Discovery of low molecular weight protein-binding fragments from bead-displayed libraries: application to monomeric proteins
Fragment-based drug discovery (FBDD) is a powerful workflow for the development of drug candidates and probe molecules that begins with the discovery of one or more low affinity, low molecular weight ligands for a protein of interest (POI). Recently, we developed a simple fragment discovery platform in which TentaGel beads displaying many copies of the fragment are incubated with a fluorescently labeled multimeric protein. Association of two or more bead-displayed ligands with the multimeric target stabilizes the complex sufficiently to allow its detection even after rigorous washing. However, this simple “pull-down” assay cannot be used for monomeric protein targets since these complexes are too kinetically labile to survive being taken out of equilibrium. Here we explore various strategies to multimerize monomeric targets, thus allowing them to be used as targets in this fragment discovery platform. We find that a particularly effective and convenient strategy is to express a fusion of the POI with FOLDON, a 30-residue, homo-trimeric peptide.