PDF(Pubmed)
Abstract：
NanoLuc-mediated bioluminescence resonance energy transfer (NanoBRET) has gained popularity for its ability to homogenously measure ligand binding to G protein-coupled receptors (GPCRs), including the subfamily of chemokine receptors. These receptors, such as ACKR3, CXCR4, CXCR3, play a crucial role in the regulation of the immune system, are associated with inflammatory diseases and cancer, and are seen as promising drug targets. The aim of this study was to optimize NanoBRET-based ligand binding to NLuc-ACKR3 and NLuc-CXCR4 using different fluorescently labeled chemokine CXCL12 analogs and their use in a multiplex NanoBRET binding assay of two chemokine receptors at the same time. The four fluorescent CXCL12 analogs (CXCL12-AZD488, -AZD546, -AZD594, -AZD647) showed high-affinity saturable binding to both NLuc-ACKR3 and NLuc-CXCR4, with relatively low levels of non-specific binding. Additionally, the binding of all AZDye-labeled CXCL12s to Nluc receptors was inhibited by pharmacologically relevant unlabeled chemokines and small molecules. The NanoBRET binding assay for CXCL10-AZD488 binding to Nluc-CXCR3 was also successfully established and successfully employed for the simultaneous measurement of the binding of unlabeled small molecules to NLuc-CXCR3 and NLuc-CXCR4. In conclusion, multiplexing the NanoBRET-based competition binding assay is a promising tool for testing unlabeled (small) molecules against multiple GPCRs simultaneously.
摘要：
NanoLuc介导的生物发光共振能量转移（NanoBRET）因其能够均匀地测量配体与G蛋白偶联受体（GPCRs）的结合而受到欢迎。包括趋化因子受体亚家族.这些受体，如ACKR3，CXCR4，CXCR3，在免疫系统的调节中起着至关重要的作用，与炎症性疾病和癌症有关，并被视为有希望的药物靶标。这项研究的目的是使用不同的荧光标记的趋化因子CXCL12类似物优化基于NanoBRET的配体与NLuc-ACKR3和NLuc-CXCR4的结合，以及它们在同时两种趋化因子受体的多重NanoBRET结合测定中的用途。四种荧光CXCL12类似物(CXCL12-AZD488、-AZD546、-AZD594、-AZD647)显示与NLuc-ACKR3和NLuc-CXCR4的高亲和力可饱和结合,具有相对低水平的非特异性结合。此外,所有AZDye标记的CXCL12s与Nluc受体的结合被药理学相关的未标记趋化因子和小分子抑制。还成功建立了CXCL10-AZD488与Nluc-CXCR3结合的NanoBRET结合测定，并成功用于同时测量未标记的小分子与NLuc-CXCR3和NLuc-CXCR4的结合。总之,多路复用基于NanoBRET的竞争结合测定是用于同时针对多个GPCRs测试未标记(小)分子的有前途的工具。
