Department of Pharmaceutics, University of Minnesota, 308 Harvard Street SE, Minneapolis, MN 55455, USA.
Drug Metab Dispos. 2011 Sep;39(9):1478-85. doi: 10.1124/dmd.111.039370. Epub 2011 May 31.
Breast cancer resistance protein (BCRP), an ATP-dependent efflux transporter, confers drug resistance to many chemotherapy agents. BCRP is overexpressed in tumors exposed to an acidic environment; therefore, it is important to establish the effect of low pH on BCRP transport activity. It has recently been reported that BCRP transports substrates more efficiently in an acidic microenvironment. In the study presented here, we examine the pH dependence of BCRP using methothrexate (MTX), pemetrexed (PMX), and estrone sulfate (ES) as model substrates. Our study revealed an increase of approximately 40-fold in the BCRP-mediated transport of PMX and MTX when the pH was decreased from 7.4 to 5.5. In contrast, only a 2-fold increase was observed for ES. These results indicate a mechanism of transport that is directly dependent on the effective ionization state of the substrates and BCRP. For ES, which retains a constant ionization state throughout the applied pH, the observed mild increase in activity is attributable to the overall changes in the effective ionization state and conformation of BCRP. For MTX and PMX, the marked increase in BCRP transport activity was likely due to the change in ionization state of MTX and PMX at lowered pH and their intermolecular interactions with BCRP. To further rationalize the molecular basis of the pH dependence, molecular modeling and docking studies were carried out using a homology model of BCRP, which has previously been closely examined in structural and site-directed mutagenesis studies (Am J Physiol Cell Physiol 299:C1100-C1109, 2010). On the basis of docking studies, all model compounds were found to associate with arginine 482 (Arg482) by direct salt-bridge interactions via their negatively charged carboxylate or sulfate groups. However, at lower pH, protonated MTX and PMX formed an additional salt-bridge interaction between their positively charged moieties and the nearby negatively charged aspartic acid 477 (Asp477) carboxylate side chain. The formation of this "salt-bridge triad" is expected to increase the overall electrostatic interactions between MTX and PMX with BCRP, which can form a rational basis for the pH dependence of the observed enhanced binding selectivity and transport activity. Removal of Arg482 in site-directed mutagenesis studies eliminated this pH dependence, which lends further support to our binding model. These results shed light on the importance of electrostatic interactions in transport activity and may have important implications in the design of ionizable chemotherapeutics intended for tumors in the acidic microenvironment.
乳腺癌耐药蛋白（BCRP）是一种依赖于 ATP 的外排转运体，它使许多化疗药物产生耐药性。BCRP 在暴露于酸性环境的肿瘤中过度表达；因此，确定低 pH 值对 BCRP 转运活性的影响非常重要。最近有报道称，BCRP 在酸性微环境中更有效地转运底物。在本研究中，我们使用氨甲蝶呤（MTX）、培美曲塞（PMX）和雌酮硫酸酯（ES）作为模型底物，研究了 BCRP 的 pH 依赖性。我们的研究表明，当 pH 从 7.4 降低到 5.5 时，BCRP 介导的 PMX 和 MTX 的转运增加了约 40 倍。相比之下，ES 的增加只有 2 倍。这些结果表明，转运机制直接依赖于底物和 BCRP 的有效离解状态。对于 ES，其在整个应用 pH 值范围内保持恒定的离解状态，观察到的活性轻度增加归因于 BCRP 的有效离解状态和构象的整体变化。对于 MTX 和 PMX，BCRP 转运活性的显著增加可能是由于 MTX 和 PMX 在较低 pH 值下的离解状态变化以及它们与 BCRP 的分子间相互作用所致。为了进一步阐明 pH 依赖性的分子基础，我们使用同源 BCRP 模型进行了分子建模和对接研究，该模型在结构和定点突变研究中已得到了密切研究（Am J Physiol Cell Physiol 299:C1100-C1109, 2010）。基于对接研究，所有模型化合物都被发现通过其带负电荷的羧基或硫酸盐与 Arg482（Arg482）形成直接盐桥相互作用。然而，在较低的 pH 值下，质子化的 MTX 和 PMX 之间形成了额外的盐桥相互作用，其带正电荷的部分与附近带负电荷的天冬氨酸 477（Asp477）羧基侧链之间形成了盐桥。形成这种“盐桥三联体”预计会增加 MTX 和 PMX 与 BCRP 之间的整体静电相互作用，这可以为观察到的增强的结合选择性和转运活性的 pH 依赖性提供合理的基础。定点突变研究中 Arg482 的去除消除了这种 pH 依赖性，这进一步支持了我们的结合模型。这些结果揭示了静电相互作用在转运活性中的重要性，并且可能对设计用于酸性微环境中的肿瘤的可电离化疗药物具有重要意义。
