Nare B, Liu Z, Prichard R K, Georges E
Institute of Parasitology, McGill University, Ste. Anne de Bellevue, Québec, Canada.
Biochem Pharmacol. 1994 Dec 16;48(12):2215-22. doi: 10.1016/0006-2952(94)00427-7.
P-glycoprotein is though to mediate the energy-dependent efflux of many structurally and functionally unrelated lipophilic compounds. Presently, the molecular mechanism underlying the binding and efflux of drugs by P-glycoprotein is not well understood. However, it has been suggested that two planar benzene ring structures and a cationic charge are commonly found in many drugs that interact with P-glycoprotein. The benzimidazoles (BZs) are potent anti-tumour, anti-fungal and anti-parasitic agents, whose mode of action is thought to result from their inhibition of microtubule functions. Although other classes of microtubule inhibitors, such as colchicine and vinblastine, have been studied extensively with respect to their interaction and efflux by P-glycoprotein, the BZ group of drugs has not been characterized. In this study, we have characterized the interaction of BZ with multidrug-resistant cells and found that resistant cells accumulated substantially less BZ compared with drug-sensitive cells. Furthermore, BZ was more toxic to sensitive than to drug-resistant cells, suggesting that BZ is likely to be a substrate for the P-glycoprotein drug efflux pump. In addition, we used a photoactive analogue of BZ ([125I]ASA-BZ) to demonstrate a direct binding between BZ and P-glycoprotein. Results showing that a molar excess of vinblastine, unmodified BZ, verapamil and rhodamine 123, but not colchicine, inhibited the photoaffinity labelling of P-glycoprotein by [125I]ASA-BZ confirmed the binding specificity of BZ to P-glycoprotein. Protease digestion of [125I]ASA-BZ photoaffinity labelled P-glycoprotein yielded two peptides that were similar to those obtained with other P-glycoprotein-associated drugs, e.g. azidopine and iodoaryl azidoprazosin. Taken together, these results demonstrate a direct and specific interaction between P-glycoprotein and BZ in a manner that is probably similar to other previously characterized P-glycoprotein-associated drugs.
P-糖蛋白被认为可介导许多结构和功能不相关的亲脂性化合物的能量依赖性外排。目前，P-糖蛋白结合和外排药物的分子机制尚未完全清楚。然而，有人提出，在许多与P-糖蛋白相互作用的药物中通常会发现两个平面苯环结构和一个阳离子电荷。苯并咪唑类（BZs）是有效的抗肿瘤、抗真菌和抗寄生虫药物，其作用方式被认为是由于它们对微管功能的抑制。尽管其他类别的微管抑制剂，如秋水仙碱和长春碱，已就它们与P-糖蛋白的相互作用和外排进行了广泛研究，但BZ类药物尚未得到表征。在本研究中，我们表征了BZ与多药耐药细胞的相互作用，发现耐药细胞积累的BZ比药物敏感细胞少得多。此外，BZ对敏感细胞的毒性比对耐药细胞的毒性更大，这表明BZ可能是P-糖蛋白药物外排泵的底物。此外，我们使用了BZ的光活性类似物（[125I]ASA-BZ）来证明BZ与P-糖蛋白之间的直接结合。结果表明，摩尔过量的长春碱、未修饰的BZ、维拉帕米和罗丹明123，但不是秋水仙碱，抑制了[125I]ASA-BZ对P-糖蛋白的光亲和标记，证实了BZ与P-糖蛋白的结合特异性。对[125I]ASA-BZ光亲和标记的P-糖蛋白进行蛋白酶消化产生了两个肽段，它们与用其他与P-糖蛋白相关的药物（如叠氮平碘芳基叠氮哌唑嗪）获得的肽段相似。综上所述，这些结果表明P-糖蛋白与BZ之间存在直接和特异性相互作用，其方式可能与其他先前表征的与P-糖蛋白相关的药物相似。
