PROJECT SUMMARY/ABSTRACT There is a critical need for effective pharmacotherapy for the management of non-alcoholic fatty liver disease (NAFLD). The proposed research is to conduct early-stage preclinical validation of new therapeutic leads that target thioesterase superfamily member 1 (Them1; synonym Acyl-CoA thioesterase 11), a key enzyme of fatty acid metabolism that becomes maladaptive in NAFLD. The long-term goal is to develop inhibitors of Them1 as a therapeutic modality in the management of human NAFLD. The objective of this research is to optimize the drug-like properties of small molecule inhibitors in order to create lead compounds, and to demonstrate their safety, efficacy and specificity in cell-based assays and in experimental NAFLD using mice. Targeting Them1 with optimized small molecule inhibitors is expected to mitigate NAFLD by: 1) Increasing energy expenditure in thermogenic brown and beige adipose tissue; 2) Decreasing hepatic steatosis, as well as glucose production by the liver; and 3) Reducing inflammation in white adipose tissue. The rationale is that a lead compound that addresses these three independent pathogenic contributions of Them1 should prove effective in NAFLD. We have completed a high-throughput small molecule screen that has identified promising inhibitors targeting the fatty acyl-CoA thioesterase activity of Them1. Motivated by extensive mechanistic data on the contributions of Them1 to NAFLD pathogenesis, the development of inhibitors into lead compounds will be accomplished in three specific aims: 1) To optimize the potency and specificity of Them1 small molecule inhibitors; 2) To establish drug-like properties and efficacy in cell culture of early lead compounds; and 3) To evaluate therapeutic lead compounds in experimental NAFLD using mice. In Aim 1, small molecule inhibitors will be optimized by medicinal chemistry strategies to improve potency, specificity and drug-like properties. These efforts will be guided by structure-activity relationships based on in vitro enzymatic assays, as well as biophysical and structural determinants of Them1-inhibitor interactions. Aim 2 will evaluate early lead compounds for drug-like properties, as well as cytotoxicity. Selected early leads will then be tested for efficacy in primary cultured mouse and human cells for their capacities to increase fatty acid oxidation in brown adipocytes and hepatocytes, to reduce hepatic glucose production in hepatocytes and to decrease production of inflammatory mediators by white adipocytes. Specificity will be assessed using cells cultured from Them1-/- mice. In Aim 3, lead compounds will be tested in mice for pharmacokinetics and tolerability. Upon establishing dosing routes and schedules, efficacy to prevent and to reverse NAFLD will be assessed in mouse models. Off-target effects will be evaluated using Them1-/- mice. The expected outcome of these studies is the development and pre-clinical validation of optimized lead Them1 inhibi...