Screening potential P-glycoprotein inhibitors by combination of a detergent-free membrane protein extraction with surface plasmon resonance biosensor.
Screening potential P-glycoprotein inhibitors by combination of a detergent-free membrane protein extraction with surface plasmon resonance biosensor.
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无去污剂膜蛋白提取与表面等离子共振生物传感器相结合筛选潜在的 P-糖蛋白抑制剂
DOI：
10.1016/j.apsb.2022.03.016
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发表时间：
2022-07
影响因子：
14.5
通讯作者：
Chai, Yifeng
中科院分区：
文献类型：
--
作者：
Cao, Yuhong;Fang, Jiahao;Shi, Yiwei;Wang, Hui;Chen, Xiaofei;Liu, Yue;Zhu, Zhenyu;Cao, Yan;Hong, Zhanying;Chai, Yifeng
关键词：
P-糖蛋白（P-gp）在肿瘤细胞中的高表达可导致肿瘤细胞产生多药耐药（MDR），P-gp抑制剂与抗癌药物的联合应用已成为肿瘤治疗中逆转MDR的有效策略。本研究将表面等离子体共振（SPR）生物传感器与苯乙烯马来酸（SMA）聚合物膜蛋白（MPs）稳定技术相结合，建立了一种无标记、无洗涤剂的P-gp抑制剂筛选系统。首先，使用SMA聚合物从MCF-7/ADR细胞中提取P-gp以形成SMA脂质体（SMALPs）。然后将SMALPs固定在SPR生物传感器芯片上，建立P-gp抑制剂筛选系统，测定P-gp与小分子配体的亲和力。方法学考察表明，该筛选体系具有良好的特异性和稳定性。从50种天然产物中筛选出9种P-gp配体，并测定了它们与P-gp的亲和常数。体外细胞验证实验表明粉防己碱、防己诺林碱、白花前胡素B、新黄芩素和淫羊藿苷能显著增加MCF-7/ADR细胞对阿霉素（Adr）的敏感性。粉防己碱、白花前胡素B和新黄芩素可通过抑制P-gp的功能逆转MCF-7/ADR细胞的多药耐药。这是首次将SMALPs稳定策略应用于SPR分析系统。SMA聚合物可以将P-gp保留在天然脂双层的环境中，从而维持P-gp的正确构象和生理功能。该系统可以快速、准确地筛选出复合MP的小分子配体，并在不需要蛋白纯化的情况下获得复合MP与小分子配体之间的亲和性。将SMALPs固定在表面等离子体共振生物传感器芯片上，建立P-gp抑制剂筛选系统，并计算P-gp与配体的亲和力。
P-glycoprotein (P-gp) highly expressed in cancer cells can lead to multidrug resistance (MDR) and the combination of anti-cancer drugs with P-gp inhibitor has been a promising strategy to reverse MDR in cancer treatment. In this study, we established a label-free and detergent-free system combining surface plasmon resonance (SPR) biosensor with styrene maleic acid (SMA) polymer membrane proteins (MPs) stabilization technology to screen potential P-gp inhibitors. First, P-gp was extracted from MCF-7/ADR cells using SMA polymer to form SMA liposomes (SMALPs). Following that, SMALPs were immobilized on an SPR biosensor chip to establish a P-gp inhibitor screening system, and the affinity between P-gp and small molecule ligand was determined. The methodological investigation proved that the screening system had good specificity and stability. Nine P-gp ligands were screened out from 50 natural products, and their affinity constants with P-gp were also determined. The in vitro cell verification experiments demonstrated that tetrandrine, fangchinoline, praeruptorin B, neobaicalein, and icariin could significantly increase the sensitivity of MCF-7/ADR cells to Adriamycin (Adr). Moreover, tetrandrine, praeruptorin B, and neobaicalein could reverse MDR in MCF-7/ADR cells by inhibiting the function of P-gp. This is the first time that SMALPs-based stabilization strategy was applied to SPR analysis system. SMA polymer can retain P-gp in the environment of natural lipid bilayer and thus maintain the correct conformation and physiological functions of P-gp. The developed system can quickly and accurately screen small molecule ligands of complex MPs and obtain affinity between complex MPs and small molecule ligands without protein purification. SMALPs were immobilized on a surface plasmon resonance biosensor chip to establish a P-gp inhibitor screening system and calculated the affinity between P-gp and ligands.
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影响因子：
4
作者：
Jamshad M;Charlton J;Lin YP;Routledge SJ;Bawa Z;Knowles TJ;Overduin M;Dekker N;Dafforn TR;Bill RM;Poyner DR;Wheatley M
通讯作者：
Wheatley M
影响因子：
3.5
作者：
Al-Majdoub ZM;Achour B;Couto N;Howard M;Elmorsi Y;Scotcher D;Alrubia S;El-Khateeb E;Vasilogianni AM;Alohali N;Neuhoff S;Schmitt L;Rostami-Hodjegan A;Barber J
通讯作者：
Barber J
影响因子：
2.9
作者：
Logez, Christel;Damian, Marjorie;Baneres, Jean-Louis
通讯作者：
Baneres, Jean-Louis
影响因子：
4.7
作者：
Maynard, Jennifer A;Lindquist, Nathan C;Sutherland, Jamie N;Lesuffleur, Antoine;Warrington, Arthur E;Rodriguez, Moses;Oh, Sang-Hyun
通讯作者：
Oh, Sang-Hyun
影响因子：
3.4
作者：
Gulamhussein, Aiman A.;Uddin, Romez;Rothnie, Alice J.
通讯作者：
Rothnie, Alice J.