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

• Published in: Journal of medicinal chemistry 2023-05, Vol.66 (10), p.6743-6755
• Main Authors: He, Rong-Jing, Tian, Zhen-Hao, Huang, Jian, Sun, Meng-Ru, Wei, Feng, Li, Chun-Yu, Zeng, Hai-Rong, Zhang, Feng, Guan, Xiao-Qing, Feng, Yan, Meng, Xiang-Ming, Yang, Hui, Ge, Guang-Bo
• Format: Article
• Language: English
• Subjects: Cytochrome P-450 CYP3A - metabolism; Drug Interactions; Fluorescent Dyes - pharmacology
• ISSN: 0022-2623; 1520-4804
• DOI: 10.1021/acs.jmedchem.3c00101

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.