Fragment‐Based Screening to Identify Thioesterase Superfamily Member 2 (Them2) Inhibitors for the Management of Obesity‐Related Disorders

Abstract

BackgroundThe prevalence of obesity‐related disorders continues to increase, but current management options remained limited. In the setting of chronic overnutrition, excessive tissue concentrations of free fatty acid (FFA) contribute to the pathogenesis of obesity‐related disorders. Thioesterase superfamily member 2 (Them2) is an acyl‐CoA thioesterase (Acot) that hydrolyzes long‐chain fatty acyl‐CoAs into free fatty acids and CoASH. Them2 is enriched in oxidative tissues, and Them2−/− mice are protected from diet‐induced obesity, diabetes, and non‐alcoholic fatty liver disease. These findings suggest that chemical inhibition of Them2 could be leveraged in the management of obesity‐related disorders.AimThis study was designed to develop a small molecule inhibitor that targets the enzymatic activity of Them2.MethodsRecombinant His‐tagged human Them2 was expressed in E. coli, and purified by affinity chromatography and size exclusion chromatography. Two fragment‐based screens (FBS) were performed: 1) A 1056 fragment library was screened by differential scanning fluorimetry (DSF), and 2) a 500 19F‐labeled fragment library was screened by 19F‐nuclear magnetic resonance (19F‐NMR). Secondary orthogonal biochemical (microscale thermophoresis; MST) assays confirmed binding interactions between fragment compound hits and Them2. A microplate‐based fluorometric Acot activity assay was used to measure the Acot activity of Them2.Results30 DSF‐derived fragment compounds (2.8% hit rate) that decreased the melting temperature of Them2 by greater than 4°C (i.e. Them2 de‐stabilizers) were identified. Deconvoluted NMR chemical shifts revealed binding of 15 19F‐labeled fragment compounds to Them2 (3% hit rate). The most promising fragment compound hits exhibited Kd values in the μM range by MST. The most potent fragment compound hits were those that suppressed Acot activity.ConclusionsComplementary biochemical approaches identify fragment compounds capable of both binding and inhibiting Them2. Following structural characterization of ligand binding by x‐ray crystallography, medicinal chemistry strategies can be leveraged to improve potency. Lead compounds that inhibit Them2 should prove attractive in the management of obesity‐related disorders.Support or Funding InformationThis work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01 DK056626, R37 DK048873, and T32 DK116970 (to D.E.C).