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

描述(由申请人提供)：微管细胞骨架是癌症化疗的有效和有效的靶点。紫杉醇已被FDA批准用于治疗几种人类恶性肿瘤，包括乳腺癌和卵巢癌，其临床疗效明显证明了这一点。紫杉醇是一种抗有丝分裂药物，具有稳定微管防止解聚的能力。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/分解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。虽然已经有了α/β-微管蛋白二聚体和与紫杉醇络合的微管的结构模型，但分辨率不足以揭示药物结合如何改变微管/原丝结构。氢/氢交换(HDX)-质谱学(MS)已成为研究蛋白质结构/动力学许多方面的一种快速而有力的实验工具。这项技术为我们提供了一条获得微管蛋白结构/动力学的关键知识的途径，这是电子结晶学无法获得的，而且在不久的将来也不太可能。在这一应用中，我们建议开发HDX-MS技术来分析由于聚合而改变的微管中人微管蛋白的溶剂可及性。每个微管蛋白单体中溶剂可及性的改变将局限于每个亚基的特定多肽区域，并可能局限于单个残基。结合现有的微管/微管的电子结晶学数据，这项技术将在以下情况下定位微管结构的变化：(A)在紫杉醇或非水解性GTP类似物存在或不存在的情况下聚合；(B)通过环氧丙酮类、盘状或月桂胺类化合物稳定微管；以及(C)引入不同的微管蛋白同型组成或单点突变。

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.