Center of Excellent in Natural Products for Ageing and Chronic Diseases, Chulalongkorn University, Bangkok 10330, Thailand; Pharmaceutical Sciences and Technology Program, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand.
Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
Life Sci. 2022 Aug 15;303:120661. doi: 10.1016/j.lfs.2022.120661. Epub 2022 May 26.
The C-X-C chemokine-receptor type 4 (CXCR4) is an emerging target for cancer drug discovery due to its high expression in cancer cells. The present study aimed to produce CXCR4 overexpressing HEK293T cells for a non-radioactive binding assay as a platform to identify drug candidates targeting CXCR4.
HEK293T cells stably expressing human CXCR4 were constructed by transfection of CXCR4 plasmids from the human CXCR4 gene. The CXCR4 overexpressing HEK293T cells were obtained by fluorescence-activated sorting and verified by conducting the competition binding assay of a known CXCR4 inhibitor, AMD3100 (plerixafor), to determine the IC value against monoclonal anti-human CD184 (hCD184) antibody tagged with fluorescence probe, phycoerythrin (PE). The non-radioactive binding assay using CXCR4 overexpressing HEK293T cells and PE-anti hCD184 was applied as a platform for identifying the target of natural compounds that exhibited cytotoxicity against cancer cell lines.
The CXCR4 overexpressing HEK293T cells were produced with high expression (99.8%). The IC value of plerixafor determined by fluorescence tagged antibody-based competition assay using our developed cells agree with previously reported values using a radioligand binding assay. We observed no significant displacement of bound PE-anti-hCD184 by the test natural compounds which could be due to non-specific binding to other functional targets or organelles, low potency of the natural compounds, or binding to CXCR4 at deeper pockets.
The verified non-radioactive binding assay can serve as an alternative screening tool for anticancer lead compounds targeting CXCR4 and an essential tool for proof of mechanism study in the drug discovery.
趋化因子受体 4（CXCR4）在癌细胞中高表达，因此成为癌症药物发现的新兴靶点。本研究旨在构建过表达 CXCR4 的人胚肾 293T（HEK293T）细胞，用于非放射性结合测定，以此作为鉴定 CXCR4 靶向药物候选物的平台。
通过转染人 CXCR4 质粒构建稳定表达人 CXCR4 的 HEK293T 细胞。通过荧光激活细胞分选获得 CXCR4 过表达的 HEK293T 细胞，并通过已知的 CXCR4 抑制剂 AMD3100（plerixafor）与荧光探针藻红蛋白（PE）标记的单克隆抗人 CD184（hCD184）抗体的竞争结合测定来验证，以确定针对单克隆抗人 CD184（hCD184）抗体的 IC 值。使用 CXCR4 过表达的 HEK293T 细胞和 PE-抗 hCD184 的非放射性结合测定作为平台，用于鉴定对癌细胞系具有细胞毒性的天然化合物的靶标。
高表达（99.8%）的 CXCR4 过表达 HEK293T 细胞得以生成。使用我们开发的细胞进行的基于荧光标记抗体的竞争测定确定的 plerixafor 的 IC 值与使用放射性配体结合测定法先前报道的值一致。我们观察到测试的天然化合物没有显著置换结合的 PE-抗 hCD184，这可能是由于与其他功能靶点或细胞器的非特异性结合、天然化合物的低效力或与 CXCR4 更深的结合口袋结合。
验证的非放射性结合测定可作为针对 CXCR4 的抗癌先导化合物的替代筛选工具，也是药物发现中证明作用机制研究的重要工具。
