N-Heterocyclic Carbene Capture by Cytochrome P450 3A4

Abstract

Cytochrome P450 3A4 (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 ritonavir, widely used as a pharmacoenhancer. 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, whereas 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 pK a of 7.4. Hence, this fragment of N-methylritonavir is expected to be readily deprotonated under physiologic conditions to yield a thiazol-2-ylidene, which is an N-heterocyclic carbene that has highaffinity 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 tightest-binding azaheterocycles found in P450 inhibitors and could offer new avenues for inhibitor development.

Computation Sheds Insight into Iron Porphyrin Carbenes’ Electronic Structure, Formation, and N–H Insertion Reactivity

“…23 In addition to accomplishing many reactions, iron porphyrin carbenes can accomplish reactions under many different conditions: for instance, water-soluble iron porphyrins mediate cyclopropanation 13 and N-H insertion. 44,45 To the best of our knowledge, an iron porphyrin carbene has never been spectroscopically characterized inside the artificial metalloenzymes where it has been proposed to catalyze reactions. 24 Propelled by a drive to expand the reactivity of iron carbenes, Arnold, Fasan, Hauer, and their colleagues have studied and engineered P450 enzymes, as well as other heme enzymes, which host putative iron porphyrin carbenes.…”

“…Though experimentalists have postulated the existence of the iron porphyrin carbene inside of modified cytochrome P450 and myoglobin enzymes, such complexes have only rarely been spectroscopically characterized inside wild type enzymes and involve carbenes quite different from that studied in this work. 44,45 To the best of our knowledge, an iron porphyrin carbene has never been spectroscopically characterized inside the artificial metalloenzymes where it has been proposed to catalyze reactions. Arnold and Fasan proposed that Fe(II) is the oxidation state of the iron porphyrin reacting with EDA.…”

Mechanism-Based Inactivation of Cytochrome P450 Enzymes: Computational Insights

“…During the past decades, carbenes have attracted an ever-growing interest in medicinal/therapeutic scopes, − and the chemistry of carbenes has been studied both experimentally and computationally. − Due to high nucleophilicity, carbenes are among compounds with toxic effects in living organisms. For instance, the P450-mediated metabolism of some chemicals can generate carbenes which lead to the inhibition of enzymes and further undesired effects such as drug–drug interactions and hepatotoxicity in the human body. ,, Methylenedioxyphenyl (MDP) derivatives are the best-known examples for the MBI of P450s. , MDP is an integral part of the chemical structure of several drugs, pesticides, and flavoring agents (Scheme ). − The highly reactive intermediate of oxidized MDP (a carbene molecule) produces a MIC with the heme-iron, which leads to a quasi-irreversible inhibition of the enzyme.…”

“…For instance, the P450-mediated metabolism of some chemicals can generate carbenes which lead to the inhibition of enzymes and further undesired effects such as drug−drug interactions and hepatotoxicity in the human body. 48,133,134 Methylenedioxyphenyl (MDP) derivatives are the best-known examples for the MBI of P450s. 135,136 MDP is an integral part of the chemical structure of several drugs, pesticides, and flavoring agents (Scheme 3).…”

Noncovalent interactions dominate dynamic heme distortion in cytochrome P450 4B1

“…3A4 was expressed and purified as described previously (55). Rabbit 4B1 was expressed in Escherichia coli strain DH5␣ transformed with pCW-4B1#7 (54) and purified as described previously (20).…”