Bai R, Roach M C, Jayaram S K, Barkoczy J, Pettit G R, Ludueña R F, Hamel E
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Biochem Pharmacol. 1993 Apr 6;45(7):1503-15. doi: 10.1016/0006-2952(93)90051-w.
Dolastatin 10 is a potent antimitotic peptide isolated from the marine mollusk Dolabella auricularia. Four of its five residues are modified amino acids (in sequence, dolavaline, valine, dolaisoleuine, dolaproine, dolaphenine). Besides inhibiting tubulin polymerization, dolastatin 10 non-competitively inhibits vinca alkaloid binding to tubulin, inhibits nucleotide exchange and formation of the beta s cross-link, and stabilizes the colchicine binding activity of tubulin. To examine the mechanism of action of dolastatin 10 we prepared six chiral isomers, one tri- and one tetrapeptide segment, and one pentapeptide analog of dolastatin 10, all of which differ little from dolastatin 10 as inhibitors of tubulin polymerization. However, only two of the chiral isomers were similar to dolastatin 10 in their cytotoxicity for L1210 murine leukemia cells and in their effects on vinblastine binding, nucleotide exchange, beta s cross-link formation, and colchicine binding. These were isomer 2, with reversal of configuration at position C(19a) in the dolaisoleuine moiety, and isomer 19, with reversal of configuration at position C(6) in the dolaphenine moiety. The pentapeptides with reduced cytotoxicity and reduced effects on tubulin interactions with other ligands were all modified in the dolaproine moiety at positions C(9) and/or C(10). The tripeptide and tetrapeptide segments which inhibited polymerization but not ligand interactions were the amino terminal tripeptide (lacking dolaproine and dolaphenine) and the carboxyl terminal tetrapeptide (lacking dolavaline). We speculate that strong inhibition of other ligand interactions with tubulin requires stable peptide binding to tubulin (i.e. slow dissociation), but that inhibition of polymerization requires only rapid binding to tubulin.
多拉司他汀10是一种从海洋软体动物耳状芋螺中分离出的强效抗有丝分裂肽。其五个残基中有四个是修饰氨基酸（依次为多拉缬氨酸、缬氨酸、多拉异亮氨酸、多拉脯氨酸、多拉苯丙氨酸）。除了抑制微管蛋白聚合外，多拉司他汀10还非竞争性抑制长春花生物碱与微管蛋白的结合，抑制核苷酸交换和βs交联的形成，并稳定微管蛋白的秋水仙碱结合活性。为了研究多拉司他汀10的作用机制，我们制备了六种手性异构体、一个三肽和一个四肽片段以及一个多拉司他汀10的五肽类似物，所有这些在作为微管蛋白聚合抑制剂方面与多拉司他汀10差异不大。然而，只有两种手性异构体在对L1210小鼠白血病细胞的细胞毒性以及对长春碱结合、核苷酸交换、βs交联形成和秋水仙碱结合的影响方面与多拉司他汀10相似。它们是异构体2，在多拉异亮氨酸部分的C(19a)位置构型反转；以及异构体19，在多拉苯丙氨酸部分的C(6)位置构型反转。细胞毒性降低且对微管蛋白与其他配体相互作用影响降低的五肽在多拉脯氨酸部分的C(9)和/或C(10)位置均有修饰。抑制聚合但不抑制配体相互作用的三肽和四肽片段是氨基末端三肽（缺少多拉脯氨酸和多拉苯丙氨酸）和羧基末端四肽（缺少多拉缬氨酸）。我们推测，对微管蛋白与其他配体相互作用的强烈抑制需要肽与微管蛋白稳定结合（即解离缓慢），而抑制聚合仅需要与微管蛋白快速结合。
