CellFy: A Cell-Based Fragment Screen against C-Type Lectins,ACS Chemical Biology

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CellFy: A Cell-Based Fragment Screen against C-Type Lectins
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2018-11-27 00:00:00 , DOI: 10.1021/acschembio.8b00875
Jessica Schulze _{1,

2} , Hannes Baukmann _{1,

2} , Robert Wawrzinek _{1,

2} , Felix F. Fuchsberger _{1,

2} , Edgar Specker ₃ , Jonas Aretz _{1,

2} , Marc Nazaré ₃ , Christoph Rademacher _{1,

2}

Affiliation

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.

中文翻译：

CellFy：针对C型凝集素的基于细胞的片段筛选

基于片段的药物发现是常规高通量筛选的有力补充，特别是对于困难的目标。筛选低分子量片段通常需要高度敏感的生物物理方法，因为已鉴定的配体通常亲和力较低。在这里，我们开发了一种基于细胞的片段筛选测定法（cellFy），与在溶液中进行分离的靶标筛选相比，它可以在生理上更相关的环境中灵敏地鉴定片段命中。为此，使用了荧光标记的多价报道分子，可以直接测量低分子量片段的位移，而无需酶促反应或受体活化。我们应用了这项技术来确定针对C型凝集素受体（CLR）家族两个具有挑战性的目标的匹配：树突状细胞特异的细胞间粘附分子3-劫持非整联蛋白（DC-SIGN）和朗格林。两种受体都参与病原体识别和免疫应答的启动，这使其成为有吸引力的免疫调节靶标。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的药物样配体稀疏。筛选片段文库，然后进行命中验证，确定了一些有希望的候选物，可用于DC-SIGN的进一步片段进化。另外，开发了用于同时针对多个CLR进行筛选的多重测定形式，从而可以进行选择性反向筛选。总体而言，这种基于细胞的灵敏片段筛选测定法提供了强大的工具，可快速识别生物活性片段，甚至用于困难的目标。

更新日期：2018-11-27

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