Multiscale Simulations on the Mechanism and Inhibition of the AAA Protein p97

AAA 蛋白 p97 的机制和抑制的多尺度模拟

• 批准号: 7912463
• 负责人: Jeffery Wereszczynski
• 金额: $ 4.76万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7912463
• 关键词: ATP Hydrolysis; Active Sites; Address; Affinity; Apoptosis; C-terminal; Cells; Complex; Computer Simulation; Couples; Cytosol; Degradation Pathway; Development; Docking; Down-Regulation; Drug Design; Enzymes; Family; Family member; Free Energy; Genetic Recombination; Hydrolysis; Ions; Lead; Length; Maintenance; Malignant Neoplasms; Mechanics; Methodology; Methods; Motion; N-terminal; Organelles; Pathway interactions; Play; Process; Proteins; Public Health; Resolution; Role; Scheme; Screening procedure; Shapes; Solutions; Structure; Testing; Time; Water; Work; base; cancer cell; cost; drug development; improved; in vivo; inhibitor/antagonist; interest; member; molecular dynamics; molecular mechanics; monomer; novel; nucleoside triphosphate; protein degradation; public health relevance; quantum; research study; simulation; small molecule; valosin-containing protein; virtual

DESCRIPTION (provided by applicant): The mechanocoupling of energy stored in triphosphate nucleosides provide the power necessary for many in vivo processes. One major class of enzymes, the AAA family, couples ATP hydrolysis to mechanical motions essential in a variety of cellular pathways including (but not limited to) protein degradation, organelle maintenance, replication and recombination. The protein p97 (also known as valosin-containing protein) is one of the most widely studied members of this family and has therefore become a representative member from which general principles of AAA proteins may be inferred. First discovered in 1990, p97 is highly abundant in the cell (composing nearly 1% of the cytosol), hexamerizes, and forms a stacked-ring shaped complex in solution. p97 is also believed to play a key role in the degradation pathway of IkBa, which results in the down-regulation of apoptosis in cancer cells and explains the observation of increased p97 presence in numerous cancer lines. Structurally, each monomer is composed of two hydrolysis domains (D1 and D2 with only D2 being catalytically active under standard cellular conditions), an N-terminal domain that interacts with effector proteins, a C-terminal domain, and linker regions between them. In this proposal we suggest computational experiments to expand our understanding of the structure and function of p97 in addition to developing small molecule inhibitors that target its active site. Initial in silico work will focus on the conformational structures and motions inherent to the major hydrolysis states through the use of long-times scale molecular dynamics (MD) simulations. In addition, discrepancies between low and high resolution experimental structures will be addressed. In an attempt to discover small molecules that inhibit p97, virtual screening will then be performed against structures resulting from the MD simulations using docking methods in conjunction with the relaxed complex scheme. Top candidate molecules will then be experimentally tested by our collaborators and their results may then be used in guiding further screening calculations. Molecules identified as top inhibitors will then be refined through lead optimization, which will be assisted through the development of a novel lead optimization methodology. Finally, hydrolysis pathways will be analyzed through free energy calculations with combined quantum mechanical/molecular mechanics calculations to further our understanding of residues, water molecules, and ions in the active site. Results of simulations will advance our understanding of p97 structure and function on multiple time and length scales while also developing new small molecule inhibitors that target this highly important protein. Additionally, the lead optimization methods developed herein will allow for increased accuracy at a reduced cost in structure based drug design. PUBLIC HEALTH RELEVANCE: Results from simulations proposed here might potentially have a significant impact on public health through advancing our understanding of the structure, function, and hydrolysis mechanism of the highly abundant enzyme p97. The importance to various cellular pathways makes p97 an interesting chemotherapeutic target, and one project described here aims to discover high-affinity inhibitors of this target (of which there are currently none) and could potentially lead to drug development targeting cancer cells. Additionally, the development of lead optimizations methods could result in improved structure based drug design methods for a variety of enzyme targets.

描述（由申请人提供）：三磷酸核苷中存储的能量的机械耦合为许多体内过程提供了必要的功率。一类主要的酶，即AAA家族，将ATP水解与各种细胞途径中必不可少的机械运动相结合，包括（但不限于）蛋白质降解，细胞器维持，复制和重组。蛋白质p97（也称为含瓣膜蛋白）是该家族中研究最广泛的成员之一，因此已成为代表成员，可以从中推断出AAA蛋白的一般原理。 P97在1990年首次发现，在细胞中高度丰富（构成了近1％的细胞质），六聚体，并在溶液中形成堆叠的环形复合物。 p97还被认为在IKBA的降解途径中起着关键作用，这导致癌细胞中凋亡的下调，并解释了观察到众多癌细胞系中p97存在的观察。从结构上讲，每个单体由两个水解结构域（在标准细胞条件下仅具有D2具有催化活性的D1和D2），这是一个与效应蛋白相互作用的N末端结构域，与效应蛋白相互作用，C末端结构域和它们之间的接头区域。在此提案中，我们建议进行计算实验，以扩展我们对p97的结构和功能的理解，除了开发针对其活性位点的小分子抑制剂。最初的计算机工作将通过使用长时间尺度分子动力学（MD）模拟来关注主要水解状态固有的构象结构和运动。此外，将解决低分辨率实验结构之间的差异。为了发现抑制p97的小分子，将对使用对接方法与松弛的复杂方案结合使用的MD模拟产生的结构进行虚拟筛选。然后，我们的合作者将对顶级候选分子进行实验测试，然后可以将其结果用于指导进一步的筛选计算。然后将通过铅优化来完善被识别为顶级抑制剂的分子，通过开发新的铅优化方法，这将有助于。最后，将通过使用量子力学/分子力学合并的自由能计算来分析水解途径，以进一步了解我们对活性位点中残基，水分子和离子的理解。模拟的结果将提高我们对p97结构和功能多数时间和长度尺度的理解，同时还会开发针对这种非常重要的蛋白质的新的小分子抑制剂。此外，此处开发的铅优化方法将允许以降低基于结构的药物设计成本的成本提高准确性。 公共卫生相关性：此处提出的模拟结果可能会通过促进我们对高度丰富酶P97的结构，功能和水解机制的理解来对公共卫生产生重大影响。对各种细胞途径的重要性使P97成为一个有趣的化学治疗靶标，此处描述的一个项目旨在发现该靶标的高亲和力抑制剂（目前没有），并且可能导致针对癌细胞的药物开发。此外，铅优化方法的开发可能会导致改善基于结构的药物设计方法，用于各种酶靶标。