Structure/Function of Potential Human Cytochrome P450 Drug Targets

University Of Michigan At Ann Arbor, Ann Arbor MI

Investigators

Abstract

PROJECT SUMMARY Our laboratory focuses on the structure and function of human membrane cytochrome P450 enzymes using a set of integrated structural and biochemical techniques. These monooxygenases play important roles in either the metabolism of drugs or as drug targets for prevalent diseases such as cancers, diabetes, and cardiovascular conditions, as well as rare diseases such as neuromuscular spastic paraplegia and Bietti's Crystalline Dystrophy. Many drug-metabolizing and steroidogenic P450s have been well characterized functionally and structurally by our lab, supporting improved drug design, but many other P450 drug targets are very poorly characterized. In some diseases inappropriate P450 action is implicated and inhibitors are likely to be an advantageous treatment, but the active site knowledge to design them is unknown until we determine structures. In other cases P450 mutations cause a disease, but not knowing whether the defect is in protein stability or some aspect of catalytic function similarly impedes effective progress toward developing effective therapeutics. We have a 20- year track record defining P450 interactions with ligands for various drug-metabolizing and steroidogenic human enzymes. For most we generated the first structures by X-ray crystallography. The overall goal of the proposed research for the next five years is to generate initial X-ray structures and corresponding biochemical information of a selected set of validated disease-related P450 enzymes to enable viable pursuit of drug design A subset of poorly-characterized human P450 enzymes with significant evidence supporting their roles in one or more disease states were identified and will be pursued using an overall strategy including recombinant expression and purification, identification of new ligands as necessary, evaluation of clinical mutations when appropriate, followed by integrated determination of substrate and inhibitor binding affinities, substrate metabolism, inhibitor efficacy, and active site topology, primarily by X-ray crystallography and/or pharmacophore generation. These P450/ligand interaction studies, along with proposed studies of P450 interactions with their catalytic partner proteins and membranes, are expected to provide critical foundational knowledge facilitating broad impacts across a number of major and rare diseases. The feasibility of this work rests not only on the strong premise for the selected P450 enzymes, but the investigator track record, laboratory expertise, and access to all of the required instrumentation and resources in an exceedingly strong research environment. With this combination of resources we have previously accomplished similar feats for a number of other human membrane P450 enzymes. Our overall vision is to creatively employ multiple techniques to answer some of the most critical questions about how this diverse enzyme superfamily functions and can be modulated to improve human health.