Identification of Binding Sites in the Nucleotide-Binding Domain of P‑Glycoprotein for a Potent and Nontoxic Modulator, the Amine-Containing Monomeric Flavonoid FM04

• Published in: Journal of medicinal chemistry 2023-05, Vol.66 (9), p.6160-6183
• Main Authors: Liu, Zhen, Wong, Iris L. K., Sang, Jingcheng, Liu, Fufeng, Yan, Clare S. W., Kan, Jason W. Y., Chan, Tak Hang, Chow, Larry M. C.
• Format: Article
• Language: English
• Subjects: ATP Binding Cassette Transporter, Subfamily B - metabolism; ATP Binding Cassette Transporter, Subfamily B, Member 1 - metabolism; Binding Sites; Chromatography, Liquid; Flavonoids - chemistry; Flavonoids - pharmacology; Humans; Molecular Docking Simulation; Nucleotides - metabolism; Tandem Mass Spectrometry
• ISSN: 0022-2623; 1520-4804
• DOI: 10.1021/acs.jmedchem.2c02005

We have previously discovered an amine-containing flavonoid monomer FM04 as a potent P-glycoprotein (P-gp) inhibitor (EC50 = 83 nM). Here, a series of photoactive FM04 analogues were synthesized and used together with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the FM04-binding sites on P-gp. Point mutations around the photo-crosslinked sites were made for verification. Together with the results from mutational studies, molecular docking, and molecular dynamics simulations, it was found that FM04 can interact with Q1193 and I1115 in the nucleotide-binding domain 2 (NBD2) of human P-gp. It was proposed that FM04 can inhibit P-gp in 2 novel mechanisms. FM04 can either bind to (1) Q1193, followed by interacting with the functionally critical residues H1195 and T1226 or (2) I1115 (a functionally critical residue itself), disrupting the R262-Q1081-Q1118 interaction pocket and uncoupling ICL2–NBD2 interaction and thereby inhibiting P-gp. Q1118 would subsequently be pushed to the ATP-binding site and stimulate ATPase.