CellFy: A Cell-Based Fragment Screen against C-Type Lectins.
CellFy: A Cell-Based Fragment Screen against C-Type Lectins.
复制标题
DOI：
10.1021/acschembio.8b00875
复制
发表时间：
2018-11
影响因子：
4
通讯作者：
J. Schulze;H. Baukmann;Robert Wawrzinek;Felix F. Fuchsberger;E. Specker;Jonas Aretz;M. Nazaré;C. Rademacher
中科院分区：
文献类型：
--
作者：
J. Schulze;H. Baukmann;Robert Wawrzinek;Felix F. Fuchsberger;E. Specker;Jonas Aretz;M. Nazaré;C. Rademacher
基于片段的药物发现是对传统高通量筛选的有力补充，特别是对于困难的靶标。由于所鉴定的配体的亲和力通常较低，筛选低分子量片段通常需要高度灵敏的生物物理方法。在这里，我们开发了一种基于细胞的片段筛选测定（cellFy），与溶液中的孤立靶点筛选相比，它可以在生理上更相关的环境中灵敏地识别片段命中。为此，采用了荧光标记的多价报告基因，能够直接测量低分子量片段的位移，而不需要酶促反应或受体激活。我们应用该技术来识别针对 C 型凝集素受体 (CLR) 家族的两个具有挑战性的靶标的命中：树突状细胞特异性细胞间粘附分子-3-抓取非整联蛋白 (DC-SIGN) 和 Langerin。这两种受体都参与病原体识别和免疫反应的启动，这使得它们成为免疫调节的有吸引力的靶标。由于其浅且亲水的主要结合位点，CLR 的命中鉴定具有挑战性，并且 CLR 的药物样配体很少。筛选片段库，然后进行命中验证，确定了几个有前途的候选者，可用于 DC-SIGN 的进一步片段进化。此外，还开发了一种多重检测格式，用于同时筛选多种 CLR，从而实现选择性反筛选。总体而言，这种敏感的基于细胞的片段筛选分析为快速鉴定生物活性片段（甚至是困难的靶标）提供了强大的工具。
Fragment-based drug discovery is a powerful complement to conventional high-throughput screening, especially for difficult targets. Screening low-molecular-weight fragments usually requires highly sensitive biophysical methods, because of the generally low affinity of the identified ligands. Here, we developed a cell-based fragment screening assay (cellFy) that allows sensitive identification of fragment hits in a physiologically more relevant environment, in contrast to isolated target screenings in solution. For this, a fluorescently labeled multivalent reporter was employed, enabling direct measurement of displacement by low-molecular-weight fragments without requiring enzymatic reactions or receptor activation. We applied this technique to identify hits against two challenging targets of the C-type lectin receptor (CLR) family: Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin (DC-SIGN) and Langerin. Both receptors are involved in pathogen recognition and initiation of an immune response, which renders them attractive targets for immune modulation. Because of their shallow and hydrophilic primary binding site, hit identification for CLRs is challenging and druglike ligands for CLRs are sparse. Screening of a fragment library followed by hit validation identified several promising candidates for further fragment evolution for DC-SIGN. In addition, a multiplexed assay format was developed for simultaneous screening against multiple CLRs, allowing a selectivity counterscreening. Overall, this sensitive cell-based fragment screening assay provides a powerful tool for rapid identification of bioactive fragments, even for difficult targets.