Cytochrome P450-mediated metabolism of K31, a Ko143-derived ATP-binding cassette subfamily G member 2 inhibitor

Ko143 is a potent inhibitor of ATP-binding cassette subfamily G member 2, but its development is limited by poor metabolic stability due to rapid ester hydrolysis. To address this limitation, K31, an amide analog of Ko143, was recently developed. However, the metabolic profile of K31 in human liver microsomes (HLMs) remains suboptimal, and its underlying metabolic pathways are unclear. In this study, we systematically investigated the metabolism of K31 using HLMs, recombinant cytochrome P450 (P450) enzymes, chemical inhibition assays, and metabolomic analysis. Three P450-mediated metabolites of K31 were identified, with M1 as the predominant metabolite formed via O-demethylation. P450 phenotyping revealed that CYP2J2 and CYP4F3B contribute to M1 formation, an unusual finding given their primary roles in endobiotic metabolism and the predominantly extrahepatic expression of CYP2J2. Similar CYP2J2- and CYP4F3B-mediated O-demethylation pathways were also observed for Ko143 and its analog, K2. Chemical inhibition studies in HLMs further indicated that CYP4F3B plays a more prominent role than CYP2J2 in mediating O-demethylation of all 3 compounds, suggesting that this pathway is primarily driven by CYP4F3B in the liver and by CYP2J2 in extrahepatic tissues. Collectively, these findings provide new insights into the metabolism of Ko143 derivatives and inform the rational design of next-generation ATP-binding cassette subfamily G member 2 inhibitors with improved metabolic stability, reduced off-target metabolism, and minimized drug-endobiotic interactions. SIGNIFICANCE STATEMENT: This study highlights the central role of CYP4F3B in mediating the O-demethylation of Ko143 and its analogs in the liver. This study also identified a contribution from CYP2J2, the predominant cytochrome P450 isoform in the heart, suggesting potential extrahepatic metabolism of these compounds. These findings provide a foundation for the rational design of next-generation Ko143-derived ATP-binding cassette subfamily G member 2 inhibitors with enhanced metabolic stability, reduced off-target metabolism, and minimized drug-endobiotic interactions.