Speaker
Description
Background: Cytochrome P450 1A2 (CYP1A2) is a critical isozyme involved in drug metabolism and drug-drug interactions. Developing functional probes that combine high selectivity and suitability for both cell imaging and high-throughput screening (HTS) is crucial for accurately assessing CYP1A2 activity and inhibitor discovery.
Methods: In this study, a series of CYP1A2-specific fluorescent probes were designed and synthesized based on the mechanism of P450-mediated sulfoxidation. Using 7-nitrobenz-2-oxa-1,3-diazole (NBD) as the fluorophore scaffold, six distinct sulfur-containing substituents were introduced at the reaction site. Candidate probes underwent systematic characterization and validation, including determination of spectral properties and fluorescence quantum yields, evaluation of metabolic phenotypes, and assessment of their applicability for live-cell imaging and HTS of CYP1A2 inhibitors.
Results: Screening and optimization identified NBTM as a highly selective CYP1A2 fluorescent probe. NBTM exhibited excitation/emission wavelengths of 500/550 nm, while its S-oxidized metabolite, NBTMO, displayed excitation/emission maxima at 485/550 nm. NBTM demonstrated a characteristic enzyme-activated "off–on" response: fluorescence was significantly enhanced following CYP1A2-mediated sulfoxidation, with an approximately 8-fold increase in fluorescence quantum yield. The probe enabled real-time imaging of endogenous CYP1A2 in live cells and was successfully applied to the high-throughput screening of CYP1A2 inhibitors.
Conclusion: We successfully developed NBTM, an enzyme-activated "off-on" fluorescent probe based on the sulfoxidation mechanism of CYP1A2. This probe combines high sensitivity with excellent biocompatibility, enabling real-time imaging of intracellular CYP1A2 activity and proving suitable for screening and evaluating its inhibitors.
