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Fluorescent Ligand Enables Target Engagement Studies for the Intracellular Allosteric Binding Site of the Chemokine Receptor CXCR2
ChemRxiv Pub Date : 2023-03-21 , DOI: 10.26434/chemrxiv-2023-d9d87
Max Huber 1 , Silas Wurnig 2 , Corinna Weiler 2 , Nicole Merten 2 , Lara Toy 1 , Evi Kostenis 2 , Finn Hansen 2 , Matthias Schiedel 1
Affiliation  

Fluorescently labeled ligands are versatile molecular tools to study G protein-coupled receptors (GPCRs) and can be used for a range of different applications, including bioluminescence resonance energy transfer (BRET) assays and fluorescence microscopy. Herein, we report the structure-based development of a fluorescent ligand targeting the intracellular allosteric binding site (IABS) of the CXC chemokine receptor 2 (CXCR2), a class A GPCR that has been pursued as a drug target in oncology and inflammation. Starting from the co-crystallized intracellular CXCR2 antagonist 00767013 (1), a tetramethylrhodamine (TAMRA)-labeled CXCR2 ligand was designed, synthesized, and tested for its suitability as fluorescent reporter to probe binding to the IABS of CXCR2. By means of these studies, we developed Mz438 (9) as a high affinity fluorescent CXCR2 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a non-isotopic and high-throughput manner. Further, we show that 9 can be used as a tool to visualize intracellular target engagement for CXCR2 via fluorescence microscopy. Thus, our small molecule-based fluorescent CXCR2 ligand 9 represents a promising tool for future studies of CXCR2 pharmacology.

中文翻译:

荧光配体使趋化因子受体 CXCR2 的细胞内变构结合位点的目标参与研究成为可能

荧光标记的配体是研究 G 蛋白偶联受体 (GPCR) 的多功能分子工具,可用于一系列不同的应用,包括生物发光共振能量转移 (BRET) 测定和荧光显微镜。在此,我们报告了针对 CXC 趋化因子受体 2 (CXCR2) 的细胞内变构结合位点 (IABS) 的荧光配体的基于结构的开发,CXC 趋化因子受体 2 (CXCR2) 是一种 A 类 GPCR,已被用作肿瘤学和炎症的药物靶点。从共结晶的细胞内 CXCR2 拮抗剂 00767013 (1) 开始,设计、合成了四甲基罗丹明 (TAMRA) 标记的 CXCR2 配体,并测试了其作为荧光报告基因的适用性,以探测与 CXCR2 IABS 的结合。通过这些研究,我们开发了 Mz438 (9) 作为高亲和力荧光 CXCR2 配体,以非同位素和高通量方式进行无细胞和基于细胞 NanoBRET 的结合研究。此外,我们表明 9 可以用作一种工具,通过荧光显微镜观察 CXCR2 的细胞内靶点参与。因此,我们基于小分子的荧光 CXCR2 配体 9 代表了未来 CXCR2 药理学研究的有前途的工具。
更新日期:2023-03-21
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