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.