PROJECT SUMMARY The goal of this project is to develop small molecule mitochondrial uncouplers for the treatment of non- alcoholic steatohepatitis (NASH), an advanced form of non-alcoholic fatty liver disease (NAFLD). Approximately 26 million people (~6-8% of US population) are affected by NASH, and more than 30% of patients with NASH- related cirrhosis will die from liver failure or hepatocellular cancer. There are currently no FDA-approved drugs to treat NASH and the top drugs in clinical trials suffer from ineffectiveness or have unwanted side effects. NASH is characterized by fat accumulation in the liver, inflammation, and tissue remodeling. Mitochondrial uncouplers have strong potential as NASH therapeutics by the nature of their mode of action targeting fat accumulation and oxidative stress. Mitochondrial uncouplers transport protons from the mitochondrial intermembrane space into the matrix bypassing ATP synthase. This process increases fat oxidation and decreases mitochondrial reactive oxygen species production; thereby, directly targeting two pathways that underlie NASH pathophysiology that drive inflammation and the fibrogenic response. Our laboratories have recently discovered mitochondria- selective, safe, and orally bioavailable mitochondrial uncouplers that have wide therapeutic window. Proof-of- principle studies in an accelerated mouse model of NASH (STAM model) achieved the FDA benchmark for approval of a NASH therapeutic by decreasing the NAFLD Activity Score (NAS) by 2 points, with at least one point coming from two different NAS criteria, and with no worsening of fibrosis. These strong preliminary data support a medicinal chemistry program to improve the drug-like properties of the molecules (Aim 1) and validate molecules in a more physiologically and clinically relevant mouse model of NASH involving Amylin diet (Aim 2). The major innovation of our program is the discovery of novel chemical scaffolds that have different levels of mitochondrial uncoupling capacity that are safe and self-limiting. As there are currently no drugs with a mitochondrial uncoupling as the primary mode of action in the clinic, our program could be the first to deliver mitochondrial uncouplers for the treatment of NASH. Indeed, this project addresses a critical need and will make significant contribution to data supporting the validity of modulating mitochondrial uncoupling for safe and efficacious treatment of NASH.