Rationally Engineered CYP3A4 Fluorogenic Substrates for Functional Imaging Analysis and Drug–Drug Interaction Studies
Rong-Jing He, Zhenhao Tian, Jian Huang, Mengru Sun, Wei Feng, Chunyu Li, Hairong Zeng, Feng Zhang, Xiao‐Qing Guan, Yan Feng, Xiangming Meng, Hui Yang, Guang‐Bo Ge
Abstract
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.