Computational Investigation of Ligand Binding to the Peripheral Site in CYP3A4: Conformational Dynamics and Inhibitor Discovery

Human cytochrome P450 3A4 (CYP3A4) is a major drug-metabolizing enzyme responsible for the metabolism of ∼50% of clinically used drugs and is often involved in drug–drug interactions. It exhibits atypical binding and kinetic behavior toward many ligands. Binding of ligands to CYP3A4 is a complex process. Recent studies from both crystallography and biochemistry suggested the existence of a peripheral ligand-binding site at the enzyme surface. However, the stability of the ligand bound at this peripheral site and the possibility of discovering new CYP3A4 ligands based on this site remain unclear. In this study, we employed a combination of molecular docking, multiparalleled molecular dynamics (MD) simulations, virtual screening, and experimental bioassay to investigate these issues. Our results revealed that the binding mode of progesterone (PGS), a substrate of CYP3A4, in the crystal structure was not stable and underwent a significant conformational change. Through Glide docking and MD refinement, it was found that PGS was able to stably bind at the peripheral site via contacts with Phe215, Phe219, Phe220, and Asp214. On the basis of the refined peripheral site, virtual screening was then performed against the Enamine database. A total of three compounds were finally found to have inhibitory activity against CYP3A4 in both human liver microsome and recombinant human CYP3A4 enzyme assays, one of which showed potent inhibitory activity with IC 50 lower than 1 μM and two of which exhibited moderate inhibitory activity with IC 50 values lower than 10 μM. The findings not only presented the dynamic behavior of PGS at the peripheral site but also demonstrated the first indication of discovering CYP3A4 inhibitors based on the peripheral site.