H/D Exchange Coupled to MS to Study Drug-Induced Change in Microtuble Structure

描述（由申请人提供）：微管细胞骨架是一种有效且经过验证的癌症化疗靶点。紫杉醇的临床疗效已经被FDA批准用于治疗包括乳腺癌和卵巢癌在内的几种人类恶性肿瘤，这一点很明显。紫杉醇是一种抗有丝分裂剂，具有稳定微管防止解聚的能力。深入了解紫杉醇如何稳定微管并影响微管动力学，对于设计旨在改变微管组装/拆卸动力学的新药以及制定预防或克服耐药性的策略具有重要意义。虽然α / β -微管二聚体和微管与紫杉醇络合的结构模型都是可用的，但分辨率不足以揭示药物结合如何改变微管/原丝结构。氢/氘交换(HDX)-质谱（MS）已成为一种快速而强大的实验工具，用于研究蛋白质结构/动力学的许多方面。这项技术为我们提供了获取微管蛋白结构/动力学关键知识的途径，这是电子晶体学无法获得的，而且在不久的将来不太可能实现。在这个应用中，我们建议开发HDX-MS技术来分析由于聚合而改变的人微管中微管蛋白的溶剂可及性。每个微管蛋白单体内溶剂可及性的改变将定位于每个亚基的特定肽区域，并可能定位于单个残基。结合现有的微管/微管的电子晶体学数据，该技术将定位微管结构在以下情况下的变化：(a)在存在或不存在紫杉醇或不可水解GTP类似物的情况下的聚合；(b)微管被epothilones， disdermolide或laulimalide稳定；(c)引入不同的微管异型组成或单点突变。

DESCRIPTION (provided by applicant): The microtubule cytoskeleton is an effective and validated target for cancer chemotherapy. This is clearly evident by the clinical effectiveness of Taxol that has been approved by the FDA for the treatment of several human malignancies, including breast and ovarian cancers. Taxol is an antimitotic agent that has the capacity to stabilize microtubules against depolymerization. Insight into how Taxol stabilizes microtubules and influences microtubule dynamics is important in the design of new drugs directed at altering the dynamics of microtubule assembly/disassembly and in the development of strategies to prevent or overcome drug resistance. Although structural models for both the alpha/beta-tubulin dimer and microtubules complexed with Taxol are available, the resolution is not sufficient to reveal how tubulin/protofilament structure is altered by drug binding. Hydrogen/Deuterium exchange (HDX)-Mass Spectrometry (MS) has emerged as a rapid and powerful experimental tool to investigate many aspects of protein architecture/dynamics. This technology provides us with an avenue to obtain crucial knowledge of tubulin structure/dynamics that is not available from electron crystallography, and is not likely to be in the near future. In this application, we propose to develop HDX-MS technologies for analysis of the altered solvent accessibility of human tubulin in microtubules as a result of polymerization. Alterations in solvent accessibility within each tubulin monomer will be localized to specific peptide regions of each subunit and potentially to individual residues. In combination with the existing electron crystallography data on tubulin/microtubules, this technology will localize changes in tubulin structure upon (a) polymerization in the presence or absence of Taxol or a nonhydrolyzable GTP analog (b) stabilization of microtubules by the epothilones, discodermolide or laulimalide and (c) the introduction of different tubulin isotype compositions or single point mutations.