Using CYP3A4 NMR derived structural information to mitigate time‐dependent inhibition due to diltiazem metabolism

Abstract

Human CYP3A4 metabolizes a significant fraction of clinically relevant drugs. CYP3A4‐mediated metabolism of the vasodilator diltiazem leads to time‐dependent inhibition (TDI) and clinically relevant drug‐drug interactions. 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. A model of diltiazem in the CYP3A4 active site was produced using distance restraints generated from longitudinal T1 NMR relaxation experiments. CYP3A4‐ligand interactions were also monitored using spectral binding titrations. Evaluation of diltiazem‐mediated CYP3A4 TDI was performed using the reporter substrate 7‐ethoxy‐4‐(trifluoromethyl)coumarin. The diltiazem model revealed that the dimethylamino group that undergoes CYP3A4‐mediated N‐demethylation is oriented toward the heme iron with the aromatic ring of the benzothiazepine core near active site residues I369 and L373, the ester moiety near L216, and the p‐hydroxyphenyl group near S119. Diltiazem caused a Type II spin shift upon binding to CYP3A4, indicating interaction of the amine with the heme iron. Substitution of I369 with a larger residue decreased CYP3A4 TDI, and an NMR derived model of diltiazem in complex with CYP3A4 I369W revealed that the p‐hydroxyphenyl group was toward the heme iron instead of the dimethylamino group. In summary, a hybrid NMR‐computational approach was used to rapidly generate a model of diltiazem in complex with CYP3A4. Introduction of steric bulk into the CYP3A4 active site by mutagenesis altered the orientation of diltiazem and mitigated TDI. The next step is to add steric bulk to the cognate part of the compound. 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.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.