PROJECT SUMMARY Metabolic dysfunction-associated steatohepatitis (MASH, formerly NASH) is a liver disease characterized by the unnatural accumulation of lipids within hepatic cells, inflammation, and ultimately fibrosis. It is frequently connected to obesity and estimated to affect up to 5% of adults in the U.S., and despite huge investments by the pharmaceutical industry, there are not (yet) any FDA-approved drugs for MASH. Anti-inflammatory compounds are relatively under investigated for this disease, and may have the potential to minimize the dangerous liver fibrosis that occurs as MASH progresses. This project will investigate a novel approach to the treatment of MASH: the induction of anti-inflammatory signals in liver tissue via biased small molecule modulators of protease-activated receptor 1 (PAR1), called parmodulins. Published and unpublished results using wild-type and PAR1-modified mice fed high-fat diets (HFD) suggest that inflammatory effects of MASH are lessened with a natural activator of PAR1, activated protein C (APC), and also by compounds that can block the inflammatory signaling initiated by the activation of PAR1 by thrombin (i.e., thromboinflammation). In this project, we will test the ability of orally-active parmodulins to produce protective effects in mice by tipping the balance of PAR1 signaling in liver tissue from a pro-inflammatory to an anti-inflammatory phenotype. In Aim 1, novel parmodulins will be synthesized that have been designed to lack the potential to generate toxic metabolites, and which may possess improved potency at PAR1. In Aim 2, these compounds will be tested in cellular models of PAR1 signaling and inflammation, and promising examples will be studied in a series of standard drug-like profiling assays, including cytotoxicity, solubility, plasma and metabolic stability, and off- target activity. In Aim 3, select parmodulins will undergo PK studies, and representative examples will progress to a mouse model of advanced MASH in the lab of co-investigator Dr. James Luyendyk (Michigan State Univ.). Mice with a high fat, high cholesterol, and high sugar diet will be dosed with carbon tetrachloride to induce an advanced form of MASH with significant fibrosis. Mice will then be treated with parmodulins at high and low doses to determine their ability to inhibit a range of measures of liver injury, steatosis, inflammation, and fibrosis. It is anticipated that parmodulins will have minimal bleeding risks relative to certain thrombin inhibitors and PAR1 antagonists, and this will be supported with a guinea pig hemostasis study. Successful results in these studies will justify the completion of lead optimization and the future preclinical development of parmodulins for the treatment of MASH and potentially other liver diseases.