WO2007130431A2

WO2007130431A2 - FUNCTION OF ESTROGEN RELATED RECEPTOR GAMMA (ERRγ) IN INCREASING MITOCHONDRIAL BIOGENESIS

Info

Publication number: WO2007130431A2
Application number: PCT/US2007/010592
Authority: WO
Inventors: Shamina M. Rangwala; Susan C. Stevenson; Zhidan Wu
Original assignee: Novartis Ag; Novartis Pharma Gmbh
Priority date: 2006-05-03
Filing date: 2007-05-02
Publication date: 2007-11-15
Also published as: WO2007130431A3

Abstract

The present invention relates to the novel method for identifying an ERRγ agonist that enhances the mitochondriogenesis. The ERRγ agonist can be a chemical compound or a polypeptide. The present invention further relates to the use of the ERRγ agonist in the treatment of diseases selected from diabetes, type 2 diabetes, insulin resistance, metabolic disease/metabolic syndrome, dyslipidemia, obesity/overweight, neurodegenerative diseases. Furthermore, the compound is useful for improving exercise endurance capacity in a subject.

Description

50201A

Function of Estrogen Related Receptor Gamma (£RRγ) in Increasing

Mitochondrial Biogenesis

TECHNICAL FIELD

The present invention relates to methods of identifying ERRγ agonists and the obtained agonists in the treatment of diseases whose conditions are improved by activation of ERRγ.

BACKGROUND

The estrogen-related receptors (ERR) comprise 3 members, ERRα, ERRβ, and ERRγ, that form a subfamily of orphan nuclear receptors for which natural ligands have yet to be identified. The ERRs share significant amino acid homology with the estrogen receptor (ER) within their DNA binding domains (DBD) and ligand binding domains (LBD), but do not respond to estradiol (Giguere, et al., Trends Endocrinol Metab., 13:220-5 (2002); Heard, et al., MoL Endocrinol, 14:382-92(2000).). Whereas ERs are ligand-activated receptors, ERRs can activate gene transcription in a constitutive manner (Hong, et al., J. Bio. Chem., 21 A: 22618-26 (1999); Chen, et al., J. Bio. Chem., 276:28465-70 (2001).) and their activation ability may be determined by the presence of transcriptional coactivators (Kamei, et al., PNAS USA, 100:12378-83(2003); Schreiber, et al., J. Bio. Chem., 278:9013-8 (2003).). Structural studies confirm this finding by demonstrating that the ERRγ LBD can adopt a transcriptionally active conformation and interact with the steroid receptor coactivator 1 (SRC-I) in the absence of any ligand (Greschik, et al., M?/. Cell, 9:303-13 (2002).).

ERRγ is highly expressed in adult tissue such as the brain, skeletal muscle, heart, kidney and retina. It is also expressed in fetal tissue such as the placenta, brain, heart, skeletal muscle, kidney and lung (Bonnelye et al. Mot. Endo., 11: 905-16 (1997).).

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for screening/identifying a compound that binds to a polypeptide containing the ligand-binding domain (LBD) of 50201A

ERRγ (a ERRγ LBD-containing polypeptide), and upon binding to ERRy, helps recruiting a PPAR gamma coactivator 1 alpha (PGC- lα) polypeptide into contact with the ERRγ LBD-containing polypeptide. The screening is performed by contacting a compound with a biological sample containing the ERRγ LBD-containing polypeptide and the PGC- lα polypeptide and selecting the compound that binds to the ERRγ LBD- containing polypeptide and recruits the PGC- lα polypeptide. In one embodiment, the ERRγ LBD-containing polypeptide is the ERRγ polypeptide.

In another aspect, the screening assay is a cell-based screening assay. In the cell- based screening assay, a cell expressing an ERRγ LBD-containing polypeptide and a PGC- lα polypeptide, or functional fragments thereof, is contacted with a test compound, and the ability of the test compound to increase the activity of the ERRγ LBD-containing polypeptide is determined. Preferably, the activity is transcriptional activity of the ERRγ LBD-containing polypeptide. Also preferably, the ERRγ LBD-containing polypeptide is ERRγ polypeptide.

BRIEF DESCRIPTION OF FIGURES

Figure 1 shows the diagram of FRET assay configuration.

Figure 2 shows the agonist-induced FRET response.

Figure 3 shows comparison of the ability of ERRα and ERRγ to transactivate a ERE reporter in the presence and absence of PGC-I α.

Figure 4 shows adenoviral overexpression of PGC-I α and ERRγ in HeIa cells.

Figures 5 A and 5B show the expression of mitochondrial marker genes in HeIa cells over expressing PGC-lα and ERRγ.

Figure 6 shows effects of overexpression of PGC-lα and ERRγ on fatty acid oxidation in HeIa cells.

Figure 7 shows effects of overexpression of PGC-I alpha and ERR gamma on expression of PGC-I alpha and ERRγ in ERRα null MEFs. 50201A

Figure 8 shows effect of overexpression of PGC-I alpha and ERR gamma on expression of mitochondrial markers in ERR alpha null MEFs.

Figure 9 shows effect of overexpression of PGC-I alpha and ERR gamma on expression of genes of oxidative stress protection in ERR alpha null MEFs.

Figure 10 shows effects of overexpression of ERR gamma and PGC-I alpha on cytochrome c protein levels in ERR alpha null MEFs.

Figure 11 shows the human ERRγ polypeptide sequence (SEQ ID NO. 1).

Figure 12 shows the LBD domain sequence of human ERRγ sequence (SEQ ID NO. 2).

Figure 13 shows the ERRγ ERE sequence (SEQ ID NO. 3).

Figure 14 shows expression levels of genes of oxidative phosphorylation in mouse myotubes treated with Compound 1.

Figure 15 shows expression levels of genes of fatty acid oxidation in mouse myotubes treated with Compound 1.

Figure 16 shows expression levels of IDH3α and ATP-5b in mouse myotubes treated with Compound 1.

Figure 17 shows expression levels of UCP2 and UCP3 in mouse myotubes treated with Compound 1.

Figure 18 shows expression levels of ERRγ and ERRα in mouse myotubes treated with Compound 1.

Figure 19 shows expression levels of PGC-I α and PGC- lβ in mouse myotubes treated with Compound 1.

Figure 20 shows expression levels of PP ARa, PPARγ, and PPARδ in mouse myotubes treated with Compound 1. 50201A

Figure 21 shows protein expression levels of cytochrome c in mouse myotubes treated with Compound 1.

Figure 22 shows citrate synthase activity in mouse myotubes treated with Compound 1.

Figure 23 shows measurement of cellular respiration in primary mouse myotubes treated with Compound 1.

DETAILED DESCRIPTION

Definitions

As used herein, the term "agonist of ERRγ" refers to a molecule which when bound to the LBD sequence of ERRγ, increases the amount of, or prolongs the duration of, or enhances the activity of ERRγ. Agonists can include polypeptides, nucleic acids, carbohydrates, lipids, or any derivatives thereof, or any other molecules.

As used herein, the term "a sample" refers to a whole organism or a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). "A sample" further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. Most often, the sample has been removed from an animal, but the term "sample" can also refer to cells or tissue analyzed in vivo, i.e., without removal from animal. Typically, "a sample" will contain cells from the animal, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine, that can be used to measure the cancer-associated polynucleotide or polypeptides levels. "A sample" further refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules. 50201 A

As used herein, the term "polypeptide" refers to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds. It also refers to either a full-length naturally-occurring amino acid sequence or a fragment thereof between about 8 and about 500 amino acids in length. Additionally, unnatural amino acids, for example, beta-alanine, phenyl glycine and homoarginine may be included. Commonly-encountered amino acids which are not gene-encoded may also be used in the present invention. All of the amino acids used in the present invention may be either the D- or L-optical isomer. The L-isomers are preferred. A polypeptide sequence also encompasses naturally-occurring allelic variants of said polypeptide.

As used herein, the term "detectable polypeptide" refers to a polypeptide whose presence or absence can be measured directly or indirectly, quantitatively or qualitatively with standard methods well known in the art.

As used herein, the term "mitochondrial marker gene" refers to gene product (RNA or protein) of a nuclear or mitochondrial encoded DNA sequence, that forms a component of the mitochondria, and the measurement of which provides an indicator of mitochondrial function.

As used herein, the term "a significant increase in the level" or "a significant increase in the expression level" refers to either an increase of the level or the expression level from the control level by an amount greater than the standard error of the assay employed. The term also refers to an increase by at least about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 90%, about 100%, about 150%, or about 200%, or greater.

As used herein, the term "a," "an," "the" and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can 50201A

be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

The present invention is based on the surprising discovery that ERRγ agonists result in enhance mitochondriogenesis.

In one aspect, the present invention provides a method for screening/identifying a compound that binds to a polypeptide containing the ligand-binding domain (LBD) of ERRγ (a ERRγ LBD-containing polypeptide), and upon binding to ERRγ, helps recruiting a PPAR gamma coactivator 1 alpha (PGC- lα) polypeptide into contact with the ERRγ LBD-containing polypeptide. The screening is performed by contacting a compound with a biological sample containing the ERRγ LBD-containing polypeptide and the PGC-I α polypeptide and selecting the compound that binds to the ERRγ LBD- containing polypeptide and recruits the PGC- lα polypeptide. In one embodiment, the ERRγ LBD-containing polypeptide is the ERRγ polypeptide.

To assay the binding described herein, a number of methods known in the art can be employed. The binding of the compound can be tested by detecting a direct binding to the ERRγ LBD-containing polypeptide and the close contact of the ERRγ-containing polypeptide with the PGC-I α polypeptide, or by detecting the signal that indicates the direct binding to ERRγ LBD-containing polypeptide or the close contact of ERRγ LBD- containing polypeptide with the PGC- lα polypeptide.

For purposes of detecting a direct binding, competition assays provide a suitable format for identifying test compounds that specifically bind to the ERRγ LBD-containing polypeptide. In such formats, test compounds are screened in competition with a compound already known to bind to the ERRγ LBD-containing polypeptide. If the test compounds inhibit binding of the compound known to bind the ERRγ LBD-containing polypeptide, then the test compounds also bind to the ERRγ LBD-containing polypeptide. 50201A

Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (ElA), sandwich competition assay (see Stahli et al., Methods in En∑ymology 9:242-253 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol 137:3614-3619 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, "Antibodies, A Laboratory Manual '," Cold Spring Harbor Press (1988)); solid phase direct label RIA using ¹²⁵I label (see Morel et al., MoI. Immunol 25(1) :7-15 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552 (1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77-82 (1990)). Typically, such an assay involves the use of purified polypeptide bound to a solid surface or cells bearing either of these, an unlabelled test compound and a labeled reference compound. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test compound. Usually the test compound is present in excess. Test compounds identified by competition assay include compounds binding to the same epitope as the reference compound and compounds binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference compound for steric hindrance to occur. Usually, when a competing agent is present in excess, it will inhibit specific binding of a reference compound to a common target polypeptide by at least 50 or 75%.

For purposes of detecting the signal that indicates the direct binding of the compound to the ERRγ LBD-containing polypeptide and consequently the close contact of the ERRγ LBD-containing polypeptide with the PGC-lα polypeptide, fluorescence resonance energy transfer (FRET) can be employed. FRET is a quantum mechanical phenomenon that occurs between a fluorescence donor (D) and a fluorescence acceptor (A) in close proximity to each other (usually <100 A of separation) if the emission spectrum of D overlaps with the excitation spectrum of A. The molecules to be tested, e.g. the ERRγ LBD-containing polypeptide and the PGC-lα polypeptide, are labeled with a complementary pair of donor and acceptor fluorophores. While bound closely together by the ERRγ-LBD/PGC-lα interaction in the presence of the test compound, the fluorescence emitted upon excitation of the donor fluorophore will have a different wavelength than that emitted in response to that excitation wavelength when the text 50201 A

compound is not present, providing for quantitation of bound versus unbound molecules by measurement of emission intensity at each wavelength. See also Zhou et al., "Nuclear Receptors have distinct affinities for coactivators: characterization by fluorescence resonance energy transfer," MoL Endocrin., 12: 1594-1604 (1998).

In another aspect, the screening assay is a ceil-based screening assay. In the cell- based screening assay, a cell expressing an ERRγ LBD-containing polypeptide and a PGC- lα polypeptide, or functional fragments thereof, is contacted with a test compound, and the ability of the test compound to increase the activity of the ERRγ LBD-containing polypeptide is determined. Preferably, the activity is transcriptional activity of the ERRγ LBD-containing polypeptide. Also preferably, the ERRγ LBD-containing polypeptide is ERRγ polypeptide.

Determining the ability of the test compound to increase the activity of the ERRγ LBD-containing polypeptide can be accomplished by assessing induction of a reporter gene expressed in the cell and comprising a sequence that is recognized by the ERRγ DNA-binding domain (DBD) wherein the reporter gene encodes a detectable polypeptide. The level of the detectable polypeptide is significantly increased in the presence of the test compound relative to that in the absence of the test compound. The detectable polypeptide can be any kind of polypeptide that is detected through standard methods. Preferably, the detectable polypeptide is selected from chloramphenicol acetyl transferase, green fluorescent protein, or yellow fluorescent protein, or luciferase, β- galactosidase, secreted alkaline phosphatase, etc. The cell can be a mammalian cell, an insect cell, a bacterial cell, or a yeast cell, etc..

Alternatively, determining the ability of the test compound to increase the activity of the ERRγ LBD-containing polypeptide can be accomplished by assessing the increase in the expression levels of the mitochondrial marker genes in cells/tissues. The increased expression levels of mitochondrial marker genes in cells/tissues indicate increased mitochondrial functions, which positively correlate with insulin sensitivity and improvement of anti-diabetic properties of the cells/tissues. See Goodpaster BH et al, 50201A

Diabetes, 52: 2191-2197, 2003; Menshikova et al, American J Physiol Endocrinol Metab 288: E818 - E825, 2005.

Another aspect of the present invention pertains to the compound obtained from the above screening assays. The compound which is ERRγ agonist, can be a chemical compound or a polypeptide. The compound is useful for the prevention, delay of progression or the treatment of diabetes preferably type 2 diabetes, insulin resistance, metabolic disease/metabolic syndrome, dyslipidemia, obesity/weight loss, Neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease or Huntington's disease. Furthermore, the compound is useful for improving exercise endurance capacity in a subject.

EXAMPLES

The examples below are non-limiting and are merely representative of various aspects and features of the present invention.

Example 1

FRET Assay to Detect Binding

The FRET assay is designed to detect agonist-induced activation of ERRγ in the presence of the coactivator peptide PGC- lα. The following components were added in a final volume of 50 μL: (His)₆-hERRγ LBD or GST-hERRy LBD, Europium-labeled anti- (His)β antibody or Europium-labeled anti-GST antibody and Cy5 -labeled PGC-I α peptide (Cy5-RPCSELLKYLTT (SEQ ID NO. 4) with C-terminal acid, custom made and labeled at AnaSpec). Mix 1 contained antibody, hERRγ LBD in buffer in a volume of 19 μL and was added to 30 μL of Mix 2 which contained Cy5-peptide in buffer (1. 6xHis- ERRγ-LBD, 3.6mg/ml, MW 27KDa; 2. Cy5-PGC-I peptide from AnaSpec; 3. Other buffer components from Sigma; 4. EU-antiHisβ-Ab from Perkin Elmer; and 5. Test compounds.). The assays were carried out in black 384-well plates and incubated at room temperature for 3hrs before FRET signals were measured using a Wallac Victor 2 (Perkin Elmer) plate reader. The ratio of emission signals (665 nm/615 ran) was used to determine the FRET assay response. In some cases, the FRET signal to background ratio 50201A

was used and this is simply defined as the FRET ratio in the presence of protein or FRET ratio in the absence of protein. The test compounds were dissolved in DMSO at 10 mM and used as indicated.

Table 1 Reagent List

Reagent Vendor Catalog #

Europium-labeled anti-(His)₆ antibody Perkin Elmer ADOI lO Europium-labeled anti-GST antibody Perkin Elmer AD0254 Cy5-labeled PGC- lα peptide AnaSpec Custom Order

(Cy5-RPCSELLKYLTT (SEQ ID NO. 4); C-terminal acid of PGC-I α)

Dithiothreitol Sigma D-0632

Tris Sigma T-2194

384-well plates Corning 3654

The ability of agonist compounds to induce an increased FRET response between the Europium-labeled anti-(His)₆ antibody/(His)₆-hERRγ LBD complex and Cy5- RPCSELLKYLTT (SEQ ID NO. 4) was assessed by using two compounds described in the literature as ERRγ agonists, GSK4716 (Glaxo-Smith-Kline; Compound 1) and GSK9089 (Glaxo-Smith-Kline; Compound 2). See Zuercher WJ₅ Gaillard S, Miller- Orband LA, et al. (2005), "Identification and structure-activity relationship of phenolic acyl hydrazones as selective agonists for the estrogen-related orphan nuclear receptors ERRβ and ERRγ," J Med Chem; 48:3107-9.).

Compound 1 Compound 2

The compounds were solubilized in 10 mM DMSO and the concentration range of the titration was from 100 μM down to 2 nM. The final DMSO concentration was 2%. A DMSO control (no compound) at 2% was also prepared. All samples were made in duplicate. As shown in Figure 2, Compound 1 was able to induce up to a 60% increase in 50201A

the FRET signal at the highest concentration tested. The FRET response is dose- dependent and an EC₅0 of 2.1 μM was derived from the data. Similarly, Compound 2 also induced a dose-dependent FRET response having a maximal response of 70% at saturating concentrations of compound with an EC5₀ of 0.54 μM. Both EC₅₀'s were similar to the reported values of 1.3 μM and 0.13 μM for the Compound 1 and Compound 2 equivalents, respectively.

Example 2

Cell-Based Assay

Materials and Methods

Mouse embryonic fibroblasts

Mouse embryonic fibroblast cells (MEFs) were isolated from ERRα null mice. Briefly, timed heterozygote matings were set up. On day 13 and a half post coiturn, pregnant females was euthanized and the embryos isolated into PBS. Each embryo was individually dissected to remove liver and eye tissue. These tissues were saved for genotyping. The embryos were then individually homogenized in PBS using 3 ml syringes and 18 and a half gauge needles. These crude suspensions were the.n plated in 10 cm cell culture dishes containing DMEM (Gibco, catalog number 11965-092), 10% FBS (Gibco catalog number 10082-147) and 1% Penicillin / Streptomycin (Gibco catalog number 15140-122). After two days in culture, cells were passaged. At the second passage, similar genotypes were pooled, and cryopreserved in FBS containing 10% DMSO (Sigma, catalog number D2650).

Transfection

HeLa cells were transfected as follows. A pre-made transfection reaction mixture was prepared according to manufacturer's instructions. For each well of cells to be transfected, 40 ng of ERE-luciferase reporter (firefly luminescence measurement, Promega), 3 ng of phRL-SV40 (for Renilla luminescence measurement, Promega), 120 ng of total receptor cDNA plasmid (mouse PGC- lα or human ERRγ or control plasmid, 50201 A

and 0.325 μl of Fugene 6 transfection reagent (Roche catalog number NC9167392) were added to 10 μl of Opti-MEM (Gibco catalog number 31985-062). When a single receptor plasmid was transfected, the total amount was made up with control plasmid to 120 ng. This mixture was added into each well of a 96 well assay plate (Fisher catalog number 07200566). Cells (8000/well) were subsequently added to each well. The cells were incubated at 37°C for 72 hours.

Luciferase assay

Luciferase activity was measured with the Dual-Glo luciferase assay system (Promega catalog number PRE2940) according to manufacturer's instructions. Briefly, 72 hours after transfection, one volume of Dual-Glo luciferase reagent equal to the culture medium volume (90ul per cell for 96 well plate ) was added to each well. After 10 minutes of gently mixing, the firefly luminescence was measured with En Vision (Envision ™ 2100 multilabel reader, Perkin Elmer, Wellesley, MA) at 0.1 sec/well. Subsequently, one volume of Dual-Glo Stop Sc GIo reagent was added to each well. Renilla luminescence was measured at the same speed after 10 minutes mixing. The ratio of firefly luminescence over renilla luminescence (for transfection efficiency control) was calculated.

Adenoviral transduction

HeLa cells or MEFs were trypsinized and counted using a hemocytometer and plated in a 6 well plate at 0.5 x 10⁶ cells per well. Cells were then transduced with adenovirus expressing 1) GFP, 2) mouse PGC- lα, 3) human ERRγ or 4) human ERRγ with mouse PGC- lα. The ERRγ and PGC-I α adenoviruses expressed GFP as a secondary transcript through an internal ribosomal entry site in the adenoviral vector.

The transduction medium consisted of DMEM (Gibco, catalog number 11965- 092) and 2% FBS (Gibco, catalog number 10082-147). For each adenovirus, HeLa cells were transduced 4000 viral particles/cell, while MEFs were infected at a viral titer of 30,000 viral particles/cell. When using two adenoviruses in combination, the adenoviral 50201 A

titer was doubled, resulting in 8000 viral particles/cell in the case of HeLa cells and 60,000 viral particles/cell in the case of MEFs. The media was replenished 24 hrs after infection. The percent of GFP-positive cells in each well was determined visually with a fluorescence microscope and served as an index for transduction efficiency.

JRNA preparation and real time quantitative PCR analysis of gene expression

Total RNA was extracted from HeLa or MEF cells using Trizol (Invitrogen, catalog number 15596-026) according to the instructions provided by the supplier. The RNA concentration and purity were determined using an Agilent 2100 Bioanalyzer according to the manufacturer's manual. A 2 μg aliquot of total RNA was used for cDNA synthesis performed with Superscript III RNaseH Reverse Transcriptase (Invitrogen, catalog number 18080-044) according to the manufacturer's manual. The synthesized cDNA was diluted 1 :4 in water to a final volume of 200 μl and stored at -20⁰C until used. Quantitative real time PCR was performed using Taqman (Applied Biosystems) as well known in the art. (Jemiolo and Trappe, BBRC, 320: 1043-1050, 2004). Relative mRNA expression levels were calculated comparing the level of expression of target genes to that of B2M of the infected cells to the cells infected with adenoviral GFP. Samples were assayed in quadruplicate and expressed as the mean ± SEM of the fold change relative to the control (set at 100%).

Protein extraction

Infected cells were lysed using Cell Extraction Buffer (Biosource, catalog number FNNOOl 1) supplemented with Complete Mini protease inhibitor cocktail (Roche, catalog number 1836153) for 30 minutes on ice. The cell lysate was centrifuged at 14,000 rpm for 20 minutes. The supernatant was transferred to a fresh tube and stored at -20⁰C until use. The protein was quantified using the Bio-RAD DC protein Assay kit (Biorad, Hercules, CA, Catalog number 500-0116) according to the manufacturer's instructions. 50201A

Cytochrome CELISA

The Cytochrome C protein level was measured by ELISA using an immunoassay kit from R&D Systems (catalog number DCTCO) according to the manufacturer's instructions. Samples were assayed in triplicate and expressed as the mean ± SEM.

Results

We investigated whether ERRγ could increase transactivation of an ERE- luciferase reporter in HeLa cells (Figure 3). In comparison to ERRγ, ERRγ is a stronger inducer of transcription on the ERE in this cell line, with an approximate 50-fold induction with ERRγ as compared to 6 fold induction with ERRα. On addition of exogenous PGC-lα, this induction further increased (174 fold in the presence of PGC-lα compared to 50 fold without). PGC-lα induced ERRγ more strongly than ERRα (174 fold as compared to 34 fold).

We wanted to examine the effects of overexpression of ERRγ on mitochondrial function in HeLa cells. We infected HeLa cells with adenoviral vectors expressing either PGC-lα alone, ERRγ alone or ERRγ and PGC-lα together. As expected, PGC-lα was levels were induced in cells that were infected with PGC-lα virus, and ERRγ levels were increased in cells infected with ERRγ virus (Figure 4). In the presence of both ERRγ and PGC-lα, PGC-lα was more highly expressed than in cells transduced with PGC-lα alone. This is possibly due to an enhancement in the feed forward mechanism exerted by the presence of both exogenous ERRγ and PGC-lα on the endogenous mouse PGC-lα gene. The levels of adenoviral ERRγ were significantly lower in the cells transduced with both ERRγ and PGC-lα.

We next examined the levels of mitochondrial gene expression in HeLa cells infected with either PGC-I α alone, ERRγ alone or ERRγγ and PGC-lα together. Interestingly, while the levels of cytochrome C, ERRα, COX-4 and IDH3 were induced in cells that were infected with PGC-lα virus, they were further enhanced in cells containing both ERRγ and PGC-lα. ERRγ overexpression alone did not change levels of 5020 IA

these genes in HeLa cells (Figures 5 A and 5B). This could be due to low levels of PGC- lα expression in HeLa cells.

Rates of fatty acid oxidation were also increased in the presence of either PGC-I α alone or both ERRγ and PGC- lα, implying that the changes in gene expression can also translate into an improvement in mitochondrial function (Figure 6).

Thus, we have shown that ERRγ increases mitochondrial gene expression as well function in HeLa cells. However, there is a concurrent increase in of ERRα levels in these cell. It is possible that the effects of ERRγ are secondary to this effect. Therefore, we performed a series of similar experiments in ERRα null MEFs. Interestingly, we found that ERRγ in combination with PGC-I α could increase mitochondrial gene expression (COX-4 and IDH3) in ERRα null MEFs (Figure 8). To verify that these cells overexpressed PGC-I α and ERRγ, we measured the expression of these transcripts as well (Figure T). Both of these transcripts were several fold overexpressed in the appropriate cells. Further, we found that the expression of genes of the oxidative stress protection pathway (SOD2, PRX3, PRX5, TXN2 and TXNRD2) were also induced in the presence of ERRγ and PGC-Iq together (Figure 9).

Overexpression of ERRγ as well as ERRγ in combination with PGC-lα increased cytochrome c protein levels in ERRα null MEFs (Figure 10).

Example 3

Characterization of Compound 1 on mitochondrial function in primary mouse mvotubes

Primary mouse myoblasts from FVB mice were isolated and maintained as described in.. Megeney, L.A., B. Kablar, K. Garrett, J.E. Anderson, and M.A. Rudnicki. 1996. MyoD is required for myogenic stem cell function in adult skeletal muscle. Genes Dev. 10: 1173-1183 For experimentation, mouse myoblasts were grown to 80% confluence in F-10/Ham's media containing 20% fetal bovine serum, 1% Penicillin/Streptomycin and2.5 ng/mL bFGF (human recombinant). The cells were then plated to 700,000 cells per well in 6-well plates, and allowed to differentiate into myo tubes for 36 to 48 hours in DMEM containing 5% equine serum and 50201A

Penicillin/Streptomycin. The cells were treated with the ERRγ/β agonist Compound 1 for 24 hours. Assays performed included analysis of gene expression by real time quantitative PCR (RT-PCR), cytochrome c ELISA, citrate synthase assay, fatty acid oxidation assay, and a respiration assay. For the RT-PCR, cytochrome c ELISA and citrate synthase assay, myotubes were treated with Compound 1 at the following concentrations: 1 μM, 3 μM, 10 μM, and 30 μM. For the fatty acid oxidation assay, myotubes were treated with Compound 1 at the following concentrations: 10 μM, and 30 μM. For the respiration assay, myotubes were treated with Compound 1 at a concentration of 30 μM. Each dose was tested in trplicate, and a set of wells treated with an equivalent volume of DMSO used was included in every experimental readout for control purposes.

For the purpose of assessing gene expression by RT-PCR, RNA was isolated from cell lysates, and cDNA was subsequently synthesized from this RNA. For RNA isolation, cells were homogenized in TRIzol (Invitrogen, catalog number 15596-026, Carlsbad, CA), arid total RNA was isolated following the manufacturer's instructions. RNA was quantified using the spectrophotometer.

Reverse transcription was performed using the BD Sprint™ PowerScript™ kit from BD Biosciences (catalog number 639562). Quantitative real time PCR for the following genes was performed using Assay-on-Demand primer probes from Applied Biosystems (See Post-text Table 8-1 for catalog numbers): B2M, ERRγ, ERRβ, ERRα, PGC-lα, PGC-lβ, PPARα, PPARγ, PPARδ, COX-4, cytochrome c, UQCRB, CPT-Ib, LCAD, MCAD, lDH3a, ATP-5b, UCP-2, and UCP-3.

Taqman real time quantitative PCR was performed and analyzed following the manufacturer's instructions (Applied Biosystems). Specifically, amplification was performed in triplicate, in a 10 μl reaction mixture. The reaction mixture included: IX TaqMan® Universal PCR Master Mix (Applied Biosystems, catalog number 4304437), IX of Assay-on-Demand primer probe, and 2 μl of cDNA sample. Gene expression was calculated by normalizing to total cDNA as measured by B2M endogenous control (Applied Biosystems, catalog number Mm00437762_ml). The samples were initially 50201A

incubated for 2 min at 50⁰C for optimum uracyl-N-glycosylase activity. The PCR program started with a 95⁰C denaturing for 10 min, followed by 40 cycles of 95°C/15 sec and 60°C/l min in a 384-well thermal cycler (Perkin-Elmer Applied Biosystems). Each amplification run contained "No Template" controls (buffer and primers only). Amplification data were collected by the 7700 Sequence Detector and analyzed using the Sequence Detection System software developed by Perkin-Elmer (Applied Biosystems). The fractional cycle number reflecting a positive PCR result is called the cycle threshold (Ct).

Average gene expression values were calculated for each group relative to B2M, using 2^'&ACt (as described by Applied Biosystems, Foster City, CA) with the expression in the vehicle treated cells (0 μM) normalized to a value of 1. The data are expressed as mean ± SEM (n = 3 replicate wells). Statistical significance was measured using Student' s t test.

The cytochrome c enzyme-linked immunosorbent assay (ELISA) was performed using the Rat/Mouse Cytochrome c Immunoassay kit from R&D Systems (catalog number MCTCO, R&D Systems, Minneapolis, MN) following the manufacturer's instructions.

The citrate synthase assay was performed following the procedure described in above in this application.

Respiration was measured in myotubes incubated with Compound 1 for 24 hrs according to the published method with modifications (St-Pierre, et al., JBC, 278(29): 26597-603 (2003)). Cells were washed once with phosphate-buffered saline (PBS) and treated for 5 min at 37^°C with 2 ml of trypsin (Mediatech, catalog number 25-052-CI). Without removing trypsin, 10 ml of DMEM plus 10% FBS was added into each well. The cells were transferred to a 15 ml tube and centrifuged for 5 min at 1000 rpm. The cells were washed once with the medium and pelleted before being resuspended in the assay buffer containing 25 mM glucose (Sigma, catalog number G-5400), 1 mM pyruvate (Invitrogen, catalog number 11360-070) and 2% bovine serum albumin (BSA) (MP Biomedicals, catalog number 103703) in D-PBS (Invitrogen, catalog number 14040-133). 50201A

The cell suspension was diluted to IxIO⁶ cells per ml in the assay buffer and kept at 37⁰C until used. Oxygen consumption was measured with a Clark electrode according to the instructions provided by Hansatech (Norfolk, UK). One half of the cell suspension was used for each measurement. The concentrations of oligomycin (MP Biomedicals, catalog number 151786) and FCCP (4-(trifluoromethoxy) carbonyl cyanide phenyl hydrazone) (Sigma, catalog number C2920) were 2 μg/ml and 2 to 5 μM, respectively. The experiments were performed in triplicate. The rate of respiration was measured by calculating the slope of of the flux of oxygen consumption using the software provided by the manufacturer.

Data manipulations and graph generation were performed using Microsoft Excel and GraphPad Prism 4 software. Data are presented as mean ± SEM. In all experiments, each dose was tested in triplicate. The only exception to this were the respiration measurements where the experiment was repeated three times. Statistical analysis was preformed using a two-tailed Student's t test.

Results

We examined the expression of various markers of mitochondrial gene expression in differentiated mouse myotubes treated with Compound 1 at the following concentrations: 30 μM, 10 μM, 3 μM and 1 μM. Stock solutions were prepared such that a constant volume of drug solution was added to each well of cells. All effects were compared to that vehicle (DMSO) alone. Mitochondrial pathways/genes examined included oxidative phosphorylation, fatty acid oxidation, Krebs cycle, ATP synthase, and uncoupling proteins. Additionally, expression of transcriptional regulators functionally related to ERRγ were examined. For almost all of the genes examined, we found a dose- dependent increase in gene expression following treatment with Compound 1. All gene expression results are from 24-hour Compound 1 treatment.

Genes of the oxidative phosphorylation demonstrated an elevated expression with increasing concentrations of Compound 1. COX-4 expression was increased by 2-fold, cytochrome c by 3.7-fold, and UQCRB by 2-fold when treated with Compound 1 at a concentration of 30 μM (Figure 14). 50201A

Similarly, three genes examined that are associated with fatty acid oxidation, were shown to be upregulated in a dose-dependent manner following 24-hour treatment with Compound 1. CPT-Ib expression was increased by over 3-fold, LCAD by 2.8-fold, and MCAD by 1.6-fold following 30 μM treatment of AJQ710 (Figure 15).

Two other mitochondrial genes examined, IDH3α, a component of the Krebs cycle, and ATP-5b, an ATP-synthesizing enzyme, demonstrated a dose-dependent induction in expression with increasing doses of Compound 1. IDH3α expression was increased by over 2-fold, and ATP-5b was increased by 1.6-fold after 24 hour treatment with 30 μM of Compound 1 (Figure 16).

We measured mRNA expression for the uncoupling proteins UCP-2 and UCP-3. UCP-2 showed marginal upregulation in expression with all doses of Compound 1, with its highest expression, 1.5-fold, following 10 μM treatment. UCP-3 showed a dose- dependent increase in expression, with an increase of over 3-fold following 30 μM treatment of Compound 1 (Figure 17).

We measured the expression of the ERR family in response to treatment of mytoubes with agonist. Compound 1 purportedly activates both ERRγ and ERRβ, but does not activate ERRα and the ERs (Zuercher, et al., J. Med. Chem., 48(9): 3107-9 (2005)). The expression of ERRα was increased to a greater extent than ERRβ or ERRγ following treatment with Compound 1. ERRγ was elevated to 1.4-fold expression, ERRβ showed a 1.5-fold increase in expression, and ERRα showed a 3.7-fold increase in expression with 30 μM treatment of Compound 1 (Figure 18).

The co-activators PGC- lα and PGC- lβ were also examined for gene expression. The expression of these transcripts showed a similar dose-dependent increase in expression, with PGC-I α elevated to 1.9-fold expression and PGC-I β elevated to 1.8-fold (Figure 19).

We further examined gene expression for the PPARs, a family of nuclear receptors that play an important role in lipid metabolism. PP ARa and PPARδ showed no change in expression following Compound 1 treatment. PPARγ showed a dose- dependent increase in expression with a maximal 2.2-fold expression following 30 μM Compound 1 treatment (Figure 20).

To assess mitochondrial activity at the protein level, a cytochrome c ELISA was performed. Cytochrome c is a critical element of the electron transport chain, and quantities of the protein serve as a biomarker for mitochondrial number and oxidative phosphorylation activity. We observed a dose-dependent increase of cytochrome c with compound treatment, with an increase of 88% when myotubes were treated with 30 μM Compound 1 for 24 hours (Figure 21).

To measure mitochondrial activity, citrate synthase activity was determined. This enzyme is often used as an indicator of mitochondrial content or activity in human muscle (Kelley, et al., Diabetes, 51(10): 2944-50 (2002)). Citrate synthase catalyzes the initial step of the Krebs cycle, which supplies substrate for oxidative phosphorylation. Myotubes treated with 30 μM Compound 1 for 24 hours showed a 28% increase in citrate synthase activity, while 10 μM and 3 μM treatment showed an increase of 8% and 9% respectively (Figure 22).

We further investigated whether induction of mitochondrial gene expression has an impact on oxidative phosphorylation. Cellular respiration was measured in mouse primary myotubes treated with Compound 1 for 24 hr. Consistent with the induction of expression of the respiratory chain components, basal respiration was increased by 37% in Compound 1 -treated muscle cells, compared with the vehicle treated controls (Figure 23). Oligomycin-ϊnsensitive respiration, the proton leak, was increased by 36%, although this increase was not statistically significant. In the presence of FCCP, respiration rate was increased by 32% by Compound 1, as compared to the vehicle-treated control cells (Figure 23). These results indicate that an ERRγ agonist can increase mitochondria] oxidative capacity through coupled respiration.

OTHER EMBODIMENTS

Other embodiments will be evident to those of skill in the art. It should be understood that the foregoing detailed description is provided for clarity only and is

- 20 - 5020 IA

merely exemplary. The spirit and scope of the present invention are not limited to the above examples, but are encompassed by the following claims.

Claims

50201A

The Claims

1 A method of identifying an ERRγ-binding compound comprising:

a) contacting a compound with a sample comprising (i) a polypeptide comprising

ERRγ LBD sequence and (ii) a PGC- let polypeptide;

b) selecting the compound that binds to said polypeptide and increases said polypeptide's interaction with PGC-I α. polypeptide.

2 A method of identifying an ERRγ agonist comprising:

a) contacting a candidate agonist with a cell expressing a polypeptide comprising an

ERRγ LBD sequence and an ERRγ DBD sequence and a PGC-Ia₉ wherein the cell comprises a reporter gene that is recognized by the ERRγ DBD sequence, and wherein the reporter gene encodes a detectable polypeptide;

b) measuring the level of the detectable polypeptide relative to that in the absence of the candidate agonist; and

c) selecting the candidate agonist that induces significant increase in the level of the detectable polypeptide.

3 A method of identifying an ERRγ agonist comprising:

a) contacting a candidate agonist with a cell expressing (i) a polypeptide comprising an ERRγ polypeptide sequence and a PGC- lα polypeptide, wherein the cell comprises a reporter gene that is recognized by the ERRγ DBD sequence, and wherein the reporter gene encodes a detectable polypeptide;

c) selecting the candidate agonist that induces significant increase in the level of the detectable polypeptide. 50201A

4 The method of claim 2, wherein the agonist is a chemical compound, or a polypeptide.

5 The method of claim 3, wherein the agonist is a chemical compound, or a polypeptide.

6 The method of claim 2, wherein the detectable polypeptide comprises luciferase, β- galactosidase, or secreted alkaline phosphatase.

7 The method of claim 3, wherein the detectable polypeptide comprises luciferase β- galactosidase, or secreted alkaline phosphatase.

8 A method of identifying an ERRγ agonist comprising:

ERRγ LBD sequence and an ERRγ DBD sequence and a PGC- lα polypeptide;

b) measuring the expression level of a mitochondrial marker gene in the cell relative to that in the absence of the candidate agonist; and

c) selecting the candidate agonist that induces significant increase in the expression level of the mitochondrial marker gene.

9 The method of claim S, wherein the mitochondrial marker gene is one selected from the group consisting of PGC-I α, PGC- lβ, cytochrome c, citrate synthase, isocitrate dehydrogenase, NADH oxidase, UCP-3, ERRα, ERRγ, SOD2, cytochrome oxidase IV, ATP synthase, carnitine palmitoyl transferase -Ib.

10 The method of claim 8, wherein the agonist is a chemical compound, or a polypeptide.

11 A method of identifying an ERRγ agonist comprising:

a) contacting a candidate agonist with a cell expressing a polypeptide comprising an ERRγ polypeptide sequence and a PGC-I α polypeptide; 50201A

12 The method of claim 11, wherein the mitochondrial marker gene is one selected from the group consisting of PGC-I α, PGC- lβ, cytochrome c, citrate synthase, isocitrate dehydrogenase, NADH oxidase, UCP-3, ERRa₅ ERRγ, SOD2, cytochrome oxidase IV, ATP synthase, carnitine palmitoyl transferase -Ib

13 The method of claim 11, wherein the agonist is a chemical compound, or a polypeptide.