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学疗法的有效且有效的靶标。紫杉醇的临床有效性已被FDA批准用于治疗几种人类恶性肿瘤，包括乳腺癌和卵巢癌。紫杉醇是一种抗魔法剂，具有稳定微管抗聚合的能力。深入了解紫杉醇如何稳定微管和影响微管动力学对旨在改变微管组装/拆卸动力学的新药物的设计以及预防或克服耐药性的策略。尽管可以使用α/β-微管蛋白二聚体和与紫杉醇复合的微管的结构模型，但该分辨率不足以揭示小管蛋白/原丝结构如何通过药物结合而改变。氢/氘交换（HDX） - 质谱法（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一条途径，以获取电子晶体学无法获得的小管蛋白结构/动力学知识，并且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，以分析由于聚合而导致人小管蛋白在微管中的溶剂可及性的变化。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，并可能与单个残基。结合了微管蛋白/微管上的现有电子晶体学数据，该技术将在（a）在存在或不存在紫杉醇或不可用的GTP类似物（b）稳定微蛋白酶的情况下（b）单个epothilones，discothilide is和contripe indorment is（c）的稳定（c）在（a）在（a）在（a）聚合的情况下定位的变化。突变。