Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites.
Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites.
复制标题
DOI：
10.1016/s0021-9258(17)44880-0
复制
发表时间：
1990-10
期刊：
影响因子：
--
通讯作者：
R. Bai;George R. Pettit;E. Hamel
中科院分区：
文献类型：
--
作者：
R. Bai;George R. Pettit;E. Hamel
Dolastatin 10是一种来自海洋动物的强效抗有丝分裂肽，能强烈抑制微管组装、微管蛋白依赖性GTP水解和长春花生物碱与微管蛋白的结合。在研究[3H]长春新碱与蛋白质结合的过程中，以长春新碱作为竞争性抑制的对照（Ki值，6.6微米），我们发现大环内酯类抗有丝分裂药物美坦辛和根瘤素也是竞争性抑制剂（Ki值，3.1和12微米）。Dolastatin 10和一种不相关的抗有丝分裂肽phomopsin A是更有效但非竞争性的抑制剂（Ki值为1.4和2.8微米）。由于美坦辛和vinblastine在较小程度上干扰了微管蛋白上的核苷酸交换，因此所有药物都被检查了对可交换GTP位点上核苷酸相互作用的影响。根瘤素的作用介于长春碱和美坦辛之间。两种多肽对放射性标记GTP与微管蛋白结合的抑制作用甚至比美坦辛更强，但没有药物取代微管蛋白上的核苷酸。评价药物对微管蛋白秋水仙碱结合活性的稳定作用。肽阻止了这种活性的丧失，长春碱提供了部分保护，而根毒素和美坦辛不能稳定微管蛋白。dolastatin 10的三肽段也有效抑制微管蛋白聚合和GTP水解。该三肽对长春新碱结合和核苷酸交换均无明显抑制作用，对秋水仙碱结合也无稳定作用。这些发现是合理的，根据一个模型，两个不同的药物结合位点在物理上彼此接近，并在β -微管蛋白上的可交换GTP位点。
Dolastatin 10, a potent antimitotic peptide from a marine animal, strongly inhibits microtubule assembly, tubulin-dependent GTP hydrolysis, and the binding of vinca alkaloids to tubulin. In studies of the binding of [3H]vincristine to the protein, with vinblastine as a control for competitive inhibition (Ki, 6.6 microM), we found that the macrolide antimitotic agents maytansine and rhizoxin were also competitive inhibitors (Ki values, 3.1 and 12 microM). Dolastatin 10 and an unrelated peptide antimitotic, phomopsin A, were more potent but noncompetitive inhibitors (Ki values, 1.4 and 2.8 microM). Since maytansine and, to a much lesser extent, vinblastine interfere with nucleotide exchange on tubulin, all drugs were examined for effects on nucleotide interactions at the exchangeable GTP site. Rhizoxin had effects intermediate between those of vinblastine and maytansine. Both peptides inhibited binding of radiolabeled GTP to tubulin even more strongly than did maytansine, but no drug displaced nucleotide from tubulin. The drugs were evaluated for stabilizing effects on the colchicine binding activity of tubulin. The peptides prevented loss of this activity, and vinblastine provided partial protection, while rhizoxin and maytansine did not stabilize tubulin. A tripeptide segment of dolastatin 10 also effectively inhibits tubulin polymerization and GTP hydrolysis. The tripeptide did not significantly inhibit either vincristine binding or nucleotide exchange, nor did it stabilize colchicine binding. These findings are rationalized in terms of a model with two distinct drug binding sites in close physical proximity to each other and to the exchangeable GTP site on beta-tubulin.