Project Summary Hepatocellular carcinoma (HCC) is a highly lethal cancer, and obesity is associated with an 89% increased risk of HCC. Because of the need for safer and more effective HCC drugs, the increasing prevalence of obesity, and the complex relationship between obesity and HCC, obesity-associated HCC is an emerging, challenging issue. The level and composition of bile acids (BAs) play a key role in metabolic diseases and HCC. Cytochrome P450 family 8 subfamily B polypeptide 1 (CYP8B1), a protein expressed only in the liver, is vital in forming cholic acid (CA), the 12α-hydroxylated (12α-OH) primary BA. Hence, CYP8B1 activity increases the ratio of 12α-OH to non- 12α-OH BAs, which is linked to metabolic diseases (e.g., obesity, diabetes, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis (NASH)) and HCC in rodents and humans. High levels of deoxycholic acid (DCA), a secondary BA derived from CA by gut microbiota, promotes obesity-associated HCC in mice and are present in the hepatocytes of obesity-induced HCC mice and livers of patients with NASH, which drastically increases the risk of HCC. Currently, there are no reported selective inhibitors of CYP8B1. This proposal is based on our substantial preliminary data. Using an oncogene-induced HCC mouse model, we determined that Cyp8b1 knockout (KO) mice develop less aggressive HCC relative to wild-type (WT) mice. We also identified multiple promising CYP8B1 inhibitors via a high-throughput screening (HTS) using a novel biochemical assay and selectivity assays. Based on the structures of the promising CYP8B1 inhibitors from the HTS and the CYP8B1/inhibitor binding modes from our molecular docking studies, I designed and synthesized several novel compounds displaying selective and potent inhibition of CYP8B1 activity in our biochemical assay, cell-based assay, selectivity assays, and preliminary mouse studies. Additionally, we successfully obtained diffraction quality crystals of CYP8B1 and CYP8B1/inhibitor to determine the binding modes with greater accuracy, which will allow me to develop more effective CYP8B1 inhibitors. I hypothesize that CYP8B1 plays a crucial role in the development of obesity-associated HCC and that novel CYP8B1 inhibitors will significantly decrease the ratio of 12α-OH to non-12α-OH BAs and dramatically reduce the levels of DCA, thereby suppressing the development of obesity-associated HCC. I propose two aims to test my hypothesis. In Aim 1, I will determine the role of CYP8B1 by examining the HCC growth in obesity-associated HCC WT and Cyp8b1 KO mice and by evaluating the HCC suppression in the obesity-associated HCC WT and Cyp8b1 KO mice treated with a novel CYP8B1 inhibitor. In Aim 2, I will perform further chemical modifications and structure- activity relationship studies in order to develop lead candidates with greater potency, selectivity, and efficacy. By determining the role of CYP8B1 in obesity-associated HCC and developing novel CYP8B1 inhibit...