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）：储存在三磷酸核苷中的能量的机械偶联提供了许多体内过程所需的动力。一个主要类别的酶，AAA家族，将ATP水解与多种细胞途径中必不可少的机械运动偶联，所述细胞途径包括（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valosin-containing蛋白质）是该家族中研究最广泛的成员之一，因此已成为可从中推断AAA蛋白质的一般原理的代表性成员。1990年首次发现，p97在细胞中高度丰富（占细胞质的近1%），六聚化，并在溶液中形成堆叠的环状复合物。p97还被认为在IkBa的降解途径中起关键作用，这导致癌细胞中细胞凋亡的下调，并解释了在许多癌细胞系中观察到的p97存在增加。在结构上，每个单体由两个水解结构域（D1和D2，只有D2在标准细胞条件下具有催化活性）、与效应蛋白相互作用的N末端结构域、C末端结构域以及它们之间的接头区域组成。在这个建议中，我们建议计算实验，以扩大我们的理解的结构和功能的p97除了开发小分子抑制剂，针对其活性位点。最初的计算机工作将集中在构象结构和运动固有的主要水解状态，通过使用长时间尺度的分子动力学（MD）模拟。此外，低和高分辨率的实验结构之间的差异将得到解决。在试图发现抑制p97的小分子时，然后将使用对接方法结合松弛复合物方案对MD模拟产生的结构进行虚拟筛选。然后，我们的合作者将对最佳候选分子进行实验测试，其结果可用于指导进一步的筛选计算。然后将通过先导优化对确定为顶级抑制剂的分子进行精制，这将通过开发新的先导优化方法来辅助。最后，水解途径将通过结合量子力学/分子力学计算的自由能计算进行分析，以进一步了解活性位点中的残留物，水分子和离子。模拟的结果将促进我们对p97在多个时间和长度尺度上的结构和功能的理解，同时还将开发针对这种高度重要的蛋白质的新的小分子抑制剂。此外，本文开发的先导优化方法将允许在基于结构的药物设计中以降低的成本提高准确性。 公共卫生相关性：本文提出的模拟结果可能会对公众健康产生重大影响，通过推进我们对高度丰富的酶p97的结构，功能和水解机制的理解。对各种细胞通路的重要性使p97成为一个有趣的化疗靶点，这里描述的一个项目旨在发现该靶点的高亲和力抑制剂（目前还没有），并可能导致靶向癌细胞的药物开发。此外，铅优化方法的发展可能会导致改进的基于结构的药物设计方法的各种酶的目标。