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.
J Pharm Sci. 2024 Jul;113(7):1960-1974. doi: 10.1016/j.xphs.2024.03.014. Epub 2024 Mar 23.
The efficacy of many cancer drugs is hindered by P-glycoprotein (Pgp), a cellular pump that removes drugs from cells. To improve chemotherapy, drugs capable of evading Pgp must be developed. Despite similarities in structure, vinca alkaloids (VAs) show disparate Pgp-mediated efflux ratios. ATPase activity and binding affinity studies show at least two binding sites for the VAs: high- and low-affinity sites that stimulate and inhibit the ATPase activity rate, respectively. The affinity for ATP from the ATPase kinetics curve for vinblastine (VBL) at the high-affinity site was 2- and 9-fold higher than vinorelbine (VRL) and vincristine (VCR), respectively. Conversely, VBL had the highest K (ATP) for the low-affinity site. The dissociation constants (Ks) determined by protein fluorescence quenching were in the order VBL < VRL< VCR. The order of the Ks was reversed at higher substrate concentrations. Acrylamide quenching of protein fluorescence indicate that the VAs, either at 10 µM or 150 µM, predominantly maintain Pgp in an open-outward conformation. When 3.2 mM AMPPNP was present, 10 µM of either VBL, VRL, or VCR cause Pgp to shift to an open-outward conformation, while 150 µM of the VAs shifted the conformation of Pgp to an intermediate orientation, between opened inward and open-outward. However, the conformational shift induced by saturating AMPPNP and VCR condition was less than either VBL or VRL in the presence of AMPPNP. At 150 µM, atomic force microscopy (AFM) revealed that the VAs shift Pgp population to a predominantly open-inward conformation. Additionally, STDD NMR studies revealed comparable groups in VBL, VRL, and VCR are in contact with the protein during binding. Our results, when coupled with VAs-microtubule structure-activity relationship studies, could lay the foundation for developing next-generation VAs that are effective as anti-tumor agents. A model that illustrates the intricate process of Pgp-mediated transport of the VAs is presented.
许多癌症药物的疗效受到 P-糖蛋白（Pgp）的阻碍，Pgp 是一种从细胞中清除药物的细胞泵。为了改善化疗，必须开发能够逃避 Pgp 的药物。尽管结构相似，但长春碱类药物（VAs）显示出不同的 Pgp 介导的外排比率。ATPase 活性和结合亲和力研究表明，VAs 至少有两个结合位点：高亲和性和低亲和性位点，分别刺激和抑制 ATPase 活性速率。从高亲和力位点的长春碱（VBL）ATPase 动力学曲线得到的 ATP 亲和力分别比长春新碱（VRL）和长春碱（VCR）高 2 倍和 9 倍。相反，VBL 对低亲和力位点的 K（ATP）最高。通过蛋白质荧光猝灭测定的解离常数（Ks）的顺序为 VBL<VRL<VCR。在较高的底物浓度下，Ks 的顺序发生逆转。丙烯酰胺猝灭蛋白质荧光表明，VAs 无论是在 10 μM 还是 150 μM 下，主要使 Pgp 保持开放外向构象。当存在 3.2 mM AMPPNP 时，10 μM 的 VBL、VRL 或 VCR 使 Pgp 转变为开放外向构象，而 150 μM 的 VAs 将 Pgp 的构象转变为中间取向，介于开放内向和开放外向之间。然而，在存在 AMPPNP 的情况下，与 VBL 或 VRL 相比，饱和 AMPPNP 和 VCR 条件诱导的构象变化较小。在 150 μM 时，原子力显微镜（AFM）显示 VAs 将 Pgp 群体转变为主要的开放内向构象。此外，STD-NMR 研究表明，VBL、VRL 和 VCR 中的可比基团在结合过程中与蛋白质接触。当与 VAs-微管结构-活性关系研究相结合时，我们的结果可以为开发作为抗肿瘤剂有效的下一代 VAs 奠定基础。提出了一个说明 Pgp 介导的 VAs 转运复杂过程的模型。
