Rational design of a fluorescent probe for selective sensing of human cytochrome P450 2C19 and its application in exploration of enzymatic inactivation.

Human cytochrome P450 2C19 (CYP2C19) is a drug-metabolizing enzyme frequently implicated in adverse drug reactions. However, the identification of inhibitors, especially irreversible inactivators, that underlie CYP2C19-mediated drug interactions remains challenging, chiefly owing to the paucity of robust molecular probes capable of quantification of enzymatic inactivation profiles in biospecimens. In the present study, we propose a molecular modification strategy that fine-tunes unfavorable substrate-aromatic interactions within the CYP2C19 pocket by rationally modifying a key junction domain of B1, a substrate that currently suffers from sub-optimal selectivity and sensitivity. The optimized probe BCPM exhibits 20-fold selectivity over other CYP isoforms and a detection limit of 0.012 nM. A dual-mode visual platform was developed that enables screening and mechanistic elucidation of reversible inhibitors and inactivators of CYP2C19 among drugs and natural products. Using this platform, we thoroughly validated the multifaceted inhibitory effects of representative compounds on CYP2C19, as well as the potential for adverse drug interactions resulting from enzyme inactivation. Collectively, our rationally fine-tuned structural strategy generated a robust tool for CYP2C19 with broad utility in biomedical and pharmaceutical research.