Postdoctoral Associate Weill Cornell Medical College New York, New York, United States
Christopher Krumm (Weill Cornell Medical College)| Lavoisier Ramos-Espiritu (The Rockefeller University)| Carolina Adura (The Rockefeller University)| Matthew Tillman (Emory University)| Renée Landzberg (Weill Cornell Medical College)| J. Fraser Glickman (The Rockefeller University)| Eric Ortlund (Emory University)| David Cohen (Weill Cornell Medical College)
Background: Thioesterase superfamily member 1 (Them1; synonyms acyl-CoA thioesterase 11 and StarD14) is a fatty acyl-CoA thioesterase that hydrolyzes long-chain fatty acyl-CoAs into free fatty acids plus CoASH. Genetic disruption of Them1 in mice leads to increased energy expenditure, along with resistance to high fat diet-induced obesity, diabetes and non-alcoholic fatty liver disease. Them1 is comprised of a N-terminal enzymatic domain linked to a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain that can allosterically modulate enzymatic activity. Taken together, these findings suggest that chemical inhibition of Them1 could be leveraged in the management of obesity-related disorders.
Aim: This study was designed to develop a small molecule inhibitor that targets Them1 activity.
Methods: Recombinant Them1 protein was expressed in E. coli and purified using a N-terminal His-tag. A fluorescence-based assay to detect free CoASH liberated by the activity of Them1 was optimized and miniaturized into 384-well plates. A high-throughput screen utilizing a highly diverse small molecule library (360,705 compounds) was conducted to identify small molecule inhibitors targeting Them1 activity. The threshold for small molecule inhibitors was set to normalized percent inhibition > 30 and z-score < -3. To select Them1-specific small molecule inhibitors, a counter screen was conducted targeting other Acot isoforms, as well as a truncated Them1 containing only the enzymatic domain (i.e. lacking the START domain). To further improve the potency of small molecules to inhibit Them1 activity, initial structure activity relationship (SAR) expansion was performed. Biomolecular microscale thermophoresis assays were utilized to determine binding affinities of small molecule inhibitors to Them1.
Results: 330 small molecules were identified from the high-throughput screen (hit rate 0.09%). Ten small molecules derived from the counter screen showed specificity toward inhibiting Them1 activity either through the enzymatic (3 compounds; IC50: 4 – 10 μM) or START (7 compounds; IC50: 6 – 20 μM) domains. SAR analyses revealed molecular features that influenced the IC50 values for selected small molecules. Three small molecules exhibited Kd values in the low μM range.
Conclusions: We have identified small molecule inhibitors targeting Them1 activity. An optimized small molecule inhibitor targeting Them1 activity should become an attractive candidate for the pharmacologic management of obesity-related disorders.
Support or Funding Information
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01 DK103046 and T32 DK116970 (to D.E.C.)