Toxicological importance of CYP3A4 catalysis and inhibition

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Program Director/Principal Investigator (Sevrioukova, Irina F.): Project Summary Human cytochrome P450 3A4 (CYP3A4) is the major and most clinically relevant drug-metabolizing enzyme, notoriously known for its extreme substrate promiscuity and allosteric behavior. Drugs and other xenobiotics can also stimulate and inhibit CYP3A4 activity, which frequently leads to undesired drug-drug interactions (DDIs), chemical toxicity and therapeutic failures. Despite extensive investigations, the CYP3A4 inhibitory and activation mechanisms remain incompletely understood. This proposal centers on using structural biology approaches to address key issues in both areas of CYP3A4 research. Aim 1 is set to investigate the CYP3A4 inhibitory mechanism via rational structure-based design of analogues of ritonavir, an HIV protease inhibitor whose ability to potently inhibit CYP3A4 was purely coincidental. We will identify structural determinants required for potent inhibition by rationally designing and investigating structure-activity relations of ritonavir-like compounds and, based on our findings, build a 3D-pharmacophore model for a potent CYP3A4-specific inhibitor that can be used for early prediction/elimination of the inhibitory potential in drug candidates and for development of more effective pharmacoenhancers. Aim 2 will utilize an integrated biochemical, chemical labeling, structural and computational approach to investigate the CYP3A4 substrate binding cooperativity and allosterism. Our recent structural findings confirmed the importance of the previously mapped peripheral area and identified three novel inner sites that could serve for substrate/effector docking. We will evaluate the role and relative importance of these areas by assessing how their modification/disruption affects CYP3A4 conformation, substrate binding cooperativity, stoichiometry and metabolism. The research outlined in this proposal is important from both the basic and translational science perspectives, because it will fill the knowledge gaps and provide fundamental insights into plasticity and adaptability of CYP3A4 to structurally diverse ligands, clarify molecular mechanisms underlying the complex ligand binding behavior and oxidative kinetics, and help develop better tools for in silico prediction of protein-ligand contacts, metabolic stability and DDI potential in drug candidates to improve their efficacy and reduce off-target effects. OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015) Page Continuation Format Page