The effects of anthracycline drugs on the conformational distribution of mouse P-glycoprotein explains their transport rate differences.
The effects of anthracycline drugs on the conformational distribution of mouse P-glycoprotein explains their transport rate differences.
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DOI：
10.1016/j.bcp.2020.113813
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发表时间：
2020-04
影响因子：
5.8
通讯作者：
Roberts AG
中科院分区：
文献类型：
--
作者：
Nguyen PH;Sigdel KP;Schaefer KG;Mensah GAK;King GM;Roberts AG
P-糖蛋白（P-glycoprotein，Pgp）是一种ATP依赖的外排转运蛋白，通过从肿瘤组织中泵出多种化学性质的细胞毒药物，在肿瘤的耐药性中发挥重要作用。尽管对转运蛋白进行了大量研究，但驱动抗癌药物外排的分子特征尚未得到很好的理解。即使抗癌药物分子结构的细微差异也会导致其转运速率的巨大差异。为了揭示这些结构差异，本研究集中在两种密切相关的蒽环类药物柔红霉素（DNR）和阿霉素（DOX）与小鼠Pgp。虽然DNR仅与DOX的羟基官能团不同，但DNR的转运速率比DOX高4至5倍。它们都非竞争性地抑制Pgp介导的ATP水解低于基础水平。从动力学曲线中提取的Pgp介导的ATP水解的Km对于DOX比DNR低。然而，通过荧光猝灭测定的这些药物的解离常数（KD）几乎相同。丙烯酰胺淬灭Pgp色氨酸荧光以探测Pgp的三级结构，表明DNR将Pgp转变为“闭合”构象，而DOX将Pgp转变为“中间”构象。这些药物对Pgp在脂质双层中构象分布的影响也通过原子力显微镜（AFM）进行了研究。AFM图像分析表明，柔红霉素和阿霉素引起的Pgp的构象分布的明显和显着的变化。结合上述结果，建立了蒽环类药物通过Pgp转运的构象分布模型。
P-glycoprotein (Pgp) is an ATP-dependent efflux transporter and plays a major role in anti-cancer drug resistance by pumping a chemically diverse range of cytotoxic drugs from cancerous tumors. Despite numerous studies with the transporter, the molecular features that drive anti-cancer drug efflux are not well understood. Even subtle differences in the anti-cancer drug molecular structure can lead to dramatic differences in their transport rates. To unmask these structural differences, this study focused on two closely-related anthracycline drugs, daunorubicin (DNR), and doxorubicin (DOX), with mouse Pgp. While only differing by a single hydroxyl functional group, DNR has a 4 to 5-fold higher transport rate than DOX. They both non-competitively inhibited Pgp-mediated ATP hydrolysis below basal levels. The Km of Pgp-mediated ATP hydrolysis extracted from the kinetics curves was lower for DOX than DNR. However, the dissociation constants (KDs) for these drugs determined by fluorescence quenching were virtually identical. Acrylamide quenching of Pgp tryptophan fluorescence to probe the tertiary structure of Pgp suggested that DNR shifts Pgp to a “closed” conformation, while DOX shifts Pgp to an “intermediate” conformation. The effects of these drugs on the Pgp conformational distributions in a lipid bilayer were also examined by atomic force microscopy (AFM). Analysis of AFM images revealed that DNR and DOX cause distinct and significant shifts in the conformational distribution of Pgp. The results were combined to build a conformational distribution model for anthracycline transport by Pgp.
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