Drug-Mediated Alteration of Cytochrome P450

药物介导的细胞色素 P450 改变

• 批准号: 7889001
• 负责人: YOICHI OSAWA
• 金额: $ 38.63万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7889001
• 关键词: Address; Adverse drug effect; Adverse effects; Affect; Antihypertensive Agents; Aryl Hydrocarbon Hydroxylases; Binding; Binding Proteins; Biochemical; Biological; Biological Process; Cells; Cerebral Palsy; Chemicals; Cleaved cell; Complex; Cytochrome P450; Cytochromes; Decision Making; Dose; Drug toxicity; Drug usage; Enzymes; Functional disorder; Gastrointestinal Motility; Gastrointestinal tract structure; Grant; Guanabenz; Health; Heme; Hemeproteins; In Vitro; Injury; Knockout Mice; Knowledge; Laboratories; Lead; Liver; Mass Spectrum Analysis; Mediating; Metabolism; Methods; Modeling; Molecular; Molecular Chaperones; Neurodegenerative Disorders; Nitric Oxide Synthase; Pathway interactions; Patients; Peristalsis; Pharmaceutical Preparations; Phenotype; Physiological Processes; Play; Process; Prosthesis; Proteins; Pterins; Published Comment; Quality Control; Rattus; Reagent; Regulation; Research; Role; Safety; Scheme; Small Interfering RNA; Sorting - Cell Movement; Stomach; Stroke; Suicide; Supplementation; System; Talents; Techniques; Therapeutic Uses; Time; Tobacco; Toxic effect; Triage; Ubiquitination; Work; Xenobiotics; base; design; drug mechanism; drug metabolism; experience; human NOS3 protein; inhibitor/antagonist; man; novel; oxidation; prevent; protein degradation; protein function; protein metabolite; public health relevance; repaired; response; tetrahydrobiopterin; therapeutic protein; ubiquitin ligase

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to elucidate the mechanisms of drug- and xenobiotic- mediated inactivation, degradation, and turnover of cytochrome P450 enzymes. Nitric oxide synthase (NOS), the most highly regulated cytochrome P450 enzyme, plays a key role in a variety of biological processes, including regulation of gastrointestinal motility and liver drug metabolism. We have discovered that drugs, such as guanabenz and tobacco, are metabolism-based inactivators of NOS and cause the covalent alteration, enhanced turnover, and loss of NOS protein. In that the loss of NOS function and protein may explain some of the toxicities associated with these drugs, we wondered how drugs cause the enhanced turnover of NOS. We have established that the drugs cause prosthetic heme alteration or tetrahydrobiopterin oxidation and that such alterations are triggers for degradation of NOS. Furthermore, we discovered that these alterations labilize NOS for ubiquitination and proteasomal degradation by a chaperone-dependent mechanism involving hsp90 and hsp70. We have also uncovered a potential repair pathway where cellular proteins, including chaperones, facilitate insertion of heme into heme-deficient apo-NOS. We can now utilize the discoveries to date to determine how chaperones select for repair or ubiquitination of drug-altered NOS. Thus, we propose the following specific aims: (1) To characterize the interaction of labilized forms of NOS with hsp70- and hsp90- chaperones and cochaperones, (2) To determine the role of hsp70 and hsp90 in the ubiquitination of NOS, (3) To isolate and characterize the heme insertion machinery that facilitates heme entry into apo-NOS. We will utilize siRNA, immunopurification, biochemical, and LC-MS/MS techniques in a variety of in vitro and cellular systems to address these aims. We will show how the molecular interactions between NOS and chaperones lead to defined and predictable biological responses that ultimately determine the pharmacological and toxicological profiles of drugs. This will aid in the design of safe and effective drugs to control NOS as well as strategies to decrease adverse drug effects related to NOS. We also address the fundamental biological processes of how the heme prosthetic group is inserted into NOS and how cells maintain NOS protein quality control. Taking advantage of this quality control mechanism may provide a new method to specifically remove proteins for therapeutic benefit. Overall, this work furthers our understanding of how the metabolism of drugs, especially those used chronically, can alter the normal biological processes to give rise to adverse as well as beneficial drug effects. Ultimately, these studies may provide a way to predict, evaluate, and refine, the efficacy and safety of drugs and other xenobiotics. PUBLIC HEALTH RELEVANCE: This research furthers our understanding of how metabolism of drugs alters biological processes that lead to adverse as well as beneficial drug effects. Ultimately, these studies may provide a way to predict, evaluate, and refine, the efficacy and safety of drugs and other xenobiotics. The discoveries made through this line of study indicate that a new way to target proteins for degradation may have important therapeutic uses.

描述(申请人提供)：拟议研究的长期目标是阐明药物和异物介导的细胞色素P450酶失活、降解和周转的机制。一氧化氮合酶(NOS)是细胞色素P450中最受调控的酶，在多种生物学过程中发挥着关键作用，包括调节胃肠动力和肝脏药物代谢。我们发现，药物，如愈创木酚和烟草，是基于代谢的一氧化氮合酶失活剂，并导致一氧化氮合酶蛋白的共价改变、周转增加和丢失。由于一氧化氮合酶功能和蛋白的丧失可能解释了与这些药物相关的一些毒性，我们想知道药物是如何导致一氧化氮合酶周转增加的。我们已经确定，这些药物引起人工血红素改变或四氢生物蝶呤氧化，并且这种改变是一氧化氮合酶降解的触发因素。此外，我们还发现，这些改变通过涉及HSP90和HSP70的伴侣依赖的机制，使一氧化氮合酶的泛素化和蛋白酶体降解合法化。我们还发现了一种潜在的修复途径，在这种途径中，包括伴侣在内的细胞蛋白有助于将血红素插入到缺乏血红素的apo-NOS中。我们现在可以利用迄今为止的发现来确定伴侣如何选择修复或泛素化药物改变的一氧化氮合酶。因此，我们提出了以下具体目标：(1)表征标记形式的NOS与HSP70和HSP90-伴侣和辅伴侣的相互作用；(2)确定HSP70和HSP90在一氧化氮合酶泛素化中的作用；(3)分离和表征促进血红素进入apo-NOS的血红素插入机制。我们将在各种体外和细胞系统中利用siRNA、免疫提纯、生化和LC-MS/MS技术来实现这些目标。我们将展示一氧化氮合酶和伴侣之间的分子相互作用如何导致明确和可预测的生物反应，最终决定药物的药理学和毒理学特征。这将有助于设计安全有效的药物来控制一氧化氮合酶，以及减少与一氧化氮合酶相关的药物不良反应的策略。我们还讨论了如何将血红素修复基团插入到NOS中以及细胞如何维持NOS蛋白质质量控制的基本生物学过程。利用这种质量控制机制，可能会提供一种新的方法来特异性地去除蛋白质，从而达到治疗的目的。总体而言，这项工作加深了我们对药物代谢的理解，特别是那些长期使用的药物，如何改变正常的生物过程，产生不利和有益的药物效果。最终，这些研究可能提供一种方法来预测、评估和改进药物和其他外源药物的有效性和安全性。 与公共卫生相关：这项研究加深了我们对药物新陈代谢如何改变生物过程的理解，这些过程导致了药物的不利和有益的影响。最终，这些研究可能提供一种方法来预测、评估和改进药物和其他外源药物的有效性和安全性。通过这一系列研究取得的发现表明，一种针对蛋白质降解的新方法可能具有重要的治疗用途。