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首页> 外文期刊>Journal of mass spectrometry: JMS >Competitive mass spectrometry binding assay for characterization of three binding sites of tubulin
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Competitive mass spectrometry binding assay for characterization of three binding sites of tubulin

机译：竞争质谱结合测定法表征微管蛋白的三个结合位点

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Tubulin is an attractive and established target for anticancer therapy. To date, the only method to determine the binding of inhibitor to tubulin has been competitive radioligand binding assays. We developed a non-radioactive mass spectrometry (MS) binding assay to study the tubulin binding of colchicine, vinblastine and paclitaxel and to identify which of these three binding sites that a novel inhibitor binds. The method involves a very simple step of separating the unbound ligand from macromolecules using ultrafiltration. The unbound ligand in the filtrate can be accurately determined using highly sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) mode. The assay was validated using podophyllotoxin, vincristine and docetaxel, drugs that compete to the colchicine-, vinblastine- and paclitaxel-binding sites in tubulin, respectively. This competitive binding assay allowed the reliable detection of interactions of these drugs with three binding sites on tubulin. This method was subsequently applied to determine the tubulin-binding site of 4-substituted methoxylbenzoyl-aryl-thiazoles (SMART-H), a potent antitubulin agent developed in our laboratory. The results indicated that SMART-H specifically and reversibly bound only to the colchicine-binding site, but not to vinblastine- or paclitaxel sites. This new non-radioligand binding method to determine the binding site on tubulin will function as a useful tool to study the binding sites of tubulin inhibitors.
机译：微管蛋白是抗癌治疗的有吸引力且确定的靶标。迄今为止，确定抑制剂与微管蛋白结合的唯一方法是竞争性放射性配体结合测定法。我们开发了一种非放射性质谱（MS）结合测定法，以研究秋水仙碱，长春碱和紫杉醇的微管蛋白结合，并确定新型抑制剂结合的这三个结合位点中的哪一个。该方法涉及使用超滤从大分子中分离未结合配体的非常简单的步骤。可以使用多反应监测（MRM）模式的高灵敏度和特异性液相色谱串联质谱（LC-MS / MS）方法，准确确定滤液中未结合的配体。使用鬼臼毒素，长春新碱和多西他赛（分别与微管蛋白中的秋水仙碱，长春碱和紫杉醇结合位点竞争的药物）验证了该测定法。这种竞争性结合测定可以可靠地检测这些药物与微管蛋白上三个结合位点的相互作用。该方法随后用于确定4-取代的甲氧基苯甲酰基-芳基-噻唑（SMART-H）的微管蛋白结合位点，这是我们实验室开发的一种有效的抗微管蛋白剂。结果表明，SMART-H仅可逆地仅与秋水仙碱结合位点结合，而不与长春碱或紫杉醇位点结合。确定微管蛋白上结合位点的这种新的非放射性配体结合方法将作为研究微管蛋白抑制剂的结合位点的有用工具。

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