A Yeast Model for Determining Tubulin-drug Interactions

描述（由申请人提供）：本研究的总体目标是确定微管蛋白和微管稳定抗有丝分裂剂之间相互作用的位点。紫杉醇是最著名的微管稳定化合物，但其他如埃博霉素显示出作为抗肿瘤剂的巨大潜力。紫杉醇和其他具有类似紫杉醇样活性的结构多样的抗有丝分裂化合物所共有的结合位点的知识将有助于合理设计与微管蛋白的紫杉醇结合区结合的抗肿瘤药物。我们的方法是使用酵母微管蛋白的诱变。虽然来自芽殖酵母酿酒酵母的微管蛋白不结合紫杉醇，但我们已经能够通过突变酵母和哺乳动物β-微管蛋白之间不同的124个氨基酸中的5个来产生与酵母微管蛋白的强紫杉醇结合。我们现在计划对紫杉醇结合区进行更全面的突变分析，以全面了解紫杉醇、埃博霉素和其他化合物与β-微管蛋白这一区域结合所需的相互作用。我们计划确定我们所做的五个突变中哪一个对紫杉醇结合最负责，以及其他哪些氨基酸也参与了紫杉醇结合，实际上做出了贡献。我们将确定我们创造的突变是否影响紫杉醇结合，微管稳定性，或两者兼而有之。我们的研究将确定微管稳定抗有丝分裂剂紫杉醇，埃博霉素，eleutherobins，和discodermolide之间的微管蛋白的结合相互作用的差异。通过使用紫杉醇光亲和类似物，我们将确定紫杉醇分子的哪一部分可能与β-微管蛋白的突变区域接触。最后，我们将研究裂殖酵母裂殖酵母微管蛋白的紫杉醇结合特性，其中紫杉醇结合区域似乎更接近哺乳动物微管蛋白。

DESCRIPTION (provided by applicant): The overall goal of this research is to determine the sites of interaction between tubulin and microtubule-stabilizing anti-mitotic agents. Taxol is the best known of the microtubule-stabilizing compounds but others such as the epothilones show great potential as anti-tumor agents. Knowledge of the binding site shared by taxol and other structurally diverse anti-mitotic compounds with similar taxol-like activity wilt be a great aid in the rational design of anti-tumor agents that bind to the taxol binding region of tubulin. Our approach is to use mutagenesis of yeast tubulin. Although tubulin from the budding yeast Saccharomyces cerevisiae does not bind taxol, we have been able to create strong taxol binding to yeast tubulin by mutating just five of the 124 amino acids that are different between yeast and mammalian beta-tubulin. We now plan a more comprehensive mutational analysis of the taxol-binding region to provide a thorough understanding of the interactions required for taxol, the epothilones, and other compounds to bind to this region of beta-tubulin. We plan to determine which of the five mutations we made are most responsible for taxol binding, and what other amino acids that have also been implicated in taxol binding, actually make a contribution. We will determine whether the mutations we create affect taxol binding, microtubule stability, or both. Our studies will determine the differences in binding interactions with tubulin amongst the microtubule-stabilizing antimitotic agents taxol, the epothilones, the eleutherobins, and discodermolide. By using taxol photoaffinity analogues we will determine what part of the taxol molecule is likely to make contact with the mutated regions of beta-tubulin. Finally, we will study the taxol binding properties of tubulin from the fission yeast Schizosaccharomyces pombe in which the taxol binding region appears to more closely resemble that of mammalian tubulin.