Raccor Brianne S, Vogt Andreas, Sikorski Rachel P, Madiraju Charitha, Balachandran Raghavan, Montgomery Kia, Shin Youseung, Fukui Yoshikazu, Jung Won-Hyuk, Curran Dennis P, Day Billy W
Departments of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Mol Pharmacol. 2008 Mar;73(3):718-26. doi: 10.1124/mol.107.042598. Epub 2007 Dec 11.
Compounds that bind to microtubules (MTs) and alter their dynamics are highly sought as a result of the clinical success of paclitaxel and docetaxel. The naturally occurring compound (-)-dictyostatin binds to MTs, causes cell cycle arrest in G(2)/M at nanomolar concentrations, and retains antiproliferative activity in paclitaxel-resistant cell lines, making dictyostatin an attractive candidate for development as an antineoplastic agent. In this study, we examined a series of dictyostatin analogs to probe biological and biochemical structure-activity relationships. We used a high-content multiparameter fluorescence-based cellular assay for MT morphology, chromatin condensation, mitotic arrest, and cellular toxicity to identify regions of dictyostatin that were essential for biological activity. Four analogs (6-epi-dictyostatin, 7-epi-dictyostatin, 16-normethyldictyostatin, and 15Z,16-normethyldictyostatin) retained low nanomolar activity in the cell-based assay and were chosen for analyses with isolated tubulin. All four compounds were potent inducers of MT assembly. Equilibrium binding constant (K(i)) determinations using [(14)C]epothilone B, which has a 3-fold higher affinity for the taxoid binding site than paclitaxel, indicated that 6-epi-dictyostatin and 7-epi-dictyostatin displaced [(14)C]epothilone B with K(i) values of 480 and 930 nM, respectively. 16-Normethyldictyostatin and 15Z,16-normethyldictyostatin had reduced affinity (K(i) values of 4.55 and 4.47 muM, respectively), consistent with previous reports showing that C16-normethyldictyostatin loses potency in paclitaxel-resistant cell lines that have a Phe270-to-Val mutation in the taxoid binding site of beta-tubulin. Finally, we developed a set of quantitative structure-activity relationship equations correlating structures with antiproliferative activity. The equations accurately predicted biological activity and will help in the design of future analogs.
由于紫杉醇和多西他赛在临床上取得的成功，能够与微管（MT）结合并改变其动力学的化合物备受关注。天然存在的化合物（-）-网抑素能与微管结合，在纳摩尔浓度下使细胞周期停滞于G（2）/M期，并且在耐紫杉醇的细胞系中保持抗增殖活性，这使得网抑素成为一种有吸引力的抗肿瘤药物开发候选物。在本研究中，我们研究了一系列网抑素类似物，以探究生物学和生物化学结构-活性关系。我们使用基于多参数荧光的高内涵细胞分析法来检测微管形态、染色质凝聚、有丝分裂停滞和细胞毒性，以确定网抑素中对生物活性至关重要的区域。四种类似物（6-表-网抑素、7-表-网抑素、16-去甲基网抑素和15Z,16-去甲基网抑素）在基于细胞的分析中保持低纳摩尔活性，并被选用于与分离的微管蛋白进行分析。所有四种化合物都是微管组装的有效诱导剂。使用对紫杉烷结合位点的亲和力比紫杉醇高3倍的[（14）C]埃坡霉素B进行平衡结合常数（K（i））测定，结果表明6-表-网抑素和7-表-网抑素取代[（14）C]埃坡霉素B的K（i）值分别为480和930 nM。16-去甲基网抑素和15Z,16-去甲基网抑素的亲和力降低（K（i）值分别为4.55和4.47 μM），这与先前的报道一致，即16-去甲基网抑素在β-微管蛋白的紫杉烷结合位点发生苯丙氨酸270到缬氨酸突变的耐紫杉醇细胞系中失去效力。最后，我们建立了一组将结构与抗增殖活性相关联的定量结构-活性关系方程。这些方程准确地预测了生物活性，并将有助于未来类似物的设计。
