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: 7472512
• 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/7472512
• 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过验证的靶标。紫杉醇的临床疗效清楚地证明了这一点，紫杉醇已被 FDA 批准用于治疗多种人类恶性肿瘤，包括乳腺癌和卵巢癌。紫杉醇是一种抗有丝分裂剂，能够稳定微管以防止解聚。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非常重要。尽管α/β-微管蛋白二聚体和与紫杉醇复合的微管的结构模型都可用，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) 已成为一种快速而强大的实验工具，可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得微管蛋白结构/动力学的重要知识的途径，这是电子晶体学无法获得的，而且在不久的将来也不太可能获得。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，该技术将在以下情况下定位微管蛋白结构的变化：(a) 在存在或不存在紫杉醇或不可水解的 GTP 类似物的情况下聚合 (b) 通过埃博霉素、discodermolide 或月桂马内酯和(c)引入不同的微管蛋白同种型组合物或单点突变。