Toxicological importance of CYP3A4 catalysis and inhibition

 描述(申请人提供)：人细胞色素P450 3A4(CYP3A4)是人类体内含量最丰富和临床上最相关的异物代谢P450，以其在底物专一性和变构行为上的极端混杂而闻名。除了药物和内源性化合物的代谢外，CYP3A4还参与农药、杀虫剂、除草剂、致癌物和环境污染物的氧化和生物激活。细胞色素P3A4也可以被外源物质抑制和激活，这可能会导致不希望看到的药物相互作用、毒性和人类死亡。尽管CYP3A4在药物代谢中起着核心作用，但其抑制和激活机制仍不清楚。这项建议旨在解决CYP3A4研究的两个领域的关键问题，并集中在我们在CYP3A4与配体结合过程的结构和功能研究方面的进展。目标1的目标是优化CYP3A4抑制剂药效团模型，该模型是基于我们对利托那韦类似物的研究建立的，利托那韦是迄今为止市场上最有效的药物增强剂。我们将使用新颖、合理设计的化合物准确地识别药效决定因素，并将根据结构和功能解释进一步优化。基于结构的CYP3A4特异性抑制剂药效团可以帮助在开发用于农业和公共卫生的药物和其他化学品的过程中识别和早期消除潜在的CYP3A4灭活剂，以及设计更有效的药物增强剂。该提案的第二部分集中于CYP3A4的变构行为，被认为是由多底物结合引起的。CYP3A4可以被各种外源物质和天然饮食化合物激活，但潜在的机制仍存在争议。我们最近的发现表明，底物-通道而不是先前发现的外围区域是高亲和力效应器结合的SIE。目标2的目标是通过比较外周和通道内位点在类黄酮类分子激活CYP3A4中的相对重要性来检验这一假设。使用结构、生化和生物物理的综合方法，我们将评估这些区域的破坏如何影响蛋白质动力学、底物结合化学计量学、动力学和新陈代谢。这些研究将对CYP3A4激活、底物协同作用和药物-药物相互作用的机制提供新的见解，这对于预测/预防化学毒性和了解药物、饮食化合物和环境化学品代谢的个体差异是必不可少的。

 DESCRIPTION (provided by applicant): Human cytochrome P450 3A4 (CYP3A4) is the most abundant and clinically relevant xenobiotic-metabolizing P450 in humans and is well known for its extreme promiscuity in substrate specificity and allosteric behavior. In addition to the metabolism of drugs and endogenous compounds, CYP3A4 is implicated in oxidation and bioactivation of pesticides, insecticides, herbicides, carcinogens and environmental pollutants. CYP3A4 can also be inhibited and activated by xenobiotics, which may lead to undesired drug-drug interactions, toxicity and human death. Despite the central role of CYP3A4 in drug metabolism, its mechanism of inhibition and activation are still poorly understood. This proposal is designed to address key issues in both areas of the CYP3A4 research and centers on our advances in the structural and functional investigation of the CYP3A4- ligand binding process. The goal of Aim 1 is to optimize the CYP3A4 inhibitor pharmacophore model that was built based on our studies of analogs of ritonavir, the most potent pharmacoenhancer marketed to date. We will accurately identify the pharmacophoric determinants using novel, rationally designed compounds, which will be further optimized based on structural and functional interpretations. The structure-based CYP3A4-specific inhibitor pharmacophore could assist in identification and early elimination of potential CYP3A4 inactivators during development of drugs and other chemicals used in agriculture and public health, as well as designing more potent pharmacoenhancers. The second part of the proposal focuses on the allosteric behavior of CYP3A4, thought to arise from the multiple substrate binding. CYP3A4 can be activated by various xenobiotics and natural dietary compounds, such as plant flavonoids, but the underlying mechanism is still under debate. Our recent findings suggest that the substrate-access channel rather than the previously identified peripheral area serves as a high affinity effector binding sie. The goal of Aim 2 is to test this hypothesis by comparing the relative importance of the peripheral and intra-channel sites in the CYP3A4 activation by flavonoid-like molecules. Using an integrated structural, biochemical and biophysical approach, we will assess how disruption of these areas affects protein dynamics, substrate binding stoichiometry, kinetics and metabolism. These studies will provide novel insights into the mechanism of CYP3A4 activation, substrate cooperativity and drug-drug interactions, which is essential for prediction/prevention of chemical toxicity and understanding interindividual differences in metabolism of drugs, dietary compounds and environmental chemicals.