Bio-organic Studies on Inhibition of Tubulin Assembly by Mitosis Inhibitors

该研究计划旨在了解微管蛋白和有丝分裂抑制剂之间的相互作用，并阐明微管蛋白的结构和功能。之前我们已经用猪脑微管蛋白证明了微管蛋白上存在着与秋水仙碱和长春花碱不同的根毒素/美登素结合位点。在本研究项目中，我们可以证明：EBI与牛脑β-微管蛋白上的半胱氨酸残基交联形成β^s，与美登素一样被根毒素强烈抑制，而秋水仙碱则不抑制EBI，而秋水仙碱仅部分抑制EBI与牛脑β-微管蛋白上的半胱氨酸残基交联形成β^s。长春花碱。这与之前用猪脑微管蛋白获得的发现一致。在多种真菌中，对根瘤菌素和安丝菌素 P-3（一种美登木素生物碱）的抗性不会与对苯并咪唑的抗性交叉。这也与上述结果一致，因为已知苯并咪唑与秋水仙碱位点结合。在构巢曲霉中，测定了野生型（根瘤素/美登素敏感）和根瘤素（和美登素）抗性菌株的β-微管蛋白的氨基酸序列。它们的序列差异仅在于第100个氨基酸；敏感微管蛋白具有Asn^<100>，而抗性微管蛋白具有Ile^<100>。有趣的是，粟酒裂殖酵母和酿酒酵母的β-微管蛋白中分别含有Ile^<100>和Val^<100>，并且它们对根瘤菌素不敏感。通过定点诱变将这些氨基酸替换为Asn^<100>赋予了根瘤素敏感性。另一方面，修饰根瘤素的侧链结构以研究与微管蛋白结合的结构要求。制备了多种衍生物，其中选择一种衍生物用于微管蛋白的光亲和标记。同样，也从美登木素生物碱制备了另一种用于对根瘤素/美登素位点进行光亲和标记的衍生物。

This research program was aimed to understand the interaction between tubulin and the mitosis inhibitors, and also to clarify structure and function of tubulin. Previously we have demonstrated with porcine brain tubulin that there exists the rhizoxin/maytansine binding site on tubulin which is different from those for colchicine and for vinblastine.In this research program, we could show the followings: EBI cross-linking to cystein residues on bovine brain beta-tubulin to form beta^s was strongly inhibited by rhizoxin as maytansine, whereas it was not inhibited by colchicine and was only partially inhibited by vinblastine. This coincides with the previous finding obtained with porcine brain tubulin. In a variety of fungi, the resistance to rhizoxin and ansamitocin P-3 (a maytansinoid) did not cross to the resistance to benzimidazoles. This also agrees with the above results, since benzimidazoles are known to bind to the colchicine site. In Aspergillus nidulans, amino acid sequences of beta-tubulins were determined for wild type (rhizoxin/maytansine sensitive) and rhizoxin (and maytansine) resistant strains. The difference between their sequences lied only in the 100th amino acid; the sensitive tubulin had Asn^<100> and the resistant tubulin had Ile^<100>. Interestingly, Schizosacchromyces pombe and Saccharomyces cervisiae had Ile^<100> and Val^<100> respectively in their beta-tubulins, and they are insensitive to rhizoxin. Replacement of these amino acids to Asn^<100> by site-directed mutagenesis conferred rhizoxin sensitivity.On the other hand, the side chain structure of rhizoxin was modified to study the structural requirement for binding to tubulin. A variety of derivatives were prepared and among them, a derivative was chosen for photo-affinity labeling to tubulin. Likewise, another derivative for photo-affinity labeling to rhizoxin/maytansin site was prepared also from maytannsinoid.