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

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为许多体内过程提供了必要的能量。一类主要的酶，AAA家族，将ATP水解与各种细胞途径中必需的机械运动结合起来，包括（但不限于）蛋白质降解，细胞器维持，复制和重组。蛋白p97（也称为含缬草蛋白）是该家族中研究最广泛的成员之一，因此已成为可以推断AAA蛋白一般原理的代表性成员。p97于1990年首次被发现，在细胞中含量丰富（占细胞质的近1%），六聚体化，并在溶液中形成堆叠环状复合物。p97也被认为在IkBa降解途径中发挥关键作用，导致癌细胞凋亡下调，并解释了在许多癌细胞中观察到p97的存在增加。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）、一个与效应蛋白相互作用的n端结构域、一个c端结构域和它们之间的连接区域组成。在本提案中，我们建议通过计算实验来扩大我们对p97结构和功能的理解，同时开发针对其活性位点的小分子抑制剂。最初的硅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分子动力学（MD）模拟。此外，将解决低分辨率和高分辨率实验结构之间的差异。为了发现抑制p97的小分子，然后将使用对接方法结合松弛复合体方案对MD模拟产生的结构进行虚拟筛选。然后，我们的合作者将对候选分子进行实验测试，他们的结果可能会用于指导进一步的筛选计算。确定为顶部抑制剂的分子将通过先导优化来细化，这将通过开发一种新的先导优化方法来辅助。最后，通过结合量子力学/分子力学计算的自由能计算来分析水解途径，进一步了解活性位点的残基、水分子和离子。模拟结果将促进我们在多个时间和长度尺度上对p97结构和功能的理解，同时也将开发针对这种高度重要蛋白质的新小分子抑制剂。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更低的成本提高准确性。

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.