Project Summary/Abstract Metabolic syndrome (MetS), a constellation of medical conditions that includes obesity, type 2 diabetes, fatty liver, hypertension, cardiovascular disease, and dyslipidemia, creates a major burden for individuals and society. MetS is caused by insulin resistance and obesity, and a drug that could reverse these two conditions would transform its treatment. Unfortunately, no safe and effective medication with such properties exists. Equator Therapeutics is developing a first-in-class drug that will increase the resting metabolic rate by activating the native pathways for mitochondrial H+ leak and thermogenesis. This drug will correct the root cause of metabolic disorders—the imbalance between excess caloric intake and limited energy expenditure— and is expected to be highly effective at reversing obesity and insulin resistance. Until recently, development of such drug was impossible due to a very limited understanding of the mechanisms of the mitochondrial H+ leak. Instead, protonophores that indiscriminately increase H+ leak not only in mitochondria but all cell membranes, were used, leading to undesirable side effects. In contrast, our drug will activate H+ leak specifically in mitochondria via the physiologically regulated native H+ leak pathways mediated by uncoupling proteins and is expected to be much safer. The development of this drug is enabled by our recent discoveries concerning the molecular mechanisms of the mitochondrial H+ leak. In Specific Aim 1, we will identify a set of diverse drug-like compounds that selectively activate a protein target natively responsible for the mitochondrial H+ leak. Importantly, these compounds will have no protonophoric activity. We have selected a library of ~67,000 small molecules with desirable chemical properties specific to our target. We will screen this library for hits that increase mitochondrial H+ leak using new high-throughput mitochondrial assays consisting of a primary screen and counter-screen to remove false positives. Compounds that pass both rounds of high- throughput screening will be validated by direct measurement of the H+ current via our target protein using mitochondrial patch-clamp electrophysiology. Additional patch-clamp studies in the plasma membrane will further select only those of them that lack any protonophoric activity. In Specific Aim 2, we will determine the effects of the novel activators of the native mitochondrial H+ leak pathways on the bioenergetics of intact human cells in vitro. We will primarily focus on the ability of our compounds to stimulate uncoupled respiration, the parameter reflecting their ability to activate mitochondrial H+ leak and thermogenesis in intact cells. Other parameters such as the respiration capacity of the cell, activity of the glycolytic pathways, and the intracellular ATP level will also be assessed. Finally, cytotoxicity assays will be performed. Successful accomplishment of these aims will generate a set of che...