A Biophysical Rationalization of Type II and Reverse Type I Inhibitor Interactions in CYP450 with Implications for Enzyme Function

Abstract

This work is a characterization of small molecule azole-based inhibitor interactions in cytochrome P450 (CYP) using a complement of biophysical methodologies to provide molecular level details of the underappreciated structural complexity of low-spin (Type II and associated reverse Type I) CYP-ligand complexes. Specifically, the results herein demonstrate the tenuous structural interpretations of `type II' ligand-induced heme spin state changes, associated UΔvis absorbance spectral signatures, and derived expectations of metabolic fate. The aim of this thesis is to challenge the historical binary classification paradigm of `high-spin substrate' and `low-spin inhibitor.' Chapter 1 presents an overview of the range of low-spin CYP-ligand interactions that occur within this diverse enzyme family, with an emphasis on the importance of H2O to ligand binding and oxidative catalysis. Additionally, select literature examples are provided that demonstrate that the current understanding of type II binding and inhibition mechanism is incomplete. Chapter 2 presents evidence for several non-canonical `type II' ligand species of diverse CYPs that were identified using a range of biophysical approaches, including electron paramagnetic resonance (EPR). During the course of characterization of the molecular details for these interactions, efforts were made to understand the catalytic properties of this category of low-spin CYP-ligand complexes. Chapter 3 presents our findings that molecular addition of 1H-1,2,3-triazole fragment--a popular drug-like azole moiety that we have determined to favor non-canonical low-spin binding structure--to a well-documented CYP3A4 substrate 17α-ethynylestradiol, dramatically alters the binding mechanism while `steering' the regioselectivity of oxidative turnover. Lastly, chapter 4 presents an analog azole library study that was conducted using two diverse CYPs: the promiscuous mammalian liver isoform CYP3A4, often considered a source of problematic drug-drug interactions; and the paradigmatic drug-target CYP, CYP19 (aromatase). The data suggest a large degree of variability in the importance of azole-heme interactions in the context of overall ligand binding energetics, even for high-affinity binders. Furthermore, we demonstrate by EPR and magnetic circular dichroism (MCD) that significant structural heterogeneity can be identified in the low-spin enzyme fraction in complex with tight-binding drug-like imidazoles. This heterogeneity is examined as a possible source of metabolic susceptibility and as a more accurate reporter of low-spin equilibrium conversion. The functional ramifications of heterogeneous type II interactions within this compound library will be presented in the context of a steady-state turnover analysis and metabolite characterization in CYP3A4.