Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering , Tokyo Medical and Dental University (TMDU) , 2-3-10 Kandasurugada , Chiyoda-ku, Tokyo 101-0062 , Japan.
Graduate School of Medical and Dental Sciences , Tokyo Medical and Dental University (TMDU) , 1-5-45 Yushima , Bunkyo-ku, Tokyo 113-8510 , Japan.
Bioconjug Chem. 2019 May 15;30(5):1442-1450. doi: 10.1021/acs.bioconjchem.9b00182. Epub 2019 Apr 11.
A critical part of the development of CXCR4 modulators is to have a simple and sensitive assay system to complement the search by screening and evaluating the binding affinity. Herein, a NanoBRET assay system was developed, and its feasibility as a high-throughput screening tool for potent CXCR4 ligands was ascertained. TAMRA-Ac-TZ14011, a fluorescent-labeled CXCR4 antagonist, was adopted as a fluorescent acceptor of bioluminescent energy from N-terminally fused NanoLuc-CXCR4 stably expressed in CHO cells. The ratio of fluorescence at 620 nm to the luminescence at 460 nm represents the interaction between test compounds and CXCR4. We have demonstrated in the present study that the NanoBRET assay system is applicable for the evaluation of CXCR4 ligands using the combination of TAMRA-Ac-TZ14011 as an acceptor and NanoLuc tagged to CXCR4 as a bioluminescent donor expressed in living cells. IC values of known CXCR4 ligands were determined and found to be compatible with the values obtained by other existing and sensitive methods, such as SDF-1:3.2 nM, Ac-TZ14011:15.3 nM, and FC131:4.5 nM, which confirmed the feasibility of our system ( Z' values ≥0.5). The introduction of an IL-6 secretory signaling peptide (secNluc-CXCR4) further enhanced the expression and trafficking of the tagged receptor, which, in turn, increased the dynamic range of the NanoBRET assay system. Thus, we have successfully developed a NanoBRET system in living cells, which is simple, homogeneous, and useful in multiwell plate screening of potent CXCR4 ligands.
开发 CXCR4 调节剂的一个关键部分是拥有一个简单而灵敏的测定系统，以通过筛选和评估结合亲和力来补充搜索。本文中，我们开发了一种 NanoBRET 测定系统，并确定其作为高通量筛选强 CXCR4 配体的工具的可行性。荧光标记的 CXCR4 拮抗剂 TAMRA-Ac-TZ14011 被用作融合到稳定表达于 CHO 细胞的 N 端的 NanoLuc-CXCR4 的生物发光能量的荧光供体。620nm 处的荧光与 460nm 处的发光的比值代表了测试化合物与 CXCR4 之间的相互作用。我们在本研究中证明，该 NanoBRET 测定系统适用于使用 TAMRA-Ac-TZ14011 作为受体和标记到 CXCR4 的 NanoLuc 作为生物发光供体的组合来评估 CXCR4 配体，该供体表达于活细胞中。我们测定了已知 CXCR4 配体的 IC 值，并发现与其他现有和灵敏的方法（如 SDF-1：3.2nM、Ac-TZ14011：15.3nM 和 FC131：4.5nM）获得的值相兼容，这证实了我们的系统的可行性（Z' 值≥0.5）。引入 IL-6 分泌信号肽（secNluc-CXCR4）进一步增强了标记受体的表达和转运，从而增加了 NanoBRET 测定系统的动态范围。因此，我们已经成功地在活细胞中开发了一种 NanoBRET 系统，该系统简单、均一，并且适用于高通量筛选强 CXCR4 配体。
