Fluorophore labeling enables imaging and evaluation of specific CXCR4-ligand interaction at the cell membrane for fluorescence-based screening

Development of CXCR4-specific ligands is an important issue in chemotherapy of HIV infection, cancer metastasis, and rheumatoid arthritis, and numerous potential ligands have been developed to date. However, it is difficult to assess their binding mode and specificity because of uncertainties in the structure of the CXCR4-ligand complexes. To address this problem, we have synthesized fluorophore labeled Ac-TZ14011, which is derived from T140, a powerful CXCR4 antagonist. Binding of Ac-TZ14011 to CXCR4 on the cell membrane was observed by fluorescence microscope, and analysis of the binding data produced IC50 values of several ligands comparable to those obtained in RI-based assays. This fluorescence-based assay is applicable to explore new pharmacophores of CXCR4-specific ligands with high-throughput screening and also to screening of the other GPCR binding ligands.

CXCR4特异性配体的开发是艾滋病病毒感染、癌症转移和类风湿性关节炎化学治疗中的一个重要问题，到目前为止已经开发了许多潜在的配体。然而，由于CXCR4 - 配体复合物结构的不确定性，很难评估它们的结合模式和特异性。为了解决这个问题，我们合成了荧光团标记的Ac - TZ14011，它源自T140，一种强效的CXCR4拮抗剂。通过荧光显微镜观察到Ac - TZ14011与细胞膜上的CXCR4结合，对结合数据的分析得出了几种配体的半数抑制浓度（IC50）值，与基于放射性同位素（RI）的检测方法所获得的值相当。这种基于荧光的检测方法适用于通过高通量筛选探索CXCR4特异性配体的新药效团，也适用于筛选其他G蛋白偶联受体（GPCR）结合配体。