Structure of pyrazole derivatives impact their affinity, stoichiometry, and cooperative interactions for CYP2E1 complexes

Abstract

CYP2E1 plays a critical role in detoxication and carcinogenic activation of drugs, pollutants, and dietary compounds; however, these metabolic processes can involve poorly characterized cooperative interactions that compromise the ability to understand and predict CYP2E1 metabolism. Herein, we employed an array of ten azoles with an emphasis on pyrazoles to establish the selectivity of catalytic and cooperative CYP2E1 sites through binding and catalytic studies. Spectral binding studies for monocyclic azoles suggested two binding events, while bicyclic azoles suggested one. Pyrazole had moderate affinity toward the CYP2E1 catalytic site that improved when a methyl group was introduced at either position 3 or 4. The presence of methyl groups simultaneously at positions 3 and 5 blocked binding, and a phenyl group at position 3 did not improve binding affinity. In contrast, pyrazole fusion to a benzene or cyclohexane ring greatly increased affinity. The consequences of these binding events on CYP2E1 catalysis were studied through inhibition studies with 4-nitrophenol, a substrate known to bind both sites. Most pyrazoles shared a common mixed cooperative inhibition mechanism in which pyrazole binding rescued CYP2E1 from substrate inhibition. Overall, inhibitor affinities toward the CYP2E1 catalytic site were similar to those reported in binding studies, and the same trend was observed for binding at the cooperative site. Taken together, these studies identified key structural determinants in the affinity and stoichiometry of azole interactions with CYP2E1 and consequences on catalysis that further advance an understanding of the relationship between structure and function for this enzyme.

Structural basis for cooperative binding of azoles to CYP2E1 as interpreted through guided molecular dynamics simulations

“…1) [10]. Data from spectral binding studies for monocyclic azoles were consistent with two binding events, while bicyclic azoles implicated only one.…”

“…Nevertheless, growing evidence implicates the importance of more complex cooperative mechanisms for CYP2E1 [3-10]. Those kinetic profiles deviate from the hyperbolic relationship predicted by the MichaelisMenten mechanism.…”

“…While the Hill equation is commonly used to qualify the degree of cooperativity, it reveals nothing of the mechanism underlying the observed kinetic profile. As an alternative, we have identified and validated mechanistic models involving two binding site to explain non-hyperbolic kinetic profiles for CYP2E1 substrates and inhibitors through the use of binding and catalytic experiments coupled with computational structural studies [6-10]. …”

“…1). The array of molecules included all azoles from the previously published experimental studies [20] as well as 5-methylpyrazole in order to provide insights on CYP2E1 structure activity relationships relationships. We modeled interactions between these azoles and CYP2E1 through a guided molecular dynamics approach using SYBYL-X 1.3 (Tripos, Inc.).…”

“…Complexes were then subjected to multiple cycles of molecular dynamics and energy minimization. When bound at the catalytic site, binary azoles were tethered to the heme iron based on reported Type II binding spectra [6, 10], which in the case of triazoles involved two binding orientations. Taken together, we generated structural solutions for a total of 28 CYP2E1 liganded complexes to understand the cooperative structure activity relationships observed in our previous experimental studies [10].…”