Rationally Engineered CYP3A4 Fluorogenic Substrates for Functional Imaging Analysis and Drug–Drug Interaction Studies

Cytochrome P450 3A4 (CYP3A4) is a key xenobiotic-metabolizing
enzyme-mediated
drug metabolism and drug–drug interaction (DDI). Herein, an
effective strategy was used to rationally construct a practical two-photon
fluorogenic substrate for hCYP3A4. Following two-round structure-based
substrate discovery and optimization, we have successfully constructed
a hCYP3A4 fluorogenic substrate (F8) with desirable features,
including high binding affinity, rapid response, excellent isoform
specificity, and low cytotoxicity. Under physiological conditions, F8 is readily metabolized by hCYP3A4 to form a brightly fluorescent
product (4-OH F8) that can be easily detected by various
fluorescence devices. The practicality of F8 for real-time
sensing and functional imaging of hCYP3A4 has been examined in tissue
preparations, living cells, and organ slices. F8 also
demonstrates good performance for high-throughput screening of hCYP3A4
inhibitors and assessing DDI potentials in vivo.
Collectively, this study develops an advanced molecular tool for sensing
CYP3A4 activities in biological systems, which strongly facilitates
CYP3A4-associated fundamental and applied research studies.