Gapud Eric J, Bai Ruoli, Ghosh Arun K, Hamel Ernest
Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnostics, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
Mol Pharmacol. 2004 Jul;66(1):113-21. doi: 10.1124/mol.66.1.113.
Previous work has shown that laulimalide, a sponge-derived natural product, resembles paclitaxel in enhancing tubulin assembly and in its effects on cellular microtubules. The two compounds, however, seem to have distinct binding sites on tubulin polymer. Nearly equimolar amounts of tubulin, laulimalide, and paclitaxel are recovered from microtubules formed with both drugs. In the present study, we searched for differences between laulimalide and paclitaxel in their interactions with tubulin polymer. Laulimalide was compared with paclitaxel and epothilone A, a natural product that competes with paclitaxel in binding to microtubules, for assembly properties at different temperatures and for effects of GTP and microtubule-associated proteins on assembly. Although minor differences were observed among the three drugs, their overall effects were highly similar, except that aberrant assembly products were observed more frequently with paclitaxel and that the polymers formed with laulimalide and epothilone A were more stable at 0 degrees C. The most dramatic difference observed between laulimalide and epothilone A was that only laulimalide was able to enhance assembly synergistically with paclitaxel, as would be predicted if the two drugs bound at different sites in polymer. Because stoichiometric amounts of laulimalide and paclitaxel can cause extensive tubulin assembly, maximum synergy was observed at lower temperatures under reaction conditions in which each drug alone is relatively inactive. Laulimalide-induced assembly, like paclitaxel-induced assembly, was inhibited by drugs that inhibit tubulin assembly by binding at either the colchicine- or vinblastine-binding site. When radiolabeled GTP is present in a reaction mixture with either laulimalide or paclitaxel, nucleotide hydrolysis occurs with incorporation of radiolabeled GDP into polymer.
先前的研究表明，来源于海绵的天然产物劳利霉素在促进微管蛋白组装以及对细胞微管的作用方面与紫杉醇相似。然而，这两种化合物在微管蛋白聚合物上似乎具有不同的结合位点。从用这两种药物形成的微管中回收的微管蛋白、劳利霉素和紫杉醇的量几乎等摩尔。在本研究中，我们探寻了劳利霉素和紫杉醇在与微管蛋白聚合物相互作用方面的差异。将劳利霉素与紫杉醇以及埃坡霉素A进行比较，埃坡霉素A是一种在与微管结合方面与紫杉醇竞争的天然产物，比较它们在不同温度下的组装特性以及GTP和微管相关蛋白对组装的影响。尽管在这三种药物之间观察到了细微差异，但它们的总体效果高度相似，只是紫杉醇形成异常组装产物的频率更高，并且用劳利霉素和埃坡霉素A形成的聚合物在0摄氏度时更稳定。在劳利霉素和埃坡霉素A之间观察到的最显著差异是，只有劳利霉素能够与紫杉醇协同增强组装，这正如如果两种药物在聚合物中的不同位点结合所预测的那样。由于化学计量的劳利霉素和紫杉醇可导致广泛的微管蛋白组装，在每种药物单独相对无活性的反应条件下，在较低温度下观察到了最大协同作用。劳利霉素诱导的组装，如同紫杉醇诱导的组装一样，被通过在秋水仙碱或长春花碱结合位点结合而抑制微管蛋白组装的药物所抑制。当放射性标记的GTP存在于与劳利霉素或紫杉醇的反应混合物中时，会发生核苷酸水解，放射性标记的GDP掺入聚合物中。
