High-throughput screening identifies a dual-activity inhibitor of OXCT1 for hepatocellular carcinoma therapy.

3-Oxoacid CoA-transferase 1 (OXCT1) plays a crucial role in hepatocellular carcinoma (HCC) progression through its ketolytic and succinyltransferase activities. Despite its potential as a therapeutic target, no small molecules have been developed to inhibit the dual enzymatic activities of OXCT1 specifically. In this study, our structural analysis revealed that the active sites for both enzymatic functions of OXCT1 are located in the same pocket. Targeting this pocket inhibits the binding of OXCT1 to its substrates and blocks both of its enzymatic activities. Thus, we developed two high-throughput screening systems to assess the effects of small molecules on OXCT1's distinct enzymatic activities. By combining these experimental approaches with virtual screening, we identified a compound, D574-0246 (iOXCT1), which effectively inhibits both enzymatic activities. In vitro and in vivo validation demonstrated that iOXCT1 suppresses HCC growth via OXCT1 inhibition. Collectively, our results establish OXCT1 as a promising therapeutic target and identify iOXCT1 as a novel dual-activity inhibitor, providing a foundation for developing OXCT1-targeted therapies against HCC.