P450 Protein-Protein Interactions Determined by Selective Protein Manipulation

通过选择性蛋白质操作测定 P450 蛋白质-蛋白质相互作用

• 批准号: 8229971
• 负责人: Peter M. Gannett
• 金额: $ 36.21万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8229971
• 关键词: P450; Protein; Interactions; Determined; Selective

During the past decade, it has become evident that in vitro enzyme data obtained in solution frequently fail to provide an accurate profile of in vivo enzyme kinetic parameters. This may be due to oligomer formation. Efforts to separate the effect of oligomer formation on P450 kinetics from factors that might influence kinetic parameters have been incomplete and are necessary to quantify the contribution that oligomer formation has on P450-mediated metabolism kinetics, to improve estimations of in vivo drug clearance. Our long-term goal is to accurately predict in vivo drug pharmacokinetics from in vitro data to improved assessments of drug safety and efficacy. Our overall objective here is to determine the important factors influencing in vitro P450-mediated metabolism kinetics through studies of homo- and hetero-oligomer formation using our recently developed immobilized enzyme methods. Our central hypothesis is that CYP2C9-mediated metabolism kinetics measured in vitro are critically dependent on the state of oligomerization, and to accurately predict in vivo pharmacokinetics using in vitro data requires inclusion of this variable. Our rationale for this project is that successful completion would provide a solid scientific foundation to more accurately assess in vivo kinetics of metabolism based upon in vitro data. To achieve our overall objectives, we propose to quantitate the modulatory effects of CYP2C9 homo and hetero-oligomer formation on enzyme kinetics. Successful achievement of this goal will well position us to determine the key factors that explain altered enzyme kinetics of oligomers versus monomers. The proposed research is innovative as a novel immobilized P450 methodology will be employed to unequivocally evaluate the effect of oligomerization on the kinetics of metabolism. Our expected outcomes are 1) determination of selected physical-chemical factors that affect CYP2C9 kinetics using nanoscale methods for protein manipulation and 2) to have quantitated the extent to which oligomer formation modulates CYP2C9-mediated metabolism kinetics. These outcomes are significant as they will help to further delineate the mechanistic details of P450 metabolism, an enzyme system responsible for metabolism of the majority of drugs, and should lead to improved pharmacokinetic data, necessary for determination of correct dosages and thus ultimately improve patient care.

在过去的十年中，很明显，在体外获得的酶数据 解决方案经常无法提供体内酶动力学的准确概况 参数。这可能是由于低聚物的形成。努力分离的影响 可能影响动力学参数的因素对 P450 动力学低聚物形成的影响 不完整，有必要量化低聚物的贡献 形成对 P450 介导的代谢动力学有影响，以改善体内的估计 药物清除。我们的长期目标是准确预测体内药物药代动力学 从体外数据到改进药物安全性和有效性的评估。我们的整体 这里的目的是确定影响体外 P450 介导的重要因素 通过使用我们的同源和异源寡聚体形成研究代谢动力学 最近开发了固定化酶方法。我们的中心假设是 体外测量的 CYP2C9 介导的代谢动力学主要取决于 寡聚化的状态，并准确预测体内药代动力学 体外数据需要包含该变量。我们开展这个项目的理由是 成功完成将为更准确地研究提供坚实的科学基础 根据体外数据评估体内代谢动力学。为了实现我们的整体 为了实现目标，我们建议定量 CYP2C9 同源和 异源寡聚物的形成对酶动力学的影响。成功实现这一目标 将使我们能够确定解释酶动力学改变的关键因素 低聚物与单体。所提出的研究作为一项新颖的研究具有创新性 将采用固定化 P450 方法来明确评估 寡聚对代谢动力学的影响。我们的预期结果是 1) 使用以下方法确定影响 CYP2C9 动力学的选定物理化学因素 蛋白质操作的纳米级方法和 2) 定量程度 该寡聚物的形成调节 CYP2C9 介导的代谢动力学。这些 结果意义重大，因为它们将有助于进一步描述其机制细节 P450代谢，负责大多数代谢的酶系统 药物，并应改进测定所需的药代动力学数据 正确的剂量，从而最终改善患者护理。