To Be, or Not to Be, an Inhibitor: A Comparison of Azole Interactions with and Oxidation by a Cytochrome P450 Enzyme

Abstract

The cytochrome P450 (CYP) superfamily of heme monooxygenases is involved in a range of important chemical biotransformations across nature. Azole-containing molecules have been developed as drugs that bind to the heme center of these enzymes, inhibiting their function. The optical spectrum of CYP enzymes after the addition of these inhibitors is used to assess how the molecules bind. Here we use the bacterial CYP199A4 enzyme, from Rhodopseudomonas palustris HaA2, to compare how imidazolyl and triazolyl inhibitors bind to ferric and ferrous heme. 4-(Imidazol-1-yl)­benzoic acid induced a red shift in the Soret wavelength (424 nm) in the ferric enzyme along with an increase and a decrease in the intensities of the δ and α bands, respectively. 4-(1H-1,2,4-Triazol-1-yl)­benzoic acid binds to CYP199A4 with a 10-fold lower affinity and induces a smaller red shift in the Soret band. The crystal structures of CYP199A4 with these two inhibitors confirmed that these differences in the optical spectra were due to coordination of the imidazolyl ligand to the ferric Fe, but the triazolyl inhibitor interacts with, rather than displaces, the ferric aqua ligand. Additional water molecules were present in the active site of 4-(1H-1,2,4-triazol-1-yl)­benzoic acid-bound CYP199A4. The space required to accommodate these additional water molecules in the active site necessitates changes in the position of the hydrophobic phenylalanine 298 residue. Upon reduction of the heme, the imidazole-based inhibitor Fe–N ligation was not retained. A 5-coordinate heme was also the predominant species in 4-(1H-1,2,4-triazol-1-yl)­benzoic acid-bound ferrous CYP199A4, but there was an obvious shoulder at 447 nm indicative of some degree of Fe–N coordination. Rather than inhibit CYP199A4, 4-(imidazol-1-yl)­benzoic acid was a substrate and was oxidized to generate a metabolite derived from ring opening of the imidazolyl ring: 4-[[2-(formylamino)­acetyl]­amino]­benzoic acid.

Redox Characterization of the Complex Molybdenum Enzyme Formate Dehydrogenase from Cupriavidus necator

“…A key requirement in any UV/visible spectroelectrochemistry experiment is utilizing mediators that do not mask the absorption spectrum of the chromophores of interest. We have reported a suite of redox-active transition metal (Fe and Co) complexes that exhibit negligible absorption at micromolar concentrations and undergo reversible redox reactions with both a range of proteins and electrode surfaces, , and they have been used successfully in the past to study a number of different proteins through optical spectroelectrochemistry. − ,− These mediators (see Supporting Information Figure S2) span a potential range of +0.1 to −0.55 V vs NHE, ensuring a stable and well-defined ORP (redox buffering) during the experiment. This also allows a wider potential range to be interrogated, as chemical reductants (such as dithionite) cannot reach the same reducing potentials covered by these reagents.…”

Discovery of Novel Pyrazolylpyridine Derivatives for 20-Hydroxyeicosatetraenoic Acid Synthase Inhibitors with Selective CYP4A11/4F2 Inhibition

“…To improve the CYP selectivity, the thiazole side chain of 6f was replaced with other moieties while maintaining the low lipophilicity. Imidazole and triazole rings are alternative azoles to thiazole and are less lipophilic than thiazole, but there was the concern of heteroaromatic rings showing an undesirable degree of affinity for the heme iron in the CYPs. − Thus, substituted benzene rings with a lower risk of heme iron affinity were investigated to improve the CYP selectivity (Table ).…”

Exploring acetaminophen prodrugs and hybrids: a review

“…43–44 . 109,110 It should be noted that the 1,2,3-triazole ring, like triazolylphenoles 43–44 , is not protonated at physiological pH because of their poor basicity. It thus would be metabolically stable and able to participate as a hydrogen-bond acceptor, which could be favorable in binding biomolecular targets.…”