Department of Pharmaceutical and Biomedical Science, University of Georgia, Athens, GA 30602, USA.
Department of Physics and Astronomy, University of Missouri, Columbia, MO 65211, USA.
Int J Mol Sci. 2023 Nov 7;24(22):16058. doi: 10.3390/ijms242216058.
P-glycoprotein (Pgp) plays a pivotal role in drug bioavailability and multi-drug resistance development. Understanding the protein's activity and designing effective drugs require insight into the mechanisms underlying Pgp-mediated transport of xenobiotics. In this study, we investigated the drug-induced conformational changes in Pgp and adopted a conformationally-gated model to elucidate the Pgp-mediated transport of camptothecin analogs (CPTs). While Pgp displays a wide range of conformations, we simplified it into three model states: 'open-inward', 'open-outward', and 'intermediate'. Utilizing acrylamide quenching of Pgp fluorescence as a tool to examine the protein's tertiary structure, we observed that topotecan (TPT), SN-38, and irinotecan (IRT) induced distinct conformational shifts in the protein. TPT caused a substantial shift akin to AMPPNP, suggesting ATP-independent 'open-outward' conformation. IRT and SN-38 had relatively moderate effects on the conformation of Pgp. Experimental atomic force microscopy (AFM) imaging supports these findings. Further, the rate of ATPase hydrolysis was correlated with ligand-induced Pgp conformational changes. We hypothesize that the separation between the nucleotide-binding domains (NBDs) creates a conformational barrier for substrate transport. Substrates that reduce the conformational barrier, like TPT, are better transported. The affinity for ATP extracted from Pgp-mediated ATP hydrolysis kinetics curves for TPT was about 2-fold and 3-fold higher than SN-38 and IRT, respectively. On the contrary, the dissociation constants () determined by fluorescence quenching for these drugs were not significantly different. Saturation transfer double difference (STDD) NMR of TPT and IRT with Pgp revealed that similar functional groups of the CPTs are accountable for Pgp-CPTs interactions. Efforts aimed at modifying these functional groups, guided by available structure-activity relationship data for CPTs and DNA-Topoisomerase-I complexes, could pave the way for the development of more potent next-generation CPTs.
P-糖蛋白（Pgp）在药物生物利用度和多药耐药性发展中起着关键作用。了解蛋白质的活性并设计有效的药物需要深入了解 Pgp 介导的外源性物质转运的机制。在这项研究中，我们研究了药物诱导的 Pgp 构象变化，并采用构象门控模型阐明了喜树碱类似物（CPTs）的 Pgp 介导转运。虽然 Pgp 表现出广泛的构象，但我们将其简化为三种模型状态：“开放向内”、“开放向外”和“中间”。利用 Pgp 荧光的丙烯酰胺猝灭来检查蛋白质的三级结构，我们观察到拓扑替康（TPT）、SN-38 和伊立替康（IRT）在蛋白质中引起了明显的构象变化。TPT 引起了类似于 AMPPNP 的大幅度变化，表明 ATP 非依赖性“开放向外”构象。IRT 和 SN-38 对 Pgp 构象的影响相对较小。实验原子力显微镜（AFM）成像支持了这些发现。此外，ATP 水解的速率与配体诱导的 Pgp 构象变化相关。我们假设核苷酸结合域（NBDs）之间的分离为底物转运创造了构象障碍。降低构象障碍的底物，如 TPT，更容易转运。从 Pgp 介导的 ATP 水解动力学曲线中提取的 TPT 对 ATP 的亲和力约为 SN-38 和 IRT 的 2 倍和 3 倍。相反，荧光猝灭法测定这些药物的解离常数（）没有显著差异。与 Pgp 的 TPT 和 IRT 的饱和转移双差（STDD）NMR 显示，CPTs 的相似功能基团负责 Pgp-CPTs 相互作用。根据 CPTs 和 DNA-拓扑异构酶 I 复合物的现有结构-活性关系数据，努力修饰这些功能基团，可能为开发更有效的下一代 CPTs 铺平道路。
