Toxicological importance of CYP3A4 catalysis and inhibition

项目负责人/主要研究者（Sevrioukova，Irina F.）： 项目摘要 人细胞色素P450 3A 4（CYP 3A 4）是主要的和临床上最相关的药物代谢酶， 以其极端的底物混杂和变构行为而闻名。药物和其他外源性物质 还可刺激和抑制CYP 3A 4活性，这通常会导致不希望的药物-药物相互作用 （DDI）、化学毒性和治疗失败。尽管进行了广泛的研究，但CYP 3A 4抑制和 激活机制仍不完全清楚。这项建议的核心是利用结构生物学 方法来解决CYP 3A 4研究的两个领域的关键问题。目的1研究CYP 3A 4 HIV蛋白酶抑制剂利托那韦类似物合理结构设计及其抑制机制 其强效抑制CYP 3A 4的能力纯粹是巧合。我们将确定结构决定因素 通过合理设计和研究利托那韦样化合物的构效关系， 化合物，并根据我们的研究结果，建立一个3D药效团模型，为有效的CYP 3A 4特异性 抑制剂，其可用于早期预测/消除候选药物中的抑制潜力， 开发更有效的药物增强剂。目标2将利用一个综合的生化，化学 标记，结构和计算方法来研究CYP 3A 4底物结合协同性， 变构我们最近的结构发现证实了以前绘制的外围区域的重要性 并确定了三个新的内部网站，可以为基板/效应对接。我们将评估 通过评估这些区域的修饰/破坏如何影响CYP 3A 4， 构象、底物结合协同性、化学计量和代谢。这项研究概述了 从基础科学和转化科学的角度来看，这项提案都很重要，因为它将填补 知识差距，并提供了对CYP 3A 4的可塑性和适应性的基本见解， 不同的配体，阐明复杂的配体结合行为和氧化的分子机制 动力学，并帮助开发更好的工具，用于计算机预测蛋白质-配体接触，代谢稳定性和 DDI在候选药物中的潜力，以提高其疗效和减少脱靶效应。 OMB编号0925-0001/0002（2012年8月批准至2015年8月31日修订版）页码续页格式页码

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