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

本研究旨在了解微管蛋白与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。先前我们用猪脑微管蛋白证实了微管蛋白上存在与秋水仙碱和长春花碱不同的根毒素/美丹氨酸结合位点。本研究结果表明：EBI与牛脑β -微管蛋白上的半胱氨酸残基交联形成β ^s受到根毒素如美塔辛的强烈抑制，而秋水仙碱对其无抑制作用，长春花碱仅对其有部分抑制作用。这与先前对猪脑微管蛋白的研究结果一致。在多种真菌中，对根毒素和抗菌肽P-3（一种美坦素类）的抗性没有交叉到对苯并咪唑的抗性。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。测定了野生型（根毒素/美坦辛敏感）和耐根毒素（和美坦辛）菌株的β -微管蛋白氨基酸序列。它们序列之间的差异只在于第100个氨基酸；敏感微管蛋白Asn^<100>，耐药微管蛋白Ile^<100>。有趣的是，裂殖菌（Schizosacchromyces pombe）和酿酒菌（Saccharomyces cervisae）的β -微管蛋白分别为Ile^<100>和Val^<100>，它们对根毒素不敏感。通过定点诱变将这些氨基酸替换为Asn^<100>，赋予了根毒素敏感性。另一方面，对根毒素侧链结构进行修饰，研究其与微管蛋白结合的结构要求。制备了多种衍生物，并从中选择了一种衍生物用于微管蛋白的光亲和标记。同样地，从类美丹素制备了另一种用于根毒素/美丹素位点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.