Identification of small-molecule scaffolds for p450 inhibitors

Abstract

Mycobacterium tuberculosis cytochrome P450 enzymes (CYP) attract ongoing interest for their pharmacological development potential, driving direct screening efforts against potential CYP targets with the ultimate goal of developing potent CYP-specific inhibitors and/or molecular probes to address M. tuberculosis biology. The property of CYP enzymes to shift the ferric heme Fe Soret band in response to ligand binding provides the basis for an experimental platform for high-throughput screening (HTS) of compound libraries to select chemotypes with high binding affinities to the target. Promising compounds can be evaluated in in vitro assays or in vivo disease models and further characterized by x-ray crystallography, leading to optimization strategies to assist drug design. Protocols are provided for compound library screening, analysis of inhibitory potential, and co-crystallization with the target CYP, as well as expression and purification of soluble CYP enzymes.

Figure 1. Examples of the type I and type II difference spectra

(A) Type I spectra resulted from the titration of CYP51 of M. tuberculosis with estriol (K_D of 100 µM). (B) Type II spectra resulted from the titration with 4-phenylimidazole (K_D of 1.3 mM) (Podust et al., 2007).