Benzimidazoles, potent anti-mitotic drugs: Substrates for the P-glycoprotein transporter in multidrug-resistant cells

Abstract

P-glycoprotein is thought 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 made 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 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.