Abstract
Time‐dependent inhibition (TDI) of human CYP3A4 is a significant challenge in drug development and is the cause of multiple clinically relevant drug‐drug interactions. The vasodilator diltiazem causes TDI when metabolized by CYP3A4. We hypothesized that introduction of steric bulk into the CYP3A4 active site by site‐directed mutagenesis will disrupt active site contacts, alter diltiazem orientation in the CYP3A4 active site, and decrease TDI, as previously seen with midazolam. CYP3A4‐ligand interactions were monitored using spectral binding titrations. In parallel, we performed molecular docking of diltiazem and the N‐demethylation products into CYP3A4 models of previously tested mutants that introduce steric bulk into the enzyme active site, followed by molecular mechanics generalized Born surface area (MM‐GBSA) rescoring. An NMR‐derived model of diltiazem in the CYP3A4 active site revealed that the dimethylamino group that undergoes CYP3A4‐mediated N‐demethylation is oriented toward the heme iron. Diltiazem caused a Type II spin shift upon binding to CYP3A4, indicating interaction of the amine with the heme iron. Docking of diltiazem, N‐desmethyldiltiazem, or N, N‐didesmethyldiltiazem into wild‐type CYP3A4 resulted in the amino group of each compound oriented toward the heme iron. In contrast, docking these three compounds into CYP3A4 I120W resulted in the amino group orienting away from the heme. Furthermore, T309Y docking results showed reorientation of diltiazem, which would prevent the initial N‐demethylation event, and I301W and A370F resulted in N‐desmethyldiltiazem reorientation. MM‐GBSA rescoring of docking experiments confirmed that all compounds used orient with the amino group toward the heme in wild‐type CYP3A4, all amino groups away from the heme in I120W, and reorientation of N‐desmethyldiltiazem in I301W and A370F. Furthermore, in rescoring experiments, N‐desmethyldiltiazem was reoriented in the Y307W active site, and N, N‐didesmethyldiltiazem was reoriented in the T309Y active site in addition to reorientation of diltiazem. Substitution of I120 and T309 with a larger residue decreased CYP3A4 TDI. In summary, a hybrid NMR‐computational approach was used to rapidly generate a model of diltiazem in complex with CYP3A4. These results indicate that NMR‐derived models of protein‐ligand interactions provide a structure‐based approach for decreasing metabolic liabilities of CYP enzyme substrates.Support or Funding InformationThis work was supported by internal funding from the University of Connecticut to James Halpert.
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