WO2010007085A2

WO2010007085A2 - Use of organic compounds

Info

Publication number: WO2010007085A2
Application number: PCT/EP2009/059036
Authority: WO
Inventors: Carina Cristina Sanchez; Susan C. Stevenson; Zhidan Wu
Original assignee: Novartis Ag
Priority date: 2008-07-17
Filing date: 2009-07-15
Publication date: 2010-01-21
Also published as: WO2010007085A3

Abstract

Use of a GCN5 inhibitor or pharmaceutically acceptable salt thereof, for the prevention, delay of progression or the treatment of diabetes, insulin resistance, metabolic disease/metabolic syndrome, dyslipidemia, obesity, overweight, neurodegenerative diseases heart failure, muscle disease, or improvement of exercise endurance capacity.

Description

USE OF ORGANIC COMPOUNDS

Background

General control of amino-acid synthesis, yeast, homolog-like 2 (GCN5) is a histone acetyltransferase (HAT), which acetylates both histone H3 (Ruiz-Garcia AB, Sendra R, Pamblanco R, et al. (1997) Gcn5p is involved in the acetylation of histone H3 in nucleosomes. FEBS lett; 403:186-90) and non-histone protein substrates including c-Myc (Patel JH, Du Y, Ard PG, et al. (2004) The c-Myc oncoprotein is a substrate of the acetyltransferases hGCN5/PCAF and TIP60. MoI Cell Biol; 24:10826-34), C/EBPβ (Wiper- Bergeron N, Salem HA, Tomlinson JJ, et al. (2007) Glucocorticoid-stimulated preadipocyte differentiation is mediated through acetylation of C/EBPbeta by GCN5. Proc Natl Acad Sci USA;104: 2703-8) and Peroxisome proliferator-activated receptor-γ coactivator (PGC-1 α). GCN5 is a member of the GNAT (GCN5-related N-acetyltransferase) superfamily, which belongs to a large HAT family (Sterner DE and Berger SL (2006) Acetylation of histones and transcription-related factors. Microbiol MoI Biol Rev; 64:435-59). This target was identified as a repressor of PGC-1α, a key transcription regulator of mitochondriogenesis (Wu Z, Puigserver P, Andersson U, et al. (1999) Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell; 98:1 15-24), in a gene-by- gene cDNA screen. Simultaneously, GCN5 was identified as part of a complex with PGC-1 α in liver cells (Lerin C, Rodgers JT, Kalume DE, et al. (2006) GCN5 acetyltransferase complex controls glucose metabolism through transcriptional repression of PGC-1 α. Cell Metab; 3:428-38).

PGC-1 α is a member of a family of transcription coactivators that plays a central role in the regulation of cellular energy metabolism. PGC-1 α stimulates mitochondrial biogenesis and promotes the remodeling of muscle tissue to a fiber-type composition that is metabolically more oxidative and less glycolytic in nature, and it participates in the regulation of both carbohydrate and lipid metabolism (Liang H, Ward W (2006) PGC-1 alpha: a key regulator of energy metabolism. Adv Physiol Educ.;30(4): 145-51 ).

GCN5 physically interacts with and acetylates PGC-1 α resulting in reduced PGC-1 α activity (Lerin C, Rodgers JT, Kalume DE, et al. (2006)). Gain-of-function and loss-of-function experiments demonstrate that GCN5 reduces the activity of PGC-1 α and the expression of its downstream target genes in hepatoma (Lerin C, Rodgers JT, Kalume DE, et al. (2006)) and muscle (Gerhart-Hines Z, Rodgers J, Bare O, et al. (2007) Metabolic control of muscle mitochondrial function and fatty acid oxidation through SIRT1/PGC-1 α. EMBO J: 26:1913-23) cells. Mitochondrial mass and function are positively correlated with insulin sensitivity in human skeletal muscle.

Summary

GCN5 inhibitors inhibit GCN5 acetylation of PGC-1 α which in turn enhances PGC-1α activity, mitochondrial function and oxidative metabolism. The invention relates to the use of a GCN5 inhibitor or a pharmaceutically acceptable salt thereof for the treatment of a disease or condition selected from diabetes (e.g. type 2 diabetes), insulin resistance, metabolic disease/metabolic syndrome, dyslipidemia, obesity, overweight, neurodegenerative diseases (e.g. Parkinson's disease, Alzheimer's disease, or Huntington's disease), heart failure, muscle disease (e.g. muscle atrophy/dystrophy), or improvement of exercise endurance capacity, by administering to an animal in need of such treatment an effective dose of at least one GCN5 inhibitor or a pharmaceutically acceptable salt thereof.

Breif Description of Figures

Figure 1 panels A and C, and panels B and D are gels and bar graphs, respectively, depicting the effect of GCN5 inhibitors on PGC-1 α acetylation.

Figure 2 is a bar graph depicting the effect of GCN5 inhibitors on PGC-1 α activity in 293T cells. The graph shows mean ± SEM of FF/RL. n=6, ^* p<0.05 vs. DMSO.

Figure 3 is a schematic showing the action of GCN5 inhibitors on PGC-1 α activity, mitochondrial function, and fatty acid oxidation capacity.

Detailed Description

The term " GCN5 inhibitor" includes a molecule that exhibits inhibition of the activity of GCN5, such as from 1-100% inhibition (e.g. with an IC₅₀ less of 0.1 mM as measured by an enzymatic reaction). The inhibitors may preserve the action of substrate molecules. Inhibition of the activity of GCN5 decreases PGC-1 α acetylation and increases PGC-1α activity as well as its target gene expression including ERRα and Cytochrome c at the protein level in muscle cells.

The term "GCN5 inhibitor " includes active metabolites and prodrugs, such as active metabolites and prodrugs of GCN5 inhibitors. A "metabolite" is an active derivative of a GCN5 inhibitor produced when the GCN5 inhibitor is metabolised. A "prodrug" is a compound that is either metabolised to GCN5 inhibitor or is metabolised to the same metabolite(s) as a GCN5 inhibitor. Examples of GCN5 inhibitors include small molecules MB-3 (Biel M, Kretsovali A, Karatzali E, et al. (2004) Design, synthesis, and biological evaluation of a small-molecule inhibitor of the histone acetyltransferase GCN5. Angew Chem lnt Ed; 43:3974-6) and ethyl-3- quinolinecarboxylate (Mai A, Rotili D, Tarantino D, et al. (2006) Small molecules inhibitors of histone acetyltransferase activity: identification and biological properties. J Med Chem; 49:6897-6907).

4-methylene-5-oxo-2-propyltetrahydro-furan- ethyl 3-quinolinecarboxylate 3-carboxylic acid (MB-3)

Further examples of GCN5 inhibitors include peptide-CoA conjugate, containing CoA covalently attached through an isopropionyl linker to the lysine ε-amino group of an N- terminal 20-aa fragment of histone H3 [H3-(Me)CoA-20] (Proux A, Cebrat M, et al. (2002) Structure of the GCN5 histone acetyltransferase bound to a bisubstrate inhibitor. PNAS; 99: 14065-14070).

In one embodiment, the GCN5 inhibitors and pharmaceutical salts thereof are orally active.

In another embodiment, the GCN5 inhibitors may be used in association with a carrier.

A carrier is a tool (natural, synthetic, peptidic, non-peptidic) for example a protein which transports specific substances through the cell membrane in which it is embedded and into the cell. Different carriers (natural, synthetic, peptidic, non-peptidic) may be required to transport different substances, as each one is designed to recognize only one substance, or group of similar substances.

Any means of detection known by the person skilled in the art can be used to detect the association of the GCN5 inhibitors with a carrier, for example, by labeling the carrier.

The active ingredients or pharmaceutically acceptable salts thereof according to the present invention may also be used in form of a solvate, such as a hydrate or including other solvents, used for crystallization.

It has now been surprisingly found that GCN5 inhibitors are useful for the prevention, delay of progression or the treatment of a disease or condition selected from diabetes (e.g. type 2 diabetes), insulin resistance, metabolic disease/metabolic syndrome, dyslipidemia, obesity, overweight, neurodegenerative diseases (e.g. Parkinson's disease, Alzheimer's disease, or Huntington's disease), heart failure, muscle disease (e.g. muscle atrophy/dystrophy), or improvement of exercise endurance capacity.

The present invention thus concerns the use of a GCN5 inhibitor (e.g. MB-3 or ethyl 3-quinolinecarboxylate), or pharmaceutically acceptable salts thereof, for the manufacture of a medicament for the prevention, delay of progression or the treatment of a disease or condition selected from diabetes (e.g. type 2 diabetes), insulin resistance, metabolic disease/metabolic syndrome, dyslipidemia, obesity, overweight, neurodegenerative diseases (e.g. Parkinson's disease, Alzheimer's disease, or Huntington's disease), heart failure, muscle disease (e.g. muscle atrophy/dystrophy), or improvement of exercise endurance capacity.

The present invention relates furthermore to a method for the prevention, delay of progression or treatment of a disease or condition selected from diabetes (e.g. type 2 diabetes), insulin resistance, metabolic disease/metabolic syndrome, dyslipidemia, obesity, overweight, neurodegenerative diseases (e.g. Parkinson's disease, Alzheimer's disease, or Huntington's disease), heart failure, muscle disease (e.g. muscle atrophy/dystrophy), or improvement of exercise endurance capacity, comprising administering to a warm-blooded animal, including man, in need thereof, a therapeutically effective amount of a GCN5 inhibitor, such as MB-3 or ethyl 3-quinolinecarboxylate.

In one embodiment, the invention relates to a method of increasing PGC-1α activity, comprising administering to a patient in need thereof a GCN5 inhibitor.

In another embodiment, the invention relates to a method for modulating mitochondriogenesis, comprising administering to a subject an effective amount (e.g. an amount effective to modulate PGC1-α activity) of a GCN5 inhibitor, such that mitochondriogenesis is modulated. In yet another embodiment, the invention relates to a method for screening for modulation of PGC-1α, comprising contacting PGC-1 α with a candidate compound, measuring PGC-1α activity after contact with said candidate compound, and determining whether or not said candidate compound modulates PGC-1 α activity.

In a further embodiment, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a GCN5 inhibitor in combination with one or more pharmaceutically acceptable carriers for the treatment of a disease or condition selected from diabetes preferably type 2 diabetes, insulin resistance, metabolic disease/metabolic syndrome, dyslipidemia, obesity, overweight, Neurodegenerative diseases (e.g. Parkinson's disease, Alzheimer's disease, or Huntington's disease), heart failure, muscle disease (e.g. muscle atrophy/dystrophy), or improvement of exercise endurance capacity.

According to the present invention the treated diseases or damages are selected from type 2 diabetes, metabolic disease/metabolic syndrome, dyslipidemia, neurodegenerative diseases (e.g. Parkinson's disease, Alzheimer's disease, or Huntington's disease), heart failure, muscle disease (e.g. muscle atrophy/dystrophy), or the GCN5 is used to improve exercise endurance capacity.

In the present description, the term "treatment" includes both prophylactic or preventative treatment as well as curative or disease suppressive treatment, including treatment of patients at risk of contracting the disease or suspected to have contracted the disease as well as ill patients. This term further includes the delaying of progression of the disease.

The term "curative", as used herein, means efficacy in treating ongoing diseases.

The term "prevention" means prophylactic administration of the combination to healthy patients to prevent the outbreak of the conditions mentioned herein. Moreover, the term "prevention" means prophylactic administration of such combination to patients being in a pre-stage of the conditions, to be treated.

The term "prophylactic" means the prevention of the onset or recurrence of diseases or damages.

The term "delay of progression" as used herein means administration of the active compound to patients being in a pre-stage or in an early phase of the disease to be treated, in which patients for example a pre-form of the corresponding disease is diagnosed or which patients are in a condition, e.g. during a medical treatment or a condition resulting from an accident, under which it is likely that a corresponding disease will develop.

The term "metabolic syndrome" as used herein is a cluster of risk factors that increases greatly the risk of occurrence of a cardiovascular event: diabetes or prediabetes, abdominal obesity, changes in cholesterol and high blood pressure. While up to 80 per cent of the almost 200 million adults worldwide with diabetes will die of cardiovascular disease, people with metabolic syndrome are also at increased risk, being twice as likely to die from and three times as likely to have a heart attack or stroke compared to people without the syndrome. People with metabolic syndrome have a fivefold greater risk of developing type 2 diabetes (if not already present). It is the exact nature of the cluster which appears to bring additional risk over and above that which would be expected from each of the components (high triglycerides when measuring cholesterol, for example). For a person to be defined as having the metabolic syndrome, the definition requires they have central obesity, plus two of the following four additional factors: raised triglycerides, reduced HDL cholesterol, raised blood pressure, or raised fasting plasma glucose level. Gender and ethnicity are also factors taken into consideration in the definition of metabolic syndrome.

The term "diabetes" as used herein means Type 2 diabetes, Type 1 diabetes and latent autoimmune diabetes of adulthood (LADA), preferably diabetes is Type 2 diabetes. "Impaired Glucose Metabolism (IGM)" is defined by blood glucose levels that are above the normal range but are not high enough to meet the diagnostic criteria for type 2 diabetes mellitus. The incidence of IGM varies from country to country, but usually occurs 2- 3 times more frequently than overt diabetes. Until recently, individuals with IGM were felt to be pre-diabetics, but data from several epidemiologic studies argue that subjects with IGM are heterogeneous with respect to their risk of diabetes and their risk of cardiovascular morbidity and mortality. The data suggest that subjects with IGM, in particular IGT, do not always develop diabetes, but whether they are diabetic or not, they are, nonetheless, at high risk for cardiovascular morbidity and mortality.

Among subjects with IGM, about 58% have Impaired Glucose Tolerance (IGT), another 29% have Impaired Fasting Glucose (IFG), and 13% have both abnormalities (IFG/IGT). IGT is characterized by elevated postprandial (post-meal) hyperglycemia while IFG has been defined by the ADA (see Table below) on the basis of fasting glycemic values.

The term "weight loss" as used herein refers to loss of a portion of total body weight and may include treating/preventing/delaying overweight, and is desirable in the case of diabetes, obesity and overweight individuals. Weight loss can help to prevent many of these harmful consequences, particularly with respect to diabetes and cardiovascular disease (CVD). Weight loss may also reduce blood pressure in both overweight hypertensive and non-hypertensive individuals; serum triglycerides levels and increases the beneficial high- density lipoprotein (HDL)-form of cholesterol. Weight loss also generally reduces somewhat the total serum cholesterol and low-density lipoprotein (LDL)- cholesterol levels. Weight loss may also reduce blood glucose levels in overweight and obese persons. Weight loss and hypocaloric diets, are also a primary goals for the control of plasma glucose levels in the treatment of type 2 diabetes. Thus appetite control and weight loss are desirable for the treatment of type 2 diabetes. The term "treatment of obesity" covers e.g. body fat reduction or weight loss.

The term "body fat reduction" means loss of a portion of body fat.

The formula for Body Mass Index (BMI) is [Weight in pounds÷Height in inches÷Height in inches]^χ703. BMI cutpoints for human adults are one fixed number, regardless of age or sex, using the following guidelines: obese human adults have a BMI of 30.0 or more, overweight human adults have a BMI of 25.0 to 29.9, and underweight human adults have a BMI less than 18.5. A normal body weight range for an adult is defined as a BMI between 18.5 and 25. BMI. Cutpoints for children under 16 are defined according to percentiles: Obesity is defined as a BMI-for-age>95th percentile, Overweight is defined as a BMI for age greater than >85th percentile, and. Underweight is a BMI-for-age<5th percentile. A normal body weight range for a child is defined as a BMI above the 5th percentile and below the 85 percentile.

The term "neurodegenerative disorder" includes conditions and diseases like dementia (e.g. senile dementia, pre-senile dementia (also known as mild cognitive impairment), Alzheimer related dementia (Alzheimer type dementia)), Huntington's disease, Huntington's chorea, acute confusion disorders and especially those in which apoptotic necrocytosis plays a part, such as amyotrophic lateral sclerosis, glaucoma, multiple sclerosis, migraine, stroke, cerebral ischemia, and Parkinson's disease and especially Alzheimer's disease.

In one embodiment, the neurodegenerative disorder is selected from Alzheimer's disease and dementia, (e.g. senile dementia, mild cognitive impairment or Alzheimer type dementia).

In another embodiment, neurodegenerative disorder is Alzheimer's disease, Parkinson's disease or Huntington's disease.

The invention also relates to a pharmaceutical composition comprising, as active ingredients a GCN5 inhibitor, in free form or in form of a pharmaceutically acceptable salt thereof.

Another aspect of the present invention is the use of a pharmaceutical composition comprising, as active ingredients a GCN5 inhibitor such as MB-3 or ethyl 3- quinolinecarboxylate, in free form or in form of a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition for the prevention, delay of progression or treatment diabetes preferably type 2 diabetes, insulin resistance, metabolic disease/metabolic syndrome, dyslipidemia, obesity, overweight, Neurodegenerative diseases (e.g. Parkinson's disease, Alzheimer's disease, or Huntington's disease), heart failure, muscle disease (e.g. muscle atrophy/dystrophy), or improvement of exercise endurance capacity.

The invention also relates to a method for the prevention, delay of progression or treatment diabetes preferably type 2 diabetes, insulin resistance, metabolic disease/metabolic syndrome, dyslipidemia, obesity, overweight, Neurodegenerative diseases (e.g, Parkinson's disease, Alzheimer's disease, or Huntington's disease), heart failure, muscle disease (e.g. muscle atrophy/dystrophy), or improvement of exercise endurance capacity, comprising administering to a warm-blooded animal, including man, in need thereof jointly therapeutically effective amounts of a pharmaceutical composition comprising, as active ingredients a GCN5 inhibitor such as MB-3 or ethyl 3-quinolinecarboxylate, in free form or in form of a pharmaceutically acceptable salt thereof.

The pharmaceutical compositions according to the invention can be prepared in a manner known per se and are those suitable for enteral, such as oral or rectal, and parenteral administration to mammals (warm-blooded animals), including man, comprising a therapeutically effective amount of the pharmacologically active compound, alone or in combination with one or more pharmaceutically acceptable carriers, especially suitable for enteral or parenteral application.

These pharmaceutical preparations are for enteral, such as oral, and also rectal or parenteral, administration to homeotherms, with the preparations comprising the pharmacological active compound either alone or together with customary pharmaceutical auxiliary substances. For example, the pharmaceutical preparations consist of from about 0.1 % to 90 %, preferably of from about 1 % to about 80 %, of the active compound. Pharmaceutical preparations for enteral or parenteral, and also for ocular administration are, for example, in unit dose forms, such as coated tablets, tablets, capsules or suppositories and also ampoules. These are prepared in a manner that is known per se, for example using conventional mixing, granulation, coating, solubulizing or lyophilising processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active compound with solid excipients, if desired granulating a mixture which has been obtained, and, if required or necessary, processing the mixture or granulate into tablets or coated tablet cores after having added suitable auxiliary substances.

The dosage of the active compound can depend on a variety of factors, such as mode of administration, homeothermic species, age and/or individual condition.

In one embodiment patients for the uses or methods according to the present invention are patients or animals suffering from diabetes (e.g. type 2 diabetes), IGM (e.g. IGT), obesity or overweight, metabolic disease dyslipidemia, neurodegenerative disease or low exercise endurance capacity.

In a further embodiment the present invention concerns the compositions, uses, or methods according to the present invention for the prevention, or delay of progression, of diabetes (e.g. type 2 diabetes), in a patient suffering from IGM (e.g. IGT).

The invention also concerns a method for the prevention or delay of progression of type 2 diabetes, comprising administering to a warm-blooded animal, including man, suffering from IGM, preferably IGT, a therapeutically effective amount of a GCN5 inhibitor. In another embodiment, the invention concerns a pharmaceutical composition comprising a therapeutically effective amount of a GCN5 inhibitor in combination with one or more pharmaceutically acceptable carriers for the prevention or delay of progression of type 2 diabetes in a patient suffering from IGM, including IGT. In yet another embodiment, the invention also relates to use of a GCN5 inhibitor or pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention or delay of progression of type 2 diabetes in a patient suffering from IGM, including IGT.

Examples of dosages, for those active ingredients of the pharmaceutical combination according to the present invention that are commercially available, are especially therapeutically effective commercially available dosages. The dosage of the active compound can depend on a variety of factors, such as mode of administration, homeothermic species, age and/or individual condition. The corresponding active ingredient or a pharmaceutically acceptable salt thereof may also be used in form of a hydrate or include other solvents used for crystallization.

For the indications of the present invention, the exact dosage will of course vary depending upon the compound employed, mode of administration and treatment desired. The compound may be administered by any conventional route including non-oral or orally. Expected therapeutic results are obtained when administered at a daily dosage of from about 0.01 mg/kg to about 100mg/kg, in other embodiments doses range from about 0.1 mg/kg to about 50mg/kg.

For the larger mammals, an indicated total daily dosage is in the range from about 0.01 mg/kg to about 100mg/kg of the compound, conveniently administered in divided doses 2 to 4 times a day in unit dosage form containing for example from about 10 to about 100 mg of the compound in sustained release form.

In yet another embodiment, the daily oral dosage in humans is between about 1 mg and about 1 g, between about 10 mg and about 500 mg (e.g. about 10 mg, or between about 10 mg and about 200 mg). Appropriate unit doses for oral administration contain for example about 10 mg to about 500 mg of the active ingredient i.e. GCN5 inhibitors. Appropriate doses for parenteral administration contain for example between about 10 mg to about 500 mg or between about 10 mg to about 200 mg of the compound.

The compounds of the invention may be administered in similar manner to known standards for uses in these utilities. The suitable daily dosage for a particular compound will depend on a number of factors such as its relative potency of activity. A person skilled in the pertinent art is fully enabled to determine the therapeutically effective dosage.

The compounds of the invention may be administered in free base form or as a pharmaceutically acceptable acid addition or quaternary ammonium salt. Such salts may be prepared in conventional manner and exhibit the same order of activity as the free forms. If these compounds have, for example, at least one basic center, they can form acid addition salts. Corresponding acid addition salts can also be formed having, if desired, an additionally present basic center. The compounds having an acid group (for example COOH) can also form salts with bases. For example, the compounds to be combined can be present as a sodium salt, as a maleate or as a dihydrochloride. The active ingredient or a pharmaceutically acceptable salt thereof may also be used in form of a hydrate or include other solvents used for crystallization.

The pharmaceutical compositions according to the invention can be prepared in a manner known per se and are those suitable for enteral, such as oral or rectal, and parenteral administration to mammals (warm-blooded animals), including man, comprising a therapeutically effective amount of the pharmacologically active compound, alone or in combination with one or more pharmaceutically acceptable carries, especially suitable for enteral or parenteral application.

The structure of the active agents identified by code nos., generic or trade names may be taken from the actual edition of the standard compendium "The Merck Index" or from databases, e.g. Patents International (e.g. IMS World Publications). The corresponding content thereof is hereby incorporated by reference. Any person skilled in the art is fully enabled to identify the active agents and, based on these references, likewise enabled to manufacture and test the pharmaceutical indications and properties in standard test models, both in vitro and in vivo.

The pharmacological activity may, for example, be demonstrated in a clinical study or in the test procedure as essentially described hereinafter in a manner known to the skilled person. Experimental

The following examples are carried out with GCN5 inhibitors to show their claimed activity.

Examples

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

Example 1 : Adenoviral-mediated overexpression of PGC-1alpha and GCN5 in C2C12 myoblasts

C2C12 mouse myoblasts are obtained from ATCC (Cat. # CRL-1222) and cultured in DMEM (Gibco, Cat. # 11965-092) supplemented with 10% fetal bovine serum (Invitrogen), 100 IU/ml of penicillin and 100 μg/ml of streptomycin (Gibco, Cat.# 15140-122). The cells are plated in 10 cm dishes at a density of 100,000 cells/dish. The following day, the cells are transduced with 5x109 viral particles/dish of an adenovirus expressing Flag-PGC-1 α, and either GFP or GCN5 (5x109 viral particles/dish) in the growth medium. The medium is replaced the next day. After 48 h of transduction, the cells are treated with 0.1 mM of MB-3 or ethyl 3-quinolinecarboxylate. DMSO is used as a control. The cell pellets are harvested after 24 h of treatment.

Example 2: Protein extraction, immunoprecipitation and Western blot analysis

Total cell extracts are obtained by lysing the cells with extraction buffer (20 mM Hepes, pH 7.9, 125 mM NaCI, 0.2% Triton X-100, 1 mM EDTA, 1 mM DTT, containing protease inhibitor (Roche Cat.# 1836153), 20 mM of fresh nicotinamide (NAM) (Sigma Cat.# N0636) and 5 μM trichostatin A (TSA) (Cell Signaling, Cat. # 9950). Cells are left on ice for 20 min then centrifuged at maximum speed for 15 min at 4°C. For immunoprecipitation, 1 mg of total protein is added in a final volume of 800 μl of extraction buffer. Twenty μl of anti-Flag M2 affinity beads (Sigma Cat. # A2220) are added, and the lysate and the mixture are rotated at 4°C overnight. The next day, the beads are washed four times with the extraction buffer containing fresh NAM and TSA. To elute the protein from the beads, 20 μl of reducing sample buffer (Boston Bioproducts, Cat.# BP1 11 R) is added and the samples are boiled for 4 min before being loaded on a 7.5% Tris-glycine gel (BioRad, Cat. # 161-1 154). Western blot analysis is carried out in a standard manner. To detect the GCN5 protein, a rabbit polyclonal antibody is used at 1 :500 dilution (Biolegend, Cat.# 607201 ). To detect acetylated and total PGC-1 α protein, the rabbit polyclonal antibodies for acetyl-lysine (Cell Signaling Cat.# 9441 ) and PGC-1α (Chemicon Cat.# ab3242) are used at 1 :1000 dilution. Example 3: PGC-1alpha activity reporter gene assay

Human 293T cells (1.4x 104 cells/well in 96-well plates) are transfected in suspension culture. For each transfection, 164 ng of DNA mixture and 0.48 μl of Fugene 6 tranfection reagent (Roche, Cat.# NC9167392) are used. The DNA mixture containing 40 ng of pGL4- UAS-luciferase construct, 60 ng of pCMV-Gal4-PGC-1α, 4 ng of pRL-SV40-renilla luciferase (a control for transfection efficiency), 60 ng of pcDNA or GCN5 is mixed with 10 μl of Opti- MEM and incubated for 20 min at RT. Cells are added to this mixture and the cell suspension is plated in one well of a 96-well plate. Cells are incubated at 37⁰C for 24 h followed by treatment with 2 mM of ethyl 3-quinolinecarboxylate or MB-3 (1 mM). The luciferase activity is measured after 24 h of treatment as carried out in a standard manner. The firefly luciferase activity is normalized to the renilla luciferase activity. The assay is run with 6 replicates, a p- value smaller than 0.05 in two-tailed, paired t-test is considered significant.

Results

Example 4: GCN5 inhibitors decrease PGC-1alpha acetylation in C2C12 myoblasts

To determine the effect of GCN5 inhibitors on PGC-1 α acetylation, C2C12 myoblasts are transduced with a Flag-PGC-1 α adenovirus along with either a Flag-GCN5 or GFP adenovirus. Cells are treated with ethyl-3-quinolinecarboxylate (2 mM) (Figure 1 , Panel A), MB-3 (0.1 mM) (Figure 1 , Panel C), or DMSO at 48 h post-transduction. Cells are harvested at 24 h post-treatment. Total cell lysates are immunoprecipated with a flag antibody. The immunoprecipitated materials are then subjected to Western blot analysis using various antibodies to determine the acetylation status of PGC-1 α as well as total PGC-1α and GCN5 protein levels as described in Example 2. Adenoviral transduction results in elevated levels of GCN5 and PGC-1 α protein in the cells. PGC-1 α acetylation is markedly increased by GCN5 (lane 2 vs. 1 in Figure 1 , Panels A and C) while treatment with either inhibitor attenuates this effect (lane 4 vs. 3 in Figure 1 , Panels A and C). The percentage of acetylated PGC-1 α protein in each condition is quantitated via densitometry (Figure 1 , Panels B and D). The level of total PGC-1 α protein is increased when GCN5 is co-expressed (lane 2 vs. 1 and 4 vs. 3).

Example 5: GCN5 inhibitor increases PGC-1 alpha activity

Reduction of PGC-1 α acetylation by knockdown of GCN5 in cells is shown to increase PGC-1 α activity as measured by a luciferase-reporter gene assay (Lerin, et al 2006). To determine the effect of inhibition of GCN5 with a small molecule inhibitor, 293T cells are transiently transfected with the pCMV-Gal4-PGC1α, pGL4-UAS-Luciferase and Renilla luciferase plasmids. Cells are treated with 2 mM of ethyl-3-quinolinecarboxylate or 1 mM of MB-3 for 24 h. Luciferase activity, indicative of PGC-1 α activity, is measured in the cell lysates. Treatment with both inhibitors significantly increases PGC-1 α activity (Figure 2).

The invention has been described above by reference to various embodiments but, as those skilled in the art will appreciate, many additions, omissions and modifications are possible all within the scope of the claims below.

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 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

We claim:

1 ) A method for the prevention, delay of progression or treatment of a disease or condition selected from diabetes, insulin resistance, metabolic disease/metabolic syndrome, dyslipidemia, obesity, overweight, neurodegenerative diseases, heart failure, muscle disease (e.g. muscle atrophy/dystrophy), or improvement of exercise endurance capacity, comprising: administering to a warm-blooded animal, in need thereof, a therapeutically effective amount of a GCN5 inhibitor, such that said disease or disorder is prevented, delayed, or treated.

2) The method of claim 1 , wherein said GCN5 inhibitor is selected from MB-3 and ethyl 3-quinolinecarboxylate.

3) Use of a GCN5 inhibitor or pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention, delay of progression or the treatment of a disease or condition selected from diabetes, insulin resistance, metabolic disease/metabolic syndrome, dyslipidemia, obesity, overweight, neurodegenerative diseases, heart failure, muscle disease (e.g. muscle atrophy/dystrophy), or improvement of exercise endurance capacity.

4) A method of increasing PGC-1 α activity, comprising administering to a patient in need thereof a GCN5 inhibitor.

5) A method for modulating mitochondriogenesis, comprising administering to a subject an effective amount of a GCN5 inhibitor, such that mitochondriogenesis is modulated.

6) The method of claim 5, wherein said effective amount is effective to modulate PGC1- α activity.

7) A method for screening for modulation of PGC-1α, comprising contacting PGC-1α with a candidate compound, measuring PGC-1 α activity after contact with said candidate compound, and determining whether or not said candidate compound modulates PGC-1α activity.