CellFy: A Cell-Based Fragment Screen against C-Type Lectins

Abstract

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.

Application of Fragment-Based Drug Discovery to Versatile Targets

“…Weak affinity chromatography was developed as a tool to screen hits in FBDD ( Duong-Thi et al, 2011 ). Fragment-based screening was also carried out using cell-based assays ( Szõllõsi et al, 2015 ; Schulze et al, 2018 ). A study showed that fragments reacting with cysteine residues were able to identify proteins that formed interactions with these compounds ( Backus et al, 2016 ).…”

Molecular Recognition in C‐Type Lectins: The Cases of DC‐SIGN, Langerin, MGL, and L‐Sectin

“…Although multivalence is in general the most employed strategy to overcome the poor intrinsic affinities of C-type lectins, computational and screening studies have gradually directed the attention to other novel DC-SIGN ligands. [147,148] Indeed, previous works have already found nonsugar fragments with remarkably low affinities compared to monosaccharides (μM; Figure 6A and B), [149] and some of them are able to efficiently trigger cell signaling when conjugated to a protein scaffold. [150] Fragment screening analyses have identified other potentially druggable sites in the CRD architecture, [151] which could be exploited for the design of more potent DC-SIGN modulators ( Figure 6C).…”

“…Recently, a promising screening method has been developed and simultaneously tested with DC-SIGN and langerin. [148] The method enables a better evaluation of the binding potencies under physiological conditions, directly on the CLR-containing cells ( Figure 10A). Detection of hits is achieved by direct competition between a reference fluorescent ligand (FITC-dextran) and the tested fragments.…”

One pot synthesis ofthio-glycosidesviaaziridine opening reactions

“…2 The development of functional carbohydrate mimics has been tackled either by structure-based design, 1,[3][4][5][6][7][8][9][10][11][12][13] or by discovery campaigns, often planned taking advantage of microarray technology. [14][15][16] The latter approach has been frustrated by the cumbersome synthesis of oligosaccharides, that severely limits the diversity and the number of compounds that can be rapidly generated.…”