Computational and Vibrational Probes of CYP3A4 Solution Dynamics

 描述（由申请方提供）：毫无疑问，细胞色素P450 CYP 3A 4是人体异生物质代谢中最重要的单一蛋白质。CYP 3A 4在药物代谢中的重要性导致每年对数千种分子作为该酶底物的活性进行常规研究。已经开发了许多计算方法来预测CYP 3A 4代谢。基于配体的方法已经考虑了一系列令人困惑的潜在底物的结构-活性关系，具有一组复杂的相当不可靠的预测。关键的问题是，CYP 3A 4的构象灵活性或可塑性还没有得到，因此没有被考虑到这些预测中。这项研究计划的长期目标是开发和应用新的方法，大大扩展我们对P450酶机制的理解。本申请的目的是结合联合收割机最先进的计算化学，化学生物学和分子光谱学的工具，以深入了解血红素反应性和蛋白质动力学的耦合，以及与氧化还原伙伴的相互作用与CYP 3A 4配体的适应的影响。为了实现这一目标，我们有三个具体的目标，我们将：1）描绘CYP 3A 4在溶液中的构象状态的范围和这些构象之间的转换途径。能够采样低频蛋白质运动的分子动力学方法将提供CYP 3A 4溶液构象的描述，到目前为止，已经难以捉摸的其他手段。2)定义配体和氧化还原伴侣相互作用对CYP 3A 4血红素动力学的功能后果。共振拉曼光谱将用于探测CYP 3A 4、细胞色素P450还原酶和细胞色素b5之间相互作用导致的构象位移和电子结构变化，这些相互作用预先组织活性位点以促进电子转移。3)通过选择性掺入拉曼活性振动探针绘制CYP 3A 4静电的变化。氨基酸类似物与振动探针的选择性掺入将允许通过由配体结合、蛋白质-蛋白质与氧化还原伙伴的相互作用以及由此产生的构象互换诱导的溶剂化变色位移直接观察局部静电变化。为了提供准确的代谢预测CYP 3A 4代谢，构象可塑性和构象和血红素动力学之间的相互作用，必须了解。我们建议使用一套交互式实验设计来接近我们目前理解的这些漏洞。这组研究的意义是一个更清晰的洞察配体和氧化还原伴侣诱导的P450动力学和血红素反应性的变化和这些迄今研究不足的因素在适应新的底物结构的CYP 3A 4的影响的承诺。

 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.