Human Cytochrome P450 3A4 (CYP3A4) is an important member of the cytochrome P450 superfamily with responsibility for metabolizing ~50% of clinical drugs. Experimental evidence showed that CYP3A4 can adopt multiple substrates in its active site to form a cooperative binding model, accelerating substrate metabolism efficiency. In the current study, we constructed both normal and cooperative binding models of human CYP3A4 with antifungal drug ketoconazoles (KLN). Molecular dynamics simulation and free energy calculation were then carried out to study the cooperative binding mechanism. Our simulation showed that the second KLN in the cooperative binding model had a positive impact on the first one binding in the active site by two significant pi-pi stacking interactions. The first one was formed by Phe215, functioning to position the first KLN in a favorable orientation in the active site for further metabolism reactions. The second one was contributed by Phe304. This pi-pi stacking was enhanced in the cooperative binding model by the parallel conformation between the aromatic rings in Phe304 and the dioxolan moiety of the first KLN. These findings can provide an atomic insight into the cooperative binding in CYP3A4, revealing a novel pi-pi stacking mechanism for drug-drug interactions.
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