at least 25% relative to the second sample. In some embodiments, a significantly higher level includes upregulating the level by at least 25% relative to the second sample. In some embodiments, the metabolic response includes a) modified level of a biomarker that can be selected from among the biomarkers listed in Table 1; b) modified thermogenesis in adipose cells; c) modified basal respiration or uncoupled respiration; d) modified whole body oxygen consumption; e) modified obesity; f) modified diabetes; or g) modified activity of UCP1 protein. In some embodiments, a significantly lower level includes downregulating the level by at least 25% relative to the second sample. In some embodiments, the amount of the marker is compared. In some embodiments, the amount of the marker is determined by mass spectrometry. In some embodiments, the metabolic response is upregulated. In some embodiments, the metabolic response is downregulated. In some embodiments, the selenium is comprised by an organic molecule. In some embodiments, the selenium is comprised by selenomethionine.

Brief Description of the Drawings

Fig. 1A - Fig. 1C show characteristic features of the selenocysteine containing peptide of Gpxl. Fig. 1A shows an MSI spectrum of Gpxl tryptic peptide containing selenocysteine displaying isotope signature reflecting the natural isotope abundance of selenium. Fig. IB and Fig. 1C show a fragment spectrum of UCP1 peptide localizing selenocysteine to the expected locus.

Fig. 2A - Fig. 2G show selenocysteine incorporation into 253 locus of UCP1. Fig. 2A shows an MSI spectrum of UCP1 tryptic peptide spanning position 253 with a mass shift corresponding to the exact mass difference between cysteine and selenocysteine. Fig. 2B shows the MS criteria for identification of a facultative selenocysteine site. Top — presence of MSI selenium isotope signature. Middle — Mass shift corresponding to addition of selenocysteine derivatization agents. Bottom — fragment mass spectrum localizing selenium mass to cysteine locus. Fig. 2C shows that presence of selenium stabile isotope signature on mass-shifted UCP1 peptide spanning position 253. Fig. 2D shows fragment spectrum of UCP1 peptide localizing the mass difference between sulfur and selenium to position 253. Fig. 2E shows that presence of selenium stabile isotope signature on methionine oxidized form of mass-shifted UCP1 peptide spanning position 253. Fig. 2F shows fragment spectrum of UCP1 methionine oxidized peptide localizing the mass difference between sulfur and selenium to position 253. Fig. 2G shows fragment spectrum of synthetic AQUA peptide with selenocysteine at position 253.

Fig. 3A - Fig. 3L show peptide features of UCP1 cysteine containing peptides. Fig. 3A shows MSI spectrum of UCP1 tryptic peptide containing cysteine 25. Fig. 3B and Fig. 3C show fragment spectrum of UCP1 tryptic peptide containing cysteine 25. Fig. 3D shows MSI spectrum of UCP1 tryptic peptide containing cysteine 188. Fig. 3E and Fig.

3F show fragment spectrum of UCP1 tryptic peptide containing cysteine 188. Fig. 3G shows MSI spectrum of UCP1 tryptic peptide containing cysteine 213 and 224. Fig. 3H and Fig. 31 show fragment spectrum of UCP1 tryptic peptide containing cysteine 213 and cysteine 224. Fig. 3J shows MSI spectrum of UCP1 tryptic peptide containing cysteine 307. Fig. 3K and Fig. 3L show a fragment spectrum of UCP1 tryptic peptide containing cysteine 307

Fig. 4A - Fig. 4E show identification of UCP1 selenocysteine 253 using AQUA peptide. Fig. 4A and Fig. 4B show MSI spectrum of synthesized AQUA peptide containing selenocysteine and a heavy tyrosine (+ — 10.03 Da). Fig. 4C shows TMT labeling strategy to multiplex synthetic AQUA peptide with biological samples. Fig. 4D shows co-elution of endogenous (red) and synthetic heavy (blue) UCP1 selenocysteine 253 peptide. Fig. 4E shows treatment of brown adipocytes with sodium selenite during differentiation increases selenoprotein abundance.

Fig. 5A - Fig. 5F show characterization of selenocysteine incorporation into the UCP1 253 locus. Fig. 5A shows proportion of selenocysteine 253 form of UCP1 estimated using internal standard AQUA peptides. Fig. 5B shows relative abundance of the selenic acid and sulfenic acid forms of UCP1. N = 3. Fig. 5C shows autoradiogram of brown adipocyte lysates treated with sodium 75-selenite throughout differentiation. Fig. 5D shows relative changes in the content of selenocysteine form of UCP1 in brown adipocytes treated with sodium selenite. N = 3. Fig. 5E shows relative changes in the content of selenocysteine form of UCP1 in brown adipocytes treated with selenomethionine. N = 3. Fig. 5F shows relative changes in the content of selenocysteine form of UCP1 in brown adipocytes treated with selenocysteine. N = 3

Fig. 6A - Fig. 6D show that dietary selenium differentially modifies canonical and facultative selenation of proteins. Fig. 6A shows expression of canonical selenoproteins in BAT are modulated by 0.1 to 0.4 ppm dietary sodium selenite. N = 5. Fig. 6B shows identification of selective facultative selenocysteine insertion into BAT metabolic proteins. N 5. Fig. 6C shows identification of selective facultative selenomethionine insertion into BAT metabolic proteins. N 5. Fig. 6D shows that 2.25 ppm dietary selenium increases the amount of Sec253 UCP1. N = 5.

Fig. 7A - Fig. 7J show that elevated dietary selenium enhances BAT energy expenditure and protects against obesogenesis induced by high fat feeding. Fig. 7A shows (A) Weight of mice prior to intervention with defined selenium-containing chow diets. N = 24-25. Fig. 7B shows weight gain of mice on chow diets containing defined selenium concentrations. N = 24-25. Fig. 7C shows cumulative food intake of mice on chow diets containing defined selenium concentrations. N 24-25. Fig. 7D shows whole body oxygen consumption upon acute 1 mg/kg CL of mice following 8 weeks on chow diets containing defined selenium concentrations. N = 7. Fig. 7E shows whole body C02 release upon acute 1 mg/kg CL of mice following 8 weeks on chow diets containing defined selenium concentrations. N = 7. Fig. 7F shows whole body respiratory exchange quotient (RER) upon acute 1 mg/kg CL of mice following 8 weeks on chow diets containing defined selenium concentrations. N = 7. Fig. 7G shows weight of mice prior to intervention with defined selenium-containing HFD. N = 12-15. Fig. 7H shows weight gain of mice on HFD containing 0.1 ppm or 2.25 ppm sodium selenite. N = 12-15. Fig. 71 shows whole body composition of mice following 8 weeks HFD containing either 0.1 ppm of 2.25 ppm sodium selenite. N = 12-15. Fig. 7J shows cumulative food intake of mice on chow diets containing either 0.1 ppm of 2.25 ppm sodium selenite. N = 12-15.

Detailed Description of the Invention

The present invention is based, at least in part, on the discovery that selenium can be incorporated into certain proteins at certain locations ( e.g ., a particular cysteine of a protein) in a manner that does not rely on a selenocysteine insertion sequence (SECIS). In some embodiments, because selenium-containing proteins can be particularly reactive with reactive oxygen species, facultative selenium incorporation can be used to increase energy expenditure by a cell or a subject, to thereby facilitate, for example, weight loss.

Accordingly, the present invention provides methods of identifying a selenium- containing amino acid of a selenylated peptide, methods of selecting a subject for modulation of energy expenditure in the subject’s brown adipose tissue via selenium supplementation, methods for determining suitability of a subject for weight-loss treatment via selenium supplementation, compositions including particular biomarkers, agents that modulate selenium for use in modulating a metabolic response in a subject, methods of modulating a metabolic response in a cell, methods of preventing or treating a metabolic disorder in a subject, cell-based assays for screening for agents that modulate a metabolic response in a cell by modulating selenium, and methods for assessing the efficacy of an agent that modulates selenium uptake.

The disclosure provides a mass spectrometric method to interrogate incorporation of selenols into proteins. Unexpectedly, this approach revealed facultative incorporation of selenium into proteins that have no canonical encoding for selenium-containing amino acids. Selenium was selectively incorporated into regulatory sites on key metabolic proteins, including selenocysteine at locus 253 in UCP1. This facultative utilization of selenium was initiated by increasing cellular levels of organic, but not inorganic, forms of selenium. Remarkably, dietary selenium supplementation elevated facultative incorporation into UCP1 and coincided in elevated energy expenditure through thermogenic adipose tissue, while protecting against obesity. Together, these findings reveal the existence of facultative protein selenation, which impacts thermogenic adipocyte function, and, presumably, other biological processes as well.

These findings reveal that dietary selenium can elevate energy expenditure in brown adipose tissue and can be a simple dietary modality to reverse weight gain on high fat diet.

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The term “amino acid” is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing. The names of the natural amino acids are abbreviated herein in accordance with the recommendations of IUPAC-IUB.

The term “antisense” nucleic acid refers to oligonucleotides which specifically hybridize ( e.g ., bind) under cellular conditions with a gene sequence, such as at the cellular mRNA and/or genomic DNA level, so as to inhibit expression of that gene, e.g. , by inhibiting transcription and/or translation. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.

The terms “beige fat” or “brite (brown in white) fat” or “iBAT (induced brown adipose tissue)” or “recruitable BAT (brown adipose tissue)” or “wBAT (white adipose BAT)” refer to clusters of UCP1 -expressing adipocytes having thermogenic capacity that develop in white adipose tissue (WAT). Beige fat can develop in subcutaneous WAT, such as in inguinal WAT, or in intra-abdominal WAT such as in epididymal WAT. Similar to adipocytes in brown adipose tissue (BAT), beige cells are characterized by a) multilocular lipid droplet morphology, b), high mitochondrial content, and/or c) expression of a core set of brown fat-specific genes, such as Ucpl, Cidea, Pgcla, and other listed in Table 1. BAT and beige fat both are able to undergo thermogenesis, but these are distinct cell types since beige cells do not derive from Myf5 precursor cells like BAT cells, beige fat express thermogenic genes only in response to activators like beta-adrenergic receptor or PPARgamma agonists unlike constitutive expression in BAT cells (Harms and Seale (2013) Nat. Med. 19:1252-1263).

The term “binding” or “interacting” refers to an association, which may be a stable association, between two molecules, e.g. , between a polypeptide of the invention and a binding partner, due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen- bond interactions under physiological conditions. Exemplary interactions include protein- protein, protein-nucleic acid, protein-small molecule, and small molecule-nucleic acid interactions.

The term “biological sample” when used in reference to a diagnostic assay is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

The term “isolated polypeptide” refers to a polypeptide, in certain embodiments prepared from recombinant DNA or RNA, or of synthetic origin, or some combination thereof, which (1) is not associated with proteins that it is normally found within nature, (2) is isolated from the cell in which it normally occurs, (3) is isolated free of other proteins from the same cellular source, (4) is expressed by a cell from a different species, or (5) does not occur in nature.

The terms “label” or “labeled” refer to incorporation or attachment, optionally covalently or non-covalently, of a detectable marker into a molecule, such as a polypeptide. Various methods of labeling polypeptides are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes, fluorescent labels, heavy atoms, enzymatic labels or reporter genes, chemiluminescent groups, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). Examples and use of such labels are described in more detail below. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

The terms “metabolic disorder” and “obesity related disorders” are used interchangeably herein and include a disorder, disease or condition which is caused or characterized by an abnormal or unwanted metabolism (i.e., the chemical changes in living cells by which energy is provided for vital processes and activities) in a subject. Metabolic disorders include diseases, disorders, or conditions associated with aberrant or unwanted (higher or lower) thermogenesis or aberrant or unwanted levels (high or low) adipose cell ( e.g ., brown or white adipose cell) content or function. Metabolic disorders can be characterized by a misregulation (e.g., downregulation or upregulation) of PGC-1 activity. Metabolic disorders can detrimentally affect cellular functions such as cellular proliferation, growth, differentiation, or migration, cellular regulation of homeostasis, inter- or intra cellular communication; tissue function, such as liver function, muscle function, or adipocyte function; systemic responses in an organism, such as hormonal responses (e.g, insulin response). Examples of metabolic disorders include obesity, insulin resistance, type II diabetes, hypertension, hyperuricemia, fatty liver, non-alcoholic fatty liver disease, polycystic ovarian syndrome, acanthosis nigricans, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Lawrence-Moon syndrome, Prader- Labhart-Willi syndrome, anorexia, and cachexia.

In some embodiments, “pain” is included within the term “metabolic disorder.”

Pain is a sensation and a perception that is comprised of a complex series of mechanisms. Pain can be experienced both acutely and chronically. Acute pain is the instantaneous onset of a painful sensation in response to a noxious stimulus. It is considered to be adaptive because it can prevent an organism from damaging itself in some instances. Unlike acute pain (e.g, the transient protective physiology pain), persistent pain (also called chronic pain) usually has a delayed onset but can last for hours to days, or even months or years. Persistent pain may involve an amalgamation of physical, social, and psychologic factors. Persistent pain occurs in a variety of forms including, but not limited to, spontaneous pain (painful sensation without an external stimulus), allodynia (painful sensation in response to a normally innocuous stimulus) and hyperalgesia (strong painful sensation to a mildly painful stimulus). Persistent pain can be caused by many different factors. For example, persistent pain can be caused by conditions that accompany the aging process (e.g, conditions that may affect bones and joints in ways that cause persistent pain). In some embodiments, persistent pain can be caused by inflammation or nerve injury (for example, damage to or malfunction of the nervous system). In some embodiments, persistent pain can be inflammatory pain or neuropathic pain (for example, peripheral neuropathic pain and central neuropathic pain). In some embodiments, persistent pain is mediated by hyper-excitable pain-processing neurons in peripheral and central nervous system (e.g, peripheral sensitization or central sensitization). Surrogate indicators of pain are well-known in the art and can be assayed using routine methods, such as hot plate or tail immersion assays to determine thermally-induced pain, electronic von Frey apparatus assays to determine mechanically -induced pain, acetic acid assays to determine chemically- induced pain, adjuvant injection assays to determine inflammatory pain, and the like.

As used herein, “obesity” refers to a body mass index (BMI) of 30 kg/m2 or more (National Institute of Health, Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults (1998)). However, the present invention is also intended to include a disease, disorder, or condition that is characterized by a body mass index (BMI) of 25 kg/m2 or more, 26 kg/m2 or more, 27 kg/m2 or more, 28 kg/m2 or more, 29 kg/m2 or more, 29.5 kg/m2 or more, or 29.9 kg/m2 or more, all of which are typically referred to as overweight (National Institute of Health, Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults (1998)).

The obesity described herein may be due to any cause, whether genetic or environmental. Examples of disorders that may result in obesity or be the cause of obesity include overeating and bulimia, polycystic ovarian disease, craniopharyngioma, the Prader-Willi Syndrome, Frohlich's syndrome, Type II diabetics, GH-deficient subjects, normal variant short stature, Turner's syndrome, and other pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat- free mass, e.g., children with acute lymphoblastic leukemia.

As used herein, the term “selenium” includes the salt forms such as sodium selenite, other salt forms, as well as organic forms such as seleneocysteine and/or selenomethionine, and derivatives thereof unless otherwise specified.

The term “salts thereof’ may refer to relatively non-toxic, inorganic and organic base addition salts of selenium (e.g., of an anionic form, such as selenite). These salts can likewise be prepared in situ during the final isolation and purification of selenium, or by separately reacting the purified selenium in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a metal cation, with ammonia, or with an organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

In other embodiments, selenium can be administered to subjects. Accordingly, in some embodiments, selenium in its various forms or derivatives thereof, can be prepared for oral administration, for example, aqueous or non-aqueous solutions or suspensions, preferably as an aqueous formulation to be given ad libitum. In yet other embodiments the aqueous formulation comprises 0.5% to 4% selenium or absolute equivalent amounts thereof in a relevant formulation, such as a solid dosage form. Selenium in the aqueous formulations disclosed herein can be at a concentration of about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, or 4.0%, or any range in between, such as at a concentration of 1.0% to 1.5% inclusive. Absolute equivalent amounts can be determined according to well-known metrics, such as mg/kg/day. For example, absolute equivalent amounts for the aqueous formulations described above can be 50 pg/day, 100 pg/day, 200 pg/day, 300 pg/day, 4000 pg/day, 500 pg/day, 600 pg/day, 700 pg/day, 800 pg/day, 900 pg/day, 1,000 pg/day, 1,100 pg/day, 1,200 pg/day, 1,300 pg/day, 1,400 pg/day, 1,500 pg/day, 1,600 pg/day, 1,700 pg/day, 1,800 pg/day, 1,900 pg/day, 2,000 pg/day, 2,100 pg/day, 2,200 pg/day, 2,300 pg/day, 2,400 pg/day, 2,500 pg/day, 2,600 pg/day, 2,700 pg/day, 2,800 pg/day, 2,900 pg/day, 3,000 pg/day, 3,100 pg/day, 3,200 pg/day, 3,300 pg/day, 3,400 pg/day, 3,500 pg/day, 3,600 pg/day, 3,700 pg/day, 3,800 pg/day, 3,900 pg/day, 4,000 pg/day, or any range in between, such as 1,000 pg/day to 2,800 pg/day.

In some embodiments, selenium in its various forms ( e.g ., salts, organic compounds), or derivatives thereof, can be produced and administered which have enhanced biological properties (e.g., pro-drugs). In addition, the selenium can be modified according to well-known pharmacological methods in the art (e.g, pegylation, glycosylation, oligomerization, etc.) in order to further enhance desirable biological activities, such as increased bioavailability and decreased proteolytic degradation. In preferred embodiments, selenium is not a substituted selenium, for example dimethyl- or diethyl- selenium. In some embodiments, the selenium can be modified to be an inhibitor, such as a competitive inhibitor that prevents or impedes uptake of extracellular selenium.

It will be appreciated that specific sequence identifiers (SEQ ID NOs) have been referenced throughout the specification for purposes of illustration and should therefore not be construed to be limiting. Any marker of the invention, including, but not limited to, the markers described in the specification and markers described herein are well-known in the art in their non-selenylated forms, and can be used in the embodiments of the invention. An important and well-known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

In view of the foregoing, the nucleotide sequence of a DNA or RNA coding for a fusion protein or polypeptide of the present invention (or any portion thereof) can be used to derive the fusion protein or polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence. Likewise, for a fusion protein or polypeptide amino acid sequence, corresponding nucleotide sequences that can encode the fusion protein or polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence). Thus, description and/or disclosure herein of a nucleotide sequence which encodes a fusion protein or polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, description and/or disclosure of a fusion protein or polypeptide amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

In various embodiments, a reference is made to certain biomarkers. Some exemplary biomarkers are provided below in Table 1 and Table 2.

Table 1

Table 2 * Both Table 1 and Table 2 list mouse proteins, peptides from those proteins, and positions in those proteins that can be selenylated (e.g, with selenocysteine for Table 2, with selenomethionine for the last 22 rows of Table 1). The residue numberings are as in standard databases (e.g, UniProt); in some databases, the residue numberings may slightly differ, but such numberings can alternatively deduced from the provided peptide sequences. For example, the position 254 given above for UCP1 corresponds to position 253 for UCP1 throughout most of the rest of this disclosure (e.g, Examples, Figures), with the exception of the Claims section, in which position 254 given above for UCP1 is recited as position 254. Table 2 is defined here to also include the information in Fig. 6B, and Table 1 is defined here to also include the information in Fig. 6B and Fig. 6C.

* Both Table 1 and Table 2 are hereby defined to also include the corresponding human orthologs of the provided mouse proteins and peptides with the corresponding selenylation positions. Such positions can be deduced from the known homology between the mouse and human proteins (e.g, by aligning the mouse and human proteins with each other). As an example of that, below is the human orthologue of UCP1, which has a similar cysteine also at position 254.

SEQ ID NO: 1 = sp|P25874|UCP1_HUMAN sapiens OX=9606 GN=UCP1 PE=1 SV=3

MGGLTASDVHPTLGVQLFSAGIAACLADVITFPLDTAKVRLQVQGECPTSSVIRYKG VLG

TITAW KTEGRMKLYSGLPAGLQRQISSASLRIGLYDTVQEFLTAGKETAPSLGSKILAG

LTTGGVAVFIGQPTEW KVRLQAQSHLHGIKPRYTGTYNAYRIIATTEGLTGLWKGTTPN

LMRSVIINCTELVTYDLMKEAFVKNNILADDVPCHLVSALIAGFCATAMSSPVDW KTRF

INSPPGQYKSVPNCAMKVFTNEGPTAFFKGLVPSFLRLGSWNVIMFVCFEQLKRELS KSR

QTMDCAT

* When referred to as biomarkers in the claims, the proteins or peptides of Table 1 and Table 2 are defined to be in selenylated form (e.g, having at least one selenocysteine or a selenomethionine). In addition, Table 1 and Table 2 are hereby defined to include peptide fragments of the included proteins other than the exemplary peptides shown. For example, for UCP1, peptide fragments can include 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 residues around (e.g, including, although not necessarily at the center) the cysteine at position 254. The biomarkers include the full proteins as well as the potential peptide fragments of these proteins, both for mice and humans.

T _ Nucleic Acids

Also provided herein are compositions comprising one or more nucleic acids comprising or capable of expressing at least 1, 2, 3, 4, 5, 10, 20 or more small nucleic acids or antisense oligonucleotides or derivatives thereof, wherein said small nucleic acids or antisense oligonucleotides or derivatives thereof in a cell specifically hybridize (e.g, bind) under cellular conditions, with cellular nucleic acids ( e.g ., small non-coding RNAS such as miRNAs, pre-miRNAs, pri-miRNAs, miRNA*, piwiRNA, anti-miRNA, a miRNA binding site, a variant and/or functional variant thereof, cellular mRNAs or a fragments thereof). Such nucleic acids are useful, for example, in modulating the expression of brown/beige fat genes, especially those associated with selenium, such as SDH. In one embodiment, expression of the small nucleic acids or antisense oligonucleotides or derivatives thereof in a cell can enhance or upregulate one or more biological activities associated with the corresponding wild-type, naturally occurring, or synthetic small nucleic acids. In another embodiment, expression of the small nucleic acids or antisense oligonucleotides or derivatives thereof in a cell can inhibit expression or biological activity of cellular nucleic acids and/or proteins, e.g., by inhibiting transcription, translation and/or small nucleic acid processing of, for example, one or more biomarkers of the present invention, including one or more biomarkers listed in Table 1, the Figures, and the Examples, or fragment(s) thereof. In one embodiment, the small nucleic acids or antisense oligonucleotides or derivatives thereof are small RNAs (e.g, microRNAs) or complements of small RNAs. In another embodiment, the small nucleic acids or antisense oligonucleotides or derivatives thereof can be single or double stranded and are at least six nucleotides in length and are less than about

1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 24, 23, 22, 21,20, 19, 18

17, 16, 15, or 10 nucleotides in length. In another embodiment, a composition may comprise a library of nucleic acids comprising or capable of expressing small nucleic acids or antisense oligonucleotides or derivatives thereof, or pools of said small nucleic acids or antisense oligonucleotides or derivatives thereof. A pool of nucleic acids may comprise about 2-5, 5-10, 10-20, 10-30 or more nucleic acids comprising or capable of expressing small nucleic acids or antisense oligonucleotides or derivatives thereof.

In one embodiment, binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general, “antisense” refers to the range of techniques generally employed in the art, and includes any process that relies on specific binding to oligonucleotide sequences.

It is well-known in the art that modifications can be made to the sequence of a miRNA or a pre-miRNA without disrupting miRNA activity. As used herein, the term “functional variant” of a miRNA sequence refers to an oligonucleotide sequence that varies from the natural miRNA sequence, but retains one or more functional characteristics of the miRNA. In some embodiments, a functional variant of a miRNA sequence retains all of the functional characteristics of the miRNA. In certain embodiments, a functional variant of a miRNA has a nucleobase sequence that is a least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the miRNA or precursor thereof over a region of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases, or that the functional variant hybridizes to the complement of the miRNA or precursor thereof under stringent hybridization conditions. Accordingly, in certain embodiments the nucleobase sequence of a functional variant is capable of hybridizing to one or more target sequences of the miRNA. miRNAs and their corresponding stem-loop sequences described herein may be found in miRBase, an online searchable database of miRNA sequences and annotation, found on the world wide web at microrna.sanger.ac.uk. Entries in the miRBase Sequence database represent a predicted hairpin portion of a miRNA transcript (the stem-loop), with information on the location and sequence of the mature miRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miRNAs), and may in some instances include the pre-miRNA and some flanking sequence from the presumed primary transcript. The miRNA nucleobase sequences described herein encompass any version of the miRNA, including the sequences described in Release 10.0 of the miRBase sequence database and sequences described in any earlier Release of the miRBase sequence database. A sequence database release may result in the re-naming of certain miRNAs. A sequence database release may result in a variation of a mature miRNA sequence.

In some embodiments, miRNA sequences of the present invention may be associated with a second RNA sequence that may be located on the same RNA molecule or on a separate RNA molecule as the miRNA sequence. In such cases, the miRNA sequence may be referred to as the active strand, while the second RNA sequence, which is at least partially complementary to the miRNA sequence, may be referred to as the complementary strand. The active and complementary strands are hybridized to create a double-stranded RNA that is similar to a naturally occurring miRNA precursor. The activity of a miRNA may be optimized by maximizing uptake of the active strand and minimizing uptake of the complementary strand by the miRNA protein complex that regulates gene translation. This can be done through modification and/or design of the complementary strand. In some embodiments, the complementary strand is modified so that a chemical group other than a phosphate or hydroxyl at its 5' terminus. The presence of the 5' modification apparently eliminates uptake of the complementary strand and subsequently favors uptake of the active strand by the miRNA protein complex. The 5' modification can be any of a variety of molecules known in the art, including NIL·, NHCOCH3, and biotin.

In another embodiment, the uptake of the complementary strand by the miRNA pathway is reduced by incorporating nucleotides with sugar modifications in the first 2-6 nucleotides of the complementary strand. It should be noted that such sugar modifications can be combined with the 5' terminal modifications described above to further enhance miRNA activities.

In some embodiments, the complementary strand is designed so that nucleotides in the 3' end of the complementary strand are not complementary to the active strand. This results in double-strand hybrid RNAs that are stable at the 3' end of the active strand but relatively unstable at the 5' end of the active strand. This difference in stability enhances the uptake of the active strand by the miRNA pathway, while reducing uptake of the complementary strand, thereby enhancing miRNA activity.

Small nucleic acid and/or antisense constructs of the methods and compositions presented herein can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of cellular nucleic acids ( e.g ., small RNAs, mRNA, and/or genomic DNA). Alternatively, the small nucleic acid molecules can produce RNA which encodes mRNA, miRNA, pre- miRNA, pri-miRNA, miRNA*, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof. For example, selection of plasmids suitable for expressing the miRNAs, methods for inserting nucleic acid sequences into the plasmid, and methods of delivering the recombinant plasmid to the cells of interest are within the skill in the art. See, for example, Zeng etal. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat. Biotechnol, 20:446-448; Brummelkamp etal. (2002), Science 296:550-553; Miyagishi etal. (2002), Nat. Biotechnol. 20:497-500; Paddison etal. (2002), Genes Dev. 16:948-958; Lee etal. (2002), Nat. Biotechnol. 20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, the entire disclosures of which are herein incorporated by reference.

Alternatively, small nucleic acids and/or antisense constructs are oligonucleotide probes that are generated ex vivo and which, when introduced into the cell, results in hybridization with cellular nucleic acids. Such oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, and are therefore stable in vivo. Exemplary nucleic acid molecules for use as small nucleic acids and/or antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Krol et al. (1988) BioTechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668.

Antisense approaches may involve the design of oligonucleotides (either DNA or RNA) that are complementary to cellular nucleic acids (e.g, complementary to biomarkers listed in Table 1, the Figures, and the Examples,). Absolute complementarity is not required. In the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with a nucleic acid (e.g, RNA) it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5’ end of the mRNA, e.g, the 5’ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3’ untranslated sequences of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well (Wagner, R. (1994) Nature 372:333). Therefore, oligonucleotides complementary to either the 5’ or 3’ untranslated, non-coding regions of genes could be used in an antisense approach to inhibit translation of endogenous mRNAs. Oligonucleotides complementary to the 5’ untranslated region of the mRNA may include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could also be used in accordance with the methods and compositions presented herein. Whether designed to hybridize to the 5’, 3’ or coding region of cellular mRNAs, small nucleic acids and/or antisense nucleic acids should be at least six nucleotides in length, and can be less than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, or 10 nucleotides in length. Regardless of the choice of target sequence, it is preferred that in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. In one embodiment these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. In another embodiment these studies compare levels of the target nucleic acid or protein with that of an internal control nucleic acid or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide. It is preferred that the control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.

Small nucleic acids and/or antisense oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded. Small nucleic acids and/or antisense oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc., and may include other appended groups such as peptides (e.g, for targeting host cell receptors), or agents facilitating transport across the cell membrane (see, e.g., Letsinger etal. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre etal. (1987) Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. W088/09810, published December 15, 1988) or the blood-brain barrier (see, e.g, PCT Publication No. W089/10134, published April 25, 1988), hybridization-triggered cleavage agents. (See, e.g, Krol etal. (1988) BioTechniques 6:958-976) or intercalating agents. (See, e.g, Zon (1988), Pharm. Res. 5:539-549). To this end, small nucleic acids and/or antisense oligonucleotides may be conjugated to another molecule, e.g, a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

Small nucleic acids and/or antisense oligonucleotides may comprise at least one modified base moiety which is selected from the group including but not limited to 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- acetylcytosine, 5-(carboxyhydroxytiethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5’-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,

2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-

3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Small nucleic acids and/or antisense oligonucleotides may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

In certain embodiments, a compound comprises an oligonucleotide ( e.g ., a miRNA or miRNA encoding oligonucleotide) conjugated to one or more moieties which enhance the activity, cellular distribution or cellular uptake of the resulting oligonucleotide. In certain such embodiments, the moiety is a cholesterol moiety (e.g., antagomirs) or a lipid moiety or liposome conjugate. Additional moieties for conjugation include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. In certain embodiments, a conjugate group is attached directly to the oligonucleotide. In certain embodiments, a conjugate group is attached to the oligonucleotide by a linking moiety selected from amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g, double or triple bonds), 8-amino-3,6- dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1- carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), substituted Cl -CIO alkyl, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl. In certain such embodiments, a substituent group is selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain such embodiments, the compound comprises the oligonucleotide having one or more stabilizing groups that are attached to one or both termini of the oligonucleotide to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the oligonucleotide from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap), or at the 3'-terminus (3'- cap), or can be present on both termini. Cap structures include, for example, inverted deoxy abasic caps. Suitable cap structures include a 4',5'-methylene nucleotide, a l-(beta-D- erythrofuranosyl) nucleotide, a 4'-thio nucleotide, a carbocyclic nucleotide, a 1,5- anhydrohexitol nucleotide, an L-nucleotide, an alpha-nucleotide, a modified base nucleotide, a phosphorodithioate linkage, a threo-pentofuranosyl nucleotide, an acyclic 3',4'-seco nucleotide, an acyclic 3,4-dihydroxybutyl nucleotide, an acyclic 3,5- dihydroxypentyl nucleotide, a 3 '-3 '-inverted nucleotide moiety, a 3 '-3 '-inverted abasic moiety, a 3'-2'-inverted nucleotide moiety, a 3'-2'-inverted abasic moiety, a 1,4-butanediol phosphate, a 3'-phosphoramidate, a hexylphosphate, an aminohexyl phosphate, a 3'- phosphate, a 3'-phosphorothioate, a phosphorodithioate, a bridging methylphosphonate moiety, and a non-bridging methylphosphonate moiety 5'-amino-alkyl phosphate, a 1,3- diamino-2-propyl phosphate, 3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1,2- aminododecyl phosphate, a hydroxypropyl phosphate, a 5'-5'-inverted nucleotide moiety, a 5'-5'-inverted abasic moiety, a 5'-phosphoramidate, a 5'-phosphorothioate, a 5'-amino, a bridging and/or non-bridging 5'-phosphoramidate, a phosphorothioate, and a 5'-mercapto moiety.

It is to be understood that additional well-known nucleic acid architecture or chemistry can be applied. Different modifications can be placed at different positions to prevent the oligonucleotide from activating RNase H and/or being capable of recruiting the RNAi machinery. In another embodiment, they may be placed such as to allow RNase H activation and/or recruitment of the RNAi machinery. The modifications can be non natural bases, e.g. universal bases. It may be modifications on the backbone sugar or phosphate, e.g., 2'-0-modifications including LNA or phosphorothioate linkages. As used herein, it makes no difference whether the modifications are present on the nucleotide before incorporation into the oligonucleotide or whether the oligonucleotide is modified after synthesis.

Preferred modifications are those that increase the affinity of the oligonucleotide for complementary sequences, i.e. increases the tm (melting temperature) of the oligonucleotide base paired to a complementary sequence. Such modifications include 2'- O-flouro, 2'-0-methyl, 2'-0-methoxy ethyl. The use of LNA (locked nucleic acid) units, phosphoramidate, PNA (peptide nucleic acid) units or INA (intercalating nucleic acid) units is preferred. For shorter oligonucleotides, it is preferred that a higher percentage of affinity increasing modifications are present. If the oligonucleotide is less than 12 or 10 units long, it may be composed entirely of LNA units. A wide range of other non-natural units may also be build into the oligonucleotide, e.g, morpholino, 2'-deoxy-2'-fluoro-arabinonucleic acid (FANA) and arabinonucleic acid (ANA). In a preferred embodiment, the fraction of units modified at either the base or sugar relatively to the units not modified at either the base or sugar is selected from the group consisting of less than less than 99%, 95%, less than 90%, less than 85% or less than 75%, less than 70%, less than 65%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, and less than 5%, less than 1%, more than 99%, more than 95%, more than 90%, more than 85% or more than 75%, more than 70%, more than 65%, more than 60%, more than 50%, more than 45%, more than 40%, more than 35%, more than 30%, more than 25%, more than 20%, more than 15%, more than 10%, and more than 5% and more than 1%.

Small nucleic acids and/or antisense oligonucleotides can also contain a neutral peptide-like backbone. Such molecules are termed peptide nucleic acid (PNA)-oligomers and are described, e.g, in Perry-O’Keefe etal. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:14670 and in Eglom etal. (1993) Nature 365:566. One advantage of PNA oligomers is their capability to bind to complementary DNA essentially independently from the ionic strength of the medium due to the neutral backbone of the DNA. In yet another embodiment, small nucleic acids and/or antisense oligonucleotides comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In a further embodiment, small nucleic acids and/or antisense oligonucleotides are a-anomeric oligonucleotides. An a-anomeric oligonucleotide forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier etal. (1987) Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2’-0-methylribonucleotide (Inoue etal. (1987) Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue etal. (1987) FEBS Lett. 215:327-330).

Small nucleic acids and/or antisense oligonucleotides of the methods and compositions presented herein may be synthesized by standard methods known in the art, e.g, by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988) Nucl. Acids Res. 16:3209, methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc. For example, an isolated miRNA can be chemically synthesized or recombinantly produced using methods known in the art. In some instances, miRNA are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), Cruachem (Glasgow, UK), and Exiqon (Vedbaek, Denmark).

Small nucleic acids and/or antisense oligonucleotides can be delivered to cells in vivo. A number of methods have been developed for delivering small nucleic acids and/or antisense oligonucleotides DNA or RNA to cells; e.g, antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g, antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically.

In one embodiment, small nucleic acids and/or antisense oligonucleotides may comprise or be generated from double stranded small interfering RNAs (siRNAs), in which sequences fully complementary to cellular nucleic acids (e.g, mRNAs) sequences mediate degradation or in which sequences incompletely complementary to cellular nucleic acids (e.g, mRNAs) mediate translational repression when expressed within cells. In another embodiment, double stranded siRNAs can be processed into single stranded antisense RNAs that bind single stranded cellular RNAs (e.g, microRNAs) and inhibit their expression. RNA interference (RNAi) is the process of sequence-specific, post- transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene in vivo , long dsRNA is cleaved by ribonuclease III to generate 21- and 22-nucleotide siRNAs. It has been shown that 21 -nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells (Elbashir et al. (2001) Nature 411 :494-498). Accordingly, translation of a gene in a cell can be inhibited by contacting the cell with short double stranded RNAs having a length of about 15 to 30 nucleotides or of about 18 to 21 nucleotides or of about 19 to 21 nucleotides. Alternatively, a vector encoding for such siRNAs or short hairpin RNAs (shRNAs) that are metabolized into siRNAs can be introduced into a target cell (see, e.g., McManus etal. (2002) RNA 8:842; Xia etal. (2002) Nature Biotechnology 20:1006; and Brummelkamp et al. (2002) Science 296:550). Vectors that can be used are commercially available, e.g, from OligoEngine under the name pSuper RNAi System™

Ribozyme molecules designed to catalytically cleave cellular mRNA transcripts can also be used to prevent translation of cellular mRNAs and expression of cellular polypeptides, or both (See, e.g, PCT International Publication WO90/11364, published October 4, 1990; Sarver etal. (1990) Science 247:1222-1225 and U.S. Patent No. 5,093,246). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy cellular mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5’-UG-3\ The construction and production of hammerhead ribozymes is well-known in the art and is described more fully in Haseloff and Gerlach (1988) Nature 334:585-591. The ribozyme may be engineered so that the cleavage recognition site is located near the 5’ end of cellular mRNAs; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

The ribozymes of the methods and compositions presented herein also include RNA endoribonucleases (hereinafter “Cech-type ribozymes”) such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al. (1984) Science 224:574-578; Zaug, et al. (1986) Science 231:470-475; Zaug, et al. (1986) Nature 324:429-433; published International patent application No. W088/04300 by University Patents Inc.; Been, et al. (1986) Cell 47:207-216). The Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place. The methods and compositions presented herein encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in cellular genes.

As in the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g, for improved stability, targeting, etc.). A preferred method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous cellular messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription of cellular genes are preferably single stranded and composed of deoxyribonucleotides. The base composition of these oligonucleotides should promote triple helix formation via Hoogsteen base pairing rules, which generally require sizable stretches of either purines or pyrimidines to be present on one strand of a duplex.

Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules may be chosen that are purine-rich, for example, containing a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in CGC triplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so called “switchback” nucleic acid molecule. Switchback molecules are synthesized in an alternating 5’-3’, 3’-5’ manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizable stretch of either purines or pyrimidines to be present on one strand of a duplex.

Small nucleic acids ( e.g miRNAs, pre-miRNAs, pri-miRNAs, miRNA*, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof), antisense oligonucleotides, ribozymes, and triple helix molecules of the methods and compositions presented herein may be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well-known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Moreover, various well-known modifications to nucleic acid molecules may be introduced as a means of increasing intracellular stability and half-life. One of skill in the art will readily understand that polypeptides, small nucleic acids, and antisense oligonucleotides can be further linked to another peptide or polypeptide ( e.g ., a heterologous peptide), e.g., that serves as a means of protein detection. Non-limiting examples of label peptide or polypeptide moieties useful for detection in the invention include, without limitation, suitable enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; epitope tags, such as FLAG,

MYC, HA, or HIS tags; fluorophores such as green fluorescent protein; dyes; radioisotopes; digoxygenin; biotin; antibodies; polymers; as well as others known in the art, for example, in Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999).

In addition, other nucleic acid forms are well-known in the art, such as mRNAs, cDNAs, and products thereof, and other modulatory agents (e.g, antibodies, small molecules, peptides, fusion proteins, or small nucleic acids) are contemplated and they can be incorporated into pharmaceutical compositions and administered to a subject in vivo.

The compositions may contain a single such molecule or agent or any combination of agents described herein. Based on the genetic pathway analyses described herein, it is believed that such combinations of agents is especially effective in diagnosing, prognosing, preventing, and treating a metabolic disorder. Thus, “single active agents” described herein can be combined with other pharmacologically active compounds (“second active agents”) known in the art according to the methods and compositions provided herein. It is believed that certain combinations work synergistically in the treatment of particular types of metabolic disorders. Second active agents can be large molecules (e.g, proteins) or small molecules (e.g, synthetic inorganic, organometallic, or organic molecules). For example, agents that modulate (e.g, promote) brown fat cell-like development and/or activity can be provided as combination agents. Exemplary agents include, without limitation, PRDM16 (U.S. Pat. Publ. 2011/0059051), C/EBRb (U.S. Pat. Publ. 2012/0022500), FNDC5/Irisin (U.S. Pat. 8,969,519 and PCT Publ. No. WO 2013/039996), Meteorin/Meteorin-like (PCT Publ. No. WO 2014/116556), respiration uncoupling agents (e.g, dinitrophenol, CCCP, and FCCP), and the like. II. Identification of Compounds that Modulate Selenium

The selenium and related agents described herein may be used to identify modulators of one or more of biological activities of selenium. In particular, information useful for the design of therapeutic and diagnostic molecules, including, for example, selenium and derivatives thereof is now available or attainable using the methods described herein.

In one aspect, modulators, inhibitors, or antagonists against the selenium and agents of the invention, biological complexes containing them, or orthologues thereof, may be used to treat any disease or other treatable condition of a patient (including humans and animals), including, for example, metabolic disorders.

Modulators of selenium may be identified and developed as set forth below using techniques and methods known to those of skill in the art. The modulators of the invention can be used, for example, to inhibit and treat selenium and/or thermogenesis-mediated diseases or disorders. The modulators of the invention may elicit a change in one or more of the following activities: (a) a change in the level and/or rate of formation of selenium- protein complex or product ( e.g ., naturally occurring or synthetic), such as UCP1, (b) a change in the uptake of selenium, (c) a change in the stability of selenium, (d) a change in the metabolism of a selenium, or (e) a change in the activity of at least one polypeptide contained in a selenium complex or substrate/enzyme configuration. A number of methods for identifying a molecule which modulates a selenium are known in the art. For example, in one such method, selenium is contacted with a test compound, and the activity of selenium is determined in the presence of the test compound, wherein a change in the activity of the selenium in the presence of the compound as compared to the activity in the absence of the compound (or in the presence of a control compound) indicates that the test compound modulates the activity of the selenium. Included within the term selenium modulation is selenium uptake.

Compounds to be tested for their ability to act as modulators of selenium can be produced, for example, by bacteria, yeast or other organisms (e.g. natural products), produced chemically (e.g. small molecules, including peptidomimetics), or produced recombinantly. Compounds for use with the above-described methods may be selected from the group of compounds consisting of lipids, carbohydrates, polypeptides, peptidomimetics, peptide-nucleic acids (PNAs), small molecules, natural products, aptamers and polynucleotides. In certain embodiments, the compound is a polynucleotide. In some embodiments, said polynucleotide is an antisense nucleic acid. In other embodiments, said polynucleotide is an siRNA. In certain embodiments, the compound comprises an analogue of selenium ( e.g ., a dominant negative form that binds to, but does not activate, selenium-induced activity). In other embodiments, the compound promotes (e.g., increases enzymatic activity, such as a substrate having a structure requiring a lower activation energy) or inhibits (e.g, decreases selenium activity, such as a reversible or irreversible inhibitor, like a covalent inhibitor).

A variety of assay formats will suffice and, in light of the present disclosure, those not expressly described herein may nevertheless be comprehended by one of ordinary skill in the art based on the teachings herein. Assay formats for analyzing selenium-substrate complex formation and/or activity of selenium may be generated in many different forms, and include assays based on cell-free systems, e.g. purified proteins or cell lysates, as well as cell-based assays, which utilize intact cells, such as to determine cellular uptake, cellular metabolism, and the like, of selenium. Simple binding assays can also be used to detect agents which modulate selenium, for example, by enhancing the formation of selenium metabolites, by enhancing the rate of selenium metabolism, and/or by enhancing the binding of selenium to a polypeptide or polypeptide complex. Another example of an assay useful for identifying a modulator of selenium is a competitive assay that combines selenium with a potential modulator, such as, for example, polypeptides, nucleic acids, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. Selenium can be labeled, such as by radioactivity or a colorimetric compound, such that seleniumcomplex formation and/or activity can be determined accurately to assess the effectiveness of the potential modulator.

Assays may employ kinetic or thermodynamic methodology using a wide variety of techniques including, but not limited to, microcalorimetry, circular dichroism, capillary zone electrophoresis, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and combinations thereof. Assays may also employ any of the methods for isolating, preparing and detecting selenium, selenium-complexes, polypeptide complexes comprising selenium, and the like, as described above.

Complex formation between selenium and a binding partner (e.g, a selenium binding enzyme or a metabolizer) may be detected by a variety of methods. Modulation of the complex’s formation may be quantified using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled polypeptides or binding partners, by immunoassay, or by chromatographic detection. Methods of isolating and identifying selenium-complexes described above may be incorporated into the detection methods.

In certain embodiments, it may be desirable to immobilize a selenium-binding polypeptide to facilitate separation of selenium complexes from uncomplexed forms, as well as to accommodate automation of the assay. Binding of a selenium-binding polypeptide to a binding partner may be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein may be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase/polypeptide (GST/polypeptide) fusion proteins may be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the binding partner, e.g. an ³⁵ S-labeled binding partner, and the test compound, and the mixture incubated under conditions conducive to complex formation, e.g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired. Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g. beads placed in scintillant), or in the supernatant after the complexes are subsequently dissociated. Alternatively, the complexes may be dissociated from the matrix, separated by SDS-PAGE, and the level of selenium-binding polypeptides found in the bead fraction quantified from the gel using standard electrophoretic techniques such as described in the appended examples.

Other techniques for immobilizing proteins on matrices are also available for use in the subject assay. For instance, selenium-binding polypeptide may be immobilized utilizing conjugation of biotin and streptavidin. For instance, biotinylated polypeptide molecules may be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well-known in the art (e.g, biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with the polypeptide may be derivatized to the wells of the plate, and polypeptide trapped in the wells by antibody conjugation. As above, preparations of a binding partner and a test compound are incubated in the polypeptide presenting wells of the plate, and the amount of complex trapped in the well may be quantified. Exemplary methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the binding partner, or which are reactive with the selenium-binding polypeptide and compete with the binding partner; as well as enzyme- linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity. In the instance of the latter, the enzyme may be chemically conjugated or provided as a fusion protein with the binding partner. To illustrate, the binding partner may be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of selenium trapped in the selenium complex may be assessed with a chromogenic substrate of the enzyme, e.g. 3,3'-diamino-benzadine terahydrochloride or 4-chloro-l-napthol. Likewise, a fusion protein comprising a polypeptide and glutathione-S-transferase may be provided, and selenium complex formation quantified by detecting the GST activity using 1 -chi oro-2, 4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

Antibodies against the selenium-binding polypeptide can be used for immunodetection purposes. Alternatively, the selenium-binding polypeptide to be detected may be “epitope-tagged” in the form of a fusion protein that includes, in addition to the polypeptide sequence, a second polypeptide for which antibodies are readily available (e.g, from commercial sources). For instance, the GST fusion proteins described above may also be used for quantification of binding using antibodies against the GST moiety. Other useful epitope tags include myc-epitopes (e.g., see Ellison et al. (1991) J. Biol. Chem. 266:21 ISO- 21157) which includes a 10-residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharmacia, N. J.).

In certain in vitro embodiments of the present assay, the protein or the set of proteins engaged in a protein-protein, protein-substrate, or protein-nucleic acid interaction comprises a reconstituted protein mixture of at least semi-purified proteins. By semi- purified, it is meant that the proteins utilized in the reconstituted mixture have been previously separated from other cellular or viral proteins. For instance, in contrast to cell lysates, the proteins involved in a protein-substrate, protein-protein or nucleic acid-protein interaction are present in the mixture to at least 50% purity relative to all other proteins in the mixture, and more preferably are present at 90-95% purity. In certain embodiments of the subject method, the reconstituted protein mixture is derived by mixing highly purified proteins such that the reconstituted mixture substantially lacks other proteins (such as of cellular or viral origin) which might interfere with or otherwise alter the ability to measure activity resulting from the given protein-substrate, protein-protein interaction, or nucleic acid-protein interaction.

In one embodiment, the use of reconstituted protein mixtures allows more careful control of the protein-substrate, protein-protein, or nucleic acid-protein interaction conditions. Moreover, the system may be derived to favor discovery of modulators of particular intermediate states of the protein-protein interaction. For instance, a reconstituted protein assay may be carried out both in the presence and absence of a candidate agent, thereby allowing detection of a modulator of a given protein-substrate, protein-protein, or nucleic acid-protein interaction.

Assaying biological activity resulting from a given protein-substrate, protein-protein or nucleic acid-protein interaction, in the presence and absence of a candidate modulator, may be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes.

In yet another embodiment, a selenium-binding polypeptide may be used to generate a two-hybrid or interaction trap assay (see also, U.S. Pat. No. 5,283,317; Zervos et al.

(1993) Cell 72:223-232; Madura et al. (1993) J. Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696), for subsequently detecting agents which disrupt binding of the interaction components to one another.

In particular, the method makes use of chimeric genes that express hybrid proteins. To illustrate, a first hybrid gene comprises the coding sequence for a binding domain of a transcriptional activator may be fused in frame to the coding sequence for a “bait” protein, e.g ., a selenium-binding polypeptide of sufficient length to bind to a potential interacting protein. The second hybrid protein encodes a transcriptional activation domain fused in frame to a gene encoding a “fish” protein, e.g. , a potential interacting protein of sufficient length to interact with the protein-protein interaction component polypeptide portion of the bait fusion protein. If the bait and fish proteins are able to interact, e.g. , form a protein- protein interaction component complex, they bring into close proximity the two domains of the transcriptional activator. This proximity causes transcription of a reporter gene which is operably linked to a transcriptional regulatory site responsive to the transcriptional activator, and expression of the reporter gene may be detected and used to score for the interaction of the bait and fish proteins. The host cell also contains a first chimeric gene which is capable of being expressed in the host cell. The gene encodes a chimeric protein, which comprises (a) a binding domain that recognizes the responsive element on the reporter gene in the host cell, and (b) a bait protein ( e.g ., a selenium-binding polypeptide).

A second chimeric gene is also provided which is capable of being expressed in the host cell, and encodes the “fish” fusion protein. In one embodiment, both the first and the second chimeric genes are introduced into the host cell in the form of plasmids. Preferably, however, the first chimeric gene is present in a chromosome of the host cell and the second chimeric gene is introduced into the host cell as part of a plasmid.

The binding domain of the first hybrid protein and the transcriptional activation domain of the second hybrid protein may be derived from transcriptional activators having separable binding and transcriptional activation domains. For instance, these separate binding and transcriptional activation domains are known to be found in the yeast GAL4 protein, and are known to be found in the yeast GCN4 and ADR1 proteins. Many other proteins involved in transcription also have separable binding and transcriptional activation domains which make them useful for the present invention, and include, for example, the LexA and VP 16 proteins. It will be understood that other (substantially) transcriptionally- inert binding domains may be used in the subject constructs; such as domains of ACE1, kcl, lac repressor, jun or fos. In another embodiment, the binding domain and the transcriptional activation domain may be from different proteins. The use of a LexA DNA binding domain provides certain advantages. For example, in yeast, the LexA moiety contains no activation function and has no known affect on transcription of yeast genes. In addition, use of LexA allows control over the sensitivity of the assay to the level of interaction (see, for example, PCT Publ. No. WO 1994/10300).

In certain embodiments, any enzymatic activity associated with the bait or fish proteins is inactivated, e.g., dominant negative or other mutants of a protein-protein interaction component can be used.

Continuing with the illustrative example, formation of a complex between the bait and fish fusion proteins in the host cell, causes the activation domain to activate transcription of the reporter gene. The method is carried out by introducing the first chimeric gene and the second chimeric gene into the host cell, and subjecting that cell to conditions under which the bait and fish fusion proteins and are expressed in sufficient quantity for the reporter gene to be activated. The formation of a complex results in a detectable signal produced by the expression of the reporter gene. In still further embodiments, the selenium, or substrate-complex or protein-complex thereof, of interest may be generated in whole cells, taking advantage of cell culture techniques to support the subject assay. For example, the selenium, or complex thereof, may be constituted in a prokaryotic or eukaryotic cell culture system. Advantages to generating the selenium complex, in an intact cell includes the ability to screen for modulators of the level and/or activity of selenium, or complex thereof, which are functional in an environment more closely approximating that which therapeutic use of the modulator would require, including the ability of the agent to gain entry into the cell. Furthermore, certain of the in vivo embodiments of the assay are amenable to high through put analysis of candidate agents.

The selenium can be endogenous to the cell selected to support the assay. Alternatively, some or all of the components can be derived from exogenous sources. For instance, fusion proteins can be introduced into the cell by recombinant techniques (such as through the use of an expression vector), as well as by microinjecting the fusion protein itself or mRNA encoding the fusion protein. Moreover, in the whole cell embodiments of the subject assay, the reporter gene construct can provide, upon expression, a selectable marker. Such embodiments of the subject assay are particularly amenable to high through put analysis in that proliferation of the cell can provide a simple measure of the protein- protein interaction.

The amount of transcription from the reporter gene may be measured using any method known to those of skill in the art to be suitable. For example, specific mRNA expression may be detected using Northern blots or specific protein product may be identified by a characteristic stain, western blots or an intrinsic activity. In certain embodiments, the product of the reporter gene is detected by an intrinsic activity associated with that product. For instance, the reporter gene may encode a gene product that, by enzymatic activity, gives rise to a detection signal based on color, fluorescence, or luminescence.

In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays of the present invention which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins or with lysates, are often preferred as “primary” screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or changes in enzymatic properties of the molecular target. Accordingly, potential modulators of selenium may be detected in a cell-free assay generated by constitution of selenium in a cell lysate. In an alternate format, the assay can be derived as a reconstituted protein mixture which, as described below, offers a number of benefits over lysate-based assays.

The activity of selenium or a selenium-binding polypeptide may be identified and/or assayed using a variety of methods well-known to the skilled artisan. For example, the activity of selenium may be determined by assaying for the level of expression of RNA and/or protein molecules. Transcription levels may be determined, for example, using Northern blots, hybridization to an oligonucleotide array or by assaying for the level of a resulting protein product. Translation levels may be determined, for example, using Western blotting or by identifying a detectable signal produced by a protein product ( e.g ., fluorescence, luminescence, enzymatic activity, etc.). Depending on the particular situation, it may be desirable to detect the level of transcription and/or translation of a single gene or of multiple genes.

In other embodiments, the biological activity of selenium may be assessed by monitoring changes in the phenotype of a targeted cell. For example, the detection means can include a reporter gene construct which includes a transcriptional regulatory element that is dependent in some form on the level and/or activity of selenium e Accordingly, the level of expression of the reporter gene will vary with the level of selenium.

Similarly, selenium activity can be assessed using well-known enzymatic analysis methods. For example, the rate or amount of selenium catalysis, enzyme association, enzyme dissociation, product biosynthesis, product catalysis (e.g., breakdown), and the like can be analyzed.

Moreover, in the whole cell embodiments of the subject assay, the reporter gene construct can provide, upon expression, a selectable marker. A reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein. Preferred reporter genes are those that are readily detectable. The reporter gene may also be included in the construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties. For instance, the product of the reporter gene can be an enzyme which confers resistance to an antibiotic or other drug, or an enzyme which complements a deficiency in the host cell (i.e. thymidine kinase or dihydrofolate reductase). To illustrate, the aminoglycoside phosphotransferase encoded by the bacterial transposon gene Tn5 neo can be placed under transcriptional control of a promoter element responsive to the level of a selenium present in the cell. Such embodiments of the subject assay are particularly amenable to high through-put analysis in that proliferation of the cell can provide a simple measure of inhibition of selenium.

Similarly, individual cells or analyses of phenotypes in organisms can be formed to determine effects of test agents on the modulation ( e.g ., upregulation) of one or more of the following selenium-mediated biological activities: a) brown fat and/or beige fat gene expression, such as expression of a marker selected from the group consisting of: cidea, adiponectin, adipsin, otopetrin, type II deiodinase, cig30, ppar gamma 2, pgcla, ucpl, elovl3, cAMP, Prdml6, cytochrome C, cox4il, coxIII, cox5b, cox7al, cox8b, glut4, atpase b2, cox II, atp5o, ndufb5, ap2, ndufsl, GRP109A, acylCoA-thioesterase 4, EARA1, claudinl, PEPCK, fgf21, acylCoA-thioesterase 3, dio2, fatty acid synthase (fas), leptin, resistin, and nuclear respiratory factor-1 (nrfl); b) thermogenesis in adipose cells; c) differentiation of adipose cells; d) insulin sensitivity of adipose cells; e) basal respiration or uncoupled respiration; f) whole body oxygen consumption; g) obesity or appetite; h) insulin secretion of pancreatic beta cells; i) glucose tolerance; j) modified phosphorylation of EGFR, ERK, AMPK, protein kinase A (PKA) substrates having an RRX(S/T) motif, wherein the X is any amino acid and the (S/T) residue is a serine or threonine, HSL; k) modified activity of UCP1 protein; and 1) growth and effects of metabolic disorders, such as obesity-associated cancer, cachexia, anorexia, diabetes, and obesity

III Methods of the Invention

One aspect of the present invention relates to methods of using and/or selecting agents (e.g., antibodies, fusion constructs, peptides, small molecules, small nucleic acids, and the like) which bind to, upregulate, downregulate, or modulate selenium and/or one or more biomarkers of the present invention listed in Table 1, the Figures, and the Examples, and/or a metabolic disorder. Such methods can use screening assays, including cell-based and non-cell based assays.

In any method described herein, such as a diagnostic method, prognostic method, therapeutic method, or combination thereof, all steps of the method can be performed by a single actor or, alternatively, by more than one actor. For example, diagnosis can be performed directly by the actor providing therapeutic treatment. Alternatively, a person providing a therapeutic agent can request that a diagnostic assay be performed. The diagnostician and/or the therapeutic interventionist can interpret the diagnostic assay results to determine a therapeutic strategy. Similarly, such alternative processes can apply to other assays, such as prognostic assays.

In one embodiment, the invention relates to assays for screening candidate or test compounds which bind to or modulate the expression or activity level of, one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment or ortholog thereof. Such compounds include, without limitation, antibodies, proteins, fusion proteins, nucleic acid molecules, and small molecules.

As described in detail in the section above, in one embodiment, an assay is a cell- based assay, comprising contacting a cell expressing one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof, with a test compound and determining the ability of the test compound to modulate ( e.g stimulate or inhibit) the level of interaction between the biomarker and its natural binding partners as measured by direct binding or by measuring a parameter related to a metabolic disorder.

For example, in a direct binding assay, the biomarker polypeptide, a binding partner polypeptide of the biomarker, or a fragment(s) thereof, can be coupled with a radioisotope or enzymatic label such that binding of the biomarker polypeptide or a fragment thereof to its natural (or synthetic) binding partner(s) or a fragment(s) thereof can be determined by detecting the labeled molecule in a complex. For example, the biomarker polypeptide, a binding partner polypeptide of the biomarker, or a fragment(s) thereof, can be labeled with ¹²⁵ 1, ³⁵ S, ¹⁴ C, or ³ H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, the polypeptides of interest can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

It is also within the scope of this invention to determine the ability of a compound to modulate the interactions between one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof, and its natural (or synthetic) binding partner(s) ( e.g ., naturally occurring or synthetic SDH) or a fragment(s) thereof, without the labeling of any of the interactants (e.g., using a microphysiometer as described in McConnell, H. M. etal. (1992) Science 257:1906-1912). As used herein, a “microphysiometer” (e.g, Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between compound and receptor.

In a preferred embodiment, determining the ability of blocking agents (e.g, antibodies, fusion proteins, peptides, nucleic acid molecules, and small molecules) to antagonize the interaction between a given set of nucleic acid molecules and/or polypeptides can be accomplished by determining the activity of one or more members of the set of interacting molecules. For example, the activity of one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof, can be determined by detecting induction of metabolic response, detecting catalytic/enzymatic activity of an appropriate substrate, detecting the induction of a reporter gene (comprising a target- responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g, chloramphenicol acetyl transferase), or detecting a cellular response regulated by the biomarker or a fragment thereof (e.g, modulations of biological pathways identified herein, such as modulated cellular respiration, brown/beige fat gene expression, mitochondrial biosynthesis, and the like).

In yet another embodiment, an assay of the present invention is a cell-free assay in which one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof, e.g, a biologically active fragment thereof, is contacted with a test compound, and the ability of the test compound to bind to the polypeptide, or biologically active portion thereof, is determined. Binding of the test compound to the biomarker or a fragment thereof, can be determined either directly or indirectly as described above. Determining the ability of the biomarker or a fragment thereof to bind to its natural (or synthetic) binding partner(s) or a fragment(s) thereof can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA) (Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo etal. (1995) Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” is a technology for studying biospecific interactions in real time, without labeling any of the interactants ( e.g ., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological polypeptides. One or more biomarkers polypeptide or a fragment thereof can be immobilized on a BIAcore chip and multiple agents, e.g., blocking antibodies, fusion proteins, peptides, small molecules, and the like, can be tested for binding to the immobilized biomarker polypeptide or fragment thereof.

An example of using the BIA technology is described by Fitz et al. (1997) Oncogene 15:613.

The cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of proteins. In the case of cell-free assays in which a membrane-bound form protein is used it may be desirable to utilize a solubilizing agent such that the membrane-bound form of the protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n- dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N- methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-l-propane sulfonate (CHAPS), 3-[(3- cholamidopropyl)dimethylamminio]-2-hydroxy-l -propane sulfonate (CHAPSO), orN- dodecyl=N,N-dimethyl-3-ammonio-l -propane sulfonate.

In one or more embodiments of the above described assay methods, it may be desirable to immobilize either the biomarker nucleic acid and/or polypeptide, the natural (or synthetic) binding partner(s) of the biomarker, or fragments thereof, to facilitate separation of complexed from uncomplexed forms of the reactants, as well as to accommodate automation of the assay. Binding of a test compound in the assay can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase-base fusion proteins, can be adsorbed onto glutathione Sepharose® beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound, and the mixture incubated under conditions conducive to complex formation (e.g, at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity determined using standard techniques.

In an alternative embodiment, determining the ability of the test compound to modulate the activity of one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof, or of natural (or synthetic) binding partner(s) thereof can be accomplished by determining the ability of the test compound to modulate the expression or activity of a gene, e.g., nucleic acid, or gene product, e.g, polypeptide, that functions downstream of the interaction. For example, cellular migration or invasion can be determined by monitoring cellular movement, matrigel assays, induction of invasion- related gene expression, and the like, as described further herein.

In another embodiment, modulators of one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof, are identified in a method wherein a cell is contacted with a candidate compound and the expression or activity level of the biomarker is determined. The level of expression of biomarker RNA or polypeptide or fragments thereof in the presence of the candidate compound is compared to the level of expression of biomarker RNA or polypeptide or fragments thereof in the absence of the candidate compound. The candidate compound can then be identified as a modulator of biomarker expression based on this comparison. For example, when expression of biomarker RNA or polypeptide or fragments thereof is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of biomarker expression. Alternatively, when expression of biomarker RNA or polypeptide or fragments thereof is reduced (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of biomarker expression. The expression level of biomarker RNA or polypeptide or fragments, or products thereof such as enzyme catalyzed products, thereof in the cells or produced by the cells can be determined by methods described herein for detecting biomarker mRNA or polypeptide or fragments thereof.

In yet another aspect of the present invention, a biomarker of the present invention, including one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof, can be used as “bait” in a two-hybrid assay or three-hybrid assay (see, e.g., U S. Pat. No. 5,283,317; Zervos etal. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent W094/10300), to identify other nucleic acids and/or polypeptides which bind to or interact with the biomarker or fragments thereof and are involved in activity of the biomarkers. Such biomarker-binding proteins are also likely to be involved in the propagation of signals by the biomarker polypeptides or biomarker natural (or synthetic) binding partner(s) as, for example, downstream elements of one or more biomarkers -mediated signaling pathway.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for one or more biomarkers polypeptide is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g, GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified polypeptide (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” polypeptides are able to interact, in vivo , forming one or more biomarkers -dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g, LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the polypeptide which interacts with one or more biomarkers polypeptide of the present invention, including one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof.

In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell- based or a cell-free assay, and the ability of the agent to modulate the activity of one or more biomarkers polypeptide or a fragment thereof can be confirmed in vivo , e.g., in an animal such as an animal model for cellular transformation and/or tumorigenesis.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

In other aspects of the present invention, the biomarkers described herein, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or fragments thereof, can be used in one or more of the following methods: a) screening assays; b) predictive medicine ( e.g ., diagnostic assays, prognostic assays, and monitoring of clinical trials); and c) methods of treatment (e.g., therapeutic and prophylactic, e.g, by up- or down-modulating the copy number, level of expression, and/or level of activity of the one or more biomarkers and/or modulating relevant endpoints such as selenium uptake, cellular respiration, glucose homeostasis, etc.).

The biomarkers described herein or agents that modulate the expression and/or activity of such biomarkers can be used, for example, to (a) express one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof (e.g, via a recombinant expression vector in a host cell in gene therapy applications or synthetic nucleic acid molecule), (b) detect biomarker RNA or a fragment thereof (e.g, in a biological sample) or a genetic alteration in one or more biomarkers gene, and/or (c) modulate biomarker activity, as described further below. The biomarkers or modulatory agents thereof can be used to treat conditions or disorders characterized by insufficient or excessive production of one or more biomarkers polypeptide or fragment thereof or production of biomarker polypeptide inhibitors. In addition, the biomarker polypeptides or fragments thereof can be used to screen for naturally occurring biomarker binding partner(s), to screen for drugs or compounds which modulate biomarker activity, as well as to treat conditions or disorders characterized by insufficient or excessive production of biomarker polypeptide or a fragment thereof or production of biomarker polypeptide forms which have decreased, aberrant or unwanted activity compared to biomarker wild-type polypeptides or fragments thereof (e.g, amounts in metabolic disorder samples as compared to control samples). A. Screening Assays

In one aspect, the present invention relates to a method for preventing in a subject, a disease or condition associated with an unwanted, more than desirable, or less than desirable, expression and/or activity of one or more biomarkers described herein. Subjects at risk for a disease that would benefit from treatment with the claimed agents or methods can be identified, for example, by any one or combination of diagnostic or prognostic assays known in the art and described herein (see, for example, agents and assays described above in the section describing methods of selecting agents and compositions).

B. Predictive Medicine

The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring of clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining the expression and/or activity level of biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or fragments thereof, in the context of a biological sample ( e.g. , blood, serum, cells, or tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted biomarker expression or activity. The present invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with biomarker expression or activity. For example, mutations in one or more biomarkers gene can be assayed in a biological sample.

Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with biomarker expression or activity. For example, selenium is associated with increased thermogenesis and metabolism such that upregulation of selenium predicts treatment of metabolic disorders, either alone or in combination with additional agents. Downregulation and/or reduced activity of selenium indicates reduced thermogenesis and metabolism.

Another aspect of the present invention pertains to monitoring the influence of agents (e.g, drugs, compounds, small nucleic acid-based molecules, and the like) on the expression or activity of biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or fragments thereof, in clinical trials. These and other agents are described in further detail in the following sections.

The term “altered amount” of a marker or “altered level” of a marker refers to increased or decreased copy number of the marker and/or increased or decreased expression level of a particular marker gene or genes in a test sample, as compared to the expression level or copy number of the marker in a control sample. The term “altered amount” of a marker also includes an increased or decreased protein level of a marker in a sample, e.g., a metabolic disorder sample, as compared to the protein level of the marker in a normal, control sample.

The “amount” of a marker, e.g, expression or copy number of a marker, or protein level of a marker, in a subject is “significantly” higher or lower than the normal amount of a marker, if the amount of the marker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least twice, and more preferably three, four, five, ten or more times that amount. Alternately, the amount of the marker in the subject can be considered “significantly” higher or lower than the normal amount if the amount is at least about two, and preferably at least about three, four, or five times, higher or lower, respectively, than the normal amount of the marker. In some embodiments, the amount of the marker in the subject can be considered “significantly” higher or lower than the normal amount if the amount is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, higher or lower, respectively, than the normal amount of the marker.

The term “altered level of expression” of a marker refers to an expression level or copy number of a marker in a test sample e.g, a sample derived from a subject suffering from a metabolic disorder, that is greater or less than the standard error of the assay employed to assess expression or copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times the expression level or copy number of the marker or chromosomal region in a control sample (e.g, sample from a healthy subject not having the associated disease) and preferably, the average expression level or copy number of the marker or chromosomal region in several control samples. The altered level of expression is greater or less than the standard error of the assay employed to assess expression or copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times the expression level or copy number of the marker in a control sample ( e.g ., sample from a healthy subject not having the associated disease) and preferably, the average expression level or copy number of the marker in several control samples.

The term “altered activity” of a marker refers to an activity of a marker which is increased or decreased in a disease state, e.g., in a metabolic disorder sample, as compared to the activity of the marker in a normal, control sample. Altered activity of a marker may be the result of, for example, altered expression of the marker, altered protein level of the marker, altered structure of the marker, or, e.g, an altered interaction with other proteins involved in the same or different pathway as the marker, or altered interaction with transcriptional activators or inhibitors.

The term “altered structure” of a marker refers to the presence of mutations or allelic variants within the marker gene or maker protein, e.g, mutations which affect expression or activity of the marker, as compared to the normal or wild-type gene or protein. For example, mutations include, but are not limited to substitutions, deletions, or addition mutations. Mutations may be present in the coding or non-coding region of the marker.

The term “altered cellular localization” of a marker refers to the mislocalization of the marker within a cell relative to the normal localization within the cell e.g., within a healthy and/or wild-type cell. An indication of normal localization of the marker can be determined through an analysis of cellular localization motifs known in the field that are harbored by marker polypeptides. For example, SLNCR is a nuclear transcription factor coordinator and naturally functions to present combinations of nuclear transcription factors within the nucleus such that function is abrogated if nuclear import and/or export is inhibited.

The term “body fluid” refers to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g, amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper’s fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, peritoneal fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit). In a preferred embodiment, body fluids are restricted to blood-related fluids, including whole blood, serum, plasma, and the like. The term “classifying” includes “to associate” or “to categorize” a sample with a disease state. In certain instances, “classifying” is based on statistical evidence, empirical evidence, or both. In certain embodiments, the methods and systems of classifying use of a so-called training set of samples having known disease states. Once established, the training data set serves as a basis, model, or template against which the features of an unknown sample are compared, in order to classify the unknown disease state of the sample. In certain instances, classifying the sample is akin to diagnosing the disease state of the sample. In certain other instances, classifying the sample is akin to differentiating the disease state of the sample from another disease state.

The term “control” refers to any reference standard suitable to provide a comparison to the expression products in the test sample. In one embodiment, the control comprises obtaining a “control sample” from which expression product levels are detected and compared to the expression product levels from the test sample. Such a control sample may comprise any suitable sample, including but not limited to a sample from a control patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the patient in need of metabolism modulation, cultured primary cells/tissues isolated from a subject such as a normal subject or the patient patient in need of metabolism modulation, adjacent normal cells/tissues obtained from the same organ or body location of the patient in need of metabolism modulation, a tissue or cell sample isolated from a normal subject, or a primary cells/tissues obtained from a depository. In another preferred embodiment, the control may comprise a reference standard expression product level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.) or receiving a certain treatment. It will be understood by those of skill in the art that such control samples and reference standard expression product levels can be used in combination as controls in the methods of the present invention. In one embodiment, the control may comprise normal or non-metabolic disorder cell/tissue sample. In another preferred embodiment, the control may comprise an expression level for a set of patients, such as a set of metabolic disorder patients, or for a set of metabolic disorder patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome. In the former case, the specific expression product level of each patient can be assigned to a percentile level of expression, or expressed as either higher or lower than the mean or average of the reference standard expression level. In another preferred embodiment, the control may comprise normal cells, cells from patients treated with a therapeutic and cells from patients having modulated metabolism. In another embodiment, the control may also comprise a measured value for example, average level of expression of a particular gene in a population compared to the level of expression of a housekeeping gene in the same population. Such a population may comprise normal subjects, metabolic disorder patients who have not undergone any treatment ( i.e treatment naive), or metabolic disorder patients undergoing therapy. In another preferred embodiment, the control comprises a ratio transformation of expression product levels, including but not limited to determining a ratio of expression product levels of two genes in the test sample and comparing it to any suitable ratio of the same two genes in a reference standard; determining expression product levels of the two or more genes in the test sample and determining a difference in expression product levels in any suitable control; and determining expression product levels of the two or more genes in the test sample, normalizing their expression to expression of housekeeping genes in the test sample, and comparing to any suitable control. In particularly preferred embodiments, the control comprises a control sample which is of the same lineage and/or type as the test sample. In another embodiment, the control may comprise expression product levels grouped as percentiles within or based on a set of patient samples, such as all patients with a metabolic disorder. In one embodiment a control expression product level is established wherein higher or lower levels of expression product relative to, for instance, a particular percentile, are used as the basis for predicting outcome. In another preferred embodiment, a control expression product level is established using expression product levels from metabolic disorder control patients with a known outcome, and the expression product levels from the test sample are compared to the control expression product level as the basis for predicting outcome. As demonstrated by the data below, the methods of the present invention are not limited to use of a specific cut-point in comparing the level of expression product in the test sample to the control.

The term “pre-determined” biomarker amount and/or activity measurement(s) may be a biomarker amount and/or activity measurement(s) used to, by way of example only, evaluate a subject that may be selected for a particular treatment, evaluate a response to a treatment such as an anti-immune checkpoint inhibitor therapy, and/or evaluate the disease state. A pre-determined biomarker amount and/or activity measurement(s) may be determined in populations of patients with or without a metabolic disorder. The pre determined biomarker amount and/or activity measurement(s) can be a single number, equally applicable to every patient, or the pre-determined biomarker amount and/or activity measurement s) can vary according to specific subpopulations of patients. Age, weight, height, and other factors of a subject may affect the pre-determined biomarker amount and/or activity measurement(s) of the individual. Furthermore, the pre-determined biomarker amount and/or activity can be determined for each subject individually. In one embodiment, the amounts determined and/or compared in a method described herein are based on absolute measurements. In another embodiment, the amounts determined and/or compared in a method described herein are based on relative measurements, such as ratios ( e.g ., serum biomarker normalized to the expression of a housekeeping or otherwise generally constant biomarker). The pre-determined biomarker amount and/or activity measurement s) can be any suitable standard. For example, the pre-determined biomarker amount and/or activity measurement(s) can be obtained from the same or a different human for whom a patient selection is being assessed. In one embodiment, the pre-determined biomarker amount and/or activity measurement(s) can be obtained from a previous assessment of the same patient. In such a manner, the progress of the selection of the patient can be monitored over time. In addition, the control can be obtained from an assessment of another human or multiple humans, e.g., selected groups of humans, if the subject is a human. In such a manner, the extent of the selection of the human for whom selection is being assessed can be compared to suitable other humans, e.g, other humans who are in a similar situation to the human of interest, such as those suffering from similar or the same condition(s) and/or of the same ethnic group.

Outcome measures, such as overall survival, increased thermogenesis, and weight loss can be monitored over a period of time for subjects following therapy for whom the measurement values are known. In certain embodiments, the same doses of therapeutic agents are administered to each subject. In related embodiments, the doses administered are standard doses known in the art for therapeutic agents. The period of time for which subjects are monitored can vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months or longer. Biomarker threshold values that correlate to outcome of a therapy can be determined using methods such as those described in the Examples section. Outcomes can also be measured in terms of a “hazard ratio” (the ratio of death rates for one patient group to another; provides likelihood of death at a certain time point), “overall survival” (OS), and/or “progression free survival.” In certain embodiments, the prognosis comprises likelihood of overall survival rate at 1 year, 2 years, 3 years, 4 years, or any other suitable time point. The significance associated with the prognosis of poor outcome in all aspects of the present invention is measured by techniques known in the art. For example, significance may be measured with calculation of odds ratio. In a further embodiment, the significance is measured by a percentage. In one embodiment, a significant risk of poor outcome is measured as odds ratio of 0.8 or less or at least about 1.2, including by not limited to: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 4.0, 5.0,

10.0, 15.0, 20.0, 25.0, 30.0 and 40.0. In a further embodiment, a significant increase or reduction in risk is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 98%. In a further embodiment, a significant increase in risk is at least about 50%. Thus, the present invention further provides methods for making a treatment decision for a patient in need of modulated metabolism, comprising carrying out the methods for prognosing a patient according to the different aspects and embodiments of the present invention, and then weighing the results in light of other known clinical and pathological risk factors, in determining a course of treatment for the patient in need of modulated metabolism.

A “kit” is any manufacture ( e.g ., a package or container) comprising at least one reagent, e.g., a probe, for specifically detecting or modulating the expression of a marker of the present invention. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. Kits comprising compositions described herein are encompassed within the present invention.

1. Diagnostic Assays

The present invention provides, in part, methods, systems, and code for accurately classifying whether a biological sample is associated with a metabolic disorder or a clinical subtype thereof. In some embodiments, the present invention is useful for classifying a sample (e.g, from a subject) as a sample that will respond to metabolic intervention using a statistical algorithm and/or empirical data (e.g, the presence or level of one or biomarkers described herein).

An exemplary method for detecting the level of expression or activity of one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or fragments thereof, and thus useful for classifying whether a sample is associated with a metabolic disorder, involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the biomarker ( e.g ., polypeptide or nucleic acid that encodes the biomarker or fragments thereof) such that the level of expression or activity of the biomarker is detected in the biological sample. In some embodiments, the presence or level of at least one, two, three, four, five, six, seven, eight, nine, ten, fifty, hundred, or more biomarkers of the present invention are determined in the individual's sample. In certain instances, the statistical algorithm is a single learning statistical classifier system. Exemplary statistical analyses are presented in the Examples and can be used in certain embodiments. In other embodiments, a single learning statistical classifier system can be used to classify a sample as a metabolic disorder sample, a metabolic disorder subtype sample, or a non-metabolic disorder sample based upon a prediction or probability value and the presence or level of one or more biomarkers described herein. The use of a single learning statistical classifier system typically classifies the sample as a metabolic disorder sample with a sensitivity, specificity, positive predictive value, negative predictive value, and/or overall accuracy of at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

Other suitable statistical algorithms are well-known to those of skill in the art. For example, learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (e.g., panel of markers of interest) and making decisions based upon such data sets. In some embodiments, a single learning statistical classifier system such as a classification tree (e.g, random forest) is used. In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem. Examples of learning statistical classifier systems include, but are not limited to, those using inductive learning (e.g, decision/classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning, connectionist learning (e.g, neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g, passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc.), and genetic algorithms and evolutionary programming. Other learning statistical classifier systems include support vector machines ( e.g ., Kernel methods), multivariate adaptive regression splines (MARS), Levenberg-Marquardt algorithms, Gauss-Newton algorithms, mixtures of Gaussians, gradient descent algorithms, and learning vector quantization (LVQ). In certain embodiments, the method of the present invention further comprises sending the metabolic disorder classification results to a clinician, e.g., an endocrinologist, cardiologist, or hematologist.

In another embodiment, the method of the present invention further provides a diagnosis in the form of a probability that the individual has a metabolic disorder or a clinical subtype thereof. For example, the individual can have about a 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,

95%, or greater probability of having a metabolic disorder or a clinical subtype thereof. In yet another embodiment, the method of the present invention further provides a prognosis of a metabolic disorder in the individual. For example, the prognosis can be surgery, development or progression of a metabolic disorder or a clinical subtype thereof, development of one or more symptoms, or recovery from the metabolic disorder. In some instances, the method of classifying a sample as a metabolic disorder sample is further based on the symptoms (e.g, clinical factors) of the individual from which the sample is obtained. The symptoms or group of symptoms can be, for example, those associated with the metabolic disorder. In some embodiments, the diagnosis of an individual as having a metabolic disorder of interest or a clinical subtype thereof is followed by administering to the individual a therapeutically effective amount of a drug useful for treating one or more symptoms associated with the metabolic disorder or a clinical subtype thereof.

In some embodiments, an agent for detecting biomarker RNA, genomic DNA, or fragments thereof is a labeled nucleic acid probe capable of hybridizing to biomarker RNA, genomic DNA., or fragments thereof. The nucleic acid probe can be, for example, full- length biomarker nucleic acid, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions well-known to a skilled artisan to biomarker mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the present invention are described herein. In some embodiments, the nucleic acid probe is designed to detect transcript variants (i.e., different splice forms) of a gene.

In some embodiments, selenium and derivatives thereof can be analyzed using physical separation techniques. Separation and purification in the present invention may include any procedure known in the art, such as capillary electrophoresis ( e.g ., in capillary or on-chip) or chromatography (e.g., in capillary, column or on a chip). Electrophoresis is a method which can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be conducted in a gel, capillary, or in a microchannel on a chip. Examples of gels used for electrophoresis include starch, acrylamide, polyethylene oxides, agarose, or combinations thereof. A gel can be modified by its cross-linking, addition of detergents, or denaturants, immobilization of enzymes or antibodies (affinity electrophoresis) or substrates (zymography) and incorporation of a pH gradient. Examples of capillaries used for electrophoresis include capillaries that interface with an electrospray. Capillary electrophoresis (CE) is preferred for separating complex hydrophilic molecules and highly charged solutes. CE technology can also be implemented on microfluidic chips. Depending on the types of capillary and buffers used, CE can be further segmented into separation techniques such as capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (cITP) and capillary electrochromatography (CEC). An embodiment to couple CE techniques to electrospray ionization involves the use of volatile solutions, for example, aqueous mixtures containing a volatile acid and/or base and an organic such as an alcohol or acetonitrile. Capillary isotachophoresis (cITP) is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities. Capillary zone electrophoresis (CZE), also known as free-solution CE (FSCE), is based on differences in the electrophoretic mobility of the species, determined by the charge on the molecule, and the frictional resistance the molecule encounters during migration which is often directly proportional to the size of the molecule. Capillary isoelectric focusing (CIEF) allows weakly-ionizable amphoteric molecules, to be separated by electrophoresis in a pH gradient. CEC is a hybrid technique between traditional high performance liquid chromatography (HPLC) and CE. Separation and purification techniques used in the present invention include any chromatography procedures known in the art. Chromatography can be based on the differential adsorption and elution of certain analytes or partitioning of analytes between mobile and stationary phases. Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), etc.

Biomarker metabolites, such as those shown in Table 1 can be detected in numerous ways according to well-known techniques. For example, such metabolites, as well as biomarker proteins, can be detected using mass spectrometry methods, such as MALDI/TOF (time-of-flight), SELDI/TOF, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry, nuclear magnetic resonance spectrometry, or tandem mass spectrometry ( e.g ., MS/MS, MS/MS/MS, ESI-MS/MS, etc.). See for example, U.S. Patent Application Nos: 20030199001, 20030134304, 20030077616, which are herein incorporated by reference.

Mass spectrometry methods are well known in the art and have been used to quantify and/or identify biomolecules, such as chemical metabolites and proteins (see, e.g., Li et al. (2000) Tibtech 18, 151-160; Rowley etal. (2000) Methods 20, 383-397; Kuster and Mann (1998) Curr. Opin. Structural Biol. 8, 393-400). Further, mass spectrometric techniques have been developed that permit at least partial de novo sequencing of isolated proteins (see, e.g., Chait et al. (1993) Science 262, 89-92; Keough et al. (1999) Proc. Natl. Acad. Sci. USA. 96, 7131-7136; reviewed in Bergman (2000) EXS 88, 133-44).

In certain embodiments, a gas phase ion spectrophotometer is used. In other embodiments, laser-desorption/ionization mass spectrometry is used to analyze the sample. Modem laser desorption/ionization mass spectrometry (“LDI-MS”) can be practiced in two main variations: matrix assisted laser desorption/ionization (“MALDI”) mass spectrometry and surface-enhanced laser desorption/ionization (“SELDI”). In MALDI, the analyte is mixed with a solution containing a matrix, and a drop of the liquid is placed on the surface of a substrate. The matrix solution then co-crystallizes with the biological molecules. The substrate is inserted into the mass spectrometer. Laser energy is directed to the substrate surface where it desorbs and ionizes the biological molecules without significantly fragmenting them. However, MALDI has limitations as an analytical tool. It does not provide means for fractionating the sample, and the matrix material can interfere with detection, especially for low molecular weight analytes (see, e.g, Hellenkamp et al., U.S. Pat. No. 5,118,937 and Beavis and Chait, U.S. Pat. No. 5,045,694).

In SELDI, the substrate surface is modified so that it is an active participant in the desorption process. In one variant, the surface is derivatized with adsorbent and/or capture reagents that selectively bind the protein of interest. In another variant, the surface is derivatized with energy absorbing molecules that are not desorbed when struck with the laser. In another variant, the surface is derivatized with molecules that bind the protein of interest and that contain a photolytic bond that is broken upon application of the laser. In each of these methods, the derivatizing agent generally is localized to a specific location on the substrate surface where the sample is applied (see, e.g ., Hutchens and Yip, U.S. Pat. No. 5,719,060 and Hutchens and Yip, WO 98/59361). The two methods can be combined by, for example, using a SELDI affinity surface to capture an analyte and adding matrix- containing liquid to the captured analyte to provide the energy absorbing material.

For additional information regarding mass spectrometers, see, e.g. , Principles of Instrumental Analysis, 3rd edition., Skoog, Saunders College Publishing, Philadelphia, 1985; and Kirk-Othmer Encyclopedia of Chemical Technology, 4.sup.th ed. Vol. 15 (John Wiley & Sons, New York 1995), pp. 1071-1094.

Detection of the presence of a marker or other substances will typically involve detection of signal intensity. For example, in certain embodiments, the signal strength of peak values from spectra of a first sample and a second sample can be compared (e.g, visually or by computer analysis) to determine the relative amounts of particular biomolecules. Software programs such as the Biomarker Wizard program (Ciphergen Biosystems, Inc., Fremont, Calif.) can be used to aid in analyzing mass spectra. The mass spectrometers and their techniques are well known to those of skill in the art.

Any person skilled in the art understands, any of the components of a mass spectrometer (e.g, desorption source, mass analyzer, detect, etc.) and varied sample preparations can be combined with other suitable components or preparations described herein, or to those known in the art. For example, in some embodiments a control sample may contain heavy atoms (e.g. ¹³ C) thereby permitting the test sample to be mixed with the known control sample in the same mass spectrometry run. In some embodiments, internal controls, such as phenylalanine-d8 and/or valine-d8 can be run with the samples.

In one embodiment, a laser desorption time-of-flight (TOF) mass spectrometer is used. In laser desorption mass spectrometry, a substrate with a bound marker is introduced into an inlet system. The marker is desorbed and ionized into the gas phase by laser from the ionization source. The ions generated are collected by an ion optic assembly, and then in a time-of-flight mass analyzer, ions are accelerated through a short high voltage field and let drift into a high vacuum chamber. At the far end of the high vacuum chamber, the accelerated ions strike a sensitive detector surface at a different time. Since the time-of- flight is a function of the mass of the ions, the elapsed time between ion formation and ion detector impact can be used to identify the presence or absence of molecules of specific mass to charge ratio.

In some embodiments the relative amounts of one or more biomolecules present in a first or second sample is determined, in part, by executing an algorithm with a programmable digital computer. The algorithm identifies at least one peak value in the first mass spectrum and the second mass spectrum. The algorithm then compares the signal strength of the peak value of the first mass spectrum to the signal strength of the peak value of the second mass spectrum of the mass spectrum. The relative signal strengths are an indication of the amount of the biomolecule that is present in the first and second samples.

A standard containing a known amount of a biomolecule can be analyzed as the second sample to provide better quantification of the amount of the biomolecule present in the first sample. In certain embodiments, the identity of the biomolecules in the first and second sample can also be determined.

Another preferred agent for detecting selenium biomarkers in complex with biomarker proteins is an antibody capable of binding to the biomarker, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof ( e.g Fab or F(ab')2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term “biological sample” is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject. That is, the detection method of the present invention can be used to detect biomarker mRNA, polypeptide, genomic DNA, or fragments thereof, in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of biomarker mRNA or a fragment thereof include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of biomarker polypeptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of biomarker genomic DNA or a fragment thereof include Southern hybridizations. Furthermore, in vivo techniques for detection of one or more biomarkers polypeptide or a fragment thereof include introducing into a subject a labeled anti- biomarker antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains polypeptide molecules from the test subject. Alternatively, the biological sample can contain RNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a hematological tissue ( e.g a sample comprising blood, plasma, B cell, bone marrow, etc.) sample isolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting the desired biomarker, such as selenium or selenium derivative and/or polypeptide, RNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof of one or more biomarkers listed in Table 1, the Figures, and the Examples, such that the presence of biomarker polypeptide, RNA, genomic DNA, or fragments thereof, is detected in the biological sample, and comparing the presence of biomarker polypeptide, RNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri- miRNA, miRNA*, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof in the control sample with the presence of biomarker polypeptide, RNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, piwiRNA, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof in the test sample.

The invention also encompasses kits for detecting the presence of a biomarker of interest, such as selenium, selenium derivative, etc. such as by using a polypeptide, RNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, piwiRNA, anti- miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof, of one or more biomarkers listed in Table 1, the Figures, and the Examples, in a biological sample. For example, the kit can comprise a labeled compound or agent capable of detecting one or more biomarkers polypeptide, RNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof, in a biological sample; means for determining the amount of the biomarker polypeptide, RNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof, f in the sample; and means for comparing the amount of the biomarker polypeptide, RNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof, in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect the biomarker polypeptide, RNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof.

In some embodiments, therapies tailored to treat stratified patient populations based on the described diagnostic assays are further administered, such as metabolic disorder standards of treatment, immune therapy, and combinations thereof described herein.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant expression or activity of one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof. As used herein, the term “aberrant” includes biomarker expression or activity levels which deviates from the normal expression or activity in a control.

The assays described herein, such as the preceding diagnostic assays or the following assays, can be used to identify a subject that would benefit from metabolic interventions ( e.g. , low levels of plasma selenium indicate that selenium administration would be differentially beneficial). Alternatively, the prognostic assays can be used to identify a subject having or at risk for developing a disorder associated with a misregulation of biomarker activity or expression. Thus, the present invention provides a method for identifying and/or classifying a disease associated with aberrant expression or activity of one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples. Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant biomarker expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a metabolic disorder. Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disease associated with aberrant biomarker expression or activity in which a test sample is obtained and biomarker polypeptide or nucleic acid expression or activity is detected (e.g, wherein a significant increase or decrease in biomarker polypeptide or nucleic acid expression or activity relative to a control is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant biomarker expression or activity). In some embodiments, significant increase or decrease in biomarker expression or activity comprises at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 22.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15,

16, 17, 18, 19, 20 times or more higher or lower, respectively, than the expression activity or level of the marker in a control sample.

The methods of the present invention can also be used to detect genetic alterations in one or more biomarkers of the present invention, including one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof, thereby determining if a subject with the altered biomarker is at risk for a metabolic disorder characterized by aberrant biomarker activity or expression levels. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one alteration affecting the integrity of a gene encoding one or more biomarkers, or the mis-expression of the biomarker ( e.g ., mutations and/or splice variants). For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from one or more biomarkers gene, 2) an addition of one or more nucleotides to one or more biomarkers gene, 3) a substitution of one or more nucleotides of one or more biomarkers gene, 4) a chromosomal rearrangement of one or more biomarkers gene, 5) an alteration in the level of a messenger RNA transcript of one or more biomarkers gene, 6) aberrant modification of one or more biomarkers gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of an RNA transcript of one or more biomarkers gene, 8) a non-wild type level of one or more biomarkers polypeptide, 9) allelic loss of one or more biomarkers gene, and 10) inappropriate post-translational modification of one or more biomarkers polypeptide. As described herein, there are a large number of assays known in the art which can be used for detecting alterations in one or more biomarkers gene. A preferred biological sample is a tissue or serum sample isolated by conventional means from a subject.

In certain embodiments, detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patents 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g, Landegran etal. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91 :360-364), the latter of which can be particularly useful for detecting point mutations in one or more biomarkers gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g, genomic DNA, mRNA, cDNA, small RNA, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to one or more biomarkers gene of the present invention, including the biomarker genes listed in Table 1, the Figures, and the Examples, or fragments thereof, under conditions such that hybridization and amplification of the biomarker gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self-sustained sequence replication (Guatelli, J. C. etal. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al. (1988) Bio-Technology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

In an alternative embodiment, mutations in one or more biomarkers gene of the present invention, including one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof, from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Patent 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in one or more biomarkers gene of the present invention, including a gene listed in Table 1, the Figures, and the Examples, or a fragment thereof, can be identified by hybridizing a sample and control nucleic acids, e.g., DNA, RNA, mRNA, small RNA, cDNA, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof, to high density arrays containing hundreds or thousands of oligonucleotide probes (Cronin, M. T. et al. (1996) Hum. Mutat. 7:244-255; Kozal, M. J. etal. (1996) Nat. Med. 2:753-759). For example, genetic mutations in one or more biomarkers can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin et al. (1996) supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential, overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence one or more biomarkers gene of the present invention, including a gene listed in Table 1, the Figures, and the Examples, or a fragment thereof, and detect mutations by comparing the sequence of the sample biomarker gene with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert (1977) Proc. Natl. Acad. Sci. USA 74:560 or Sanger (1977) Proc. Natl. Acad Sci. USA 74:5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W. (1995) Biotechniques 19:448-53), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen etal. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in one or more biomarkers gene of the present invention, including a gene listed in Table 1, the Figures, and the Examples, or fragments thereof, include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the art technique of “mismatch cleavage” starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digest the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397 and Saleeba etal. (1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in biomarker genes of the present invention, including genes listed in Table 1, the Figures, and the Examples, or fragments thereof, obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657- 1662). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent 5,459,039. In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in biomarker genes of the present invention, including genes listed in Table 1, the Figures, and the Examples, or fragments thereof. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766; see also Cotton (1993) Mutat. Res. 285:125-144 and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313 :495). When DGGE is used as the method of analysis, DNA will be modified to ensure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high- melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163; Saiki etal. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA. In some embodiments, the hybridization reactions can occur using biochips, microarrays, etc., or other array technology that are well-known in the art.

Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs etal. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini el al. (1992) Mol. Cell Probes 6: 1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88: 189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

The methods described herein may be performed, for example, by utilizing pre packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving one or more biomarkers of the present invention, including one or more biomarkers listed in Table 1, the Figures, and the Examples, or fragments thereof.

3. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g, drugs) on the expression or activity of one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof (e.g, the modulation of a metabolic state) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase expression and/or activity of one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof, can be monitored in clinical trials of subjects exhibiting decreased expression and/or activity of one or more biomarkers of the present invention, including one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof, relative to a control reference. Alternatively, the effectiveness of an agent determined by a screening assay to decrease expression and/or activity of one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof, can be monitored in clinical trials of subjects exhibiting decreased expression and/or activity of the biomarker of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof relative to a control reference. In such clinical trials, the expression and/or activity of the biomarker can be used as a “read out” or marker of the phenotype of a particular cell.

In some embodiments, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent ( e.g ., selenium or selenium derivative, an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression and/or activity of one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or fragments thereof in the pre administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the biomarker in the post administration samples (such as circulating level, intracellular level, cellular uptake, metabolism, etc.); (v) comparing the level of expression or activity of the biomarker or fragments thereof in the pre-administration sample with the that of the biomarker in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of one or more biomarkers to higher levels than detected (e.g., to increase the effectiveness of the agent.) Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of the biomarker to lower levels than detected (e.g, to decrease the effectiveness of the agent). According to such an embodiment, biomarker expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response. C. Methods of Treatment

The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder characterized by insufficient or excessive production of biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or fragments thereof, which have aberrant expression or activity compared to a control. Moreover, agents of the present invention described herein can be used to detect and isolate the biomarkers or fragments thereof, regulate the bioavailability of the biomarkers or fragments thereof, and modulate biomarker expression levels or activity.

1. Prophylactic Methods

In one aspect, the present invention provides a method for preventing in a subject, a disease or condition associated with an aberrant expression or activity of one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof, by administering to the subject an agent which modulates biomarker expression or at least one activity of the biomarker. Subjects at risk for a disease or disorder which is caused or contributed to by aberrant biomarker expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the biomarker expression or activity aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.

2. Therapeutic Methods

Another aspect of the present invention pertains to methods of modulating the expression or activity of, or interaction with natural (or synthetic) binding partner(s) of, one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or fragments thereof, for therapeutic purposes. The biomarkers of the present invention have been demonstrated to correlate with adipose tissue thermogenesis and modulation of metabolism. Accordingly, the activity and/or expression of the biomarker, as well as the interaction between one or more biomarkers or a fragment thereof and its natural (or synthetic) binding partner(s) or a fragment(s) thereof can be modulated in order to modulate the immune response. Modulatory methods of the present invention involve contacting a cell with one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof or agent that modulates one or more of the activities of biomarker activity associated with the cell or produced by the cell. An agent that modulates biomarker activity can be an agent as described herein, such as a nucleic acid or a polypeptide, a naturally- occurring binding partner of the biomarker, an antibody against the biomarker, a combination of antibodies against the biomarker and antibodies against other immune related targets, one or more biomarkers agonist or antagonist, a peptidomimetic of one or more biomarkers agonist or antagonist, one or more biomarkers peptidomimetic, other small molecule, or small RNA directed against or a mimic of one or more biomarkers nucleic acid gene expression product.

An agent that modulates the expression of one or more biomarkers of the present invention, including one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof includes small molecules ( e.g ., selenium, selenium derivatives, selenium modulators), as well as a nucleic acid molecule described herein, e.g., an antisense nucleic acid molecule, RNAi molecule, shRNA, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof, or other small RNA molecule, triplex oligonucleotide, ribozyme, or recombinant vector for expression of one or more biomarkers polypeptide. For example, an oligonucleotide complementary to the area around one or more biomarkers polypeptide translation initiation site can be synthesized. One or more antisense oligonucleotides can be added to cell media, typically at 200 pg/ml, or administered to a patient to prevent the synthesis of one or more biomarkers polypeptide. The antisense oligonucleotide is taken up by cells and hybridizes to one or more biomarkers mRNA to prevent translation. Alternatively, an oligonucleotide which binds double-stranded DNA to form a triplex construct to prevent DNA unwinding and transcription can be used. As a result of either, synthesis of biomarker polypeptide is blocked. When biomarker expression is modulated, preferably, such modulation occurs by a means other than by knocking out the biomarker gene. Agents which modulate expression, by virtue of the fact that they control the amount of biomarker in a cell, also modulate the total amount of biomarker activity in a cell.

In one embodiment, the agent stimulates one or more activities of one or more biomarkers of the present invention, including one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof. Examples of such stimulatory agents include active biomarker polypeptides, or a fragment thereof, such as selenium binding partners, and/or a nucleic acid molecule encoding the biomarker or a fragment thereof that has been introduced into the cell ( e.g ., cDNA, mRNA, shRNAs, siRNAs, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof, or other functionally equivalent molecule known to a skilled artisan). In another embodiment, the agent inhibits one or more biomarker activities. In one embodiment, the agent inhibits or enhances the interaction of the biomarker with its natural (or synthetic) binding partner(s). Examples of such inhibitory agents include antisense nucleic acid molecules, anti-biomarker antibodies, biomarker inhibitors, and compounds identified in the screening assays described herein.

These modulatory methods can be performed in vitro (e.g., by contacting the cell with the agent) or, alternatively, by contacting an agent with cells in vivo (e.g, by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a condition or disorder that would benefit from up- or down-modulation of one or more biomarkers of the present invention, such as selenium, a selenium derivative, and/or one or more biomarkers listed in Table 1, the Figures, and the Examples, or a fragment thereof, e.g, a disorder characterized by unwanted, insufficient, or aberrant expression or activity of the biomarker or fragments thereof. In one embodiment, the method involves administering an agent (e.g, an agent identified by a screening assay described herein), or combination of agents that modulates (e.g, upregulates or downregulates) biomarker expression or activity. In another embodiment, the method involves administering one or more biomarkers polypeptide or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted biomarker expression or activity.

Stimulation of biomarker activity is desirable in situations in which the biomarker is abnormally downregulated and/or in which increased biomarker activity is likely to have a beneficial effect. Likewise, inhibition of biomarker activity is desirable in situations in which biomarker is abnormally upregulated and/or in which decreased biomarker activity is likely to have a beneficial effect.

In addition, these modulatory agents can also be administered in combination therapy with, e.g., metabolism enhancing agents, such as transplanted brown and/or beige fat cells, hormones, and the like. The preceding treatment methods can be administered in conjunction with other forms of conventional therapy (e.g, standard-of-care treatments for metabolic disorders are well-known to the skilled artisan), either consecutively with, pre- or post-conventional therapy. For example, these modulatory agents can be administered with a therapeutically effective dose of a metabolism modulatory agent.

The methods of the present invention relate to selenium sufficient to modulate (e.g, induce or repress) brown and/or beige fat cell differentiation and/or activity, wherein increases in differentiated brown and/or beige fat cells or activity increase energy expenditure and favorably affect other metabolic processes and can therefore be used to treat metabolic disorders such as obesity, diabetes, decreased thermogenesis and subjects in need of more excersise; and, wherein decreases in differentiated brown and/or beige fat cells or activity decrease energy expenditure and can therefore be used to treat the effects of such conditions as cachexia, anorexia, and obesity-associated cancer.

The invention also relates to methods for increasing energy expenditure in a mammal comprising inducing expression and/or activity of selenium sufficient to activate brown and/or beige fat cell differentiation or activity in the mammal, wherein the differentiated and/or more active brown fat and/or beige fat cells promote energy expenditure thereby increasing energy expenditure in the mammal.

The term “sufficient to activate” is intended to encompass any increase in amount and/or activity of selenium that promotes, activates, stimulates, enhances, or results in brown fat and/or beige fat differentiation or activity.

In another aspect, the invention relates to methods for treating metabolic disorders in a subject comprising administering to the subject an agent that induces promotion and/or activity of selenium, wherein promotion and/or activity of selenium increases respiration and energy expenditure to thereby treat the metabolic disorder. In one embodiment, total respiration is increased following increase and/or upregulation of selenium. In another embodiment, uncoupled respiration is increased following increase and/or upregulation of selenium. Uncoupled respiration dissipates heat and thereby increases energy expenditure in the subject. As used herein, the term “agent” and “therapeutic agent” is defined broadly as anything that cells from a subject having a metabolic disorder may be exposed to in a therapeutic protocol. In one embodiment, the agent is selenium. In another embodiment, the agent is selenocysteine and/or selenomethionine.

The term “administering” is intended to include routes of administration which allow the agent to perform its intended function of modulating ( e.g ., increasing or decreasing) selenium. Examples of routes of administration which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc., such as in a subcutaneous injection into white, brown, and/or beige fat depots), oral, inhalation, and transdermal. The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the agent can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The agent may be administered alone, or in conjunction with a pharmaceutically acceptable carrier. Further the agent may be co administered with a pharmaceutically acceptable carrier. The agent also may be administered as a prodrug, which is converted to its active form in vivo. The agent may also be administered in combination with one or more additional therapeutic agent(s) (e.g., before, after or simultaneously therewith).

The term “effective amount” of an agent that induces modulation of selenium is that amount necessary or sufficient to modulate (e.g, increase or decrease) selenium in the subject or population of subjects. The effective amount can vary depending on such factors as the type of therapeutic agent(s) employed, the size of the subject, or the severity of the disorder.

It will be appreciated that individual dosages may be varied depending upon the requirements of the subject in the judgment of the attending clinician, the severity of the condition being treated and the particular compound being employed. In determining the therapeutically effective amount or dose, a number of additional factors may be considered by the attending clinician, including, but not limited to: the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the desired time course of treatment; the species of mammal; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the kind of concurrent treatment; and other relevant circumstances.

Treatment can be initiated with smaller dosages which are less than the effective dose of the compound. Thereafter, in one embodiment, the dosage should be increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.

The effectiveness of any particular agent to treat a metabolic disorder can be monitored by comparing two or more samples obtained from a subject undergoing anti- metabolic disorder or metabolic disorder-related disorder treatment. In general, it is preferable to obtain a first sample from the subject prior to beginning therapy and one or more samples during treatment. In such a use, a baseline of expression of cells from subjects with obesity or obesity -related disorders prior to therapy is determined and then changes in the baseline state of expression of cells from subjects with obesity or obesity- related disorders is monitored during the course of therapy. Alternatively, two or more successive samples obtained during treatment can be used without the need of a pre treatment baseline sample. In such a use, the first sample obtained from the subject is used as a baseline for determining whether the expression of cells from subjects with obesity or obesity -related disorders is increasing or decreasing.

Another aspect of the invention relates to a method for inducing brown fat and/or beige fat cell differentiation and/or activity in a mammal comprising modulating selenium in a mammal and, optionally, monitoring the differentiation of brown fat cells in the mammal. Increased brown and/or beige adipose tissue in the mammal will warm up the body and blood of the mammal resulting in an increased energy expenditure from the cells. The increased energy expenditure will increase the metabolic rate of the subject and may be used for the treatment and/or prevention of obesity and obesity related disorders. The induction of brown fat cells may be monitored by analyzing a) brown fat and/or beige fat gene expression, such as expression of a marker selected from the group consisting of: cidea, adiponectin, adipsin, otopetrin, type II deiodinase, cig30, ppar gamma 2, pgcla, ucpl, elovl3, cAMP, Prdml6, cytochrome C, cox4il, coxIII, cox5b, cox7al, cox8b, glut4, atpase b2, cox II, atp5o, ndufb5, ap2, ndufsl, GRP109A, acylCoA-thioesterase 4, EARA1, claudinl, PEPCK, fgf21, acylCoA-thioesterase 3, dio2, fatty acid synthase (fas), leptin, resistin, and nuclear respiratory factor-1 (nrfl); b) thermogenesis in adipose cells; c) differentiation of adipose cells; d) insulin sensitivity of adipose cells; e) basal respiration or uncoupled respiration; f) whole body oxygen consumption; g) obesity or appetite; h) insulin secretion of pancreatic beta cells; i) glucose tolerance; j) modified phosphorylation of EGFR, ERK, AMPK, protein kinase A (PKA) substrates having an RRX(S/T) motif, wherein the X is any amino acid and the (S/T) residue is a serine or threonine, HSL; k) modified activity of UCP1 protein; and 1) growth and effects of metabolic disorders, such as obesity-associated cancer, cachexia, anorexia, diabetes, and obesity.

Any means for the introduction of a therapeutic agent into mammals, human or non human, or cells thereof may be adapted to the practice of this invention for the delivery of the various constructs of the invention into the intended recipient. In one embodiment of the invention, small molecules and/or nucleic acids are delivered to cells by transfection, e.g ., by delivery of “naked” small molecules and/or DNA using uptake machinery by cells or in a complex that does not require cellular machinery for uptake. A colloidal system includes macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a lipid-complexed or liposome-formulated DNA. In the former approach, prior to formulation of DNA, e.g. , with lipid, a plasmid containing a transgene bearing the desired DNA constructs may first be experimentally optimized for expression (e.g, inclusion of an intron in the 5' untranslated region and elimination of unnecessary sequences (Feigner, et ak, Ann NY Acad Sci 126-139, 1995). Formulation of DNA, e.g. with various lipid or liposome materials, may then be effected using known methods and materials and delivered to the recipient mammal. See, e.g, Canonico et al, Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al, Am J Physiol 268; Alton et al., Nat Genet. 5:135-142, 1993 and U.S. patent No. 5,679,647 by Carson et al.

The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ- specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs, which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

The surface of the targeted delivery system may be modified in a variety of ways.

In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand. Naked DNA or DNA associated with a delivery vehicle, e.g ., liposomes, can be administered to several sites in a subject (see below).

Nucleic acids can be delivered in any desired vector. These include viral or non- viral vectors, including adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus). Nucleic acids can be administered in any desired format that provides sufficiently efficient delivery levels, including in virus particles, in liposomes, in nanoparticles, and complexed to polymers.

The nucleic acids encoding a protein or nucleic acid of interest may be in a plasmid or viral vector, or other vector as is known in the art. Such vectors are well-known and any can be selected for a particular application. In one embodiment of the invention, the gene delivery vehicle comprises a promoter and a demethylase coding sequence. Preferred promoters are tissue-specific promoters and promoters which are activated by cellular proliferation, such as the thymidine kinase and thymidylate synthase promoters. Other preferred promoters include promoters which are activatable by infection with a virus, such as the a- and b-interferon promoters, and promoters which are activatable by a hormone, such as estrogen. Other promoters which can be used include the Moloney virus LTR, the CMV promoter, and the mouse albumin promoter. A promoter may be constitutive or inducible.

In another embodiment, naked polynucleotide molecules are used as gene delivery vehicles, as described in WO 90/11092 and U.S. Patent 5,580,859. Such gene delivery vehicles can be either growth factor DNA or RNA and, in certain embodiments, are linked to killed adenovirus. Curiel et ah, Hum. Gene. Ther. 3:147-154, 1992. Other vehicles which can optionally be used include DNA-ligand (Wu et ah, J. Biol. Chem. 264:16985-16987, 1989), lipid-DNA combinations (Feigner et ah, Proc. Natl. Acad. Sci. USA 84:7413 7417, 1989), liposomes (Wang et al., Proc. Natl. Acad. Sci. 84:7851-7855, 1987) and microprojectiles (Williams et al., Proc. Natl. Acad. Sci. 88:2726-2730, 1991).

A gene delivery vehicle can optionally comprise viral sequences such as a viral origin of replication or packaging signal. These viral sequences can be selected from viruses such as astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus, togavirus or adenovirus. In a preferred embodiment, the growth factor gene delivery vehicle is a recombinant retroviral vector. Recombinant retroviruses and various uses thereof have been described in numerous references including, for example, Mann et al., Cell 33:153, 1983, Cane and Mulligan,

Proc. Nat'l. Acad. Sci. USA 81:6349, 1984, Miller et al., Human Gene Therapy 1:5-14, 1990, U.S. Patent Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Application Nos.

WO 89/02,468, WO 89/05,349, and WO 90/02,806. Numerous retroviral gene delivery vehicles can be utilized in the present invention, including for example those described in EP 0,415,731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 9311230; WO 9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88,

1993; Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J. Neurosurg. 79:729-735, 1993 (U.S. Patent No. 4,777,127, GB 2,200,651, EP 0,345,242 and W091/02805).

Other viral vector systems that can be used to deliver a polynucleotide of the invention have been derived from herpes virus, e.g ., Herpes Simplex Virus (U.S. Patent No. 5,631,236 by Woo et al., issued May 20, 1997 and WO 00/08191 by Neurovex), vaccinia virus (Ridgeway (1988) Ridgeway, “Mammalian expression vectors,” In: Rodriguez RL, Denhardt D T, ed. Vectors: A survey of molecular cloning vectors and their uses.

Stoneham: Butterworth,; Baichwal and Sugden (1986) “Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes,” In: Kucherlapati R, ed. Gene transfer. New York: Plenum Press; Coupar et al. (1988) Gene,

68: 1-10), and several RNA viruses. Preferred viruses include an alphavirus, a poxivirus, an arena virus, a vaccinia virus, a polio virus, and the like. They offer several attractive features for various mammalian cells (Friedmann (1989) Science, 244:1275-1281; Ridgeway, 1988, supra; Baichwal and Sugden, 1986, supra; Coupar et al., 1988; Horwich et al.(1990) J. Virol., 64:642-650). In other embodiments, target DNA in the genome can be manipulated using well- known methods in the art. For example, the target DNA in the genome can be manipulated by deletion, insertion, and/or mutation are retroviral insertion, artificial chromosome techniques, gene insertion, random insertion with tissue specific promoters, gene targeting, transposable elements and/or any other method for introducing foreign DNA or producing modified DNA/modified nuclear DNA. Other modification techniques include deleting DNA sequences from a genome and/or altering nuclear DNA sequences. Nuclear DNA sequences, for example, may be altered by site-directed mutagenesis.

IV. Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of an agent that modulates ( e.g ., increases or decreases) selenium, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.

The phrase “therapeutically-effective amount” as used herein means that amount of an agent that modulates (e.g., enhances) selenium, or expression and/or activity of a selenium enzyme complex, or composition comprising an agent that modulates (e.g, enhances) selenium, or expression and/or activity of the complex, which is effective for producing some desired therapeutic effect, e.g, weight loss, at a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable” is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

The term “pharmaceutically-acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that modulates ( e.g ., enhances) selenium, or expression and/or activity of the complex encompassed by the invention.

These salts can be prepared in situ during the final isolation and purification of the agents, or by separately reacting a purified agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, selenium, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (See, for example, Berge etal. (1977) “Pharmaceutical Salts”, J Pharm. Sci. 66:1-19).

In other cases, the agents useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically- acceptable salts with pharmaceutically-acceptable bases. The term “pharmaceutically- acceptable salts” in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of agents that modulates ( e.g ., enhances) selenium, or expression and/or activity of the complex. These salts can likewise be prepared in situ during the final isolation and purification of the agents, or by separately reacting the purified agent in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically- acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al ., supra).

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations useful in the methods of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.

Methods of preparing these formulations or compositions include the step of bringing into association an agent that modulates ( e.g ., increases or decreases) selenium with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a agent with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non- aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a agent as an active ingredient. A compound may also be administered as a bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well-known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions, which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active agent may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more agents with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of an agent that modulates ( e.g ., increases or decreases) selenium include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to a agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an agent that modulates (e.g., increases or decreases) selenium, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

The agent that modulates (e.g, increases or decreases) selenium, can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nonaqueous (e.g, fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound. Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlled delivery of a agent to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the peptidomimetic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the peptidomimetic in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more agents in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form.

Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of an agent that modulates ( e.g ., increases or decreases) selenium, in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.

When the agents of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be determined by the methods of the present invention so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.

The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:30543057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g, retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system

EXAMPLES

Example 1: Facultative Protein Selenation Controls Redox Sensitivity and Enhances Adipose Tissue Thermogenesis

Oxidation of cysteine thiols by physiological reactive oxygen species (ROS) initiates thermogenesis in brown and beige adipose tissues. Cellular selenols, where sulfur is replaced with selenium, exhibit enhanced reactivity with ROS. Despite their critical roles in physiology, methods for broad and direct detection of cellular selenols are lacking. This example demonstrates, in part, developments of a mass spectrometric method to interrogate incorporation of selenols into proteins. Unexpectedly, this approach revealed facultative incorporation of selenium into proteins that have no canonical encoding for selenium- containing amino acids. Selenium was selectively incorporated into regulatory sites on key metabolic proteins, including selenocysteine at locus 253 in UCP1. This facultative utilization of selenium was initiated by increasing cellular levels of organic, but not inorganic, forms of selenium. Remarkably, dietary selenium supplementation elevated facultative incorporation into UCP1 and coincided in elevated energy expenditure through thermogenic adipose tissue, while protecting against obesity. Together, these findings reveal the existence of facultative protein selenation, which impacts thermogenic adipocyte function, and, presumably, other biological processes as well.

Thermogenesis in brown and beige adipose tissues can combat obesity and diabetes, encouraging investigation of mechanisms that control this process. Recently, interventions that modify physiological reactive oxygen species (ROS) levels in BAT and beige fat have demonstrated an important role for redox signaling in thermogenesis in vivo (Chouchani et al. (2016) Nature 532: 112-116; Han et al. (2016) Diabetes 65: 2639-2651; Mills etal. (2018) Nature 560: 102-106; Chouchani etal. (2017) The Journal of Biological Chemistry 292: 16810-16816). Redox signaling by ROS drives activation of thermogenic gene programs (Han etal. (2016) Diabetes 65: 2639-2651), potentiates thermogenic respiration (Chouchani et al. (2016) Nature 532: 112-116; Han et al. (2016) Diabetes 65: 2639-2651; Mills etal. (2018) Nature 560: 102-106), and involves oxidative modification of sensitive cysteine (Cys) thiol residues on protein targets (Chouchani et al. (2016) Nature 532: 112- 116; Han etal. (2016) Diabetes 65: 2639-2651; Mills etal. (2018) Nature 560: 102-106). Selenocysteine (Sec) is one of the rarest proteogenic amino acids and differs from Cys by a single atom, containing a terminal selenium instead of sulfur on its side-chain. This difference renders Sec much more reactive due to a lower side-chain pKa and stronger nucleophilicity, compared to Cys (Hatfield etal. (2014) Trends Biochem Sci 39: 112-120). Sec incorporation into proteins is thought to be limited to the so-called selenoproteins, approximately 25 proteins in which Sec is exclusively encoded at a particular site by co- translational cis- and /ra//.s-acting elements (Labunskyy et al. (2014) Physiol Rev 94: 739- 777). In this small number of mammalian selenoproteins, Sec typically exists in enzymatic domains to facilitate catalytic redox reactions. The physiological action of these selenoproteins depends strictly on the presence of Sec, while mutagenesis to Cys leads to decreased or abrogated functionality (Labunskyy et al. (2014) Physiol Rev 94: 739-777). Importantly, the breadth of established selenoproteins has been determined strictly on the basis of specific nucleotide sequences in co-translational cis-elements, so-called SECIS nucleotide sequences.

Because of the enhanced redox sensitivity encoded by Sec, and the importance of redox signaling in adipose tissue thermogenesis, disclosed here are investigations into whether selenoprotein status modifies thermogenic function in BAT. To begin examining this possibility, a targeted mass spectrometry (MS) approach was developed to identify selenium insertion into proteins. In addition to identifying established obligate selenoproteins, this approach unexpectedly revealed heterogeneous but non-random incorporation of selenocysteine and selenomethionine into other proteins. These newly found selenation events are herein termed “facultative,” as they are not added to the entire pool of individual proteins and are not required for basic protein functionality. These facultative sites were found to increase in response to elevating selenomethionine levels. Sites where this heterogeneous incorporation was observed included the regulatory 253 locus of UCP1, a major effector of adipose tissue thermogenesis. This unexpected evidence of Sec incorporation into UCP1 was indicative of a distinct pool more sensitive to redox modification than the corresponding cysteine. Moreover, it was found that increasing protein selenation through dietary intervention coincided with significant elevation in whole body energy expenditure driven by thermogenic adipose tissue, and protection from obesity. Results

A mass spectrometric method for identification of selenocysteine-containing proteins

Recent examinations of redox regulation of adipose tissue thermogenesis have relied on the use of quantitative mass spectrometry (MS) to identify redox sensitive regulatory cysteine residues (Chouchani et al. (2016) Nature 532: 112-116). Selenocysteine enhances protein redox sensitivity, but the role of selenium status in thermogenesis has not been systematically investigated. Although systematic methods for assessment of selenocysteine containing peptides by MS have not been widely employed, recent studies have shown that these species should be amenable to detection using standard peptide preparations, and that selenium could potentially be more broadly incorporated into proteins than previously appreciated (Bak et al. {20\%) Cell Chem Biol 25 1157-1167 el 154; Guo etal. (2018) Cell Chem Biol 25: 1380-1388 el384). To test this, the mouse brown fat proteome was analyzed for modifications corresponding to the mass of selenocysteine. As expected, this approach successfully identified peptides corresponding to well-established selenoproteins ( e.g ., Gpx 1; Fig. 1A - Fig. 1C). Unexpectedly, also identified were a number of cysteine- encoded peptides with additional mass corresponding exactly to the mass difference between Cys and Sec. An example of this was identified on the UCP1 tryptic peptide containing Cys253, which was identified as a second form shifted in mass by +47.94445 Da (Fig. 2A). This mass shift was observed exclusively for the Cys253 peptide and was never observed in the other six UCP1 Cys-containing tryptic peptides (Fig. 3A - Fig. 3L). This intriguing finding led to the examination of whether a portion of the total pool of UCP1 and other proteins may facultatively incorporate Sec at particular cysteine sites.

First, a targeted-MS approach was developed for determination of Sec-containing peptides. Sec reacts efficiently and irreversibly with thiol derivatizing agents, such as N- ethylmaleimide (NEM) or iodoacetamide (JAM), which are typically used in redox- proteomic workflows (Fenyo and Beavis (2016) J Proteome Res 15: 677-678). Therefore, in principle, Sec containing peptides can be readily identified by a distinct mass shift corresponding to the mass of Sec + the covalent conjugate of the derivatizing agent (Fenyo and Beavis (2016) J Proteome Res 15: 677-678; Galano et al. (2013 Molecular & Cellular Proteomics: MCP 12: 2196-2204). Also, selenium exhibits a distribution of naturally occurring stable isotopes not found in any other element. In addition to the most abundant isotope, Selenium-80 (-50% total), four stable isotopes exhibit sufficient natural abundance to be distinguished by MS, thus generating a unique isotopic signature (Fig. 1A). Therefore, a peptide containing bona fide cysteine-to-selenocysteine replacement would exhibit the following distinct features upon MS analysis. They must have a fragment spectrum mass attributable to Sec at a Cys locus (Fig. 2B, bottom) that can be shifted in mass by derivatization with Sec-reactive nucleophiles to the mass of Sec + the derivatizing agent (Fig. 2B, middle). Additionally, this peptide must exhibit the unique Sec mass isotope fingerprint (Fig. 2B, top).

The analytic framework described above was first validated by MS analysis of GPxl, a classic selenoprotein that is abundant in mouse BAT. As expected, the Gpxl Sec site was readily identified by a unique mass shift and fragment spectrum attributable to Sec derivatization with NEM (Fig. 1A). The expected Sec mass isotope fingerprint was also observed (Fig. 1A). This approach was next applied to targeted MS profiling of BAT UCP1. MS identified UCP1 peptides containing six of seven UCP1 cysteine residues in the protein following derivatization with NEM (Fig. 3A - Fig. 3L). Uniquely, a UCPl-Sec253 peptide was identified that exhibited the molecular features unique to Sec-containing tryptic peptides. The major precursor ion mass corresponded to Sec alkylated with NEM (Fig.

2C), indicating Sec-derivatization. Moreover, this precursor peptide mass exhibited the unique selenium isotope fingerprint (Fig. 2C). Additionally, high accuracy peptide fragment ions demonstrated that the Sec mass addition was localized to the 253 position (Fig. 2D). As the UCP1 peptide spanning position 253 contains two methionines, the form of this peptide containing oxidized methionines can additionally be present. Importantly, the UCPl-Sec253 peptide was additionally identified in the methionine oxidized form.

This peptide also exhibited all key unique features of a Sec replacement of Cys (Fig. 2E and Fig. 2F). Notably, all of the Sec-specific features described for these peptides were not present in the Cys form of the UCP 1-253 peptide (Fig. 3A - Fig. 3L).

Validation and quantification of UCPl-Sec253

To confirm the identity of the putative UCP1 selenium-containing peptide, a synthetic peptide identical to the endogenous UCP1 Cys253 peptide with a selenocysteine replacement at position 253 and an isotopically heavy tyrosine was generated (AQUA peptide - +10.0272 Da; Fig. 4A and Fig. 4B). This molecule would allow for identification and quantification of the endogenous form. To determine the presence of UCP 1 Sec253 in the brown fat proteome, the AQUA standard was derivatized with a distinct isobaric TMT reporter. In this way the AQUA peptide would allow for endogenous peptide identification by multiplexing (Fig. 4D). IT was found that the endogenous UCP1 peptide displayed identical physiochemical properties as the AQUA standard, demonstrating unequivocally the presence of selenocysteine in position 253 of UCP1 (Fig. 4D).

Use of the AQUA internal standard allowed for estimation of the stoichiometry of the selenation replacement of cysteine at position 253. The stoichiometry of Selenation was examined in BAT of WT male mice under standard housing conditions. In this case, selenium incorporation was estimated at — 4-5% of total UCP1 (Fig. 5A). So, a small but substantial proportion of UCP1 exists in the selenated form.

It was found that UCP1 Cys253 can become oxidatively modified to a sulfenic acid upon activation of thermogenesis in vivo, a physiological stimulus that increases ROS levels in BAT. Moreover, based on mutagenesis and pharmacological manipulation of this residue, modification of this site is sufficient to affect UCP1 function. Selenocysteine exhibits extraordinary sensitivity to oxidative modification compared to cysteine, so whether selenated UCP1 was more sensitive to oxidative modification was examined. Remarkably, it was found that under baseline conditions when Cys253 is predominantly unmodified, Sec253 is substantially oxidized to a selenic acid (Fig. 5B). So, the Sec253 form of UCP1 represents a substantial and distinct pool of the protein that is sensitive to oxidation.

Facultative incorporation of selenium into proteins occurs via a non-canonical pathway

Next, how selenium could be incorporated into proteins such as UCP1 whose sequence contains no apparent coding for Sec incorporation via established co-translational pathways was examined. Co-translational incorporation of selenium as selenocysteine is facilitated by a distinct tRNA charging mechanism that utilizes inorganic selenite as the source for selenium. This mode of incorporation can be observed by protein autoradiography following supplementation with ⁷⁵ -selenite. ⁷⁵ -selenite treated brown adipocytes exhibited robust labeling of proteins with molecular masses corresponding to well-established selenoproteins, but no signal was observed at the molecular mass corresponding to UCP 1 (Fig. 5C). Despite this, Sec253 incorporation in UCP1 was readily apparent by MS of the brown adipocyte protein, suggesting that facultative Sec incorporation occurs independent of this co-translational pathway.

To test this further, primary brown adipocytes were treated with increasing concentrations of sodium selenite throughout differentiation, when UCP 1 protein is generated de novo at high levels. As expected, sodium selenite increased the abundance of classical selenoproteins for which Sec incorporation occurs co-translationally (Fig. 4E). However, under these conditions, sodium selenite had no effect on the levels of UCP 1- Sec253 (Fig. 5D). Next, whether other forms of selenium may contribute to facultative incorporation into proteins was considered. Organic forms of selenium are a major source of selenium and can be generated from selenite by systemic metabolism. Among the most prevalent forms of organic selenium is selenomethionine. When brown adipocytes were supplemented with selenomethionine throughout differentiation, the proportion of the Sec253 form of UCP 1 increased substantially (Fig. 5E). Interestingly, this was not observed when cells were supplemented with the other major organic form of selenium, selenocysteine (Fig. 5F).

Importantly, the tryptic peptide of UCP 1 that contains Sec253 also contains two methionines. Therefore, the observed increase in selenation of this peptide following selenomethioine treatment could be attributable to selenomethionine incorporation instead of selenocysteine. To distinguish between these two possibilities, the fragment spectra of the selenated UCP 1 tryptic peptide was examined. By examining the b and y ions spanning the Cys and Met locus, it was found that the selenium addition was attributable exclusively to position 253 (Fig. 4E).

Dietary selenium supplementation modifies the status of both obligatory selenoproteins and facultative selenocysteine sites in brown adipose tissue

Modulation of the classic selenoproteins can be readily achieved by dietary supplementation of sodium selenite (Novoselov et al. (2005) Oncogene 24: 8003-8011). Dietary selenite is assimilated from the diet and converted by endogenous metabolism to generate organic selenium-containing metabolites including selenomethionine and selenocysteine. The mechanisms through which this occurs are not clear but appear to involve utilization by multiple tissues including liver and the gut. Interestingly, there is evidence that dietary selenium status may play a role in modulating energy homeostasis, but the mechanisms that underlie this are unclear. Moreover, there has been no systematic determination of selenium incorporation into adipose tissue proteins following modulation of dietary selenium. Therefore, diets containing defined amounts selenite were made use of to investigate this (Novoselov et al. (2005) Oncogene 24: 8003-8011). A selenium- deficient diet (<0.03 ppm selenium) is below the minimal threshold to generate basal levels of obligate selenoproteins. 0.1 ppm selenium corresponds to the daily recommended intake of selenite and the minimal amount required to achieve maximal expression of obligate selenoproteins (Novoselov et al. (2005) Oncogene 24: 8003-8011; Burk (2002) Nutr Clin Care 5: 75-79). 0.4 ppm selenite corresponds to the common dose used in clinical studies (Novoselov et al. (2005) Oncogene 24: 8003-8011; Clark et al. (1996) JAMA 276: 1957- 1963). 2.25 ppm selenite corresponds to a safe high level, and well above the amount required for maximal obligate selenoprotein synthesis (Novoselov etal. (2005) Oncogene 24: 8003-8011). Diets were provided to mice for 8 weeks prior to isolation of BAT and analysis by MS.

As expected, we found that expression of obligate selenoproteins in BAT depended on dietary selenite, and expression was saturated by dietary supplementation between 0.1 and 0.4 ppm selenite (Fig. 6A). MS was additionally used to examine the breadth of facultative selenium incorporation across the BAT proteome. Using the criteria described in Fig. 2B, several apparent facultative selenocysteine sites were observed (Fig. 6B). These findings suggest that selenium is incorporated more broadly into proteins than previously appreciated. Remarkably, across four biological replicate experiments facultative protein selenation was highly specific to certain residues, indicating non-random incorporation. Most of these sites were localized within core metabolic proteins such as aconitase, malate dehydrogenase, and fatty acid synthase (Fig. 6B). Notably, some of these sites have established functional roles in controlling protein function. For example cysteine 451 in mitochondrial aconitase is critical for Fe-S coordination, enzymatic activity, and is known to be subject to redox modification (Temette et al. (2013) Cell Reports 3: 689-700). Numerous other sites are not known to be sites of redox regulation, but the reproducible and specific facultative selenation signature suggests that these sites may be previously unappreciated regulatory sites.

Remarkably, numerous instances of selective, non-random incorporation of selenium as selenomethionine into methionine encoding loci were also identified (Fig. 6C). The majority of targets of selenomethionine insertion were also major metabolic enzymes and fatty acid handing proteins. Moreover, some proteins, such as FABP4, exhibited separate instances of selenocysteine and selenomethionine incorporation (Fig. 6B and Fig. 6C). Next, whether transcripts encoding proteins that contain facultative selenocysteine and selenomethionine sites exhibited any structural features suggestive of co-translational regulatory elements was examined.

Dietary selenium status regulates adipose tissue thermogenesis and modifies obesogenesis

Notably, between 0.1 and 0.4 ppm dietary selenium was sufficient to maximize selenoprotein expression in BAT. However, through quantification of UCPl-Sec253 it was found that selenation of this site was elevated only by 2.25 ppm selenite (Fig. 6D). Therefore, the threshold for obligate selenoprotein synthesis and facultative selenation of UCP1 appear to be distinct.

Based on the analysis of adipose tissue protein selenation following dietary selenium modification, the findings allow distinguishing between the following physiological states. 1) low total protein selenation with low levels of obligate selenoproteins and low facultative UCP1 selenation driven by < 0.03 ppm dietary selenium (Fig. 6A and Fig. 6C); 2) high levels of obligate selenoproteins and low levels of facultative UCP1 selenation driven by 0.1-0.4 ppm dietary selenium (Fig. 6A and Fig. 6C); 3) high levels of obligate selenoproteins and high levels of facultative UCP1 selenation driven by 2.25 ppm dietary selenium (Fig. 6A and Fig. 6C). Under these conditions, whether modification of obligate or facultative selenium status affected metabolic physiology regulated by thermogenic adipose tissue was examined.

Following 8 weeks supplementation with defined selenium diets (Fig. 7A - Fig. 7C), mice were subjected to acute pharmacological stimulation of BAT and beige adipose tissue thermogenesis using the P3-agonist CL-316243 (CL; Fig. 7D - Fig. 7F). Remarkably, mice on the 2.25 ppm selenium diet exhibited enhanced b3 -adrenoreceptor stimulated oxygen consumption and CO2 release compared to all other groups (Fig. 7D - Fig. 7F). Interestingly, no difference in energy expenditure was observed between the selenium depleted group and 0.1 ppm and 0.4 ppm supplemented groups (Fig. 7D - Fig. 7F). Together, these data suggest that modification of obligate selenoprotein status do not affect BAT and beige thermogenesis. However, further elevation of selenium status at 2.25 ppm, that coincides with elevated facultative UCP1 selenation, enhances thermogenesis (Fig. 7 A - Fig. 7F).

Since elevating dietary selenium was sufficient to potentiate BAT thermogenesis by b3 -adrenoreceptor agonism, whether this intervention could modify the physiological response to high fat feeding was explored. These were compared: 1) mice ingesting high fat diet (HFD) supplemented with 0.1 ppm dietary selenium, a level sufficient for expression of selenoproteins in BAT (Fig. 6A) and no effect on BAT thermogenesis (Fig. 7D - Fig. 7F); and 2) mice ingesting high fat diet (HFD) supplemented with 2.25 ppm dietary selenium, a level sufficient to elevate facultative UCP1 selenation in BAT (Fig. 6C) and stimulate BAT thermogenesis (Fig. 7D - Fig. 7F). Remarkably, mice on 2.25 ppm containing selenium diets exhibited robust protection against weight gain upon high fat feeding over 14 weeks (Fig. 7G and Fig. 7H). This effect was specifically attributable to a decrease in fat mass (Fig. 71) Importantly, this protection against obesity occurred despite no detectable changes in food intake (Fig. 7J).

Discussion

In humans, elevated dietary selenium intake has been linked to protection against obesity and diabetes (Wang et al. (2016) Nutrients 8; Wang et al. (2017) PLoS One 12: e0174149), although the underlying mechanisms remain unknown. Because of the established role of selenium as a regulator of redox and ROS metabolism, here its role in regulating thermogenic adipose tissue function in mice was systematically investigated. Remarkably, it was found that modification of selenium status has robust and substantial effects on energy expenditure and adipose tissue thermogenesis. These effects coincide with previously unappreciated modes of selenium utilization in thermogenic adipose tissue. Specifically, shown here is that selenium is selectively encoded into redox sensitive sites of a new class of facultative (as opposed to obligate) selenoproteins. The exemplar is UCP1, which as shown here can incorporate Sec at position 253 as an alternative to Cys. Also demonstrated is that UCPl-Sec253 is highly sensitive to redox modification, and that increasing the pool of this type of facultative selenoprotein coincides with BAT-dependent energy expenditure in vivo. In addition to UCP1, other major metabolic proteins display signatures indicative of selenation at either cysteine or methioinine. It is therefore likely that physiological effects attributable to facultative protein selenation are due to effects on many of these proteins. Taken together, these findings suggest a novel paradigm for encoding of enhanced redox sensitivity and functionality into proteins. Moreover, they demonstrate that modification of selenium status in the diet could be a relatively simple means of enhancing thermogenic function in adipose tissues and modifying obesity.

Example 2: Methods for Example 1

Animal experiments

Animal experiments were performed according to procedures approved by the Institutional Animal Care and Use Committee of the Beth Israel Deaconess Medical Center. Unless otherwise stated, mice used were male C57BL/6J (8-12 weeks of age; Jackson Laboratories), and housed in a temperature-controlled (23 °C) room on a 12-h light-dark cycle. Primary brown adipocyte preparation and differentiation

Interscapular brown adipose stromal vascular fraction was obtained from 2 to 6 day old pups as described previously (Mills et al. (2018) Nature 560: 102-106). Interscapular brown adipose tissue was dissected, washed in PBS, minced, and digested for 45 min at 37 °C in PBS containing 1.5 mg/mL collagenase B, 123 mM NaCl, 5 mM KC1, 1.3 mM CaCh, 5 mM glucose, 100 mM HEPES, and 4% essentially fatty acid free BSA. Tissue suspension was filtered through a 40pm cell strainer and centrifuged at 600g for 5 min to pellet the SVF. The cell pellet was resuspended in adipocyte culture medium and plated. Cells were maintained at 37 °C in 10% CO2. Primary brown preadipocytes were counted and plated in the evening. The following morning, brown preadipocytes were induced to differentiate for 2 days with an adipogenic cocktail (1 mM rosiglitazone, 0.5 mM IB MX, 5 pM dexamethasone, 0.114 pg/mL insulin, 1 nM T3, and 125 pM indomethacin) in adipocyte culture medium. Two days after induction, cells were refed every 48 h with adipocyte culture medium containing 1 pM rosiglitazone, 1 nM T3, and 0.5 pg/mL insulin. Cells were fully differentiated by day 7 after induction.

Sample preparation for mass spectrometry

Samples were homogenized in 50 mM Tris base, containing 100 mM NaCl, 100 pM DTP A, 0.1% SDS, 0.5% sodium deoxycholate, 0.5% TritonX 100, 10 mM TCEP and 50 mM iodoacetamide or 50 mM NEM depending on the experiment. Following incubation for 15 min, SDS was added to a final concentration of 1% and samples were incubated for a further 15 min.

Protein digestion

Protein pellets were dried and resuspended in 8 M urea containing 50 mM HEPES (pH 8.5). Protein concentrations were measured by BCA assay (Thermo Scientific) before protease digestion. Protein lysates were diluted to 4 M urea and digested with LysC (Wako, Japan) in a 1/100 enzyme/protein ratio overnight. Protein extracts were diluted further to a 1.0 M urea concentration, and trypsin (Promega) was added to a final 1/200 enzyme/protein ratio for 6 h at 37 °C. Digests were acidified with 20 pi of 20% formic acid (FA) to a pH ~2, and subjected to C18 solid phase extraction (50 mg SepPak, Waters). LC — MS/MS parameters for peptide analysis

All spectra were acquired using an Orbitrap Fusion mass spectrometer (Thermo Fisher) in line with an Easy nLC 1000 (Thermo Fisher Scientific) ultrahigh pressure liquid chromatography pump. Peptides were separated onto a 100-mM inner diameter column containing 1 cm of Magic C4 resin (5 pm, 100 A, Michrom Bioresources) followed by 30 cm of Sepax Technologies GPC18 resin (1.8 pm, 120 A) with a gradient consisting of 9- 30% (ACN, 0.125% FA) over 180 min at -250 nl min ¹ . For all LC-MS/MS experiments, the mass spectrometer was operated in the data dependent mode. We collected MSI spectra at a resolution of 120,000, with an AGC target of 150,000 and a maximum injection time of 100 ms. The ten most intense ions were selected for MS2 (excluding 1 Zions).

MSI precursor ions were excluded using a dynamic window (75 s ± 10 ppm). The MS2 precursors were isolated with a quadrupole mass filter set to a width of 0.5 Th. For the MS3 based TMT quantitation, MS2 spectra were collected at an AGC of 4,000, maximum injection time of 200 ms, and CID collision energy of 35%. MS3 spectra were acquired with the same Orbitrap parameters as the MS2 method except HCD collision energy was increased to 55%. Synchronous precursor selection was enabled to include up to six MS2 fragment ions for the MS3 spectrum.

Assessment of protein thiol sulfenic acids

BAT was prepared adapting a protocol used previously to stabilize endogenous protein sulfenic acids (Klomsiri et al. (2010) Methods Enzymol 473: 77-94). Briefly, samples were homogenized in 50 mM Tris base, containing 100 mM NaCl, 100 pM DTP A, 0.1% SDS, 0.5% sodium deoxycholate, 0.5% Triton-X 100, 5 mM dimedone. To minimize lysis-dependent oxidation, buffers were bubbled with argon before use. Samples were incubated for 15 min at room temperature, at which point SDS was added to a final concentration of 1% and samples were incubated for a further 15 min. After dimedone treatment, 10 mM TCEP and 50 mM NEM were added and samples were incubated for a further 15 min at 37 °C to reduce and alkylate all non-sulfenic acid protein cysteine residues. Samples were then subjected to protein digestion and LC-MS/MS as described above.

Determination of selenocvsteine and selenomethionine containing peptides

A compendium of in-house developed software was used to convert mass spectrometric data (Raw file) to the mzXML format, as well as to correct monoisotopic m/z measurements (Elias and Gygi (2007) Nat Methods 4: 207214). All experiments used the Mouse UniProt database (downloaded March 2019) where reversed protein sequences and known contaminants such as human keratins were appended. SEQUEST searches were performed using a 50 ppm precursor ion tolerance, while requiring peptide amino/carboxy (N/C) terminus to have trypsin protease specificity and allowing up to two missed cleavages. Ten-plex TMT tags on peptide N termini and lysine residues (+ 229.162932 Da) and carbamidomethylation (+57.02146 Da) or N-ethylmaleimide (+ 125.047679) of cysteine residues were set as static modifications while methionine oxidation (+ 15.99492 Da) and selenium replaces sulfur (+ 47.9444) set as variable modifications. A MS ² spectra assignment false discovery rate (FDR) of less than 1% was achieved by applying the target- decoy database search strategy (Elias and Gygi (2007) Nat Methods 4 : 207214). Filtering was performed using an in-house linear discrimination analysis (LDA) method to create one combined filter parameter from the following peptide ion and MS ² spectra metrics: SEQUEST parameters XCorr and ACn, peptide ion mass accuracy and charge state, in solution charge of peptide, peptide lerigth and mis-cleavages. Linear discrimination scores were used to assign probabilities to each MS ² spectrum for being assigned correctly and these probabilities were further used to filter the dataset with an MS ² spectra assignment FDR of smaller than a 1% at the protein level (Huttlin et al. (2010) Cell 143: 1174-1189).

Quantification of UCP1 Sec-253 peptide using heavy standard AQUA peptides AQUA peptides employed in this study:

AQUA Peptide / Sequence / Mass (Da) Heavy UCP1 240-261/ FINSLPGQYPSVPSU*AMSMYTK / 2477.20 UCP1 240-261 / FINSLPGQYPSVPSCAMSMYTK / 2430.149 U* = Selenocysteine Y = Heavy Tyrosine 10.027228

Skyline version 19.1.0.193 was used to quantify absolute and relative amounts of UCP1 253 selenocysteine and cysteine in all experiment with heavy AQUA peptides used as a reference (MacLean et al. (2010) Bioinformatics 26: 966-968).

Determination of RNA structural features in facultatively selenated proteins Determination of selenite incorporation using sodium ⁷⁵ -selenite Sodium selenite dietary intervention and high-fat feeding

All mouse dietary and high-fat feeding experiments were performed with age matched littermate controls. At eight weeks of age, mice were switched to chow or high-fat diet containing defined amounts of sodium selenite. Dietary intervention proceeded for 8 weeks prior to molecular analyses.

Body composition analysis Body composition was examined with Echo MRI (Echo Medical Systems, Houston,

Texas) using the 3inl Echo MRI Composition Analyzer.

Metabolic phenotyping

Whole-body energy metabolism was evaluated using a Comprehensive Lab Animal Monitoring System (CLAMS, Columbia Instruments). CO2 and O2 levels were collected every 30 s. CL 316,243 (Sigma-Aldrich; 1 mg kg ¹ ) was injected i.p. into mice at the indicated times.

Incorporation by Reference All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.