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Affinity Screening of Purified GPCRs on the Automated Ligand Identification System (ALIS): Protein Expression, Characterization, and Screening Results for CXCR4

机译:在自动配体鉴定系统(ALIS)上的纯化GPCR的亲和筛选:CXCR4的蛋白质表达,表征和筛选结果

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The Automated Ligand Identification System (ALIS) is an affinity selection-based screening technology for identifying small molecule hits from combinatorial libraries binding to purified protein targets. The capabilities of ALIS can be extended to include membrane proteins, such as detergent-solubilized GPCRs (J. Biomol. Screening; 2006; 11:194-207). CXCR4 is a chemokine GPCR implicated in both oncology and as an HIV co-receptor. We engineered CXCR4 with epitope tags at the protein termini allowing receptor purification by metal-chelate affinity and/or anti-FLAGM1 affinity chromatography. The receptor was then expressed from a 293-EBNA cell line. Expression and activity was verified by flow cytometry, competition binding experiments, and FLIPR. We developed a 2-step purification method using cobalt-affinity followed by anti-FLAGM1 affinity chromatography. Since other assays for purified recombinant CXCR4 were not available, the ALIS system and known CXCR4 ligands were used to evaluate affinity-purified CXCR4 preps from differing detergent formulations. The purified CXCR4 receptor could be produced in sufficient quantity to screen over 2.75 million compounds. These screens resulted in discovery of new ligands that competed against known antagonists and were capable of antagonizing HIV-1 virus uptake in cell-based assays.
机译:自动配体识别系统(ALIS)是一种基于亲和选择的筛选技术,用于鉴定组合文库与纯化蛋白靶标的组合文库的次数。 Alis的能力可以扩展到包括膜蛋白质,例如洗涤剂溶解的GPCR(J. Biomol。筛选; 2006; 11:194-207)。 CXCR4是一种趋化因子GPCR,其涉及肿瘤学和HIV共同受体。我们在蛋白质末端的表位标签中设计了CXCR4,使受体通过金属 - 螯合亲和力和/或抗旗子亲和层析纯化。然后从293-EBNA细胞系中表达受体。通过流式细胞术,竞争结合实验和FLIPR验证表达和活性。我们开发了一种使用钴亲和的2步纯化方法,然后是抗旗子亲和层析。由于不可用的其他用于纯化的重组CXCR4的测定,因此使用Alis系统和已知的CXCR4配体来评估来自不同的洗涤剂制剂的亲和纯化的CXCR4制备方法。纯化的CXCR4受体可以以足够的量产生超过275万化合物的筛选。这些屏幕导致发现竞争着名的拮抗剂的新配体,并且能够在基于细胞的测定中拮抗HIV-1病毒吸收。

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