RT Journal Article SR Electronic T1 N-heterocyclic carbene capture by cytochrome P450 3A4 JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP mol.116.103721 DO 10.1124/mol.116.103721 A1 Gareth K. Jennings A1 Caroline M. Ritchie A1 Lisa S Shock A1 Charles E Lyons A1 John Hackett YR 2016 UL http://molpharm.aspetjournals.org/content/early/2016/04/28/mol.116.103721.abstract AB CYP3A4 is the dominant P450 enzyme involved in human drug metabolism and its inhibition may result in adverse interactions or conversely, favorably reduce the systemic elimination rates of poorly bioavailable drugs. Herein we describe a spectroscopic investigation of the interaction of CYP3A4 with N-methylritonavir, an analog of the widely used as a pharmaco-enhancer ritonavir. In contrast to ritonavir, the binding affinity of N-methylritonavir for CYP3A4 is pH-dependent. At pH < 7.4, the spectra are definitively type-I, while at pH ≥ 7.4 the spectra have split Soret bands including a red-shifted component characteristic of a P450-carbene complex. Variable-pH UV-Visible spectroscopy binding studies with molecular fragments narrows the source of this pH dependence to its N-methylthiazolium fragment. The C2 proton of this group is acidic and variable-pH resonance Raman spectroscopy tentatively assigns it a pKa of 7.4. Hence, this fragment of N-methylritonavir is expected to be readily deprotonated under physiological conditions to yield a thiazol-2-ylidene, which is an N-heterocyclic carbene that has high-affinity for and is presumed to be subsequently captured by the heme iron. This mechanism is supported by time-dependent density functional theory with an active site model that accurately reproduces distinguishing features of the experimental UV-Visible spectra of N-methylritonavir bound to CYP3A4. Finally, density functional theory calculations support that this novel interaction is as strong as the tighest-binding azaheterocycles found in P450 inhibitors and could offer new avenues for inhibitor development.