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

H/D 交换与 MS 结合研究药物引起的微管结构变化

• 批准号: 7318638
• 负责人: SUSAN BAND HORWITZ
• 金额: $ 31.54万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-19 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7318638
• 关键词: Affect; Amino Acids; Antimitotic Agents; Antineoplastic Agents; Architecture; Binding; Binding Sites; Biological Factors; Breast; Cells; Chemotherapy-Oncologic Procedure; Clinical; Clinical effectiveness; Complex; Condition; Coupled; Cytoskeleton; Data; Deuterium; Development; Distal; Docking; Drug resistance; Epothilones; Future; Goals; Guanosine Triphosphate; Human; Hydrogen; Individual; Knowledge; Lead; Ligand Binding; Localized; Location; Malignant Neoplasms; Malignant neoplasm of ovary; Maps; Mass Spectrum Analysis; Methodology; Microtubule Stabilization; Microtubule stabilizing agent; Microtubules; Modeling; Modification; Molecular Conformation; Mutate; Ovarian; Paclitaxel; Peptides; Pharmaceutical Preparations; Point Mutation; Proteins; Research; Resistance; Resolution; Site; Solvents; Staging; Structural Models; Structure; Technology; Tubulin; United States Food and Drug Administration; analog; beta Tubulin; depolymerization; design; dimer; discodermolide; electron crystallography; eleutherobin; insight; laulimalide; lung Carcinoma; mRNA Differential Displays; methylene diphosphonate; monomer; polymerization; pre-clinical; prevent; programs; reconstruction; research study; tool

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.

描述（由申请人提供）：微管细胞骨架是癌症化疗的有效且经验证的靶点。紫杉醇的临床有效性清楚地证明了这一点，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。紫杉醇是一种抗有丝分裂剂，具有稳定微管以防止解聚的能力。深入了解紫杉醇如何稳定微管和影响微管动力学是非常重要的，在设计新的药物，旨在改变微管组装/拆卸的动力学，并在发展战略，以防止或克服耐药性。虽然α/β-微管蛋白二聚体和微管与紫杉醇复合的结构模型是可用的，但分辨率不足以揭示药物结合如何改变微管蛋白/原丝结构。氢/氘交换（HDX）-质谱（MS）已经成为研究蛋白质结构/动力学的许多方面的快速和强大的实验工具。这项技术为我们提供了一个途径，以获得微管蛋白结构/动力学的关键知识，这是从电子晶体学中得不到的，也不太可能在不久的将来。在此应用中，我们建议开发HDX-MS技术，用于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。结合微管蛋白/微管的现有电子晶体学数据，该技术将定位微管蛋白结构在以下情况下的变化：（a）在存在或不存在泰素或不可水解的GTP类似物的情况下聚合（B）通过埃博霉素、discodermolide或laulimalide稳定微管和（c）引入不同的微管蛋白同种型组合物或单点突变。