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Agents targeting metabolic pathways form the backbone of standard oncology treatments, though a better understanding of differential metabolic dependencies could instruct more rationale-based therapeutic approaches. We performed a chemical biology screen that revealed a strong enrichment in sensitivity to a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic versus solid tumor origin. Differential AG-636 activity translated to the in vivo setting, with complete tumor regression observed in a lymphoma model. Dissection of the relationship between uridine availability and response to AG-636 revealed a divergent ability of lymphoma and solid tumor cell lines to survive and grow in the setting of depleted extracellular uridine and DHODH inhibition. Metabolic characterization paired with unbiased functional genomic and proteomic screens pointed to adaptive mechanisms to cope with nucleotide stress as contributing to response to AG-636. These findings support targeting of DHODH in lymphoma and other hematologic malignancies and suggest combination strategies aimed at interfering with DNA-damage response pathways.

GO analysis of proteins with at least a 1.5-fold increase in expression in the presence of AG-636 compared to DMSO

Supplementary methods, references and figures

The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) was recently implicated as a synthetic lethal target in cancers with deletion of the methylthioadenosine phosphorylase (MTAP) gene, which is adjacent to the CDKN2A tumor suppressor and codeleted with CDKN2A in approximately 15% of all cancers. Previous attempts to target MAT2A with small-molecule inhibitors identified cellular adaptations that blunted their efficacy. Here, we report the discovery of highly potent, selective, orally bioavailable MAT2A inhibitors that overcome these challenges. Fragment screening followed by iterative structure-guided design enabled >10 000-fold improvement in potency of a family of allosteric MAT2A inhibitors that are substrate noncompetitive and inhibit release of the product, S-adenosyl methionine (SAM), from the enzyme's active site. We demonstrate that potent MAT2A inhibitors substantially reduce SAM levels in cancer cells and selectively block proliferation of MTAP-null cells both in tissue culture and xenograft tumors. These data supported progressing AG-270 into current clinical studies (ClinicalTrials.gov NCT03435250).

Inhibitors of mutant isocitrate dehydrogenase (mIDH) 1 and 2 cancer-associated enzymes prevent the accumulation of the oncometabolite d-2-hydroxyglutarate (2-HG) and are under clinical investigation for the treatment of several cancers harboring an IDH mutation. Herein, we describe the discovery of vorasidenib (AG-881), a potent, oral, brain-penetrant dual inhibitor of both mIDH1 and mIDH2. X-ray cocrystal structures allowed us to characterize the compound binding site, leading to an understanding of the dual mutant inhibition. Furthermore, vorasidenib penetrates the brain of several preclinical species and inhibits 2-HG production in glioma tissue by >97% in an orthotopic glioma mouse model. Vorasidenib represents a novel dual mIDH1/2 inhibitor and is currently in clinical development for the treatment of low-grade mIDH glioma.

Inhibition of the

The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) was recently implicated as a synthetic lethal target in cancers with deletion of the methylthioadenosine phosphorylase (

Agents targeting metabolic pathways form the backbone of standard oncology treatments, though a better understanding of differential metabolic dependencies could instruct more rationale-based therapeutic approaches. We performed a chemical biology screen that revealed a strong enrichment in sensitivity to a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic versus solid tumor origin. Differential AG-636 activity translated to the in vivo setting, with complete tumor regression observed in a lymphoma model. Dissection of the relationship between uridine availability and response to AG-636 revealed a divergent ability of lymphoma and solid tumor cell lines to survive and grow in the setting of depleted extracellular uridine and DHODH inhibition. Metabolic characterization paired with unbiased functional genomic and proteomic screens pointed to adaptive mechanisms to cope with nucleotide stress as contributing to response to AG-636. These findings support targeting of DHODH in lymphoma and other hematologic malignancies and suggest combination strategies aimed at interfering with DNA-damage response pathways.

A novel series of pyrazolyltetrahydropyran N-type calcium channel blockers are described. Structural modifications of the series led to potent compounds in both a cell-based fluorescent calcium influx assay and a patch clamp electrophysiology assay. Representative compounds from the series were bioavailable and showed efficacy in the rat CFA and CCI models of inflammatory and neuropathic pain.

The discovery of a novel series of N-arylpyrroles as agonists of GPR120 (FFAR4) is discussed. One lead compound is a potent GPR120 agonist, has good selectivity for related receptor GPR40 (FFAR1), has acceptable PK properties, and is active in 2 models of Type 2 Diabetes in mice.

A novel series of 5-membered heterocycle-containing phenylpropanoic acid derivatives was discovered as potent GPR120 agonists with low clearance, high oral bioavailability and in vivo antidiabetic activity in rodents.

We have discovered a novel series of tetrahydrobenzimidazoles 3 as TGR5 agonists. Initial structure–activity relationship studies with an assay that measured cAMP levels in murine enteroendocrine cells (STC-1 cells) led to the discovery of potent agonists with submicromolar EC50 values for mTGR5. Subsequent optimization through methylation of the 7-position of the core tetrahydrobenzimidazole ring resulted in the identification of potent agonists for both mTGR5 and hTGR5 (human enteroendocrine NCI-H716 cells). While the lead compounds displayed low to moderate exposure after oral dosing, they significantly reduced blood glucose levels in C57 BL/6 mice at 30 mg/kg and induced a 13–22% reduction in the area under the blood glucose curve (AUC)0–120 min in oral glucose tolerance tests (OGTT).

The methylthioadenosine phosphorylase (MTAP) gene is located adjacent to the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor-suppressor gene and is co-deleted with CDKN2A in approximately 15% of all cancers. This co-deletion leads to aggressive tumors with poor prognosis that lack effective, molecularly targeted therapies. The metabolic enzyme methionine adenosyltransferase 2α (MAT2A) was identified as a synthetic lethal target in MTAP-deleted cancers. We report the characterization of potent MAT2A inhibitors that substantially reduce levels of S-adenosylmethionine (SAM) and demonstrate antiproliferative activity in MTAP-deleted cancer cells and tumors. Using RNA sequencing and proteomics, we demonstrate that MAT2A inhibition is mechanistically linked to reduced protein arginine methyltransferase 5 (PRMT5) activity and splicing perturbations. We further show that DNA damage and mitotic defects ensue upon MAT2A inhibition in HCT116 MTAP-/- cells, providing a rationale for combining the MAT2A clinical candidate AG-270 with antimitotic taxanes.

A novel series of pyrazolyltetrahydropyran N-type calcium channel blockers are described. Structural modifications of the series led to potent compounds in both a cell-based fluorescent calcium influx assay and a patch clamp electrophysiology assay. Representative compounds from the series were bioavailable and showed efficacy in the rat CFA and CCI models of inflammatory and neuropathic pain.

We have discovered a novel series of isothiazole-based phenylpropanoic acids as GPR120 agonists. Extensive structure–activity relationship studies led to the discovery of a potent GPR120 agonist 4x, which displayed good EC50 values in both calcium and β-arrestin assays. It also presented good pharmaceutical properties and a favorable PK profile. Moreover, it demonstrated in vivo antidiabetic activity in C57BL/6 DIO mice. Studies in WT and knockout DIO mice showed that it improved glucose handling during an OGTT via GPR120. Overall, 4x possessed promising antidiabetic effect and good safety profile to be a development candidate.

The preparation of a chemokine receptor type 2 (CCR-2) antagonist bearing a cyclopenta[b]furan core is described on a 600 g scale. Compared to our previously reported synthesis of the all-carbon core CCR-2 antagonist with a similar peripheral 3-methoxypyran appendage, our work required a redesign of the original Discovery Chemistry route and took advantage of a side product seen in the diastereoselective alkylation reaction. Elaboration by reduction and oxy-cyclization eventually led to the required N-Boc acid method. After amidation using a traditional coupling reaction, a reductive amination using enantiomerically enriched 3-methoxy-4-pyranone led to the final compound. Although several steps of the syntheses involved reagents such as selenium and chromium that would not be used in a large-scale process setting, the overall route went through intermediates that could certainly be used for future scale-up campaigns. The synthesis provided a method to make lab-scale quantities of the final succinate salt ...

The preparation of a novel chemokine receptor type 2 (CCR-2) antagonist is described on a 135 g scale. The synthesis of an all-carbon bicyclic core was accomplished using a radical cyclization strategy using chiral precursors, wherein elaboration led to N-Boc carboxylic acid in good yield. After amidation using a traditional coupling reaction, a reductive amination using enantiomerically enriched 3-methoxy-4-pyranone led to the final compound. Although several steps of the syntheses involved reagents that would not be preferred in process and chromatography was used to provide the free-base diastereomer of the final succinate salt, the overall route went through stable intermediates that could be used for future scale-up. This lab-scale synthesis struck a balance between a quick scale-up and a more thorough process review of all possible methods and routes.

Both substituted 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles and 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles have been synthesized by the 3+2 intramolecular dipolar cycloaddition of nitrilimines to alkynes. This cyclization has been extended to more versatile 3-bromo derivatives by the use of alkynylbromides as dipolarophiles.

SAR study of 5-aminooctahydrocyclopentapyrrole-3a-carboxamide scaffold led to identification of several CCR2 antagonists with potent activity in both binding and functional assays. Their cardiovascular safety and pharmacokinetic properties were also evaluated.

Two cycloaddition methods were applied to chiral protected aminocyclopentenes 2 and 9 and provided novel bicyclic products 3 and 4 in good yields. The explanation for the observed stereochemistry was based on the sterically encumbered β-face forcing the cycloadditions to occur on the α-face of the cyclopentene ring. The stereochemistry of 4 was confirmed by X-ray of the fumarate salt 10 and showed the trans-relationship between the newly formed ring and the chiral –NHBoc group.

Selective androgen receptor modulators (SARMs) are androgens with tissue-selective activity. SARMs that have anabolic activity on muscle while having minimal stimulatory activity on prostate are classified as SARM agonists. They can be used to prevent the loss of lean body mass that is associated with cancer, immunodeficiency, renal disease and aging. They may also have anabolic activity on bone; thus, unlike estrogens, they may reverse the loss of bone strength associated with aging or hypogonadism. Our in-house effort on SARM program discovers a nonsteroidal androgen receptor ligand with a unique imidazolopyrazole moiety in its structure. In vitro, this compound is a weak androgen receptor binder and a weak androgen agonist. Despite this, in orchidectomized mature rats it is an effective SARM agonist, with an ED(50) on levator ani muscle of 3.3mg/kg and an ED(50) on ventral prostate of >30mg/kg. It has its maximal effect on muscle at the dose of 10mg/kg. In addition, this compound has mixed agonistic and antagonistic activities on prostate, reducing the weight of that tissue in intact rats by 22% at 10mg/kg. The compound does not have significant effect on gonadotropin levels or testosterone levels in both orchidectomized and intact male rats. It does not have notable progestin, estrogen or glucocorticoid agonistic or antagonistic activity in rats. In a female sexual behavior model, it improves the sexual desire of ovariectomized female rats for sexually mature intact males over nonsexually ovariectomized females. Overall, the imidazolopyrazole is a potent prostate-sparing candidate for development as a SARM agonist with an appropriate pharmacological profile for clinical benefit in muscle-wasting conditions and female sexual function disorders.

A novel series of 2-thio-5-thiomethyl substituted imidazoles was discovered to be potent TGR5 agonists that possessed glucose-lowering effects while inhibiting gall bladder emptying in mice.

We have discovered a novel series of 4-azetidinyl-1-aryl-cyclohexanes as CCR2 antagonists. Divergent SAR studies on hCCR2 and hERG activities led to the discovery of compound 8d, which displayed good hCCR2 binding affinity (IC50, 37 nM) and potent functional antagonism (chemotaxis IC50, 30 nM). It presented an IC50 of >50 μM in inhibition of the hERG channel and had no effect on the QTc interval up to 10 mg/kg (i.v.) in anesthetized guinea pig and dog CV studies. It also displayed high selectivity over other chemokine receptors and GPCRs, and amendable oral bioavailability in dogs and primates. In a thioglycollate-induced inflammation model in hCCR2KI mice, it had ED50 of 3 mg/kg on inhibition of the influx of leukocytes, monocytes/macrophages, and T-lymphocytes.

Selective blockers of the N-type calcium channel have proven to be effective in animal models of chronic pain. However, even though intrathecally delivered synthetic ω-conotoxin MVIIA from Conus magnus (ziconotide [Prialt®]) has been approved for the treatment of chronic pain in humans, its mode of delivery and narrow therapeutic window have limited its usefulness. Therefore, the identification of orally active, small-molecule N-type calcium channel blockers would represent a significant advancement in the treatment of chronic pain. A novel series of pyrazole-based N-type calcium channel blockers was identified by structural modification of a high-throughput screening hit and further optimized to improve potency and metabolic stability. In vivo efficacy in rat models of inflammatory and neuropathic pain was demonstrated by a representative compound from this series.

As a result of further SAR studies on a piperidinyl piperidine scaffold, we report the discovery of compound 44, a potent, orally bioavailable CCR2 antagonist. While having some in vitro hERG activity, this molecule was clean in an in vivo model of QT prolongation. In addition, it showed excellent efficacy when dosed orally in a transgenic murine model of acute inflammation.

A novel series of 4-azetidinyl-1-aryl-cyclohexanes containing indazole or benzoisoxazole moiety have been identified as potent CCR2 antagonists with high selectivity versus hERG.

A series of 4-azetidinyl-1-aryl-cyclohexanes as potent CCR2 antagonists with high selectivity over activity for the hERG potassium channel is discovered through divergent SARs of CCR2 and hERG.

A series of indazoles have been discovered as KHK inhibitors from a pyrazole hit identified through fragment-based drug discovery (FBDD). The optimization process guided by both X-ray crystallography and solution activity resulted in lead-like compounds with good pharmaceutical properties.

AG-881 is an orally available, brain penetrant, potent, small-molecule inhibitor of isocitrate dehydrogenase (IDH) 1 and IDH2 mutant proteins. Small-molecule inhibition of the mutant IDH (mIDH) protein represents a targeted approach to cancer treatment for patients who harbor an IDH1 and/or an IDH2 mutation. Direct inhibition of the gain-of-function activity of the mIDH protein is intended to inhibit the production of the oncogenic metabolite D-2-hydroxyglutarate (2-HG) and induce tumor cell differentiation. Biochemical studies of AG-881 demonstrate that it has low nanomolar potency inhibition (IC50) against multiple mIDH homodimer and heterodimer enzymes. It is a rapid-equilibrium inhibitor of mIDH1-R132H and mIDH2-R172K homodimer enzymes and is a slow-binding inhibitor of mIDH2-R140Q homodimer and wild type (wt) IDH1/mIDH1-R132H, wtIDH2/mIDH2-R140Q, and wtIDH2/mIDH2-R172K heterodimers. The potency against mIDH1 and mIDH2 enzymes has also been shown in cell lines and primary human patient samples. The IC50 range for 2-HG inhibition by AG-881 was 0.04-22 nM in cells expressing mIDH1-R132C, mIDH1-R132G, mIDH1-R132H, or mIDH1-R132S mutations and was 7-14 nM and 130 nM in cells expressing mIDH2-R140Q and mIDH2-R172K mutations, respectively. The treatment of these mIDH cell lines or primary human acute myeloid leukemia samples with AG-881 led to the onset of cellular differentiation. The pharmacokinetics of AG-881 are characterized by rapid oral absorption and low total body plasma clearance in mice (0.406 L/hr/kg) and rats (0.289 L/hr/kg). Because of these favorable properties, a series of in vivo pharmacology studies were conducted with AG-881 in mouse xenograft models. In these studies, dose-response relationships for the reduction in 2-HG in tumor by AG-881 were established. Twice-daily dosing of AG-881 in the HT1080 (mIDH1-R132C) and U87 (mIDH2-R140Q) mouse models reduced tumor 2-HG levels by >96% at doses ≥30 mg/kg. In the orthotopic glioma model (mIDH1-R132H), brain tumor 2-HG levels were reduced by >97% at doses ≥0.1 mg/kg. Based on in vivo exposure-response analyses, plasma AG-881 AUC0-24hr values of 402 hr•ng/mL and 45,200 hr•ng/mL are projected to result in sustained 97% reduction in tumor 2-HG levels in the glioma indication and the non-glioma solid and liquid tumor indications, respectively. AG-881 also exhibits excellent brain penetration, with brain-to-plasma ratios ranging from 0.62 to 1.96 in mice and 1.11 to 1.48 in rats (based on AUC0-24hr), and has an acceptable preclinical safety profile that supports clinical testing. Taken together, these data show that AG-881 is a potent inhibitor of the mIDH1 and mIDH2 proteins and suppresses 2-HG production in enzymatic, cell-based, and in vivo systems. Pharmacology studies support that suppression of 2-HG levels by AG-881 results in alterations of cellular downstream markers, leading to a release from blockage of tumor cell differentiation. AG-881 is currently in phase 1 clinical development in patients with an IDH1 and/or IDH2-mutation who have advanced solid tumors including gliomas (ClinicalTrials.gov NCT02481154), and advanced hematologic malignancies (ClinicalTrials.gov NCT02492737). Citation Format: Katharine Yen, Zenon Konteatis, Zhihua Sui, Erin Artin, Lenny Dang, Kimberly Straley, Erica Tobin, Carl Campos, Hua Yang, Raj Nagaraja, Yue Chen, Hyeryun Kim, Camelia Gliser, Brandon Nicolay, Andrew Olaharski, Lee Silverman, Scott Biller, Shinsan M. Su, Ingo Mellinghoff, Janeta Popovici-Muller. AG-881, a brain penetrant, potent, pan-mutant IDH (mIDH) inhibitor for use in mIDH solid and hematologic malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B126.

The androgens testosterone and its more potent tissue metabolite 5-alpha-dihydrotesterone regulate diverse physiological process involving both reproductive and non-reproductive functions. Most of the signaling effects of androgens are mediated through the androgen receptor (AR), a member of the nuclear receptor superfamily of transcription factors. The AR has been a target for drug development focused on the treatment of pathological conditions arising from abnormal androgen levels or altered target tissue responsiveness, the improvement of physical performance and the regulation of male fertility. The primary focus for drug design has been the synthesis of chemicals to regulate the transcriptional activity of AR based on the structural and functional properties of the ligands, with a recent preference for selectively anabolic non-steroidals. A new class of molecules targeting androgen receptors called selective androgen receptor modulators is being developed, analogous to the clinically successful and at present marketed selective estrogen receptor modulators.This article highlights and reviews research advances in this field that have been published in patent literature since 2003.The structural diversity of selective androgen receptor modulators has dramatically increased. Several compounds have emerged as clinical and preclinical candidates.

Starting from 4-amino-2-(trifluoromethyl)benzonitrile (6), an efficient and nonchromatographic process was developed for multihundred gram production of 4,5-diamino-2-(trifluoromethyl)benzonitrile (1) in 73% yield and 98 HPLC area% purity over four synthetic steps. The same synthetic strategy was applied to 4-bromo-2-(trifluoromethyl)aniline (7) that afforded 5-bromo-3-(trifluoromethyl)benzene-1,2-diamine (5) in 81% overall yield and 99% HPLC area% purity.

Monocyte chemoattractant protein-1 (MCP-1) is a major chemoattractant for monocytes and memory T cells by means of their binding to its specific cell-surface receptor, CC-chemokine receptor-2 (CCR2). CCR2 belongs to the G-protein-coupled seven-transmembrane receptor superfamily. The evidence in favor of CCR2 and MCP-1 having dominant roles in monocyte chemotaxis and chronic inflammation was provided by CCR2 and MCP-1 knockout mice. It has been recognized that CCR2 antagonists are potential therapeutic agents in preventing, treating, or ameliorating a CCR2-mediated inflammatory syndrome or disease such as psoriasis, uveitis, rheumatoid arthritis, multiple sclerosis, asthma, obesity, and chronic obstructive pulmonary disease. This review summarizes recent developments in small-molecule CCR2 antagonists disclosed by patent applications published between 2005 and 2008 and related publications.

Since the original report in 2004, phosphonium coupling has emerged as a new, mild, efficient, chemoselective and versatile methodology for the direct C–C, C–N, C–O, and C–S bond formations of unactivated and unprotected tautomerizable heterocycles. Phosphonium coupling proceeds via C–OH bond activation of a tautomerizable heterocycle with a phosphonium salt (e.g., PyBroP), and subsequent functionalization with either a nucleophile through SNAr displacement or an organometallic through transition-metal-catalyzed cross-coupling. As the first direct bond formation via C–OH bond activation, phosphonium coupling offers a powerful and practical methodology that features operational simplicity, functionality compatibility, and broad substrate scope. Its attractive protecting-group-free direct bond formation involving a domino multiple-step process in a single step provides unique and facile access to many biologically important heterocycles including macromolecules with sensitive functionalities (e.g., DNA, RNA and PNA building blocks). Consequently, the discovery of phosphonium coupling has finally enabled a single-step transformation in nucleoside chemistry, which has been an unsolved synthetic challenge in the past half-century. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

A novel series of benzopyran derivatives were synthesized and evaluated as KATP channel openers. Structure–activity relationships were investigated around 4-position of the benzopyran nucleus. Optimization of 4-substituent with some heterocyclic rings led to compound 13b bearing a benzo[d]isoxazol-3-one moiety as a potent and selective KATP channel opener in vitro. In two anesthetized rat models of myogenic bladder overactivity, compound 13b was found to inhibit spontaneous bladder contractions.

A novel and economical process was developed for recycling the undesired enantiomer, (S)-2,8-dimethoxy-5-{4-[2-(1-piperidinyl)ethoxy]phenyl}-11,12-dihydro-5H-6,13-dioxabenzo[3,4]cyclohepta[1,2-a]naphthalene (1b) obtained from chiral chromatographic separation, by refluxing 1b with HCl (4.0 equiv) in EtOH for 76 h, or with H2SO4 (2.0 equiv) in water for 68 h to afford a near racemic mixture ((R)-1a/(S)-1b = 41−42%/49−53%, chiral HPLC area%) in >96% isolated yield and good chemical purity (87−95%).

Replacement of the 7-CH2 group of natural steroid with an oxygen atom led to identification of unnatural 7-oxa-steroids as potent and selective progesterone receptor antagonists. The unnatural 7-oxa-steroids exhibited a different structure–activity relationship (SAR) from natural steroids. Molecular modeling demonstrated that the switch of carbon to oxygen in the B-ring results in a subtle conformational change of the tetracyclic skeleton and induces a remarkable spatial shift at the terminal end of the side chain of the D-ring. This shift causes the phenyl ring on the D-ring to form a perfect parallel-displaced form of π–π interaction with the phenyl ring of Phe794. The unnatural 7-oxa-steroids were orally active in a rat complement C3 assay and showed comparable pharmacokinetic and metabolic profiles to those of the natural steroid, mifepristone.

Androgens are required for the maintenance of normal sexual activity in adulthood and for enhancing muscle growth and lean body mass in adolescents and adults. Androgen receptor (AR) ligands with tissue selectivity (selective androgen receptor modulators, or SARMs) have potential for treating muscle wasting, hypogonadism of aging, osteoporosis, female sexual dysfunction, and other indications. JNJ-37654032 is a nonsteroidal AR ligand with mixed agonist and antagonist activity in androgen-responsive cell-based assays. It is an orally active SARM with muscle selectivity in orchidectomized rat models. It stimulated growth of the levator ani muscle with ED 50 0.8 mg/kg, stimulating maximal growth at a dose of 3 mg/kg. In contrast, it stimulated ventral prostate growth to 21% of its full size at 3 mg/kg. At the same time, JNJ-37654032 reduced prostate weight in intact rats by 47% at 3 mg/kg, while having no inhibitory effect on muscle. Using magnetic resonance imaging to monitor body composition, JNJ-37654032 restored about 20% of the lean body mass lost following orchidectomy in aged rats. JNJ-37654032 reduced follicle-stimulating hormone levels in orchidectomized rats and reduced testis size in intact rats. JNJ-37654032 is a potent prostate-sparing SARM with the potential for clinical benefit in muscle-wasting diseases.