1,2,3-Triazole–Heme Interactions in Cytochrome P450: Functionally Competent Triazole–Water–Heme Complexes

Abstract

In comparison to imidazole (IMZ) and 1,2,4-triazole (1,2,4-TRZ) the isosteric 1,2,3-triazole (1,2,3-TRZ) is unrepresented among CYP inhibitors. This is surprising because 1,2,3-TRZs are easily obtained via ‘click’ chemistry. To understand this underrepresentation of 1,2,3-TRZs among CYP inhibitors, thermodynamic and DFT computational studies were performed with unsusbstituted IMZ, 1,2,4-TRZ, and 1,2,3-TRZ. The results indicate that the lower affinity of 1,2,3-TRZ for the heme iron includes a large unfavorable entropy term likely originating in solvent – 1,2,3-TRZ interactions; the difference is not solely due to differences in the enthalpy of heme – ligand interactions. In addition, the 1,2,3-TRZ fragment was incorporated into a well-established CYP3A4 substrate and mechanism based inactivator, 17-α-ethynylestradiol (17EE), via click chemistry. This derivative, 17-click, yielded optical spectra consistent with low spin ferric heme iron (type II) in contrast to 17EE, which yields a high spin complex (type I). Furthermore, the rate of CYP3A4-mediated metabolism of 17-click was comparable to 17EE, and with different regioselectivity. Surprisingly, CW EPR and HYSCORE EPR spectroscopy indicate that the 17-click does not displace water from the 6th axial ligand position of CYP3A4 as expected for a type II ligand. We propose a binding model where 17-click pendant 1,2,3-TRZ hydrogen bonds with the 6th axial water ligand. The results demonstrate the potential for 1,2,3-TRZ to form metabolically labile water-bridged low spin heme complexes, consistent with recent evidence that nitrogenous type II ligands of CYPs can be efficiently metabolized. The specific case of [CYP3A4•17-click] highlights the risk of interpreting CYP-ligand complex structure on the basis of optical spectra.

N-Heterocyclic Carbene Capture by Cytochrome P450 3A4

“…thiazole. These results differ in some respects to those described by Conner et al (2012) whose study evaluated the energetics of water exchange by imidazole, 1,2,4-triazole, and 1,2,3 triazole using an identical model system, M06 functional, and the spin-state corrected 6-31G basis set (Swart et al, 2010 ). Notable differences between our results and those described in Conner et al (2012) include the 2-to 5-fold differences in exothermicities as well as the prediction that coordination of imidazole is more exothermic than 1,2,4-triazole.…”

“…The K d of thiazole is 3.4 6 1.0 mM at pH 7.4, more than 4800-fold higher than that measured for ritonavir. Additionally, the K d of thiazole is nearly one and two orders of magnitude higher than the K d values measured for 1,2,4-triazole and imidazole (Conner et al, 2012), and, as supported by density functional theory calculations, it is among the weakest azaheterocyclic ligands for thiolateligated Fe 31 -heme. NMTI was synthesized and titrated into the CYP3A4 to lend further support to the concept that the unique features of the Thiazol-2-Ylidene Coordination to CYP3A4 difference spectra were attributed to the deprotonation and coordination of the N-methylthiazolium unit of NMeR.…”

Effect of sildenafil on human aromatase activity: From in vitro structural analysis to catalysis and inhibition in cells

“…This is spectroscopically detectable as a shift of the maximum absorbance peak from 417-419 nm to 390-394 nm [31]. On the other hand, type II ligands are nitrogen-containing compounds that displace the water molecule while directly coordinating to the heme iron, or alternatively they form a hydrogen bond with the water molecule present as the sixth ligand [32][33][34]. In the case of type II ligands, the heme iron is hexa-coordinated and the maximum absorbance peak shift from 418 nm to 422-426 nm [35].…”

Substrate Specificity of Human Cytochrome P450 (CYP) 2C Subfamily and Effect of Azole Antifungal Agents on CYP2C8

“…Ketoconazole has been recognized as a weak inhibitor of CYP2C8 as listed in US FDA Guidance for Industry -Drug Interaction Studies (2012) (22), however, there were few reports in terms of the inhibition of CYP2C8 by miconazole (15). It has been established that imidazole and triazole compounds, including azole antifungals, reversibly inhibit drug-metabolizing P450s by heme-nitrogen interactions (23,24). Fluconazole can exhibit competitive, noncompetitive, or mixed-type inhibition against CYP3A4 (14,15,(25)(26)(27)(28).…”

“…Itraconazole competitively inhibited CYP3A4 (15,17,27,29). On the other hand, it has been reported that fluconazole and voriconazole exhibited mixed-type inhibition and competitive inhibition, respectively, against CYP2C9 (14,15,24,30 CYP2C9.1 (14). Thus, the inhibition type seems to be different among substrates, inhibitors (azole antifungals), P450s, and/or other experimental conditions.…”