Computational and Vibrational Probes of CYP3A4 Solution Dynamics

CYP3A4 溶液动力学的计算和振动探针

• 批准号: 9260902
• 负责人: John C Hackett
• 金额: $ 30.12万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-15 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9260902
• 关键词: Active Sites; Amino Acids; Binding Proteins; Biology; CYP3A4 gene; Chemicals; Complement; Complex; Computing Methodologies; Coupling; Critical Pathways; Crystallization; Crystallography; Cytochrome P450; Cytochromes; Cytochromes b; Cytochromes b5; Data; Drug Design; Drug Interactions; Drug Targeting; Electron Transport; Electrostatics; Engineering; Environment; Enzymatic Biochemistry; Enzyme Kinetics; Enzymes; Event; Experimental Designs; Frequencies; Goals; Heme; Human; Human Genome; Investigation; Kinetics; Knowledge; Ligands; Maps; Metabolic; Metabolism; Methods; Molecular; Molecular Conformation; Motion; NADPH-Ferrihemoprotein Reductase; Names; Nature; Oxidation-Reduction; Pathway interactions; Pharmaceutical Preparations; Phase; Propionates; Protein Dynamics; Proteins; Public Health; Raman Spectrum Analysis; Research; Risk; Role; Sampling; Spectrum Analysis; Structure; Structure-Activity Relationship; Thermodynamics; Time; Uncertainty; Xenobiotic Metabolism; Xenobiotics; alpha helix; analog; base; computational chemistry; computerized tools; conformer; drug metabolism; electronic structure; enzyme mechanism; enzyme substrate; experimental study; flexibility; insight; molecular dynamics; novel strategies; oxidation; programs; protein protein interaction; public health relevance; simulation; vibration

 DESCRIPTION (provided by applicant): There is little doubt that cytochrome P450 CYP3A4 is the single most important protein in human xenobiotic metabolism. The prominence of CYP3A4 in drug metabolism results in routine investigation of the activity of thousands of molecules annually as substrates for this enzyme. Numerous computational methods have been developed to predict CYP3A4 metabolism. Ligand-based methods have considered structure-activity relationships for a baffling array of potential substrates, with a complex set of rather unreliable predictions. The key problem is that the conformational flexibility, or plasticity, of CYP3A4 has not been available and hence, has not been factored into these predictions. The long-term goal of this research program is to develop and apply novel approaches that dramatically expand our understanding of the P450 enzyme mechanisms. The objective of this application is to combine state-of-the-art tools of computational chemistry, chemical biology, and molecular spectroscopy to gain insight into the coupling of heme reactivity and protein dynamics, and the influence of interactions with redox partners with the adaptation of CYP3A4 to ligands. To meet this objective, there are three Specific Aims, we will: 1) Delineate the range of CYP3A4 conformational states in solution and the transition pathways between these conformers. Molecular dynamics methods capable of sampling low frequency protein motions will afford a description of CYP3A4 solution conformations, that to date, have been elusive by other means. 2) Define the functional consequences of ligand and redox partner interactions on CYP3A4 heme dynamics. Resonance Raman spectroscopy will be used to probe the conformational shifts and electronic structure changes resulting from interactions between CYP3A4, cytochrome P450 reductase, and cytochrome b5 that pre-organize that active site to facilitate electron transfer. 3) Map changes in CYP3A4 electrostatics by selective incorporation of Raman-active vibrational probes. The selective incorporation of amino acid analogs with vibrational probes will permit direct observation of local electrostatic changes through solvatochromic shifts induced by ligand binding, protein-protein interactions with redox partners, and resultant conformational interchanges. To afford accurate metabolic predictions for CYP3A4 metabolism, the conformational plasticity and the interactions between conformer and heme dynamics must be understood. We propose to approach these holes in our current understanding using a suite of interactive experimental designs. The significance of this set of studies is the promise of a clearer insight into ligand- and redox-partner induced changes in P450 dynamics and heme reactivity and the impact of these heretofore understudied factors in the adaptation of CYP3A4 to new substrate structures.

 描述（由申请人提供）：毫无疑问，细胞色素 P450 CYP3A4 是人类外源代谢中最重要的单一蛋白质。 CYP3A4 在药物代谢中的突出地位导致每年对作为该酶底物的数千个分子的活性进行常规研究。已经开发了许多计算方法来预测 CYP3A4 代谢。基于配体的方法考虑了一系列令人困惑的潜在底物的结构-活性关系，以及一组复杂且相当不可靠的预测。关键问题是 CYP3A4 的构象灵活性或可塑性尚不可用，因此没有被纳入这些预测中。该研究计划的长期目标是开发和应用新方法，极大地扩展我们对 P450 酶机制的理解。该应用的目的是结合最先进的计算化学、化学生物学和分子光谱学工具，深入了解血红素反应性和蛋白质动力学的耦合，以及氧化还原伙伴相互作用对 CYP3A4 配体适应的影响。为了实现这一目标，我们将实现三个具体目标： 1) 描绘溶液中 CYP3A4 构象状态的范围以及这些构象异构体之间的转变途径。能够对低频蛋白质运动进行采样的分子动力学方法将提供 CYP3A4 溶液构象的描述，迄今为止，这是通过其他方法难以捉摸的。 2) 定义配体和氧化还原伙伴相互作用对 CYP3A4 血红素动力学的功能影响。共振拉曼光谱将用于探测 CYP3A4、细胞色素 P450 还原酶和细胞色素 b5 之间相互作用引起的构象变化和电子结构变化，这些相互作用预先组织活性位点以促进电子转移。 3) 通过选择性结合拉曼活性振动探针来绘制 CYP3A4 静电的变化图。氨基酸类似物与振动探针的选择性结合将允许通过配体结合诱导的溶剂化变色位移、与氧化还原伙伴的蛋白质-蛋白质相互作用以及由此产生的构象交换来直接观察局部静电变化。为了对 CYP3A4 代谢提供准确的代谢预测，必须了解构象可塑性以及构象异构体和血红素动力学之间的相互作用。我们建议使用一套交互式实验设计来弥补我们当前理解中的这些漏洞。这组研究的意义在于，有望更清楚地了解配体和氧化还原伙伴诱导的 P450 动力学和血红素反应性变化，以及这些迄今为止未充分研究的因素对 CYP3A4 适应新底物结构的影响。