DESCRIPTION

METHODS AND COMPOSITIONS COMPRISING CHIMERIC AND FULL- LENGTH HUMAN ACETYL-CoA CARBOXYLASE

BACKGROUND OF THE INVENTION

This application claims priority to U.S. Provisional Patent Application Serial Number 60/685,283, filed May 27, 2005, the entire contents of which is hereby specifically incorporated by reference in its entirety.

The United States Government owns rights in the present invention pursuant to National Institute of Diabetes & Digestive & Kidney Diseases STTR Grant Number DK67716.

1. Field of the Invention

The present invention relates generally to the fields of molecular biology, protein chemistry, and enzymology. More particularly, it concerns conjugates comprising a non- human acetyl-CoA carboxylase (ACC) amino acid sequence and a human ACC amino acid sequence, and nucleic acid sequences encoding these conjugate ACC sequences. The present invention also pertains to yeast cells comprising a nucleic acid sequence encoding a full- length human ACC amino acid sequence, and methods of identifying modulators of a human ACC that involve contacting a candidate substance to such a yeast cell.

2. Description of Related Art Acetyl-CoA carboxylase (ACC) catalyzes the ATP-dependent carboxylation of acetyl-

CoA to malonyl-CoA, a key intermediate in fatty acid synthesis (Roos et al, 1999; Wilson and Williamson, 1997; Gleeson, 2000). The enzyme consists of three major functional domains: the biotin carboxylase (BC) domain, the carboxyltransferase (CT) domain, and the biotin carboxyl carrier (BCC) domain containing covalently attached biotin. The first step of the ACC-catalyzed reaction is an ATP-dependent transfer of the carboxyl group from bicarbonate to the biotin residue. The carboxyl group is then transferred to acetyl-CoA, producing malonyl-CoA. Malonyl-CoA is used for de novo fatty acid biosynthesis, for regulation of fatty acid oxidation, and for malonylation reactions..

There are two types of ACC: prokaryotic ACC in which the three functional domains: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP) and carboxyltransferase (CT) are located on separable subunits (e.g., E. coli, P. aeruginosa, Anabaena, Synechococcus), and eukaryotic ACC in which all of the domains are located on one large polypeptide (e.g., rat, chicken, yeast, diatom, wheat, Toxoplasma gondii, and human).

Yeast, rat, chicken and human ACCl are cytoplasmic enzymes consisting of 250- to 280-kDa subunits. Animal ACC activity varies with the rate of fatty acid synthesis or energy requirements in different nutritional, hormonal, and developmental states.

In humans, two isoforms (ACCl and ACC2) display distinct tissue distribution and are encoded by separate genes. These isozymes display a very similar amino acid sequence over most of their length (~2400 amino acids), differing mainly by a 200-residue N-terminal extension on ACC2 that directs this form of the enzyme to mitochondria. ACCl is the primary isoform in lipogenic tissues such as liver, adipose tissue, and lactating mammary gland, whereas ACC2 is highly expressed in the skeletal muscle and heart (Barber et al, 2004). In lipogenic tissues, malonyl-CoA is the source of carbon units used in fatty acid synthesis. Malonyl-CoA, which inhibits carnitine palmitoyltransferase I (CPT I), is also considered a key regulator of fatty acid oxidation and energy metabolism (McGarry and Brown, 1997). Therefore, the activity of ACC2 contributes to reduction of fatty acid oxidation and increased storage of fat. ACCl, on the other hand, is an essential enzyme responsible for fatty acid synthesis primarily in the liver and mammary glands. Mammals cannot live without it - inactivation of the ACCl gene in mice is lethal (Abu-Elheiga et al, 2005). The level of malonyl-CoA is also controlled by malonyl-CoA decarboxylase.

It has been shown that a decrease in the level of malonyl-CoA during starvation or exercise is accompanied by increased fatty acid oxidation, but that in the fed state, increased malonyl CoA concentration results in lower fatty acid oxidation (Ruderman et al, 2003). Sustained elevation of malonyl-CoA concentration results in insulin resistance in muscle in a wide variety of hyperglycemic and/or hyperinsulinemic animals (Ruderman et al, 1999; Saha et al, 1994). In addition, elevated levels of malonyl-CoA result in elevated levels of triglyceride, diacylglycerol, and long-chain fatty acyl-CoA (McGarry, 2002). Recent reviews address a wide scope of relevant ACC-related questions including complex regulation of the animal isozymes, their possible role in hypothalamic sensing, cancer development, structural aspects of ACC inhibition by known inhibitors and the potential of ACC as a target for new pharmacotherapies (Barber et al, 2005; Dowell et al, 2005; Harwood et al, 2004; Harwood et al, 2005; Shi and Burns, 2004; Tong, 2005).

Enzymes of the fatty acid biosynthetic pathway, such as ACCase, are promising targets for inhibitors that could be used to treat various conditions. Obesity is a complex human malady, due literally to dozens of factors, some genetic, some environmental. Obesity results when more calories are consumed and stored as fat than are oxidized. Generally speaking, diet determines how many calories are taken in and physical activity (exercise) determines how many are burned, but there are other contributing factors, such as the basic metabolic rate and thermogenesis, both of which are genetically controlled by the interplay of hormones, receptors and signal cascades. There is a strong connection among diabetes, obesity, and ACC. There is evidence that malonyl-CoA probably also plays an important role in hypothalamic sensing of the energy and metabolite balance, and control of feeding behavior (Dowell et ah, 2005). Furthermore, up-regulated lipogenesis found in many tumors, e.g., breast and prostate cancers, makes exploration of the enzymes of the fatty acid pathway (including ACC) as targets of new pharmacotherapies worthwhile (Baron et ah, 2004; Brusselmans et ah, 2005). To identify new drugs for treatment of any of the conditions discussed above, one needs compounds that inhibit ACC and do nothing else. Because ACCl and ACC2 produce two separate pools of malonyl Co-A with dramatically different functions, isozyme-specific inhibitors are highly desirable.

It has been shown that aryloxyphenoxyproprionates (fops) inhibit Toxoplama gondii growth but not human host cells (Zuther et ah, 1999), and that the carboxyltransferase domain of the apicoplast T. gondii ACC is the target for this class of inhibitors (Jelenska et ah, 2002). In Jelenska et ah, the studies were conducted using yeast gene replacement strains depending for growth on the expressed T. gondii ACC derived by complementation of a yeast ACCl null mutation (Jelenska et ah, 2002). ACC has also been recognized as a potential target for therapeutic intervention in metabolic diseases, such as diabetes and obesity (Ruderman et ah, 1999; Abu-Elheiga et ah, 2001; Kim et ah, 2000, Abu-Elheiga et ah, 2003). It has also been shown that the level of malonyl-CoA in the heart decreases in response to ischemia (Dyck and Lopaschuk, 2002). This has led to a proposal to treat angina pectoris and acute myocardial infarction by targeting the enzymes controlling malonyl CoA. Targeting ACC inhibition has also been proposed in the treatment of metabolic syndrome, defined as a clustering of cardiovascular risk factors (abdominal obesity, hyperinsulinemia, atherogenic dyslipidemia, hypertension and hypercoagulability) that together increase the risk of developing coronary heart disease and type 2 diabetes (Harwood, 2004).

Inhibition of fatty acid synthase has been found to induce apoptosis in human breast cancer cells in vitro and in vivo without toxicity to proliferating normal cells. Elevation of malonyl-CoA has been implicated in this mechanism of apoptosis, and it has been found that the effect can be reproduced by simultaneous inhibition of carnitine palmitoyltransferase-1 and ACC, thus implicating ACC as a potential target for cancer chemotherapy (Thupari et al., 2001).

The current arsenal of small molecule inhibitors of human ACC is very limited, and most of these inhibitors are not promising as lead compounds for drug development (Harwood, 2004; Harwood, 2005; Lenhard and Gottschalk, 2002; Munday and Hemingway, 1999). Harwood et al. (2003) have reported that isozyme-nonselective N-substituted bipiperidylcarboxamide acetyl-CoA carboxylase inhibitors reduce tissue malonyl-CoA concentrations, inhibit fatty acid synthesis, and increase fatty acid oxidation in cultured cells and in experimental animals. MEDICA 16 (β,β'-tetramethyl-substituted hexadecanoic acid) and TOFA [5-(tetradecyloxy)-2-furoic acid] esterified with CoA make potent analogs of palmitoyl-CoA, which inhibits ACC by promoting depolymerization of the enzyme aggregates, which are the active form. However, these compounds also inhibit fatty acid synthase, the next step in the fatty acid biosynthetic pathway utilizing malonyl-CoA made by ACCl. The allosteric inhibition of ACC by TOFA proposed above may not be its real mechanism of action. A recent report describes the reduction of ACC activity in rat hepatocytes by the addition of 5-iodotubercidin (Garcia- Villafranca and Castro, 2002). Iodotubercidin is an adenosine analog and an inhibitor of adenosine kinase, also possibly an inhibitor of AMP- dependent protein kinase - one might expect iodotubercidin to increase ACC activity, but the opposite was observed. A problem with all of these inhibitors of ACC is their potential for binding to PP ARa (peroxisome proliferative activated receptor), a transcription factor that forms dimers with other factors, such as the retinoic acid receptor, and binds to elements such as the peroxisome proliferator response element (PPRE) in the promoter regions of target genes. These target genes are all involved with lipid metabolism. The transcription complexes can either promote or repress transcription. The natural ligands for the complexes are fatty acids or their derivatives, signaling a need for more or less fat metabolism. It is not yet known whether ACC gene transcription is regulated by these same complexes, but the point here is that the compounds that might be useful in regulating the activity of ACC2 will also interfere with the functioning of PPARα and therefore they will be too dangerous to use as drugs for obesity. Other inhibitors of human ACC have been reported recently: pyridoxal

phosphate (Lee et al, 2005), biotin analogs (Levert and Waldrop, 2002; Levert et al, 2002), sethoxydim (a strong herbicide targeting grass ACC) (Seng et al, 2003), and N-substituted bipiperidylcarbamides (Harwood et al, 2003).

Only the bipiperidylcarbamides (e.g., CP-640186) inhibit rat ACC with sub-micro molar IC ₅₀ leading to decreased malonyl-CoA concentration, decreased fatty acid synthesis and increased fatty acid oxidation in cultured animal cells and in whole animals (Harwood et al, 2003). It is not yet clear if CP-640186 and its homologs can be used as effective drugs against obesity and related health risks. These compounds are not isozyme selective, since both ACCl and ACC2 from rat are inhibited with similar IC ₅₀ . The structure of CP-640186 binding sites in human ACC can be deduced from x-ray crystallographic studies of the complexes of these compounds with the CT domain of yeast ACC (Zhang et al, 2004). The molecular mechanism of action differs among the ACC inhibitors described above, suggesting that different domains and functions of the ACC can be targeted.

Several tests have been described to assay or measure the ability of candidate substances to inhibit ACC. These tests include biochemical methods based on reaction kinetics (Thampy and Wakil, 1985; U.S. Patent 6,153,374), a monoclonal-based ELISA (Webb and Hall, 2000), a polyclonal-based ELISA (Webb and Hall, 2001), methods of using the crystal structure of the carboxyltransferase domain of ACC (U.S. Patent App. Pub. No. 20050009163), and assay methods that involve peptides comprising an ACC having a deleted biotin binding domain (U.S. Patent App. Pub. No. 20040086994). However, these tests are of limited value because they are laborious and expensive.

Therefore, there is the need for modulators of human ACC and novel methods of identifying modulators of human ACC. Modulators of ACC can be applied in the treatment and prevention of metabolic conditions, such as diabetes, obesity, cardiovascular disease, and cancer.

SUMMARY OF THE INVENTION

The inventors have identified novel chimeric nucleic acid sequences encoding part of a non-human ACC amino acid sequence and part of a human ACC amino acid sequence, and nucleic acid sequences expressing active proteins comprising part of a non-human ACC amino acid sequence and part of a human ACC amino acid sequence. They have also identified novel methods of identifying modulators of human ACC that involve yeast cells encoding these novel nucleic acid sequences. For example, they have created genetically- engineered yeast gene-replacement strains that depend for their growth on a chimeric ACC that includes the BC and BCCP domains from wheat ACC and the CT domain from human ACC2. They have identified mutants of these strains with improved growth. These strains respond to known modulators of human ACC2 (such as sethoxydim) and can be used in a simple test to find new modulators of human ACCs, such as by high throughput screening of large chemical libraries. These modulators of human ACC can be applied in the treatment and prevention of disease or health-related conditions that depend upon ACC activity, such as obesity, diabetes, cardiovascular disease, and cancer.

The present invention generally pertains to nucleic acid sequences encoding a non- human acetyl-CoA carboxylase (ACC) amino acid sequence and a human ACC amino acid sequence. The non-human ACC amino acid sequence can be any ACC other than a human ACC, such as a plant ACC or mammalian ACC. For example, in certain particular embodiments, the non-human ACC amino acid sequence is a wheat ACC amino acid sequence.

In some embodiments of the present invention, the chimeric nucleic acid sequence encodes a fusion protein that includes a non-human ACC amino acid sequence and human ACC amino acid sequence. In some embodiments, the ACC amino acid sequence is a wheat ACC amino acid sequence. The wheat ACC amino acid sequence can include either a portion of or the entire wheat ACC amino acid sequence. For example, in certain particular embodiments, the wheat ACC amino acid sequence includes the BC domain of wheat ACC.

The human ACC amino acid sequence may include a human ACCl amino acid sequence or a human ACC2 amino acid sequence. ACC amino acid sequences, both human and non-human, are discussed in greater detail in the specification below. In some embodiments, the human ACC amino acid sequences is a full length human ACC amino acid sequence.

The human ACC amino acid sequenc may include the CT domain of human ACC2. In one particular embodiment, the chimeric nucleic acid includes the sequence set forth in SEQ ID NO: 14. As discussed in the specification below, the chimeric nucleic acids of the present invention may include any number of additional nucleic acids that do not encode for an ACC amino acid sequence. In some embodiments of the present invention, the nucleic acid encodes a non-human ACC amino acid sequence that includes the BC domain of wheat ACC, and a human ACC amino acid sequence that includes the CT domain of human ACC2. In one particular embodiment, the conjugate includes the amino acid sequence set forth in SEQ ID NO:19. Other embodiments of the present invention generally pertain to conjugate amino acid sequences that include a non-human ACC amino acid sequence and a human ACC amino acid sequence. The non-human and human ACC amino acid acid sequences can be any of those sequences set forth above. In certain particular embodiments, the non-human acetyl-CoA carboxylase amino acid sequence is a wheat ACC amino acid sequence, such as a wheat ACC amino acid sequence that includes the BC domain of wheat ACC. The present invention also contemplates conjugates that are fusion proteins of a non-human ACC amino acid sequence and a human ACC amino acid sequence. The human ACC amino acid sequence may include a human ACCl amino acid sequence or a human ACC2 amino acid sequence. In certain particular embodiments, the human ACC amino acid sequence includes the CT domain of human ACC. In some further embodiments, the conjugate includes the BC domain of wheat ACC and the CT domain from human ACC2. In one particular embodiment, the conjugate includes the amino acid sequence set forth in SEQ ID NO:19.

The present invention also generally pertains to yeast cells that include a nucleic acid sequence encoding a human ACC amino acid sequence. Human ACC amino acid sequences are discussed in greater detail above, and elsewhere in this specification. In further embodiments, the yeast cell further includes a non-human ACC amino acid sequence. The human ACC amino acid sequence and the non-human ACC amino acid sequence may be any of the sequences set forth above. In certain particular embodiments, the non-human ACC sequence is a wheat ACC amino acid sequence, such as any of the wheat ACC sequences set forth above.

In some embodiments, the nucleic acid sequence included in the yeast cells encodes a fusion protein that includes a non-human ACC amino acid sequence and human ACC amino acid sequence. The non-human ACC amino acid sequence and human ACC amino acid sequence can be any of those sequences previously discussed. For example, in certain

particular embodiments, the non-human ACC amino acid sequence includes the BC domain of wheat ACC, and the human ACC amino acid sequence includes the CT domain of human ACC2.

The present invention also concerns methods including: (a) obtaining a candidate substance suspected of inhibiting human ACC; (b) preparing a first and second yeast cell, wherein the first and second yeast cells each include a nucleic acid sequence encoding a non- human ACC amino acid sequence and a human ACC amino acid sequence; (c) contacting the first yeast cell with the candidate substance; and (d) assaying for growth inhibition of the first yeast cell by comparing growth of the first yeast cell in the presence of the candidate substance to growth of the second yeast cell in the absence of the candidate substance. In some embodiments set forth herein, the method is further defined as a method of identifying modulators of a human ACC. For example, the modulator may be an activator or an inhibitor of a human ACC.

In some embodiments, the method further includes identifying an inhibitor of human ACC. For example, the method may further be defined as a method of identifying an inhibitor of human ACC useful for the prevention or treatment of a disease or health-related condition. Any disease or health-related condition is contemplated. For example, the disease or health-related condition may be obesity, a cardiovascular disease, diabetes or insulin resistance, and/or a hyperproliferative disease in a subject. The subject can be any subject, such as a mammal. In certain specific embodiments, the subject is a human.

The inhibitor of human ACC identified by any of the methods set forth herein can be used in the preparation of a medicament for the diagnosis, treatment, or prevention of a disease or health-related condition wherein ACC is known or suspected to be involved. For example, the disease or health-related condition may be any of the diseases or health-related conditions set forth above.

Inhibitors of human ACC and methods of identifying inhibitors of human ACC are discussed at length elsewhere in this specification, hi certain embodiments set forth herein, the inhibitor of human ACC is further defined as a small molecule. For example, the inhibitor may be a peptide, a polypeptide, a protein, or an antibody, The inhibitor of human ACC may also be a nucleic acid, such as a DNA molecule, an oligonucleotide, an RNAi or a CpG. The inhibitor may also be a ribozyme.

In further embodiments, the inhibitor of ACC is a molecule that structurally or functionally resembles a cyclohexane-l,3-dione. For example, the cyclohexane-l,3-dione may be oxydim, cycloxydim, clethodim, sethoxydim, and tralkoxydim. In further

embodiments, the inhibitor of ACC is a molecule that structurally or functionally resembles an aryloxyphenoxypropionic acid. For example, the aryloxyphenoxypropionic acid may be diclofop, fenoxaprop, fluazifop, haloxyfop, propaquizafop, or quialofop. In still further embodiments, the inhibitor of ACC is an N-substituted bipiperidylcarboxamide. The inhibitor of ACC may be a synthetic or a natural product. For example, the natural product may be a macrocyclid polyketide, or Moiramide B. In certain particular embodiments, the macrocyclide polypdtide is Soraphen A.

In certain embodiments, the methods set forth herein further include manufacturing the inhibitor of human ACC. The methods may further include administering the inhibitor of human ACC to a subject. The subject can be any subject, such as a mammal. In certain particular embodiments, the subjecdt is a human. The human may or may not be involved in a clinical trial at the time of administration. In some embodiments, the human has a disease or health-related condition. Exemplary disease or health-related conditions include obesity, or a cardiovascular disaease such as angina pectoris, myocardial infarction, or metabolic syndrome. Obesity is defined herein to refer to any body weight that is above average for height, age, and bone structure. The human may be at risk of or afflicted with diabetes or insulin resistance.

In some embodiments, the human has or is at risk of a hyperproliferative disease. For example, in certain embodiments the hyperproliferative disease is cancer. Any cancer is contemplated by the present invention. For example, the cancer may be breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, or leukemia. In certain particular embodiments, the cancer is breast cancer. In certain embodiments set forth herein, the subject is a cancer patient.

For example, the non-human acetyl-CoA carboxylase amino acid sequence may be a wheat ACC amino acid sequence. The wheat ACC amino acid sequence may be any of those wheat ACC amino acid sequences discussed above.

In some embodiments, the nucleic acid sequence may encode a fusion protein that includes a non-human ACC amino acid sequence and human ACC amino acid sequence. The non-human ACC amino acid sequence, for example, can be a wheat ACC amino acid sequence, such as a wheat ACC amino acid sequence that includes the BC domain of wheat ACC. The human ACC amino acid sequence may be any of those human ACC amino acid sequences discussed above, such as a human ACC2 or ACC2 amino acid sequence. In certain

embodiments, the human ACC amino acid sequence includes the CT domain of human ACC2.

In particular embodiments, the human ACC amino acid sequence includes the BC domain of wheat ACC, and the human ACC amino acid sequence includes the CT domain of human ACC2.

In some embodiments, the method is further defined as a method to identify substances that prevent or reduce obesity in a subject. In certain embodiments of the methods set forth herein, the nucleic acid sequence encodes a fusion protein that includes a non-human ACC amino acid sequence and human ACC amino acid sequence. The non-human and human ACC amino acid sequences may be any of those sequences discussed above. For example, in certain particular embodiments, the non-human ACC amino acid sequence includes the BC domain of wheat ACC, and the human ACC amino acid sequence includes the CT domain of human ACC2.

As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 An overview of the subunit structure of the multi-domain ACC found in eukaryotes such as human, wheat and Toxoplasma gondii, and structure of some chimeric genes constructed for human ACC expression in yeast. The wheat cytosolic ACC, the wheat

cytosolic/plastid chimera and the apicoplast T. gondii ACCl complementing the yeast ACCl null mutation and other similar chimeric genes for wheat and toxoplasma ACCs were previously described, along with the effect of known herbicides on the growth of yeast gene- replacement strains carrying such chimeric genes (Jelenska et al, 2001; Jelenska et al, 2002; Joachimiak et al. 1997; Nikolskaya et al, 1999; Zagnitko et al, 2001). Only the coding part of the artificial genes in yeast pRS shuttle vectors carrying a GALlO promoter, 5'UTR and 3"UTR from the yeast ACCl gene are shown. The circles indicate an approximate position of amino acid differences, due to PCR-related errors and construction design (D->V change in ACCl and addition of two amino acids at the end of the ACC2 gene), between our coding sequences and those deduced for sequences deposited in GenBank for human ACCl form 2 (accession number NP_942133) and human ACC2 (accession number NP_001084). Open circles indicate amino acid changes that were repaired in the final constructs. The grey circle indicates an amino acid change repaired in one variant of the construct. The diamonds indicate approximate positions of small deletions in the coding sequences due to alternative splicing ("missing exons"). Conctructs with and without these deletions were prepared and analyzed..

FIG. 2. Growth inhibition of the gene-replacement yeast strain, ACC2-col5, carrying a wheat cytosolic/human ACC2 chimera (FIG. 1) by sethoxydim. FIG. 3 and FIG. 4 below illustrate how well such gene-replacement yeast strains are suited for an inexpensive and simple screening of chemical libraries to identify new ACC inhibitors — strong inhibitors in such a screen can be scored by a significant or complete growth inhibition. All tests were performed in a 96-well format compatible with high-throughput robotic screening technology.

FIG. 3. Growth inhibition of one of the gene-replacement yeast strains carrying a wheat cytosolic/plastid ACC chimera (Fig. 1, and described in Nikolskaya et al, 1999) by haloxyfop, a known potent inhibitor of wheat plastid ACC (used as a herbicide). The results shown illustrate how well the growth phenotype of the gene-replacement strain reflects sensitivity of the ACC to the inhibitor. There is only small well-to-well variation for parallel tests on the same plate (error bars). FIG. 4 Growth inhibition of one of the gene-replacement yeast strains carrying a wheat cytosolic/plastid ACC chimera (Fig. 1, and described in Nikolskaya et al, 1999) by sethoxydim, a known potent inhibitor of wheat plastid ACC (used as a herbicide). The results shown illustrate how well the growth phenotype of the gene-replacement strain reflects

sensitivity of the ACC to a representative of another class of inhibitors. There is only small well-to-well variation for parallel tests on the same plate (error bars).

FIG. 5. Inhibitors targeting acetyl-CoA carboxylase.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The inventors have discovered certain novel methods of identifying modulators of human ACC that involve chimeric polynucleotides encoding a non-human ACC amino acid sequence and a human ACC amino acid sequence. For example, they have genetically engineered yeast gene-replacement strains that depend for their growth on a chimeric ACC that includes the BC and BCC domains from wheat ACC and the CT domain from human ACC2. These strains respond to known modulators of human ACC2, such as sethoxydim. Further, these strains can be utilized in simple tests, such as growth inhibition assays, to find new modulators of human ACCs. The available assays for ACC inhibitors rely on laborious and expensive enzymatic tests. The ACC chimeric genes of the present invention can produce active enzyme in yeast, complementing a yeast ACCl null mutation, resulting in viable yeast cells that can be applied in simple growth inhibition asssays. A simple expression of full-length human ACCl or ACC2 in yeast does not work. A yeast strain dependent for growth on a human ACC makes it possible to screen for specific modulators, such as inhibitors, of human ACC in an quick, inexpensive, and reproducible manner. The identified modulators of human ACC can be aapppplliieedd aass ppootteennttiiaall nncovel therapies of metabolic disorders, such as obesity, diabetes, and cardiovascular disease.

A. Nucleic Acid Sequences

Certain embodiments of the present invention generally pertain to nucleic acid sequences encoding a non-human ACC amino acid sequence and a human ACC amino acid sequence, such as a full-length human ACC amino acid sequence.

1. Definitions

The term "nucleic acid sequence" is well known in the art. A "nucleic acid sequence" as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising one or more nucleobases. A nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C). The term "nucleic acid sequence" encompass the terms "oligonucleotide" and "polynucleotide," each as a subgenus of the term "nucleic acid." The term "oligonucleotide" refers to a molecule of between about 3 and about 100 nucleobases in length. The term "polynucleotide" refers to at least one molecule of greater than about 100 nucleobases in length.

Non-human ACC amino acid sequences and human ACC amino acid sequences are discussed in greater detail elsewhere in the specification. The nucleic acid sequence encoding the full-length amino acid sequence of human ACCl is set forth in SEQ ID NO:1. Additional human ACCl cDNA sequence isoforms include GenBank Accession Nos. NM 198836 (SEQ ID NO:3), Ul 9822 (SEQ ID NO:4), X68968 (SEQ ID NO.5). The nucleic acid sequence encoding the full-length amino acid sequence of human ACC2 is set forth in SEQ ID NO:6. Additional human ACC2 cDNA sequence isoforms include GenBank Accession Nos. AJ575592 (SEQ ID NO:8) and NM 001093 (SEQ ID NO:.9). As discussed elsewhere in this specification, the non-human ACC amino acid sequence may be an ACC amino acid sequence from any source other than human. Thus, for example, the non-human ACC amino acid sequence may be derived from a plant ACC, a bacterial ACC, or a mammalian ACC other than a human ACC. In certain particular embodiments, the non-human ACC is a wheat ACC, such as a wheat cytosolic ACC. Nucleic acid sequence isoforms encoding the full-length ACC amino acid sequence of wheat cytosolic ACC include SEQ ID NOS :10 and 11.

An exemplary chimeric ACC coding sequence that includes a human ACC2 polynucleotide sequence and a wheat cytosolic ACC polynucleotide sequence is set forth in SEQ ID NO:14. The definition of nucleic acid sequence as used herein generally refers to a single- stranded molecule, but in specific embodiments will also encompass an additional strand that is partially, substantially or fully complementary to the single-stranded molecule. The term "vector" is used to refer to a carrier into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. The term "expression vector" or "nucleic

acid vector" refers to a vector containing a nucleic acid sequence or "cassette" coding for at least part of a gene product capable of being transcribed and "regulatory" or "control" sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, expression vectors may contain nucleic acid sequences that serve other functions as well.

It is well understood by the skilled artisan that, inherent in the definition of a non- human or human ACC amino acid sequence is the concept that there is a limit to the number of changes that may be made within a defined sequence of the native ACC amino acid sequence and still result in a molecule with an acceptable level of equivalent biological activity, e.g., ability to function as an ACC. "An ACC amino acid sequence equivalent" is thus defined herein as any ACC amino acid sequence in which some, or most, of the amino acids may be substituted so long as the polypeptide retains substantially similar activity in the context of the uses set forth herein. Thus, the present invention includes polynucleotides encoding non-human ACC amino acid sequences and human ACC amino acid sequences that are equivalent ACC amino acid sequences. A person of ordinary skill in the art would understand that commonly available experimental techniques can be used to identify or synthesize polynucleotides encoding ACC amino acid sequence equivalents. The present invention also encompasses chemically synthesized mutants of these sequences.

Another kind of sequence variant results from codon variation. Because there are several codons for most of the 20 normal amino acids, many different DNAs can encode a particular ACC amino acid sequence. Reference to the following table will allow such variants to be identified.

TABLE l

Amino Acids Codons

Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic acid Asp D GAC GAU

Glutamic acid GIu E GAA GAG

Phenylalanine Phe F UUC UUU

Glycine GIy G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine He I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU

Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCA CCC CCG CCU

Glutamine GIn Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Valine VaI V GUA GUC GUG GUU

Tryptophan Trp W UGG

Tyrosine Tyr Y UAC UAU

Allowing for the degeneracy of the genetic code, sequences that have between about 50% and about 75%, or between about 76% and about 99% of nucleotides that are identical to the nucleotides disclosed herein will be preferred. Sequences that are within the scope of the present invention are those that are capable of base-pairing with a polynucleotide segment set forth above under intracellular conditions.

The nucleic acid sequences of the present invention may include any number of additional nucleic acids other than those encoding the non-human ACC amino acid sequence and the human ACC amino acid sequence. Furthermore, there may be any number of additional nucleic acids interposed between the section of the nucleic acid encoding the non- human ACC amino acid sequence and the human ACC amino acid sequence. The polynucleotide encoding the ACC amino acid sequences may include the full-length human or non-human amino acid sequences, or any fragment thereof.

2. Promoters and Enhancers

Expression of the nucleic acid sequence in a host cell may be under the control of a promoter. A "promoter sequence" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic

elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. Together, an appropriate promoter or promoter/enhancer combination, and a gene of interest, comprise an expression cassette. One or more expression cassettes may be present in a given nucleic acid vector or expression vector. In certain aspects, one expression cassette may encode a transactivator that interacts with a promoter of a second expression cassette. The one or more expression cassettes may be present on the same and/or different expression vector.

A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating a portion the 5' non-coding sequences located upstream of the coding segment or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. In certain aspects of the invention a heterologous promoter may be a chimeric promoter, where elements of two or more endogenous, heterologous or synthetic promoter sequences are operatively coupled to produce a recombinant promoter. . .

A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Patent 4,683,202, U.S. Patent 5,928,906, each incorporated herein by reference).

A promoter and/or enhancer will typically be used that effectively directs the expression of the DNA segment in an organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al., (2001), incorporated herein by reference. The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct expression of the introduced DNA segment, such as is advantageous in the production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous or a combination thereof. In certain embodiments, a yeast GALlO promoter is utilized, which allows for regulation of expression by varying the concentration of glucose and galactose in the growth medium. Additional information regarding the GALlO promoter can be found in Hovland et al. (1989), which is herein specifically incorporated by reference in its entirety.

3. Selectable Markers In certain embodiments of the invention, a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker. Examples of selectable and screenable markers are well known to one of skill in the art.

4. Polyadenylation Signals One may include a polyadenylation signal in the expression construct to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. Also contemplated as an element of the expression cassette is a transcriptional termination site. These elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences.

5. Termination Signals

The vectors or constructs of the present invention may comprise at least one termination signal. A "termination signal" or "terminator" is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.

6. Origins of Replication In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast. In certain embodiments, pRS shuttle vectors with E. coli and yeast origins of replication will be used to propagate vectors in host cells (see Joachimiak et al. 1997, herein incorporated by reference in its entirety).

B. Amino Acid Sequences 1. Definitions

Certain embodiments of the present invention generally pertain to conjugates comprising a non-human ACC amino acid sequence and a human ACC amino acid sequence. A "conjugate" is defined herein to refer to an amino acid sequence of any length that includes a non-human ACC amino acid sequence and a human ACC amino acid sequence. For example, the amino acid sequence may include about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, or more consecutive amino acids of the full length sequence, or any range of amino acids within any of these recited numbers. In some particular embodiments, the ACC amino acid sequence is the full-length amino acid sequence. There may or may not be additional non-ACC amino acid sequences interposed between the non-human ACC amino acid sequence and the human ACC amino acid sequence. In certain particular embodiments, the conjugate includes a non-human ACC and a human ACC in which there are no amino acids interposed between the non-human ACC amino acid sequence and the human ACC amino acid sequence. Such a conjugate is defined herein to be a "fusion protein."

A "human ACC amino acid sequence" is an amino acid sequence derived from human ACC. The full-length amino acid sequence of human ACCl is provided herein as SEQ ID NO:2. Amino acid sequence isoforms of human ACCl include GenBank Accession No. NP 942133 (SEQ ID NO:15) and AAC50139 (SEQ ID NO:16). The full-length amino acid sequence of human ACC2 is provided herein as SEQ ID NO:7. Amino acid sequence isoforms of human ACC2 include GenBank Accession Nos. CAE01471 (SEQ ID NO:17) and NP 001084 (SEQ ID NO: 18).

A "non-human ACC amino acid sequence" can be derived from any source other than from a human. For example, the non-human ACC amino acid sequence may be an amino acid sequence from a plant ACC, an animal ACC, a microorganism ACC, and so forth. In certain particular embodiments, the non-human ACC amino acid sequence is a wheat ACC amino acid sequence. The full-length amino acid sequence of wheat ACC is provided herein as SEQ ID NO:20. Amino acid sequence isoforms of wheat ACC include those sequences set forth in GenBank Accession No. U10187 (SEQ ID NO:12) and U39321 (SEQ ID NO:13). An exemplary chimeric ACC protein that includes a wheat cytosolic ACC amino acid sequence and a human ACC2 amino acid sequence is set forth in SEQ ID NO: 19.

An "ACC amino acid sequence" is a consecutive amino acid segment of an ACC that is of any length, including the full length sequence of the parent ACC molecule. For example, the ACC amino acid sequence can be a polypeptide that include about 4, 5, 10, 15, 20, 25, 30, 50, 100, 200, 300, 400, 500, 1000 or any number of consecutive amino acids of the full-length ACC. One of ordinary skill in the art would understand how to generate an ACC amino acid sequence using any of a number of experimental methods well-known to those of skill in the art.

It is well understood by the skilled artisan that, inherent in the definition of a "ACC amino acid sequence," is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity, i.e., ACC activity. Thus, an ACC amino acid sequence is an amino acid sequence in which some, or most, of the amino acids may be substituted so long as the polypeptide retains substantially similar activity in the context of the uses set forth herein.

An amino acid sequence of any length is contemplated within the definition of a conjugate, so long as the polypeptide retains an acceptable level of equivalent biological activity. The conjugates set forth herein may comprise any number of additional non-ACC amino acids at the C-terminal and/or N-terminal end of the ACC amino acid sequences. Thus,

for example, the conjugate may include a total of greater than about 1000, 500-1000, 400-499, 300-399, 200-299, 100-199, 80-99, 60-79, 50-59, 40-49, 30-39, 20-29, 10-19, 9, or 8 amino acid residues, as long as the conjugate includes at least about 4 residues of a non-human ACC and at least about 4 residues of a human ACC. The ACC amino acid sequences may have an amino acid identity of about 40% with the native ACC amino acid sequence, and a chemical identity (presence of identical or chemically similar amino acids) of about 60-70%, indicating that they are biologically equivalent amino acid sequences. Therefore, these ACC amino acid sequences are equivalent sequences because only certain amino acids are substituted relative to the native ACC amino acid sequence.

The conjugates of the present invention may utilize ACC amino acid sequences obtained from a natural source or from recombinantly-produced material. One of ordinary skill in the art would know how to produce these amino acid sequences from recombinantly- produced material. The conjugates of the present invention may or may not be purified. Generally,

"purified" will refer to an amino acid sequence that has been subjected to fractionation to remove various other proteins, polypeptides, or peptides, and which composition substantially retains its activity. Purification may be substantial, in which the or equivalent is the predominant species, or to homogeneity, which purification level would permit accurate degradative sequencing.

The component ACC amino acid sequences of the conjugates set forth herein may be composed of ACC amino acid sequence mutants. Amino acid sequence mutants can be substitutional mutants or insertional mutants. Insertional mutants typically involve the addition of material at a non-terminal point in the peptide. This may include the insertion of a few residues, an immunoreactive epitope, or simply a single residue. The added material may be modified, such as by methylation, acetylation, and the like. Alternatively, additional residues may be added to the N-terminal or C-terminal ends of the peptide.

2. Methods of Conjugate Synthesis In certain embodiments of the present invention, the conjugate is encoded by a recombinant nucleic acid sequence using recombinant techniques.

a. Recombinant Techniques

In certain embodiments of the present invention, the conjugate is encoded by a recombinant polynucleotide using recombinant techniques well-known to those of ordinary skill in the art. The polynucleotide may include a sequence of additional nucleic acids that direct the expression of the conjugate in appropriate host cells.

Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence, may be used in the practice of the invention of the cloning and expression of the conjugate. Such DNA sequences include those capable of hybridizing to the chimeric sequences or their complementary sequences under stringent conditions. In one embodiment, the phrase "stringent conditions" as used herein refers to those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with a 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42 ⁰ C, with washes at 42 ⁰ C in 0.2 x SSC and 0.1% SDS. In order to express a conjugate, the nucleotide sequence coding for the conjugate must be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing the conjugate coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Sambrook et al. , 2001.

A variety of host-expression vector systems may be utilized to express the conjugate coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the chimeric protein coding sequence; yeast transformed with recombinant yeast expression vectors containing the chimeric protein coding sequence; insect cell systems infected with recombinant virus expression vectors {e.g., baculovirus) containing the chimeric

protein coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the chimeric protein coding sequence; or animal cell systems. In certain particular embodiments, the conjugate is expressed in yeast cells, as discussed in greater detail below.

The expression elements of each system vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. One of ordinary skill in the art would be familiar with expression elements for directing expression of a particular coding sequence.

Specific initiation signals may also be required for efficient translation of the inserted coding sequences. These signals include the ATG initiation codon and adjacent sequences. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et α/., 1987).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the conjugate. The presence of consensus N- glycosylation sites in a chimeric protein may require proper modification for optimal chimeric protein function.

For long-term, high-yield production of recombinant chimeric polypeptides, stable expression is preferred. For example, cell lines which stably express the chimeric polypeptide may be engineered. Rather than using expression vectors which contain viral originals of replication, host cells can be transformed with a chimeric coding sequence controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.

b. Purification

In certain embodiments of the present invention, the conjugate has been purified. Generally, "purified" will refer to a conjugate composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the polypeptide or peptide forms the major component of the composition, such as constituting about 50% to about 99.9% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of the conjugate will be known to those of skill in the art in light of the present disclosure. Exemplary techniques include high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like. The actual conditions used to purify a particular conjugate will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those having skill in the art.

C. Yeast

1. Yeast Cells

The present invention further concerns yeast cells comprising a nucleic acid sequence encoding a non-human ACC amino acid sequence and a human ACC amino acid sequence or, alternatively, a full-length human ACC sequence.

Yeast are unicellular fungi whose mechanisms of cell-cycle control are remarkably similar to that of humans. In the context of the present invention, a "yeast cell" is any unicellular fungus. One of ordinary skill in the art would be familiar with the range of yeast cells that are encompassed by this definition. Various characteristics are used to classify yeast cells. The precise classification is a field that uses the characteristics of the cell, ascospore and colony. Physiological characteristics are also used to identify species. One of the more well known characteristics is the ability to ferment sugars for the production of ethanol. Budding yeasts are true fungi of the phylum Ascomycetes, class Hemiascornycetes. The true yeasts are separated into one main order, Saccharomycetales. Yeasts are characterized by a wide dispersion of natural habitats, and are common on plant leaves and flowers, soil and salt water. Yeasts are also found on the skin surfaces and in the intestinal tracts of warm-blooded animals, where they may live symbiotically or as parasites.

Yeasts multiply as single cells that divide by budding {e.g., Saccharomyces) or direct division (fission, e.g., Schizosaccharomyces), or they may grow as simple irregular filaments (mycelium). In sexual reproduction most yeasts form asci, which contain up to eight haploid ascospores. The awesome power of yeast genetics is partially due to the ability to quickly map a phenotype-producing gene to a region of the S. cerevisiae genome. For the past two decades, S. cerevisiae has been the model system for much of molecular genetic research because the basic cellular mechanics of replication, recombination, cell division and metabolism are generally conserved between yeast and larger eukaryotes, including mammals. It is also a straightforward matter for one of ordinary skill in the art to engineer yeast cells to express a variety of heterologous constructs, and to do so in a controlled fashion.

2. Yeast Cultures

Some yeast varieties reproduce almost as rapidly as bacteria and have a genome size less than 1% that of a mammal. They are amenable to rapid molecular genetic manipulation, whereby genes can be deleted, replaced, or altered. They also have the unusual ability to proliferate in a haploid state, in which only a single copy of each gene is present in the cell. This makes it easy to isolate and study mutations that inactivate a gene as one avoids the complication of having a second copy of the gene in the cell. The process of culturing yeast strains involves isolation of a single yeast cell, maintenance of yeast cultures, and the propagation of the yeast using techniques well-known to those of ordinary skill in the art. Pure yeast cultures are obtained from a number of sources such as commercial distributors or culture collections. Various procedures are used to collect pure cultures, including culturing from a single colony, a single cell, or a mixture of isolated cells and colonies.

The objective of propagation is to produce large quantities of yeast with known characteristics in as short a time as possible. One method is a batch system of propagation, starting with a few milliliters of stock culture and scaling up until a desired quantity of yeast has been realized. Scale-up introduces actively growing cells to a fresh supply of nutrients in order to produce a crop of yeast in the optimum physiological state.

Yeast cells that may be used in accordance with the present invention include, but are not limited to, Saccharomyses species {e.g., S. cerevisiae; S. carlsbergensis), Schizosaccharomyces species {e.g., S. pombi), Pichia species {e.g., P. pastoris), Hansenula

species {e.g., H. polymorpha), Kluyveromyces species {e.g., K. lactis), Yarrowia species {e.g., Y. lipolyticd). However, virtually any yeast cell genus can be engineered as described herein.

3. Yeast Viability and Growth Viability is defined herein to refer to the ability to increase in mass and to divide.

Yeast viability may be determined by any method known to those of ordinary skill in the art. For example, viability may be determined by the standard-culture method, flow cytometry by selective staining, or by more advanced methods such as the Slide Viability Method, flocculation tests, and fermentation tests, hi certain embodiments of the present invention, yeast cell growth rate can be correlated with human ACC activity.

The standard slide-culture method of determining viability of yeasts has three steps: perform a hemacytometer count on a suspension of cells, plate a measured quantity on a wort gelatin medium, and then incubate and count the resultant colonies. However, this method may be inaccurate due to cell clumping and the death of cells during preparation. Methylene blue remains an industry standard for viability assessment. It has also been suggested that methylene violet might provide a more accurate and reproducible assessment of viability than does methylene blue because of impurities in the latter. Other stains that may be used include fluorophore dyes, such as oxonol (DiBAC), l-anilino-8-naphtalene-sulfonic acid (MgANS), berberine, Sytox Orange, propidium iodide, FUNl, and other conventional brightfield dyes. For the most part, fluorophore staining has been perceived to be less subjective to the operator compared with brightfield dye staining because of the lack of intermediate color variations.

4. Yeast Promoters In certain embodiments, the polynucleotide encoding the chimeric ACC is under the control of a yeast promoter. Any yeast promoter known to those of ordinary skill in the art is contemplated to control expression of polynucleotide encoding the chimeric ACC.

Exemplary useful yeast promoters for the conditional expression of nucleic acids encoding chimeric ACC include those directing expression of metallothionein, 3- phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3- phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others. In certain embodiments, the yeast GALlO promoter is utilized, which allows for regulation of expression by varying the concentration of galactose in the growth medium. Vectors and promoters suitable for use in yeast expression are further described in EP

73,67 '5 A, herein incorporated by reference in its entirety. Other examples of strong yeast promoters are the alcohol dehydrogenase, lactase and triosephosphate isomerase promoters.

5. Yeast Transformation Protocols A variety of approaches are available for transforming yeast cells and include electroporation, lithium acetate and protoplasting. Any method of transforming yeast cells known to those of ordinary skill in the art is contemplated. Exemplary methods are set forth below.

a. Electroporation

In certain embodiments of the present invention, a nucleic acid is introduced into a yeast cell via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high- voltage electric discharge. In some variants of this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells (U.S. Patent No. 5,384,253, incorporated herein by reference).

In one protocol, for example, cells are first grown to a density of about 1 x 10 ⁷ /ml (OD595 ca. 0.5) in minimal medium (transformation frequency is not harmed by growth until early stationary phase (OD595 = 1.5)). Cells are harvested by spinning at 3000 rpm for 5 minutes at 20°C, followed by washing once in ice-cold water and harvesting; a second time in ice-cold IM sorbitol. It has been reported (Suga and Hatakeyama, 2001), that 15 min incubation of these cells in the presence of DTT at 25 mM increases electrocompetence. The final resuspension is in ice-cold IM sorbitol at a density of 1 - 5 x 10 ⁹ /ml. Forty ul of the cell suspension are added to chilled eppendorfs containing the DNA for transformation (100 ng) and incubated on ice for 5 minutes.

The electroporator may be set as follows: (a) 1.5kV, 200 ohms, 25uF (Biorad); (b) 1.5 kV, 132 ohms, 40 uF (Jensen/Flowgen). Cells and DNA are transferred to a pre-chilled cuvette and pulsed; 0.9 ml of ice-cold IM sorbitol is then immediately added to the cuvette; the cell suspension is then returned to the eppendorf and placed on ice while other electroporations are carried out. Cells are plated as soon as possible onto minimal selective medium. Transformants should appear in 4 - 6 days at 32 ⁰ C.

b. Lithium Acetate

Lithium acetate protocols for transforming yeast cells are well-known to those of ordinary skill in the art. One exemplary lithium acetate protocol is derived from Okazaki et al. (1990), herein specifically incorporated by reference in its entirety.

D. Methods of Identifying Modulators of a Human ACC

1. Definitions

A "modulator" of a human ACC is defined herein to refer to any molecule that reduces or enhances the activity of a particular enzyme {e.g., ACC) relative to enzymatic activity in the absence of the modulator. An "inhibitor" of a human ACC is defined herein to refer to any molecule that reduces the activity of an enzyme. An "activator" of a human ACC is defined herein to refer to any molecule that enhances the activity of an enzyme. Any method known to those of ordinary skill in the art can be used to measure enzyme activity.

2. Candidate Modulators of Human ACC a. Known Modulators

Certain classes of herbicides are known inhibitors of ACC. The aryloxyphenoxyproprionates class comprises derivatives of aryloxyphenoxypropionic acid, such as diclofop, fenoxaprop, fluazifop, haloxyfop, propaquizafop, and quialofop. This class acts by causing a rapid necrosis of the meristematic tissue of sensitive plant species (Shimabukuro et al, 1979). The mode of action of these herbicides appears to be through an inhibition of fatty acid and, hence, acetyl lipid synthesis (see Harwood, 1988). Furthermore, this action on lipid formation occurs in the absence of any detectable effects on CO ₂ fixation or carbohydrate, amino acid or nucleic acid metabolism. Several derivatives of cyclohexane- 1,3-dione {i.e., cyclohexadiones) are also important post-emergence herbicides which also selectively inhibit monocot plants. This class comprises such compounds as oxydim, cycloxydim, clethodim, sethoxydim, and tralkoxydim. Cyclohexanediones, such as sethoxydim, also appear to act in a similar manner (see Harwood et al, 1988).

It has been determined that ACC is the target enzyme for both of these classes of herbicides, at least in monocots. Dicotyledonous plants, on the other hand, such as soybean, tobacco, and sunflower, are resistant to these compounds, as are other eukaryotes and prokaryotes.

Additional exemplary ACC inhibitors include Soraphen A (Shen et al, 2004, herein specifically incorporated by reference), CP-640186 (Zhang et al, 2004, herein specifically

incorporated by reference), Moiramide B and analogs (Freiberg et al, 2004, herein specifically incorporated by reference), and N-substituted bipiperidylcarboxamides (Harwood et al, 2003, herein specifically incorporated by reference), as well as agents that structurally or functionally resemble any of these known modulators.

b. Natural and Synthetic Modulators

As used herein, the term "candidate modulator"" refers to any molecule that may potentially reduce or enhance the activity of human ACC. The candidate may be a protein or fragment thereof, a small molecule, or even a nucleic acid molecule. Candidate modulators of human ACC would include those molecules that are structurally related to any of the known ACC modulators discussed above.

It may prove to be the case that the most useful pharmacological compounds will be compounds that are structurally related to compounds which interact naturally with ACC. Creating and examining the action of such molecules is known as "rational drag design," and include making predictions relating to the structure of the target molecules and the candidate substance.

The goal of rational drug design is to produce structural analogs of biologically active target compounds. By creating such analogs, it is possible to fashion drags which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for a molecule like a human ACC, and then design a molecule for its ability to interact with these polypeptides. This could be accomplished by x- ray crystallography, computer modeling or by a combination of both approaches (see, e.g., Zhang et al, 2003, specifically incorporated by reference in its entirety). On the other hand, one may simply acquire, from various commercial sources, small molecule libraries that are believed to meet the basic criteria for useful drugs in an effort to "brute force" the identification of useful compounds. Screening of such libraries, including combinatorially generated libraries, is a rapid and efficient way to screen large number of related (and unrelated) compounds for activity. Combinatorial approaches also lend themselves to rapid evolution of potential drags by the creation of second, third and fourth generation compounds modeled of active, but otherwise undesirable compounds.

Candidate modulators may include fragments or parts of naturally-occurring compounds or may be found as active combinations of known compounds which are otherwise inactive. It is proposed that compounds isolated from natural sources, such as

animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. Yet further, the candidate substance may be a known herbicide. The term "herbicide" as used herein is defined as a substance that inhibits the growth of plants, such as weeds.

It will, of course, be understood that all the screening methods of the present invention are useful in themselves notwithstanding the fact that effective candidates may not be found.

3. Assays

In certain embodiments of the present invention, the yeast cell system of the present invention is designed such that inhibition of the human ACC fragment of the conjugate causes growth retardation and eventually yeast cell death, whereas in the absence of the inhibitor, the yeast cells proliferate. One can introduce a large number of biological peptides, proteins, small molecules, or intracellular recombinant antibodies in yeast cells bearing this heterologous ACC to directly and rapidly select/identify inhibitors of ACC.

In other embodiments, activators of human ACC increase proliferation of yeast cells compared to growth in the absence of the activator.

Any method known to those of ordinary skill in the art can be used to assay for modulators of human ACC using the yeast cells of the present invention. For example, candidate modulators can be tested by using clear bottom multi-well plates (currently, 96- well plates are commercially available). Yeast cells that are engineered to include the heterologous ACC are diluted and distributed equally in each well. Candidate modulators are distributed to each well and yeast cell growth is monitored by visual inspection or measured with a multi-well plate reader (at 580-600 nm). The presence of an inhibitor of human ACC will lead to yeast cell growth inhibition and decreased turbidity in a well.

E. Therapeutic Targets

Certain embodiments of the present invention generally pertain to methods of identifying a modulator of human ACC useful in the prevention or treatment of a disease or health-related condition.

A disease is defined herein to refer to a pathological condition of a tissue, body part, organ, or a system resulting from any cause. Exemplary causes include genetic defects and environmental stress. A health-related condition is any condition that can causes or results in

abnormal structure or function or a cell, tissue, or body part of a subject. Prevention, as the term is used herein, pertains to the administration of a modulator of human ACC to a subject for the purpose of preventing the onset of a disease or health-related condition in the subject. The subject may or may not have an underlying propensity to develop the disease or condition that is being prevented. Treatment, as the term is used herein, pertains to the administration of a modulator of human ACC to a subject for the purposes of improving or stabilizing a disease or health-related condition that is presently affecting the subject.

The disease may be any disease wherein administration of a modulator of human ACC is believed to be beneficial by one of ordinary skill in the art. For example, the disease may be a disease associated with a metabolic abnormality in a subject, such as obesity or metabolic syndrome. Metabolic syndrome is a clustering of cardiovascular risk factors, including abdominal obesity, hyperinsulinemia, atherogenic dyslipidemia, hypertension and hypercoagulability (see Harwood, 2004). Other therapeutic targets include diabetes and insulin resistance. Additional diseases or health-related conditions to be prevented or treated include cardiovascular disease, such as angina pectoris or acute myocardial infarction.

The present invention also deals with the treatment and prevention of disease states that involve hyperproliferative disorders, including benign and malignant neoplasias. Such disorders include hematological malignancies, restenosis, cancer, multi-drug resistant cancer, psoriasis, inflammatory bowel disease, rheumatoid arthritis, osteoarthritis and metastatic tumors.

The present invention is directed at the treatment and prevention of human cancers, including cancers of the prostate, lung, brain, skin, liver, breast, lymphoid system, stomach, testicular, ovarian, pancreatic, bone, bone marrow, head and neck, cervical, esophagus, eye, gall bladder, kidney, adrenal glands, heart, colon, rectum and blood.

F. Pharmaceutical Preparations

Some embodiments of the present invention pertain to the administration of a modulator of human ACC to a subject. In certain embodiments, the modulator of human ACC is comprised in a pharmaceutical composition suitable for administration to a subject. The subject can be any subject, including a mammal, such as a human, as discussed above.

Pharmaceutical compositions of the present invention comprise a therapeutically or diagnostically effective amount of a composition of the present invention, wherein the composition comprises one or more modulators of human ACC. The phrases "pharmaceutical," "pharmacologically acceptable," or "therapeutically effective" refers to

molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of therapeutically effective or diagnostically effective compositions will be well-known to those of skill in the art. Moreover, for animal and human administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

As used herein, "a composition comprising a therapeutically effective amount" or "a composition comprising a preventively effective amount" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the present compositions is contemplated. The compositions of the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The compositions of the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, intratumorally, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art.

The actual required amount of a composition of the present invention administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, the tissue to be treated, the type of disease being treated or prevented, previous or concurrent preventive or therapeutic interventions, idiopathy of the patient, and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% to about 90% by weight of ACC modulator. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 0.1 mg/kg/body weight to about 1000 mg/kg/body weight or any amount within this range, or any amount greater than 1000 mg/kg/body weight per administration.

In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including, but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

The compositions of the present invention may be formulated in a free base, neutral or salt form. Pharmaceutically acceptable salts include the salts formed with the free carboxyl groups derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising, but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

Sterile injectable solutions may be prepared using techniques such as filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze- drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to

injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.

In further embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.

G. Combinational Therapy

1. Combinatorial Therapy in General Certain aspects of the present invention pertain to methods of administering a modulator of human ACC to a subject, such as a human subject. These compositions can be applied in the prevention or treatment of diseases wherein administration of a modulator of human ACC is known or suspected by one of ordinary skill in the art to be beneficial.

For example, as set forth above, the disease or health-related condition to be treated or prevented may be obesity, a hyperproliferative disease, a cardiovascular disease, diabetes, or insulin resistance. The modulator of human ACC may be administered along with another agent or therapeutic method. For example, administration of a modulator of human ACC for the purpose of treating diabetes mellitus in a human subject may precede, follow, or be concurrent with other therapies for diabetes, such as an oral hypoglycemic acid or insulin therapy. Administration of a modulator of human ACC for the purpose of treating an acute myocardial infarction may, for example, be administered following an angioplasty or coronary artery bypass procedure. In another example, administration of a modulator of human ACC of the purpose of treating or prevent obesity may precede or follow a dietary intervention or gastric surgery for the treatment of obesity. Administration of the modulator of human ACC to a patient will follow general protocols for the administration of therapeutic agents, and will take into account other parameters, including, but not limited to, other medical conditions of the patient and other therapies that the patient is receiving. It is expected that the treatment cycles would be repeated as necessary.

Treatment with the modulator of human ACC of the present invention may precede or follow the other therapy method by intervals ranging from minutes to weeks. In embodiments where another agent is administered, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the cell. For example, it is contemplated that one may administer two, three, four or more doses of one agent substantially simultaneously {i.e., within less than about a minute) with the compositions of the present invention. In other aspects, a therapeutic agent or method may be administered within about 1 minute to about 48 hours or more prior to and/or after administering a therapeutic amount of a composition of the present invention, or prior to and/or after any amount of time not set forth herein. In certain other embodiments, the modulator of human ACC of the present invention may be administered within of from about 1 day to about 21 days prior to and/or after administering another therapeutic modality, such as surgery or medical therapy. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several weeks {e.g., about 1 to 8 weeks or more) lapse between the respective administrations.

Various combinations may be employed, the modulator of human ACC is designated "A" and the secondary therapeutic agent , which can be any other therapeutic agent or method, is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A AIBIAIK AIAIBIA

2. Secondary Therapies

a. Obesity

Obesity is a common health problem in the United States, and effective treatment is challenging. Obesity is associated with an increased mortality rate and risk factors such as hypertension, hyperlipidemia and diabetes mellitus. Numerous treatments are available for obesity. Behavioral therapy, pharmacologic treatment, surgery, and pharmacologic treatment have been used with varying degrees of success. Behavioral therapy, including regular exercise and the development of healthy eating habits, continues to be the best treatment for long-term weight loss. Older anorectic agents have significant side effects and limited

benefit, and some have even been withdrawn from the U.S. market because of a possible association with cardiovascular complications. The safety of newer agents must be extensively evaluated before widespread use is recommended. Surgical forms of therapy are becoming more common. Exemplary surgical procedures include adjustable gastric banding, vertical gastric banding, Roux en-Y gastric bypass, and biliopancreatic diversion.

2. Cardiovascular Disease

Cardiovascular disease is a very common cause of morbidity and mortality in

Americans. Heart disease is the leading cause of death for all racial and ethnic groups in the U.S. More than half of persons who die each year of heart disease are women. Exemplary cardiovascular diseases include acute myocardial infarction, atherosclerosis, and congestive heart failure.

There are many forms of therapy of cardiovascular disease, including pharmacological therapies, dietary interventions, and more invasive forms of therapy, including angioplasty and cardiovascular surgery.

Over the counter aspirin (might be beneficial for reducing the risk of future heart attacks. Use of prescription medications is directed toward any underlying causes. Drugs used may include ACE inhibitors, such as captopril, enalopril, and lisinopril; beta blockers such as atenolol, meoprolol, and propranol; and the combination of hydralazine and isosorbide dinitrate. Other medications often prescribed include the blood thinner warfarin, digoxin, nitroglycerin, and diuretics, such as hydrochlorothiazide and furosemide.

Surgical treatments, such as angioplasty, bypass surgery, valve replacement, pacemaker installation, and heart transplantation, may be recommended for severe cases. Individuals with cardiovascular disease are strongly encouraged to stop smoking.

3. Metabolic Syndrome

First-line therapy for treating metabolic syndrome is lifestyle modification that targets the root causes by reducing weight and obesity, improving the amount of physical activity, and diet modification. Overweight and obesity are defined as body mass index of 25 to 29.9 kg/m ² and >30 kg/m ² , respectively. Abdominal obesity apart from general obesity presents a particular risk factor for metabolic syndrome. Abdominal obesity is defined as a waist circumference >102 cm (40 inches) in men or >88 cm (35 inches) in women. Through behavior modification (increased physical activity and dietary modification), patients should expect to reduce their weight by 7% to 10% over 6 to 12 months.

It has been shown that physical activity can treat metabolic syndrome. Dietary modification also plays an important role in the treatment of metabolic syndrome. Dietary recommendations are the same as those for managing diabetes.

A second treatment approach addresses the metabolic risk factors — insulin resistance, dyslipidemia, hypertension, and prothrombotic state — individually through nonpharmacologic and pharmacologic therapies.

4. Insulin Resistance

Insulin resistance is believed to be the main factor of metabolic syndrome, which is why metabolic syndrome is sometimes referred to as the insulin resistance syndrome. Weight loss and increased physical activity lower insulin resistance. Diets with a lower glycemic load and increased dietary fiber are also associated with lower insulin resistance. Fiber from cereal has been shown to be inversely related to prevalence of metabolic syndrome, but the same was not found for fiber from fruit, vegetables, and legumes. If lifestyle changes do not improve insulin resistance, it can be treated with pharmacotherapy. Metformin, an insulin sensitizer that has been used for more than 40 years, improves insulin sensitivity and some of the components of the metabolic syndrome by improving glucose uptake. Pioglitazone and rosiglitazone are thiazolidinediones, which increase insulin sensitivity by improving insulin- mediated muscle glucose uptake.

5. Diabetes

Type 1 diabetes is one of the most common chronic childhood illnesses. Each year nearly 15,000 Americans are diagnosed with type 1 diabetes. Type 1 diabetes currently requires lifelong insulin therapy, and intensive control is associated with increased risks for hypoglycemia.

The goal of treatment for type 1 diabetes is to achieve tight metabolic control in order to reduce complications and mortality. Treatment for type 1 diabetes consists of a series of interrelated components, including insulin therapy, medical nutrition therapy, physical activity, and self-monitoring of blood glucose (SMBG). Treatment and prevention of Type 2 diabetes includes lifestyle changes (medical nutrition therapy and increased physical activity), and pharmacologic therapy (oral and injectable medications including sulfonylureas, meglitinides, biguanides, thiazolidinediones, alpha- glucosidase inhibitors, combination oral therapy and insulin, respectively). The pharmacology, initiation and adjustment of insulin dosing are also important considerations.

The prevalence of diabetes continues to rise as the rates of obesity and sedentary behavior increase. Diabetes is known to have a long preclinical phase of approximately 10-12 years, during which metabolic changes may already be causing microvascular and macrovascular complications. Accordingly, recent research has focused on the prevention and/or delay of Type 2 diabetes. Prevention efforts generally fall into two categories: lifestyle interventions and pharmacotherapy. Current emphasis is on lifestyle interventions since they have proven to be more effective than medications, do not cause any unwanted side effects, and confer additional benefits over and above the prevention or delay of Type 2 diabetes, such as weight reduction and reduction of cardiovascular risk.

6. Hyperproliferative Disease

In certain embodiments of the present invention, the modulator of human ACC is administered to the subject for the treatment or prevention of a hyperproliferative disease, such as cancer. Administration of the modulator of human ACC of the present invention to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of these agents. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described modulators of human ACC. These therapies include but are not limited to chemotherapy, radiotherapy, immunotherapy, gene therapy and surgery. One of ordinary skill in the art would be familiar with these therapeutic modalities.

H. EXAMPLES

EXAMPLE 1 Construction of Chimeric Genes Encoding a Human ACC Amino Acid Sequence

Construction of the chimeric genes shown in FIG. 1 consisted of two major steps: cloning full length cDNA encoding human ACCl and human ACC2 in an E. coli protein expression vector; and assembling chimeric genes in the pRS shuttle vector for yeast complementation.

Full-length cDNAs were assembled from large cDNA fragments cloned by RT-PCR. For each of those fragments, one or two gene-specific primers were designed to prepare single-strand cDNA by the action of reverse transcriptase. Human RNA was used as a template for reverse transcriptase. The single-strand cDNA was then used as a template for

PCR amplification of double-stranded cDNA using pairs of gene-specific primers. Gene- specific primers were designed based on human ACC cDNA and genomic sequences available from GenBank. All PCR-amplified fragments were cloned and sequenced completely to verify their identity and sequence. Artificial restriction sites were added at some ends of the fragments by including recognition sequences for specific restriction nucleases in the primers. Native and artificial restriction sites present in the cloned cDNA fragments were then used in a "cut-and-paste" assembly process involving digestions with specific restriction enzymes and ligation of DNA fragments with DNA ligase. The assembly process was monitored by restriction analysis and by sequencing. The recombinant genes in pRS vectors carrying the GALlO promoter and the 5'UTR and 3"UTR from the yeast ACCl gene were assembled by the same "cut-and-paste" process starting from large fragments of cDNAs described above and using native as well as PCR- engineered restriction sites. The vectors carrying the promoter and UTRs were prepared as described in Joachimiak et al, 1997, herein specifically incorporated by reference. In some cases 5'-end fragments of ACC coding sequences were deleted using PCR cloned fragments with artificially inserted restriction sites in the assembly process. In cases where the coding region of chimeric genes consist of wheat or Toxoplasma ACC coding sequences and human ACC coding sequences, the previously prepared constructs (Jelenska et al, 2002; Joachimiak et al, 1997) were modified by replacing large fragments of the wheat or Toxoplasma coding region with the corresponding coding sequence of human ACCl or human ACC2. For example, chimeric genes containing wheat cytosolic ACC coding sequences and human ACC coding sequences were prepared by modification of the chimeric gene encoding a full-length wheat cytosolic ACC (shown in FIG. 1) assembled in the pRS vector and already fused to the promoter and UTRs. The assembly process was monitored by restriction analysis and by sequencing. PCR errors were repaired by replacing restriction fragments in finished constructs with corresponding PCR-cloned restriction fragments after selection of clones without the errors that caused amino acid substitutions.

EXAMPLE 2 Testing of the Chimeric Constructs for the Ability to Complement the Yeast A CCl Null

Mutation

AU new constructs carrying chimeric genes shown in FIG. 1 were tested for their ability to complement the yeast ACCl null mutation. The process consists of the following

steps: Transformation of yeast (a heterozygous diploid strain with one of its ACCl genes deleted (Joachimiak et ah, 1997)) with the constructs using the lithium acetate method and selecting transformants on single amino acid drop-out medium (His- or Trp-, depending on the selectable marker present in the pRS vector). Sporulation of cells from patches prepared from single colonies from the selective plates, induced by transfer to sporulation medium. Dissection of ascospores obtained after sporulation (tetrad analysis). Marker analysis on selective plates to confirm the genotype of the haploid strains obtained from single spores and galactose-dependence tests to confirm functionality of the chimeric gene.

The only important variation from standard yeast genetics methods introduced in these experiments is the use of YPRG medium (yeast extract-peptone-raffinose-galactose) for tetrad analysis. Note that the chimeric genes shown in FIG. 1 all use a GALlO promoter to drive their expression. The GALlO promoter is "on" in the presence of galactose and "off in the presence of glucose (the usual carbon source in yeast media). Activity of the promoter is needed for the complementation to work, hence the galactose dependence of the haploid gene- replacement strains obtained by tetrad dissection.

As set forth in previous studies (Jelenska et ah, 2002; Nikolskaya et ah, 1999) and the results presented here, not every foreign ACCs can complement the yeast ACCl null mutation: only one gene encoding wheat cytosolic/human ACC2 chimeric ACC complemented the mutation and it was used in experiments described in FIG. 2, after selection for mutants with better growth in YPRG medium. Both variants of the gene encoding full-length human ACCl (with and without the variable exon shown in FIG. 1) complement the mutation as well. Only one variant of the full-length ACC2 gene, with the variable intron shown in FIG. 1, complements. The model for control of obesity by selective inhibition of ACC2 set forth herein requires screening for compounds that slow the growth of ACC2 strains without impairing ACCl strains.

We were able to overcome the initial lack of complementation with genes encoding full length human ACC2 and ACCl. For ACC2, we constructed a series of chimeras with wheat cytosolic ACC (FIG. 1). One of the chimeras complemented the yeast ACCl null mutation but the resulting haploid gene-replacement strains grew very poorly. Colonies on dissection plates were very small and their growth was arrested after 7-14 days. These strains grow poorly in liquid medium as well. Mutant strains with significantly improved growth properties were isolated. One such mutant (ACC2-col5) grows sufficiently better in liquid medium that it can be used in the inhibition experiments discussed below. The strain is cold-

sensitive - it grows at 30°C but not at 25 ⁰ C (secondary mutants which grew at both temperatures were isolated). The latter mutation is located on the yeast chromosome, not on the plasmid used for complementation (shown by complementation with a rescued plasmid). The slow growth phenotype of the ACC2-col5 strain, presumably caused by a limiting level of ACC activity, may be desired in screening because it makes the strain more sensitive and allows more reliable identification of weak inhibitors of ACC2 {e.g., in comparison to another mutant strain, ACC2-col3, described below). Several other mutants with even faster growth phenotype than ACC2-col5 were also isolated. One of them, ACC2-col3, results from a mutation located on the plasmid (shown by complementation with rescued plasmid). This mutation, F1498->L (FIG. 1) was identified by sequencing the mutated plasmid. This strain grows at both 3O ⁰ C and 25 ⁰ C.

It is not clear why the initial full length ACC2 construct (with and without the N- terminal signal domain) did not complement. It is possible that one or more of the three amino acid substitutions, due to PCR-related errors, affect ACC2 activity (the gene also had 6 silent mutations). The three substitutions are located in less conserved domains and at highly divergent positions in the ACC sequence. The coding sequence of this ACC2 was also shorter - it lacked one exon due to alternative splicing (both splicing forms were confirmed by additional RT-PCR experiments). This splicing form has not been previously reported so its effect on ACC2 activity is not known, but similar alternative splicing was suggested to affect modification/activity of human ACCl (Barber et al., 2005). Once the three amino acid changes (and one silent mutation) were repaired, the full-length ACC2 gene complemented the yeast ACCl null mutation, but only when the variable exon shown in FIG. 1 was present. The original ACC2 gene-replacement strains obtained by complementation grew poorly, similar to the gene-replacement strains using the wheat cytosolic/human ACC2 chimera described above.

For ACCl, the coding sequences first cloned by RT-PCR contained 7 amino acid differences from the sequence deduced from the human genome sequence (plus four silent mutations). The resulting gene did not complement the yeast ACCl null mutation. Two of the differences were located at amino acid positions conserved in eukaryotic ACCs. These changes were shown to be due to PCR-related errors and once they were corrected the resulting gene did complement. The remaining three amino acid substitutions (probably also PCR-related errors) and a small deletion due to alternative splicing described previously (Barber et al, 2005) do not prevent complementation.

EXAMPLE 3

Growth Inhibition of One of the Gene-Replacement Yeast Strains Carrying a Wheat Cytosolic/Human ACC2 Chimera or a Wheat Cytosolic/Plastid ACC Chimera

Materials, Methods, and Equipment

Yeast extract Peptone Raffinose Galactose TYPRG) medium: 1% bacto-yeast extract (Difco, Detroit, MI), 2% tryptone peptone (Difco, Detroit, MI), 2% raffinose (Difco, Detroit, MI), 2% galactose (Difco, Detroit, MI), 0.1% adenine sulfate (Sigma, St. Louis, MO).

YPRG plates. YPRG medium plus 1.5 % agar (Difco, Detroit, MI).

Sterilization. Bacto-yeast extract was dissolved/mixed with tryptone peptone and agar (for plates) in 0.5 L of water and autoclaved for 30 mins. Adenine sulfate, raffinose and galactose were dissolved in 0.5 1 of water and filter sterilized using a 0.22 μm cellulose acetate filter (e.g. Corning, Big Flats, NY). The two components were mixed, and Ampicillin (50 mg/L) was added when the temperature of the autoclaved component was about 6O ⁰ C.

96-well culture plates. Falcon/Becton Dickinson Labware (Franklin Lakes NJ) or equivalent plates from another supplier. Important characteristics of culture plates include a transparent flat bottom, a transparent low evaporation lid which allows air exchange, a total well volume of 0.4 ml, and a design preventing cross-contamination during vigorous shaking. The plates must also be sterile.

Shaker/incubator. HiGro model HGA 02 from Gene Machines/Genomis Solutions (Ann Arbour MI), equipped with oxygen tank, or similar equipment from another manufacturer.

Inhibitors. Known inhibitors of ACC, including sethoxydim and haloxyfop, were dissolved in DMSO at 1-20 mM concentration. Any candidate inhibitors can be prepared in a similar manner and provided in 384-well plates dissolved in DMSO at 1-10 mM concentration or in 50% DMSO/water at 0.5 - 5 mM , such as for manual or robotic screening techniques set forth below.

Yeast Strain Preparation. Tester strains (haploid yeast ACC gene-replacement strains obtained by complementation and ascospore dissection as described in Example 1) were revived from frozen stock by streaking/patching on a YPRG plate, and incubating the plate for about 48 hours at 3O ⁰ C. Liquid culture inoculated from the patch was then grown in YPRG medium for 36-48 hours at 3O ⁰ C with vigorous shaking. It was found that 10 ml of culture grown to an optical density (OD) of 2 was sufficient for twenty 96-well plates in the screen. A tester strain was prepared for the inhibition test by diluting the culture with YPRG plus

Ampicillin (final concentration 50 mg/L) to OD ~0.05, 20 ml for each 96-well plate in the screen

Screening Methods. The screen can be performed using a manual or robotic set up. For example, one can obtain 200 μLs of the diluted tester yeast strain culture set forth in Example 1 (with YPRG medium plus Ampicillin), which should be shaken/mixed to prevent yeast from settling to the bottom of the container during pipeting the culture from the container to multiple wells of multiple plates. Then 200 μl of yeast strain culture is combined with 2 μL of a solution of the candidate inhibitor (4 μls if compound is dissolved in 50% DMSO/water) in each well of the plates. The plates can be incubated at 3O ⁰ C with shaking, and the OD measured at 580-620 nm using a plate reader at multiple time points: once or twice after 2-4 hours, and then three to four more times between 24 and 72 hours. Wells with the culture alone (no compound) should be included as a growth control. Wells with the medium alone (no yeast, no compound) should be included as blanks for the reader. Potential inhibitors can be identified by comparing cell culture density at different time points with and without compound. The test should be repeated for all potential inhibitors to eliminate any artifacts. Each potential inhibitor should be tested at several different concentrations, each in multiple wells, to determine dose dependence and statistical significance.

Results

The results shown in FIG. 2 were obtained using a manual 96-well set-up to measure growth of the yeast gene replacement strain carrying a wheat cytosolic/human ACC2 chimera in the absence and in the presence of various concentrations of sethoxydim. FIG. 3 and FIG. 4

show results from similar experiments for yeast gene replacement strain carrying wheat cytosolic/plastid ACC chimera and two know strong inhibitors of this chimeric ACC, haloxyfop and sethoxydim.

In general, these experiments rely on growing a small (about 250 μl) yeast culture in a well of a 96-well plate with shaking and aeration for a period of up to 4 days. Culture in one well contains no compound added and culture in another well contains a compounds at a concentration up to 200 μM. Lower concentrations can be used for compounds less soluble in water (yeast medium) and to refine the screen to eliminate artifacts caused for example, by inhibition of yeast growth for reasons other than inhibition of the ACC activity (general toxic effect on yeast). The purpose of the screen was to identify strong inhibitors which show a 50% inhibition at as low concentration as possible. Industry standard for compound concentration in such screens is 10-20 μM. Culture density in both wells was measured at various time points to determine the effect of the added compound on yeast growth. Yeast growth was measured by measuring culture turbidity at 580-620 nm using a plate reader with spectrophotometric capability. The potency of an inhibitor is determined from growth curves such as those shown in FIG.2-FIG.4. Multiple compounds can be tested in this fashion in each 96-well plate. Large chemical libraries can be screened using multiple 96-well plates. For example, use of a molecular biology robotic station to dispense the culture and the chemicals, and to automatically measure culture density can be performed. Different yeast strains can be incorporated into the screen or the follow-up tests. For example, wild-type yeast can be used to eliminate compounds with general toxicity towards yeast. Such control experiments were performed for the experiments illustrated in FIG. 2- FIG. 4. Neither sethoxydim nor haloxyfop inhibits the growth of wild-type yeast. These compounds have no general yeast inhibitory properties (at concentrations shown) and are known to target ACC specifically. Wild-type yeast in combination with various yeast gene- replacement strains can be used to assess specificity of any new inhibitor identified in the screen. For example, results shown in FIG. 2 and FIG. 4 show specificity of sethoxidim towards wheat plastid ACC and human ACC2: sethoxydim is a very potent inhibitor of wheat ACC and only a moderate inhibitor of human ACC2. Other combinations of gene- replacement strains reveal other differences in inhibitor specificity: wheat versus human, yeast versus wheat, yeast versus human, etc.

EXAMPLE 4 Protocol for High-Throughput Screening of Chemical Libraries

A protocol was developed for a high-throughput screening of chemical libraries. In general, this experiment relies on growing a small (-200 μL) yeast culture in a well of a 96- well plate with shaking and aeration for a period of up to 4 days. Yeast growth is assessed by measuring culture turbidity at 580-620 nm using a plate reader with spectrophotometric capability.

FIG. 2 illustrates growth curves of the yeast strain ACC2-col5 in the presence of different concentrations of a known low-affinity inhibitor of human ACC2, Sethoxydim (Seng et al, 2003). The growth curve has a lag phase, which lasts approximately 32 hours. After the lag, the yeast culture enters into log phase, which lasts up to 72 hours in control cells. Sethoxydim reduces the rate of log phase growth and when the end point is measured, the growth inhibition is about 75% at 200 μM Sethoxydim. This experiment was conducted only to test strain ACC2-col5 in our screening system - sethoxydim is too weak an inhibitor to be useful. In the proposed screening of chemical libraries, much stronger inhibitors will be selected.

FIG. 3 and FIG. 4 illustrate growth of yeast gene replacement strains with a wheat cytosolic/plastid ACC chimera and the growth inhibition of these strains with known ACC inhibitors (herbicides) with sub-micro molar IC _5O . The data show that these yeast constructs are very well suited for a sensitive, inexpensive and simple screening of chemical libraries to identify strong ACC inhibitors. Further, it has been established that the collection of yeast gene-replacement strains could be used to determine the specificity of human ACC2 inhibitors.

A series of multi-plate experiments were conducted using the collection of yeast gene- replacement strains and several herbicides with known potency against various ACCs in order to develop a screening protocol that could be scaled up for a high-throughput screen. The results shown in FIG. 2 — FIG. 4 are from such experiments. Based on these experiments, the optimal culture volume, initial cell density, culture conditions, time points for yeast growth determination (different for different strains depending on the doubling time), and most informative compound concentration (lOOμM) allowing detection of medium strength inhibitors (~10 μM IC ₅₀ ), and the type and plate position of necessary controls have been determined. Changing conditions during culture incubation were identified as potential sources of variability causing water evaporation and condensation on the lid, affecting growth

and cell density reading, and faster growth in the outer wells affecting scoring reliability. These potential sources of variability are overcome by using incubators with proper shaking, temperature and humidity control. In addition, the positional effect can be addressed by comparing results for neighboring wells. To avoid variation caused by small amounts of condensed water on the lid, the plate lids were removed. Sterile conditions were used up to the point of the final culture density determination.

The overall well-to-well variability within the same plate estimated from multiple experiments is less than 35 %, considering variability at all steps up to the final culture density measurement. This should not affect the outcome of a screen to identify strong inhibitors of human ACCs showing inhibition well above 50%, as strong as sethoxydim and haloxyfop are for wheat plastid ACC (FIG. 3 and FIG. 4). Depending on the outcome of the screen, the threshold can be changed to 75% inhibition.

All of the nucleic acids, conjugates, yeast cells, and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the nucleic acids, conjugates, yeast cells, and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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