Modulators of the activity of a protein involved in the regulation of energy homeostasis

The present invention is based on the observation that reduced activity of a protein involved in the regulation of energy homeostasis results in body fat reduction. Thus, the present invention relates to a pharmaceutical composition comprising a compound (a) reducing or inhibiting, or a compound (b) increasing the biological activity of the protein or its expression. The present invention also relates to the use of said compounds for the prevention/treatment of diseases wherein decrease or increase of the activity of the protein has a therapeutic effect, e.g. obesity, adiposity diabetes, dyslipidemia, or an anorectic disease, anorexia or emaciation. Finally, the present invention also relates to methods for the diagnosis of diseases associated with an aberrant activity of the protein.

Obesity and diabetes are highly prevalent diseases caused by the aberrant regulation of carbohydrate and lipid metabolism. Although the biochemical pathways of carbohydrate and lipid metabolism are well known, much remains to be learned about the molecular mechanisms and signals controlling flux through these pathways, and thus homeostasis. Humoral signaling molecules such as insulin and leptin are important in both carbohydrate and lipid metabolic control. How these signals link to the metabolic genes controlling the relevant biochemical pathways is a topic of current interest (Friedman, Nutr.Rev.60 (2000), pp 1-14, discussion pp 68-84, 85-17.)

Components of the insulin signal transduction pathway have been extensively studied in the context of tissue growth

control during development. Mutations in the core components of the pathway - the insulin receptor (InR) , phosphoinositide- 3-kinase (PI3K), PDKl, Akt/PKB, Tuberous sclerosis complex (Tscl/Tsc2), Rheb, the target of Rapamycin (TOR), and ribosomal protein S6 Kinase (S6K) - all cause tissue growth abnormalities or lethality. Stimulation of the insulin receptor activates PI3K, which increases the level of phosphoinositol (3, 4, 5) triphosphate (PIP3) at the plasma membrane. PIP3 binding recruits the protein kinase Akt to the membrane, where it is activated through phosphorylation by PDKl. The connection between Akt and TOR is less well understood, but TOR is genetically downstream of Akt and is activated. TOR is a protein kinase that regulates the translational machinery of the cell via two well-characterized effectors: S6K and 4E-binding protein (4E-BP). S6K controls ribosome biogenesis and thus the biosynthetic capacity of the cell. 4E-BP binds to the translation factor eIF4E and prevents assembly of a protein complex that facilitates recruitment of the ribosome. TOR phosphorylates 4E-BP and prevents its binding to eIF4E, promoting cap dependent translation.

In addition to insulin-response, this signaling cassette also integrates information on cellular nutritional status. TOR receives information on the cellular energy levels from the heterodimeric Tscl/2 complex. Tsc2 serves as a GTPase activating protein that inactivates Rheb, and thereby reduces TOR activity. Tscl/2 mutations result in TOR hyperactivation and tissue overgrowth. Mutants in mouse Tscl or Tsc2 have benign overgrowths called harmatomas and exhibit an increased susceptibility to tumor formation. Cellular AMP levels are sensed by AMP-activated protein kinase (AMPK) which phosphorylates and activates Tsc2 to inhibit TOR. TOR activity is also regulated by oxygen, through the hypoxia-induced transcription factor, HIF. HIF controls expression of REDDl /Scylla/Charybdis, which reduce Tscl/2 activity.

Considerable evidence has accumulated to indicate that the Insulin/TOR pathway controls fat metabolism as well as tissue growth. Flies and mice with reduced InR/ IGF receptor and PI3K activity are small and have elevated fat levels. Mice mutant for Akt2 become insulin resistant and develop lipoatrophy as they age; mice mutants for Aktl have normal glucose metabolism but are growth defective. Human mutants for Akt2 also have abnormal metabolism and are 35% slimmer than average. One output-branch of the core pathway is via transcription factors of the winged helix-Forkhead family. In response to insulin, Akt phosphorylates FOXO proteins, which promotes their interaction with 14-3-3 proteins and leads to cytoplasmic retention and inactivation. Foxo transcription factors have been implicated in the control of fat metabolism and lifespan in C. elegans, flies and mice. In addition, elements of the TOR branch of the pathway are beginning to be implicated in fat metabolism. TOR phosphorylates and regulates S6K and 4E- BP. S6K mutant mice are resistant to diet induced obesity. 4E- BPl mutant mice, have a defect in fat metabolism. Recent studies have also identified the eIF4E kinase LK6 as a modulator of growth and fat metabolism. However, although there is now much known about the biochemical pathways of carbohydrate and lipid metabolism, effective therapies for diseases associated with an aberrant regulation of these pathways, e.g., obesity and diabetes, are hardly available.

Thus, the technical problem underlying the present invention is to provide means suitable for treating or preventing diseases being based on aberrant regulation of carbohydrate and/or lipid metabolism.

The solution of this technical problem is achieved by providing the embodiments characterized in the claims.

During the experiments resulting in the present invention, a novel modulator of the Insulin/TOR pathway, "melted", was identified. Flies mutant for melted show pronounced defects in fat metabolism. The melted gene encodes a PH domain protein that interacts with both Tscl and FOXO. Melted protein can recruit the Tscl/2 complex to the cell membrane and thereby modulate its output via the TOR pathway. Melted protein can also recruit FOXO to the membrane in an insulin-regulated manner and thereby influence expression of Foxo target genes. Thus, melted mutants cause reduced TOR activity and increased FOXO activity. In so doing, reduced Melted activity results in physiological changes that mimic what happens when a normal animal is placed under conditions of nutrient deprivation. Thus, a method to reduce Melted activity would be useful to treat obesity and obesity-related diseases in humans and animals .

It has been found, that Melted contains two functional domains, identified as regions of high conservation between the fly and human proteins - an N-terminal protein interaction domain and a C-terminal PH domain. The PH domain targets Melted to the cell membrane. Overexpressed PH-GFP fusions show sharp membrane localization, and subcellular fractionation assays showed that in steady state the majority of endogenous Melted protein is membrane associated. Rescue assays show that, unlike the full-length protein, Melted protein missing the PH domain cannot rescue the melt ^δl mutant phenotypes. Thus, Melted protein requires its PH domain, and presumably its membrane localization, to have biological activity in vivo.

Melted functions as an adaptor, facilitating association of the TSC complex and FOXO with their upstream signaling inputs at the membrane. Both FOXO and Tsc2 are phosphorylated in vivo by PKB, which becomes activated at the cell membrane when it binds PIP3 and is phosphorylated by PDKl. Although the

phosphorylation of Tsc2 by PKB is not strictly necessary for viability it is possible that this phosphorylation is modulatory in function. Tsc2 is also regulated via phosphorylation by AMPK. AMPK is membrane associated through its inyristoylated beta-subunit . Therefore, recruiting the Tsc complex and FOXO to the cell membrane may alter their state of activation.

The activity of signaling pathways downstream of Tscl/2 and FOXO are altered in the melted mutant. Melted mutants cause reduced TOR activity, observed by measuring the level of 4E-BP phosphorylation in adipose tissue. TOR activity is not normally limiting for 4E-BP phosphorylation. Increased 4E-BP level leads to increased phosphorylated 4E-BP in normal flies. In melted mutants, total 4E-BP protein increased, but the level of 4E-BP phosphorylation did not, indicating reduced relative TOR activity in the melted mutant. TOR activity positively regulates fat accumulation in adipose tissue (c.f. Figure 6). Therefore, the reduced TOR activity in the melted mutant adipose tissue contributes to the mutant leanness.

FOXO activity is increased in melted mutants as indicated by the increased level of 4E-BP transcription (4E-BPtranscription is a well characterized FOXO target (Junger et al . , J.Biol. 2 (2003), 20; Puig et al . , Genes Dev. 17 (2003), 2006-20). 4EBP transcription is upregulated in the melted mutant, and this upregulation is FOXO dependent, suggesting that FOXO activity is increased in the melted mutant. This was confirmed by showing that decreasing FOXO activity by removing the foxo gene in a melted mutant decreases the severity of the melted mutant fat defect. Thus, FOXO is an important effector of melted in regulation of fat metabolism.

Melt ^ύl mutant animals are lean due to reduced lipid accumulation in adipose tissue, and this phenotype is

autonomous to the adipose tissue since it can be rescued by- adipose-tissue specific expression of melted. Since the leanness phenotype is adipose-tissue autonomous, the transcriptional changes that occur in the adipose tissue upon loss of Melted function were examined. Adipose tissue undergoes a dramatic transcriptional change upon loss of Melted function. At the 99% confidence level, 720 genes had altered expression levels in the fat body, and 249 were misregulated by more than 1.5-fold. 63% of the misregulated genes for which a function is annotated are metabolism related. Although many of these are involved in lipid or carbohydrate metabolism, it was surprising to find a significant number of genes involved in proteolysis and amino acid metabolism. Recently, FOXO has been shown to promote protein turnover during fasting suggesting a possible link between those genes and FOXO.

Table 1 Misregulated lipid metabolism genes in melt ^δ1 fat body

Up-regulated

Gene Function fold δ RT-PCR

CG4757 carboxylesterase/lipase 8.5 >5000

Cyp6d5 cytochrome P450 5.7 28.7

Cyp12d1-d cytochrome P450 5.3 7.4

CG4842 alcohol dehydrogenase 2.5

CG5171 trehalose-phosphatase 2.4 11.8

CG10512 Malate/L-lactate dehydrogenase 2.4 α-Est8 lipase/esterase 2.0

Adh alcohol dehydrogenase 2.0

CG3940 carbonic anhydrase 1.9

Csat galactose transport 1.8

CG6022 holocytochrome-c synthase 1.8

Glaz lipid transport 1.7

CG 10962 short-chain dehyrogenase 1.6

Cyp28d1 cytochrome P450 1.5

CG8690 alpha-glucosidase 1.5

CG4670 glucosidase activity 1.5

Down-regulated

Gene Function fold δ RT-PCR

Cyp4p1 cytochrome P450 15.0

Cyp4p3 cytochrome P450 10.6

CG 12224 aldo/keto reductase 6.1

Cyp4d8 cytochrome P450 2.8

Cyp9b2 cytochrome p450 2.5

Sug transcription factor, sugar dependent 2 4

CG30148 glucosidase 2 2

CG3488 Lipase 2 2

CG8323 mitochondrial transport 2 1

CG 15828 lipid transport 2 0

CG5288 galactokinase 2 0

CG5295 adipose triglyceride lipase homolog 2 0 yιp2 acetyl-CoA C-acyltransferase 2 0

Cyp6a17 cytochrome P450 1 9

CG1 1963 succinate-CoA ligase 1 8

CG13890 dodecenoyl-CoA delta-isomerase 1 8 1 9

AcCoAS acetyl-CoA synthase 1 8 1 9

Acox57D-d acyl-CoA oxidase/dehyrogenase 1 8 2 9

Nes O-acyl transferase 1 8

CG2065 short-chain dehydrogenase 1 7

CG1041 carnitine O-acetyltransferase 1 7

CG6178 fatty acid ligase 1 7

Gyk glycerol kinase 1 6

Pgm phosphoglucomutase 1 6

CG 17292 triacylglyerol lipase 1 6

CG2767 alcohol dehydrogenase 1 6

CG30069 short-chain dehydrogenase 1 6

Cyp9f2 cytochrome P450 1 6

Fbp1 fat body storage protein uptake 1 5

Cyp6d2 cytochrome P450 1 5

PEPCK gluconeogenesis 1 5 1 2

Human Melted protein is highly homologous to the fly protein, and can rescue the melted mutant fly. This can be seen from the various sequences contained in Fig. 8. It can be expected that the organismal function of fat metabolism regulation is also conserved in mammals and that blocking melted activity in mammals has similar effects as in the fly - reduced triglyceride accumulation by adipose tissue. The changes in the physiology of melted mutant flies resemble the effects of nutrient deprivation in normal animals. Since melted is highly conserved between flies and humans, and because of its pronounced effects on fat metabolism, modification of its activity provides a basis for therapy. Thus, methods to reduce Melted activity are expected to be useful in treating obesity and obesity-related diseases including diabetes.

Brief description of the drawings

Figure 1 : The melted gene product regulates tissue growth

(A) Overexpression of melted in the posterior compartment (red wing; engrailed-galA , EP31685) . A normal wing is shown in green for comparison.

Note that the anterior compartments are the same size (overlap) but the posterior (lower) is larger in the red wing.

(B) Schematic representation of the melted locus. melt ^δ1 is a 22 kb deletion, including the adjacent genes CG32390 and cornetto. melt ^δ2 deleted 2.5 kb of DNA, including exon 1. The first exon of the predicted gene is non-coding, with the ATG for the open reading frame located in exon 2. RT-PCR of exons 5-7 showed that an alternate form of melt transcript was produced in the homozygous melt ^δ2 deletion flies, suggesting that it is not a null allele.

(C) Average body weight of 3-day old wild-type male flies, tubulin-GAL4 flies and flies overexpressing EP31685. n=number of flies measured. Error bars: standard deviation.

(D) Average body weight of 3-day old wild-type male flies, melt ^δ1 homozygous deletion mutant flies and melt ^δ1 homozygous mutants expressing full-length Drosophila Melted (dmelt) , dMelt lacking the PH domain (dMeltδPH) , or the full length Human Melted (hMelt) . In addition, eggs laid by melt ^δ1 homozygous females were 13% smaller than control eggs (t-test = 10-7,- rescued by expression of UAS-melt) .

* indicates statistically significant difference from wildtype . ** indicates statistically significant difference from melt ^δ1

Figure 2: Reduced fat reserves in melted mutants

(A) Total body fat (normalized to total protein) in wild-type, melt ^δ1 and melt ^δ1 male flies rescued by expression of full length Melted. Error bars: standard deviation (3 replicates).

(B) Total body fat in male flies expressing UAS-melted-RNAi .

(C) Normalized total body fat of adult males of indicated genotypes .

Figure 3 : Membrane localization of Melted, a PIP binding protein

(A, A') Immunoblots showing binding of bacterially expressed Melted protein to immobilized phosphoinositides (PIP Strip, Echelon Biosciences) at Melted protein concentrations of roughly 100 ng/mL (A) or IOng/mL (A ^' ) .

Ptdlns=phosphatidylinositol ; PE=phosphatidylethanolamine; PC=phosphatidylcholine; PS=phosphatidylserine; PA=phosphatidic acid. The binding pattern for a control using the PIP3 binding domain of GRPl was different.

(B) Immunofluorescence showing membrane localization of Melted-GFP in serum starved (-insulin) and insulin-treated S2 cells .

(C) Immunofluorescence showing membrane localization of MeltedPH-GFP in wing imaginal disc cells.

(D) Immunofluorescence showing cytoplasmic localization of Melted-GFP lacking the PH domain (MeltδPH-GFP) in wing imaginal disc cells.

(E) Immunoblot showing the distribution of Melted protein in cytosolic (c) and membrane (m) fractions of wild-type and melt ^δ2 homozygous mutant tissue. Controls are anti-Yl4, a cytoplasmic protein (Hachet and Ephrussi, Curr Biol. 11

(2001), 1666-74) and anti-PVR (PDGF/VEGF receptor a transmembrane protein (Duchek et al . , Cell 107 (2001), 17-26).

Figure 4: Melted binds Tscl/2 and regulates TOR activity

(A) Immunofluorescence showing S2 cells transfected to express Tscl, Tsc2 and Melted, as indicated. Tscl was epitope tagged with myc (green) , Tsc2 was tagged with V5 (red) . Melted was detected with anti-Melted antibody (Blue) . Nuclei were visualized with DAPI (white) .

(B) Left: Immunoblot of fat-body tissue from wild-type and melt mutant larvae. Right: Immunoblot of fat-body tissue from wild-type and larvae expressing 4E-BP under ppl-Gal4 control.

Upper panels: probed with anti-4E-BP to visualize total 4E-BP protein .

Middle panels: probed with phosphospecific antibody to visualize 4E-BP protein phosphorylated on Thr36/47. Lower panels: loading controls.

(C) Quantitative RT-PCR comparing 4E-BP transcript levels in wild-type and melted mutant fat body tissue. Error bars: standard deviation.

Figure 5 : Melted binds FOXO and regulates expression of the FOXO target 4E-BP

(A) Immunofluorescence showing S2 cells transfected to express FOXO-GFP and Melted, as indicated. FOXO-GFP was visualized with anti-GFP (green) , Melted was detected with anti-Melted antibody (red) . Nuclei were visualized with DAPI (blue) . Cells were treated with insulin (+) or serum starved (-) as indicated.

(B) Co-immunoprecipitation of Melted with HIS-tagged FOXO. S2 cells were transfected to express FOXO-HIS and/or Melted as indicated. Lysates were immunoprecipitated with anti-HIS and blots probed with anti-Melted. Melted expression in the total lysates is shown below.

(C) Quantitative PCR comparing 4E-BP transcript levels in wild-type, melt, foxo and melt/foxo double mutant flies. Fed flies have normal levels of circulating insulin and therefore low FOXO activity. 4E-BP gene expression increased after starvation for 24 hours to reduce insulin levels, and showed a further FOXO-dependent increase in the melt mutant. Error bars: standard deviation.

Figure 6: Melted acts via FOXO and TOR

(A) Total body triglyceride (normalized to total protein) in wild-type, melt ^δl and melt ^δ1 foxo double mutant 3-day old male flies. melt ^δ1 and melt ^δ1 foxo double mutant larvae were hand selected and grown without competition from heterozygous

siblings. Error bars: standard deviation (5-9 replicates each) .

(B) Normalized total body triglyceride in 3-day old male flies containing the UAS-TOR or ppl-Gal4 transgenes or both combined to express UAS-TOR under ppl-G4 control. Error bars: standard deviation. One representative example is shown. The experiment was repeated 5 times, yielding a t-test=0.03.

(C) Normalized total body triglyceride in 3-day old male wild- type, melt ^δ1 and melt ^δl mutant flies carrying the ppl-gal4 driver, UAS-TOR transgene or expressing UAS-TOR under ppl-Gal4 control .

(D) Normalized total body triglyceride in newly eclosed wild- type, melt ^δ1 and melt ^δ1 mutant flies carrying the fat body specific gal4 driver Isp2, the UAS-DpIlO transgene (the catalytic subunit of PI3K) , or expressing UAS-DpIlO under Isp2-Gal4 control.

Figure 7: Transcriptional changes in melt ^δ1 mutant fat body and resulting metabolic effects

(A) Pie-chart showing the distribution of genes misregulated >1.5 fold in the melt mutant fat body.

(B) Total circulating diacylglycide levels in hemolymph from' wild-type and melt ^δ1 mutant larvae. The reduction in circulating DAG is statistically significant (t-test = 0.02).

(C) Total circulating sugar levels in hemolymph from wild-type and mel t ^δ1 mutant larvae.

Figure 8 : Sequence information

Thus, the present invention relates to a pharmaceutical composition comprising a compound (a) reducing or inhibiting, or a compound (b) increasing the biological activity of the Melted protein or its expression, wherein said compound (a) is an antisense compound, antibody, an inactive version of the

Melted protein or a nucleic acid sequence encoding an inactive version of the Melted protein and wherein said compound (b) is a biologically active version of the Melted protein or a nucleic acid sequence encoding a biologically active version of the Melted protein.

The term "Melted protein" as used herein relates to a Melted protein from any organism, with the mammalian protein being preferred. The term "Melted protein" covers in particular the following :

1. the human Melted protein

2. the mouse Melted protein

3. the drosophila Melted protein

4. an isoform, fragment, or variant of said proteins or polypeptides

5. a polypeptide which is at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% and up to 99,6% identical to the amino acid sequence of said proteins

6. a homolog from any organism of said proteins

7. a polypeptide composed of a fusion between a polypeptide described in 1-6 with another polypeptide that may be, but is not limited to, a fluorescent protein, an epitope tag or an enzyme .

The term "biological activity of a Melted protein" as used herein includes, but is not limited to:

1. localization at the cell membrane

2. phospholipid binding

3. phosphatidylinositol ( 5 ) P binding

4. binding to a drosophila FOXO protein, a human FOXO protein, a FOXO homolog from another organism, or functional equivalents thereof

5. binding to drosophila Tscl protein, to the human homolog Hamartin, to a homolog from another organism, or to a functional equivalent thereof

The term "reducing, inhibiting or increasing the biological activity of the Melted protein" as used herein means that at least one of the biological activities of Melted is reduced, inhibited or increased, preferably the biological activity associated with the N-terminal protein interaction domain or the C-terminal PH domain.

The generation of suitable antisense oligonucleotides includes determination of a site or sites within the Melted gene for the antisense interaction to occur such that the desired effect, e.g., inhibition of expression of the protein, will result. A preferred intragenic site is (a) the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene or (b) a region of the mRNA which is a "loop" or "bulge", i.e., not part of a secondary structure. Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. In the context of this invention, "hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. "Complementary" as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions

in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound does not need to be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., in the case of therapeutic treatment .

The skilled person can generate antisense compounds according to the present invention on the basis of the known DNA sequences for Melted. In this regard reference is made to Fig.

"Oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. While antisense oligonucleotides are a preferred form of the

antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention comprise from about 8 to about 50 nucleobases (i.e. from about 8 to about 50 linked nucleosides) . Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 15 to about 25 nucleobases. Antisense compounds include RNAi, _ξ iRNA, micro _m iRNA, ribozymes, external guide sequences (EGS), oligonucleotides (oligozymes) , and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and inhibit its expression.

Alternatively, the pharmaceutical composition of the invention contains a vector allowing to transcribe an antisense oligonucleotide of the invention, e.g., in a mammalian host. Preferably, such a vector is a vector useful for gene therapy. Preferred vectors useful for gene therapy are viral vectors, e.g. adenovirus, herpes virus, vaccinia, or, more preferably, an RNA virus such as a retrovirus. Even more preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of such retroviral vectors which can be used in the present invention are: Moloney murine leukemia virus (MoMuLV) , Harvey murine sarcoma virus (HaMuSV) , murine mammary tumor virus (MuMTV) and Rous sarcoma virus (RSV) . Most preferably, a non-human primate retroviral vector is employed, such as the gibbon ape leukemia virus (GaLV) , providing a broader host range compared to murine vectors. Since recombinant retroviruses are defective, assistance is required in order to produce infectious particles. Such assistance can be provided, e.g., by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. Suitable helper cell lines are well known to those

skilled in the art. Said vectors can additionally contain a gene encoding a selectable marker so that the transduced cells can be identified. Moreover, the retroviral vectors can be modified in such a way that they become target specific. This can be achieved, e.g., by inserting a polynucleotide encoding a sugar, a glycolipid, or a protein, preferably an antibody. Those skilled in the art know additional methods for generating target specific vectors. Further suitable vectors and methods for in vitro- or in vivo-gene therapy are described in the literature and are known to the persons skilled in the art; see, e.g., WO 94/29469 or WO 97/00957.

In order to achieve expression only in the target organ, the DNA sequences for transcription of the antisense oligonucleotides can be linked to a tissue specific promoter and used for gene therapy. Such promoters are well known to those skilled in the art (see e.g. Zimmermann et al . , (1994) Neuron _12, 11-24; Vidal et al . ; (1990) EMBO J. 9_, 833-840; Mayford et al . , (1995), Cell 8JL, 891-904; Pinkert et al . , (1987) Genes & Dev. JL, 268-76) .

Within an oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5 ¹ phosphodiester linkage. Specific examples of preferred antisense compounds useful in the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified oligonucleotide backbones which can result in increased stability are known to the person skilled in the art, preferably such modification is a phosphorothioate linkage.

A preferred oligonucleotide mimetic is an oligonucleotide mimetic that has been shown to have excellent hybridization properties, and is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone (see, e.g., Nielsen et al . , Science 254 (1991), 1497-1500.)

Modified oligonucleotides may also contain one or more substituted or modified sugar moieties. Preferred oligonucleotides comprise one of the following at the 2 ' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-0-alkyl , wherein the alkyl , alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C ₂ to Ci ₀ alkenyl and alkynyl. A particularly preferred modified sugar moiety is a 2 ' -O-methoxyethyl sugar moiety .

Oligonucleotides useful in the present invention may also include nucleobase modifications or substitutions. Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil , 2- thiothymine and 2-thiocytosine etc., with 5-methylcytosine substitutions being preferred since these modifications have been shown to increase nucleic acid duplex stability.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such

moieties include lipid moieties such as a cholesterol moiety, cholic acid, a thioether, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety .

The present invention also includes antisense compounds which are chimeric compounds. "Chimeric" antisense compounds or "chimeras, " in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA: DNA or RNArRNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNArDNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers .

Antibodies useful for the present invention are polyclonal or monoclonal antibodies and their derivatives, especially chimeras, Fab-fragments, Fc- fragments, diabodies etc. The term ,,antibody", preferably, relates to antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specifities, as well as distinct monoclonal antibody preparations. Monoclonal antibodies are made from an antigen containing, e.g., a fragment of Melted by methods well known to those skilled in the art (see, e.g., Kohler et al . , Nature 256 (1975) , 495) . As used herein, the term ,,antibody" (Ab) or ,,monoclonal antibody" (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. (Wahl et al . , J. Nucl . Med. 24, 316- 325 (1983)) . Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies useful for the purposes of the present invention include chimerical, single chain, and humanized antibodies.

Alternatively, preferred compounds for the purpose of the invention are inactive versions of Melted or genes encoding inactive versions of Melted. Such inactive versions can be generated according to well known methods. The term "inactive" as used herein means that at least one of the biological activities of Melted is abolished. Such compounds can have a therapeutic effect in the human body by displacing their functionally active counterpart, in particular when applied in excess . A preferred example of an inactive version of Melted is a mutated form of Melted, wherein the PH domain has been mutated.

Further compounds suitable for the purposes of the present invention and methods how to identify/select such compounds are in more detail described below.

Preferably, in a pharmaceutical composition, such compounds as described above are combined with a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" is meant to encompass any carrier, which does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.. Such carriers can be formulated by conventional methods and the active compound can be administered to the subject at an effective dose.

An "effective dose" refers to an amount of the active ingredient that is sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology. An "effective dose" useful for treating and/or preventing these diseases or disorders may be determined using methods known to one skilled in the art (see for example, Fingl et al . , The Pharmocological Basis of Therapeutics, Goodman and Gilman, eds . Macmillan Publishing Co., New York, pp. 1-46 ((1975)).

Administration of the suitable compositions may be effected by different ways, e.g. by intravenous, intraperetoneal, subcutaneous, intramuscular, topical or intradermal administration. The route of administration, of course, depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and

other clinical factors. As is well known in the medical arts, dosages for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health and other drugs being administered concurrently.

The present invention also relates to various therapeutic uses of the compounds of the invention, e.g.,

(a) for the prevention or treatment of a disease wherein an increase or decrease of the activity of Melted has a beneficial effect, including but not limited to obesity, adiposity and diabetes;

(b) for the prevention or treatment of a disease characterized by an increased activity of Melted, including, but not limited to obesity, adiposity or diabetes; and

(c) for the prevention or treatment of a disease characterized by a decreased activity of Melted, including, but not limited to an anorectic disease, anorexia, emaciation or any other disease of low body fat accumulation.

The present invention also relates to a method for identifying a compound capable of modifying a biological activity of the Melted protein or its expression, comprising the steps of:

(a) incubating a candidate compound with a test system comprising a Melted protein or its gene,- and

(b) assaying a biological activity of the Melted protein; wherein an altered biological activity of the Melted protein compared to a test system in the absence of said test compound is indicative of the presence of a candidate compound having the desired properties .

As regards a particular biological activity, such assay may be based on the PH domain function as described in the Examples; see also the results shown in Figure 3C+D: The PH domain is

required for biological activity of melted protein in vivo. Mutant flies can be rescued by expression of a full-length Melted protein, but not by a protein lacking the PH domain. Expression of Melted protein, an epitope tagged Melted protein that can be recognised by an antibody or a GFP fusion (or any other fluorescent protein suitable for FRET, FLIM or other imaging technique based on fluorescence energy transfer) to either the full-length Melted protein or to its PH domain will show localization at the cell membrane. A tagged or GFP fusion construct to a version of Melted that is missing the PH domain is cytoplasmic. Thus, the function of the PH domain (i.e., localization of the protein at the cell membrane) can be used in an assay screening for activity of the PH domain, e.g, by transfecting cells with a Melted protein, epitope tagged Melted protein or GFP-Melted (preferably human) fusion protein, adding the candidate compound and determining the localization of the fusion protein to the membrane on the basis of GFP fluorescence, antibody labelling, fluorescence energy transfer or other suitable method. Alternately the degree of membrane localisation can be measured by subcellular fractionation, or by binding in an in vitro assay to immobilized inositol phospholipids. This can be based on use of phospholipids bound to a solid support, or any other means to measure protein-phopspholipid binding, such as Biacore, calorimetry etc.

There are also some other suitable alternative methods to screen for "Melted" function. These methods mentioned below are as well as the methods mentioned before to be understood as exemplary only:

1. Binding to phosphoinositides and/or recruitment to the cell membrane > Assay localization of a Melted protein to the cell membrane

• Said Melted protein may be a fusion to a fluorescent protein, and localization is detected by fluorescent imaging of cells

• Indirect antibody detection of a Melted protein in a cell, where said Melted protein may have an epitope tag

• FRET based assay in which a Melted protein has a donor or acceptor fluorophore, and the other fluorophore is tethered to the cell membrane. This tethering may be accomplished by fusing a fluorescent protein to a membrane localization signal such as a farnysylation signal or a CAAX domain. Alternatively, it may be fused to a protein that is constitutively membrane- localized.

> Assay recruitment of Tscl or Tsc2 to the cell membrane

• said Tsc protein may be epitope tagged or fused to a fluorescent protein or detected by antibody to the native protein

> Assay recruitment of FOXO to the cell membrane

• said FOXO protein may be epitope tagged or fused to a fluorescent protein or detected by antibody to the native protein

> Assay binding of a Melted protein to a phosphoinositide immobilized on a membrane, washing unbound Melted protein, and detection of bound Melted protein directly or indirectly.

> Assay melted binding to a phosphoinositide immobilized on a substrate suitable for detection by surface plasmon resonance .

> Assay for binding phosphoinostides in soluble form with a carrier, in micelles or inserted into lipid bilayer membranes to surface immobilized Metled protein or fragments thereof.

2. Binding to and/or affecting Tsc activity

> Assay binding of a Melted protein to a Tscl protein. This may be accomplished by:

• immobilizing one of the two proteins on a surface, allowing the other to bind, washing unbound protein, and detecting the bound protein either directly or indirectly via epitope tags or fusion to reporters such as enzymes, or fluorophores

• tagging the two proteins with fluorophores or fluorescent proteins and assaying binding by FRET or other energy transfer method such as FLIM

• any homogeneous assay known to one skilled in the art, such as the AlphaScreen, BRET2 , DELFIA, Fluorescence Polarization, LANCE, or TruPoint assays by Perkin Elmer

> Assaying modulation of Tsc activity. This may be accomplished by treating (1) cells that do express a Melted protein and (2) cells that do not express a Melted protein with a compound, detecting Tsc activity in each case, and comparing the two. Tsc activity may be detected by measuring activity of a component downstream of the Tsc signaling cascade such as Rheb GTPase or the TOR protein kinase, but not limited to these.

3. Binding to and/or affecting activity of a winged-felix Forkhead-family transcription factor, in particular a FOXO, but not limited to this subfamily.

> Assay binding of a Melted protein to a FOXO protein. This may be accomplished by:

• immobilizing one of the two proteins on a surface, allowing the other to bind, washing unbound protein, and detecting the bound protein either directly or

indirectly via epitope tags or fusion to reporters such as enzymes, or fluorophores

• tagging the two proteins with fluorophores or fluorescent proteins and assaying binding by FRET or other energy transfer method such as FLIM

• any homogeneous assay known to one skilled in the art, such as the AlphaScreen, BRET2 , DELFIA, Fluorescence Polarization, LANCE, or TruPoint assays by Perkin Elmer

> Assaying modulation of activity of a FOXO protein. This may be accomplished by treating (1) cells that do express a Melted protein and (2) cells that do not express a Melted protein with a compound, detecting FOXO activity in each case, and comparing the two. Activity of the FOXO transcription factor may be detected by monitoring expression of a FOXO target gene. Examples of such target genes are 4EBP1-3. Expression levels of such target genes may be detected by Northern blotting or by quantitative RT-PCR or by hybridization of probes derived from RNA samples from the treated cells to DNA microarrays, such as Affymetrix or related products, including spotted cDNA arrays or genomic DNA arrays.

For carrying out assays based on GFP-Melted localisation or subcellular fractionation in cells, cells may be seeded 12 to 24 hours prior to transfection, then the cells are transfected with DNA encoding the GFP-Melted-fusion protein (with the Melted protein being any Melted protein, e.g., human Melted, fly Melted or any fragment thereof.) Then, the cells are allowed to express the transfected fusion protein for 12 to 36 hours and subsequently the cells are treated with a candidate compound. GFP localization in the cells is imaged, e.g., by fluorescence microscopy.

Screening protocol using drosophila S2 cells:

(a) Drosophila cells are seeded into a 6-well dish for 4 hours ;

(b) each well is transfected with pMT-GAL4/VP16 (Cu-inducible GAL4 expression, constructed by inserting the GAL4/VP16 open reading frame between the Smol and BamHI sites of pRmHa-3, sequence provided in attached file) and pUAS-dMeltPH-GFP (c.f. Fig. 8) using 4μL of transfectin reagent (Invitrogen, Carlsbad, CA, USA) ;

(c) the reaction is stopped by adding 2 ml of complete medium and the mixture is allowed to recover for 12 hours;

(d) induction is carried out with 700 μM cupric sulphate for 12 hours ;

(e) the cells are treated with the candidate compound for a sufficient amount of time; and

(f) GFP localization is imaged by fluorescence microscopy.

Binding of Melted to the cell surface can also be assayed by use of surface plasmon resonance imaging, e.g., by the biacore method with Ptdlns(5)p (see wv7w.biac0re.com)

As regards another particular biological activity of a Melted protein, such screening assay may be based on the binding between a Melted protein and (1) a FOXO protein or (2) a Tscl protein or homolog such as human Hamartin. Such a binding assay is illustrated in examples 4, 5 and 6. Such binding can be detected in a large number of ways which a person skilled in the art can devise and for which some examples have been mentioned above.

WO 98/25146 describes further methods for screening libraries of complexes for compounds having a desired property, especially, the capacity to agonize, bind to, or antagonize a polypeptide or its cellular receptor. The complexes in such libraries comprise a compound under test, a tag recording at least one step in synthesis of the compound, and a tether

susceptible to modification by a reporter molecule. Modification of the tether is used to signify that a complex contains a compound having a desired property. The tag can be decoded to reveal at least one step in the synthesis of such a compound. Other methods for identifying compounds which interact with the melted protein or nucleic acid molecules encoding such molecules are, for example, the in vitro screening with the phage display system as well as filter binding assays or "real time" measuring of interaction using, for example, the BIAcore apparatus (Pharmacia).

Examples of candidate molecules include antibodies, oligonucleotides, proteins, peptides, or small molecules. Such molecules can be rationally designed using known techniques.

Preferably, said test system used for screening comprises substances of similar chemical and/or physical properties, most preferably said substances are almost identical. The compounds which can be tested and identified according to the method of the invention may be expression libraries, e.g., cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, peptidomimetics, PNAs or the like (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994) , 193-198 and references cited supra) . Furthermore, genes encoding a putative regulator of Melted and/or which exert their effects up- or downstream of Melted may be identified using, for example, insertion mutagenesis using, for example, gene targeting vectors known in the art. Said compounds can also be functional derivatives or analogues of known inhibitors. Such useful compounds can be for example transacting factors which bind to Melted or regulatory sequences of the gene encoding it. Identification of transacting factors can be carried out using standard methods in the art. To determine whether a protein binds to the

protein itself or regulatory sequences, standard native gel- shift analyses can be carried out. In order to identify a transacting factor which binds to the protein or regulatory sequence, the protein or regulatory sequence can be used as an affinity reagent in standard protein purification methods, or as a probe for screening an expression library. The identification of nucleic acid molecules which encode polypeptides which interact with Melted can also be achieved, for example, as described in Scofield (Science 274 (1996), 2063- 2065) by use of the so-called yeast "two-hybrid system". In this system Melted is linked to the DNA-binding domain of the GAL4 transcription factor. A yeast strain expressing this fusion polypeptide and comprising a lacZ reporter gene driven by an appropriate promoter, which is recognized by the GAL4 transcription factor, is transformed with a library of cDNAs which will express plant proteins or peptides thereof fused to an activation domain. Thus, if a peptide encoded by one of the cDNAs is able to interact with the fusion peptide comprising a peptide of Melted, the complex is able to direct expression of the reporter gene. In this way, e.g., Melted and the gene encoding Melted can be used to identify peptides and proteins interacting with Melted. It is apparent to the person skilled in the art that this and similar systems may then further be exploited for the identification of inhibitors.

It is also well known to the person skilled in the art, that it is possible to design, synthesize and evaluate mimetics of small organic compounds that, for example, can act as a substrate or ligand to the Melted protein. For example, it has been described that D-glucose mimetics of hapalosin exhibited similar efficiency as hapalosin in antagonizing multidrug resistance assistance-associated protein in cytotoxicity; see Dinh, J. Med. Chem. 41 (1998), 981-987.

All these methods can be used in accordance with the present invention to identify a compound capable of modifying the biological activity of the Melted protein.

The gene encoding the Melted protein can also serve as a target for screening inhibitors. Inhibitors may comprise, for example, proteins that bind to the mRNA of the gene encoding Melted, thereby destabilizing the native conformation of the mRNA and hampering transcription and/or translation. Furthermore, methods are described in the literature for identifying nucleic acid molecules such as an RNA fragment that mimics the structure of a defined or undefined target RNA molecule to which a compound binds inside of a cell resulting in retardation of cell growth or cell death; see, e.g., WO 98/18947 and references cited therein. These nucleic acid molecules can be used for identifying unknown compounds of pharmaceutical interest, and for identifying unknown RNA targets for use in treating a disease. These methods and compositions can be used for identifying compounds useful to reduce expression levels of Melted.

Moreover, the present invention also relates to a diagnostic composition comprising a compound which is capable of specifically (a) binding to the Melted protein or (b) hybridizing to an mRNA encoding the Melted protein. Preferably, said compounds are oligonucleotides or antibodies as already defined above.

The oligonucleotides also comprise primers for PCR. The person skilled in the art is in a position to design suitable nucleic acids probes based on the information as regards the nucleotide sequence of Melted and the description of oligonucleotides provided above (e.g., as regards complementarity, lengths etc.). For evaluating whether the concentrations of Melted encoding mRNAs (or the translated proteins) are decreased or increased,

the measured concentrations are compared with the concentration in a normal tissue. In order to restrict hybridisation of the probe to mRNA(s) , i.e. to avoid hybridisation with the corresponding DNA which lead to incorrect results, the sample can, e.g., treated with DNAse I.

The probes (nucleic acid molecules or antibodies) can be detectably labelled, for example, with a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, or an enzyme.

The expression of Melted in tissues can be studied with classical immunohistological methods. Other antibody based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA) . Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹² In), and technetium ("mTc) , and fluorescent labels, such as fluorescein and rhodamine, and biotin. In addition to assaying Melted levels in a biological sample, Melted can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or caesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labelling of nutrients for the relevant hybridoma. A protein- specific antibody or antibody fragment which has been labelled with an appropriate detectable imaging moiety, such as a radioisotope (for example, ¹³¹ I, ¹¹² In, "mTc) , a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally,

subcutaneousIy, or intraperitoneal].:/) into the patient. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of "mTc . The labelled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific DSM-protein.

Finally, the present invention relates to the use of the diagnostic compounds defined above for the preparation of a diagnostic composition for the diagnosis of a disease associated with an aberrant activity and/or level of the Melted protein or predisposition for such disease.

The below example explains the invention in more detail.

Example 1: General experimental procedures

(A) Plasmids

In the examples the melted sequences as sequenced by the inventors have been used (c.f. Fig. 8). The other melted sequences shown in Fig.8 are to be understood as possible alternatives .

A full-length melted cDNA was cloned into pUAST (c.f. Fig. 8) as an EcoRI-Notl fragment (pATl59) . This clone differs from the annotated CG8624. [www.flybase.net] due to splicing differences. Human melted was cloned by RT-PCR from HeLa cell cDNA and cloned into pUAST (pAT206) . The dMeltPH-GFP fusion was constructed by amplifying the PH domain (Primersequenzen : ccccctcgagGCTTTCTCAATGGCAGCAAT;

cgcggatcctaCGTGGGATTATCTCGGGCCTG) for cloning into a metallothionine promoter vector containing GFP (pATl95; c.f. Fig. 8) . The GFP fusion was then cloned into pUAST (pAT202) for transformation of Drosophila. His-tag Melted was made by inserting the dMelt ORF into pET23a (c.f. Fig. 8) (plasmid pAT160) . For the yeast two-hybrid screen, a bait plasmid containing dMelt without the PH domain was made by inserting a 1695 bp EcoRI-XhoI fragment from pAT159 into the EcoRI-Sall sites of pGBDU (c.f. Fig. 8) (pAT165). Myc-tagged Tscl and of V5/His-tagged Tsc2 plasmids were a kind gift of Duojia Pan. FOXO-GFP was constructed as a C-terminal GFP fusion in pUAST. Two UAS-melted-RNAi transgenes were prepared by PCR (tctagaCTCGCACGCGGACTATGTGAA and tctagaCTTGGCATGGGGTGGCTCTTC; tctagaCGCCCACACGTTGCAGCATTG and tctagaGGACTCCCGCGTGCTAAG) and cloned into pWIZ (Lee and Carthew, Methods 30 (2003), 322-9).

(B) Strains and Transgenic Lines engrailed-Gal4 and tubulin-Gal4 are described in (http://fly.bio.indiana.edu/gal4.htm) pumpless- (ppl ) -GAL4 is described in Zinke et al . , Development 126 (1999), 7275-84. chicol flies are described in Bohni et al . , Cell 97 (1999), 865-75. Flies with UAS-Tscl-myc and UAS-Tsc2-V5 were from DJ Pan.

(C) PIP-binding assay

Bacteria expressing HIS-tagged Melted were lysed by a freeze/thaw cycle, subsequent addition of 0.2 mg/ml lysozyme and 0.2 mg/ml DNAse, incubation on ice for 30 min and vigorous vortexing. No detergent was used because it inhibits binding to phosphoinositides . The lysate was cleared by centrifugation, diluted 1:1000 or 1:10,000 in TBST (10 mM Tris/HCL pH 8.0, 150 mM NaCl, 0.1% Tween-20)+3% BSA ( fatty- acid-free) +protease inhibitors and incubated for 1 h with PIP membranes (Echelon Biosciences, Salt Lake City, UT, USA),pre- blocked with 3% fatty-acid-free BSA in TBST. Strips were

washed twice with TBST for 30', incubated for 1 h RT with anti-HIS in TBST + 3% BSA followed by HRP-conjugated 2nd antibody.

(D) Y2H

A Drosophila larval cDNA library in pGAD-C was kindly provided by Grace Gill. The yeast two-hybrid screen was carried out in the strain PJ69-4α, containing reporters for lacZ expression and for growth on adenine and histidine. PJ69-4α. {ura3-, leυ.2- ) was transformed with pATl65. A dMelt expressing clone was chosen by immunoblotting. This was transformed with library plasmid DNA, plated on ura ^~ leu ^~ ade ^~ dropout synthetic medium to select for presence of the bait plasmid (URA3), the library plasmid (LEU2) and for activation of the adenine reporter. Colonies were retested for growth on ura ^~ leu ^~ ade ^~ his ^~ -medium for activation of the histidine reporter and on X-gal plates. Plasmids were isolated from clones that satisfied all these reporter-construct requirements. Sequences also identified in a second independent screen were considered non-specific and discarded.

(E) Antibodies

Anti-dMelt was raised in rats against the full-length protein expressed in E. coli. Rats were injected with 50 μg dMelt protein in RIBI adjuvant. For immunoprecipitation, cells were lysed for 5-10 minutes on ice in 50 inM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100 (with protease and phosphatase inhibitors) . Lysates were cleared by centrifugation at 4 ⁰ C at maximum speed for 15 minutes, and incubated with antibody for 2 hours at 4°C. 50 μl of 50% agarose-protein A bead slurry were washed twice in lysis buffer and added to the antibody/cell-lysate mixture for 30'. Following 3 washes with cold lysis buffer, proteins were recovered using 2x protein- gel loading buffer and heating for 5' at 95°C.

(F) Fly measurement and metabolic characterization Metabolic measurements were done on test and control flies grown under identical conditions. 50 newly-hatched 1st instar larvae were seeded per vial and adults hatching on the same day were aged for 2 or 3 days before testing. Male flies were weighed in batches of 50. Total body triglycerides were measured by homogenizing flies in 0.05% Tween-20 with protease inhibitors. After heating at 70 ⁰ C for 5' lysates were cleared by centrifugation and triglycerides measured using Infinity Triglyerides Reagent (Thermoelectron; Waltham, MA, USA. Total protein was measured on the same samples using Biorad protein assay. To measure sugars, 1 μl of hemolymph was put into 9 μl of buffer (5 mM Tris, pH 6.6, 137 mM NaCl, 2.7 mM KCl), heated for 5' at 70 ⁰ C, incubated with 1 μl of porcine trehalase (Sigma T-8778, St. Louis, MO, USA) at 37°C for 12 hr and measured using Sigma glucose (G) assay kit (GAGO-20).

Example 2: Melted modulates tissue growth

Melted was identified in a gain-of-function screen for genes affecting tissue growth during Drosophila development. EP31685 caused overgrowth when expressed in the posterior half of the wing using en-GAL4 (Fig. IA, the red wing expressed EP31685). When expressed ubiquitously with tubulin-GAL4 , EP31685 caused a small but statistically significant increase in total body weight (student t-test < 0.005; Fig. 1C). EP31685 is located ~50bp upstream of the annotated gene melted (melt, CG8624; Fig IB) . Using a UAS-melt transgene, we verified that the melted transcription unit is responsible for the tissue overgrowth phenotype (not shown) .

To generate melted mutants, deletions were prepared using P-element mediated male recombination (Preston and Engels, Genetics 144, 1611-1622,(1996)) starting from EP31685 (Fig.

δl

IB) . melt is a 22 kb deletion that removes the entire melted

δ2 gene and two adjacent genes. The second allele, melt is a 2.5

kb deletion that removes the first exon of CG8624. Flies

δI homozygous mutant for melt were semi-viable (70% viable to adult) and fertile, but were -10% smaller than control flies (Fig. ID) . Though small in magnitude, the reduction in body- size was statistically significant (t-test < 0.005). Flies

δI δ2 heterozygous mutant for melt Imelt were also significantly smaller than control flies (not shown) . Two additional lines of evidence indicate that the growth defect was due to loss of

δI melted and not the other genes deleted in melt . First, the

δI growth defect of the homozygous melt deletion flies was fully rescued by ubiquitous expression of the UAS-meIt transgene (Fig. ID) . Second, flies expressing UAS-RNAi constructs directed against two different regions of the melt transcript

δl caused tissue undergrowth, resembling the melt phenotype (not shown) .

The predicted Melted protein is conserved in C. elegans (CE25943), mice (Muto et al . , Biochimie 86, 523-531 (2004)) and humans (HGNC, KIAAl692) . Alignment of these sequences showed a conserved region at the N-terminus (76% similar in fly and human) and a conserved Pleckstrin Homology (PH) domain at the C-terminus. Ubiquitous expression of Melted lacking the

δI

PH domain did not rescue the melt size defect (Fig. ID), indicating the PH domain is required for Melted function. The truncated protein is likely to fold correctly because it was used successfully as bait in a yeast two-hybrid screen to detect proteins that also interact with full-length Melted (see below) . The human homologue of Melted also rescued the

δI size reduction of the melt mutant (Fig. ID), indicating that the molecular function of Melted is conserved.

Example 3: Melted controls fat metabolism

The next question to be answered was whether Melted might also affect fat metabolism. Drosophila stores fat mainly as

δl triglycerides. Total body triglyceride was measured for melt mutant and control flies reared under identical, controlled conditions. melt mutant flies had only 60% as much

triglyceride as control flies, when normalized to total protein to take into account the 10% reduced body size of melted mutants. This reduction in fat content was statistically significant (t-test < 0.001) and was rescued by ubiquitous expression of a UAS-melt transgene (Fig. 2A) . Further confirmation that the leanness of the mutant was due to reduced melted expression was obtained by ubiquitous expression of a melted UAS-RNAi construct in flies (25% rd leaner, Fig. 2B) . Total body triglycerides of wandering 3

δl instar melt mutant larvae were also 20% lower than controls, indicating that Melted regulates fat levels throughout development, as well as in the adult (not shown) .

Fat levels can be controlled by humoral factors, including adipokinetic hormone (AKH) , which induces mobilization of fat reserves (Lee and Park, Genetics 167, 311- 323 (2004)). Insulin-like peptides (ILPs) also control fat metabolism in the fly (Ikeya et al . , Curr . Biol. 12, 1293 (2002); Hwangbo et al . , Nature 429, 562-566 (2004)). To ask whether elevated AKH or ILP-2, -3 and -5 expression could explain the leanness of melted mutants, the inventors tested transcript levels by quantitative RT-PCR. Transcript levels were not significantly elevated. Since altered expression of the known humoral regulators did not provide an explanation for the mutant phenotype, the inventors asked if there was a defect in adipose tissue. The ^λ fat body' is the main fat- storage organ of the fly, containing over 80% of total body

δl triglycerides. The inventors isolated melt mutant and control fat body tissue from larvae and found 25% lower triglyceride

δl level in the mutant tissue. The total body leanness of melt mutants was rescued by expressing melted specifically in the fat body, using ppl-GAL4 (Fig. 2C) . In contrast, expression of melted in the nervous system using elav-GAL4 or in brain neurosecretory cells using dILP3-GAL4 did not rescue the mutant (not shown) . This indicates that Melted activity is required in adipose tissue.

Example 4: Membrane localization via the Melted PH domain

Melted contains a C-terminal PH domain. The inventors tested if bacterially expressed Melted protein binds to immobilized phosphoinositides . At higher concentration, Melted bound many phosphoinositides, but at lower concentrations showed preferential binding to PI (5) P (Fig. 3A, A')- The PH domain of Melted expressed as a GST fusion exhibited a similar binding pattern, although with reduced selectivity. Although Melted did not show strong binding to PIP3 in vitro, the inventors generated a GFP fusion to the PH domain (MeItPH-GFP) and tested it in S2 cells for insulin-induced relocalization. MeItPH-GFP was detectable predominantly at the cell membrane when cells were serum-starved to deprive them of insulin and following insulin-stimulation, without apparent difference (Fig. 3B, B'). The MeItPH-GFP fusion was also predominantly cortical in wing imaginal disc cells, whereas a Melted-GFP fusion protein lacking the PH domain was cytoplasmic (Fig. 3C, D) , suggesting that the majority of Melted protein is localized to the membrane under normal conditions. This was confirmed by subcellular fractionation of wing disc cells. The majority of the endogenous Melted protein was recovered in the membrane fraction, with a lesser amount in the cytoplasm (Fig 3E) . No signal was detected in corresponding fractions prepared from melted mutant tissue, confirming the specificity of the antibody. Control proteins fractionated predominantly as cytoplasmic (Y14) or membrane-associated (PVR). These results suggest that the endogenous Melted protein is membrane localized via its PH domain and that its localization is not regulated by insulin-dependent changes in PIP3 levels. The PH domain is required for Melted function in vivo (Fig ID) .

Example 5: Melted protein binds to Tscl and modulates TOR activity

To study the function of the N-terminal domain, the inventors performed a yeast two-hybrid screen using MeltδPH as bait to screen 2.7 million clones from a larval cDNA library.

Six interacting genes were isolated: Tscl (4 independent clones), 14-3-3e (3 independent clones), CG16719, CG5171, CG6767, and CG8242 (once each) . When expressed in S2 cells by cotransfection, Melted co-immunoprecipitated with Myc-tagged Tscl. Interaction of Melted and Tscl in S2 cells was also visualized by immunofluorescence microscopy. Tsclmyc was uniformly distributed in the cytoplasm when expressed alone (Fig. 4A row 1; S2 cells lack endogenous Melted) . Co- expression with Melted caused Tscl-myc to shift predominantly to the cell membrane (Fig. 4A row 2). When expressed in S2 cells, Tsc2 was uniformly distributed in the cytoplasm (Fig. 4 row 3) . Co-expression of Tsc2 with Melted did not cause a relocalization of Tsc2 to the membrane, suggesting that Melted and Tsc2 do not interact directly (Fig. 4A row 4) . However, when co-expressed with Melted and Tscl, Tsc2 also relocated (Fig. 4A row 6), indicating that Melted can recruit the Tscl/2 complex to the cell membrane.

Does this interaction affect TOR pathway activity? Previous reports have shown that increased PI3K or TOR signaling in the adipose tissue increased fat accumulation or reduced fat consumption (autophagy; Britton et al . , Dev Cell 2, 239-249 (2002); Rusten et al . , Dev Cell 7, 179-192 (2004)). The observation that melted mutants are lean, could be explained if TOR activity is reduced in the mutant. To test this more directly the inventors assayed the level of TOR-

Dl dependent phosphorylation of 4E-BP in control and melt mutant fat bodies (Fig. 4B). Phosphorylation of Drosophila 4E-BP at positions 36/47 depends on TOR activity and can be visualized using an antibody specific to the corresponding phosphorylated peptide from human 4E-BP (Miron et al . , Nat Cell Biol 3, 596- 601 (2001)). Although the total level of 4E-BP protein was

δl higher in melt mutant tissue, the level of phosphorylation was not correspondingly elevated (perhaps mildly reduced, Fig. 4B, left panel) . The increase in total 4E-BP level reflects a 2-3 fold increase in transcript levels in the mutant fat body (Fig. 4C) . As a control, the inventors increased 4E-BP levels by a comparable amount in wild-type fat body using ppl-Ga!4

UAS-4E-BP and observed that the level of 4E-BP phosphorylation was correspondingly elevated (Fig 4B, right panel) . This indicates that TOR activity is not limiting in wild-type fat body. Thus the observation that 4E-BP phosphorylation did not increase, despite increased total 4E-BP, provides strong evidence that TOR activity is reduced in the melted mutant adipose tissue.

Example 6: Melted binds to FOXO and modulates FOXO activity

As a second means to identify possible functions of Melted, the inventors used the Eukaryotic Linear Motif server (http://elm.eu.org/) to look for functional motifs conserved between fly and human Melted. The only conserved motifs found in the N-terminal region of these proteins were two Forkhead- associated domain ligand domains (LIG_FHA_1) . Forkhead transcription factors, FoxA2 , FoxA3 , FoxC2 and FoxOl are involved in glucose and fat metabolism. Insulin signaling activates Akt, which phosphorylates FOXO and leads to its retention in the cytoplasm. The inventors therefore asked if Melted affects the subcellular localization of a FOXO-GFP fusion protein. FOXO-GFP was predominantly nuclear in the absence of insulin stimulation in serum-starved S2 cells and increased in the cytoplasm following insulin stimulation (Fig. 5A, row 1, 2). In serum-starved cells co-transfected to express Melted, FOXO-GFP was still primarily nuclear (Fig. 5A row 3), but much of the nonnuclear protein appeared at the membrane, colocalized with Melted. Upon insulin stimulation, the inventors observed a robust increase in the level of FOXO- GFP at the cell membrane (Fig. 5A row 4) . The interaction was confirmed by co-immunoprecipitation of Melted with FOXO in insulin-stimulated S2 cells (Fig. 5B) .

The observation that insulin-stimulation induced a shift toward membrane localization of FOXO in the presence of Melted in S2 cells raised the possibility that melted regulates FOXO activity in vivo. To address this we examined expression of the FOXO target 4E-BP (Junger et al . , 2003) in wild-type and

melted mutant animals. Under fed conditions, insulin signaling is active and 4E-BP transcript levels are relatively low (Fig 5C) . In wild-type flies that were starved for 24h to reduce insulin levels and thereby activate FOXO, 4E-BP transcript increased ~4 fold (Fig 5C) . In starved flies lacking Melted, 4E-BP transcript increased over 25 fold. This increase in 4EBP transcription was absent in the starved melted/FOXO double mutant, confirming that it is FOXO dependent. Thus in the absence of Melted, FOXO activity is higher than normal, suggesting that Melted limits FOXO activity in vivo.

Example 7: Melted acts via TOR and FOXO

To ask whether the elevated FOXO activity observed in melted mutants contributes to the lean phenotype of these animals, the inventors compared the normalized triglyceride levels of melted mutant and melted foxo double mutant flies. Reducing FOXO activity suppressed the leanness of the melted mutant to a considerable degree, reaching near normal fat levels (Fig 6A) . The rescue was highly statistically significant (t-test = 10 ) . foxo mutants did not show higher than normal fat levels compared to wild-type (not shown) . These observations suggest that Melted acts by regulating FOXO activity to control expression of genes important in fat metabolism.

Melted mutants also exhibit reduced TOR activity. To ask whether TOR activity affects fat accumulation, the inventors tested the effects of increasing TOR activity in wild-type and melted mutant adipose tissue. The inventors made use of a UAS- TOR transgene that can provide TOR activity in vivo when expressed at appropriate levels (Hennig and Neufeld, Genesis 34, 107-110 (2002)). The inventors first confirmed that expression of UAS-TOR under ppl-Gal4 control in adipose tissue led to increased total body fat (Fig 6B, t-test = 0.03), as did increasing PI3K activity (not shown, see also Britton et al 2002) . In contrast, a comparable elevation of TOR expression in melted mutant flies had no effect on fat levels

(Fig 6C) . Both this result and the significant rescue caused by removal of FOXO indicate that in the melted mutant, the FOXO branch of the pathway becomes limiting for fat accumulation. In view of this finding, the inventors next asked whether elevated TOR pathway activity could increase fat levels in the melted mutant if FOXO activity was simultaneously reduced. To do so the inventors made use of the catalytic subunit of PI3K (DpIlO) to inactivate FOXO and simultaneously activate TOR. The fat body driver Isp2-Gal4 or the UAS-DpIlO transgenes had little effect on their own in the melted mutant background, but when combined, the elevated PI3K activity in the fat body increased fat levels of the melted mutant (Fig 6D) . The effect was stronger than that of removing FOXO only, increasing fat levels to above normal (compare Fig. 6A,D). Taken together these observations suggest that the TOR branch of the pathway contributes to the control of fat levels under conditions where FOXO activity levels are low. This is normally the case in feeding animals where insulin levels are relatively high (FOXO activity is elevated under starvation conditions: compare 4E-BP levels in fed vs starved wild-type and foxo mutant flies, Fig 5C) . Under conditions where insulin levels are low, or in the melted mutant, where FOXO activity is elevated, the effects of FOXO dominate.

Example 8: Transcriptional profiling of melted mutant adipose tissue

To better understand the metabolic impact of the loss of Melted function in the adipose tissue, the inventors performed microarray expression profiling of melted mutant versus control fat body. The inventors used microarrays containing 11,445 cDNA clones (DGCl and DGC2 collection) and analyzed the data using Statistical Analysis of Microarrays (SAM) in the MeV module of the TIGR TM4 microarray software suite (http://www.tigr.org/software/tm4/mev.html; Tusher et al . , PNAS 98, 5116-5121 (2001)). Allowing for a 1% false-positive rate (q value < 0.01), the inventors identified 315 genes that were upregulated and 405 genes that were downregulated in the

melted mutant. This represents 6% of all genes sampled, and reflects substantial reprogramming of the transcriptional profile of the adipose tissue. Within this set, the inventors considered genes altered by at least 1.5-fold (249 genes) and grouped them according to their functional annotation (www.flybase.org). 46% of the regulated genes are involved in metabolism (Fig. 7A) . Interestingly, many of these are involved in lipid metabolism (Table 1) . 10 are cytochrome P450 enzymes, involved in the oxidation of lipophilic molecules. We confirmed the transcriptional regulation of some of these nd genes by semi-quantitative RT-PCR (2 column, Table 1) . In addition to the genes involved in fat metabolism, a significant proportion of the misregulated metabolic genes are involved in protein degradation.

Several of the down-regulated genes are involved in the accumulation of triglycerides. One of the most highly down-

δi regulated genes in the melt mutant adipose tissue is the transcription factor sugarbabe (2.4-fold). sugarbabe was previously identified as a gene controlling the conversion of sugars to fats (Zinke et al . , EMBO J. 21, 6162-6173 (2002)). nd

It is the 2 most highly upregulated gene when flies fed sugar, but deprived of lipids, and it becomes expressed in the adipose tissue, gut and malphighian tubules. Acetyl-CoA sythase (AcCoAS) was also identified in the same screen as a gene upregulated on a sugar diet (7.3-fold), whereas in the

δl melt mutant adipose tissue it was down-regulated (1.8-fold). The down-regulation of both sugarbabe and AcCoAS suggests that

δi melt mutant adipose tissue might not be able to accumulate enough lipid. This is corroborated by the finding that glycerol kinase (Gyk) and PEPCK are among the genes most down- regulated in melted mutant adipose tissue (Table 1). In order to generate triglycerides, both free fatty acids and 3- phosphoglycerol are required by the cell. In vertebrate brown adipose tissue, 3-phosphoglycerol is made by Gyk, whereas in white adipose tissue it is made by PEPCK (Hanson and Reshef, Biochimie 85, 1199-1205(2003)). PEPCK is rate limiting in that loss of function in the mouse leads to lipodystrophy whereas overexpression in mouse adipose tissue leads to obesity.

Therefore, the finding that both Gyk and PEPCK are down-

δl regulated in the melt mutant fat-body suggests that these animals are lean because they do not accumulate enough lipid in the adipose tissue. Consistent with what is known in vertebrates, the inventors detected increased PEPCK levels by quantitative RT-PCR in other non-adipose tissues under starvation conditions and in the melted mutant, thus the transcriptional changes in adipose and non-adipose tissues do not always correlate. Recently, adipose triglyceride lipase has been reported to catalyze the initial step in triglyceride hydrolysis in mice and inhibition of this enzyme markedly decreases total adipose acyl-hydrolase activity (Zimmermann et al., Science 306, 1383-1386 (2004)). BLAST searches identified two Drosophila homologs of adipose triglyceride lipase: CG5295 and CG5560. Interestingly, CG5295 expression was markedly reduced in the melt mutant fat body. This suggests that lipid hydrolysis might be downregulated in the mutant adipose tissue .

To test experimentally these predictions from expression data, the inventors measured circulating lipids, which in the fly are mobilized from the fat body and delivered to peripheral tissues as diacylglycerides (DAG) in the hemolymph

(Canavoso et al . , Annu Rev Nutr 21, 23-46 (2001)). Hemolymph

δl

DAG was low in melt mutant larvae compared to controls (Fig 7B; t-test = 0.02). Thus, it is not likely that the reduced triglycerides in the adipose tissue can be explained by increased mobilization of fat in the form of circulating DAG. The inventors also measured the level of circulating blood sugar (trehalose + glucose) and found it was not elevated in

δl the melt mutant (Fig. 7C) . These experiments, together with the expression data, mean that the leanness observed in the mutant is likely due to reduced triglyceride accumulation in adipose tissue.

Example 9: Tissue distribution of Melted expression

By use of quantitative RT-PCR on RNA from dissected mouse tissues it was found that in addition to the already published expression domains (ventricles of the brain and the kidneys) , Melted is expressed in the hypothalamus and in the liver. This is an interesting observation since both of these organs are important for energy metabolism in mammals. Melted does not appear to be expressed in white adipose tissue (i.e. fat- WAT) and brown adipose tissue (BAT) ) .