Fig. 4
The Development of the first small molecule inhibitor of SP140 (GSK761) and investigating its affinity and selectivity. a Scheme of the three-cycle benzimidazole library and b Spotfire cube view of the SP140 selection output from the benzimidazole library. BB1, cycle 1 building blocks; BB2, cycle 2 building blocks; and BB3, cycle 3 building blocks. Each individual dot in the cube represents discrete small molecule warheads after 3 rounds of affinity selection, while the size of the dot corresponds to the number of unique instances recorded by DNA sequencing (2-24). The dots are colored by the BB3s that compose the library molecules. Library members with a single copy were removed to simplify visualization revealing a prominent line defined by a specific BB1&BB3 combination (disynthon). The most enriched trisynthon (BB1, BB2, and BB3 combination) represents the biggest dot on the line. e Biochemical characterization of the interaction between c GSK761 and recombinant SP140 in a fluorescence polarization (FP) binding assay using d a fluorophore-conjugated version of GSK761: GSK064 (generated by fluorescent labelling of GSK761). The mean binding affinity for this interaction was a Kd = 41.07 ± 1.42 nM (n = 5). f A FP-binding assay was configured using recombinant SP140 and GSK064, which was used to determine the potency of GSK761. Displacement of GSK064 from SP140 by GSK761 (circles) was achieved and determined to have a mean IC50 of 77.79 ± 8.27 nM (n=3). No effect on GSK064 motion was observed in the presence of varying concentrations of GSK761 (triangles). The data presented in d and e are representative data from a single experimental replicate, affinities, and potencies are mean values determined from multiple test occasions. g Endogenous SP140 (HuT78 nuclear extracts) and Halo-tagged SP140 (transfected HEK29 cells) were pulled down using a biotinylated version of GSK761 (GSK675) and visualized by Western blotting and gel electrophoresis. Biotinylated beads only and SP140 untransfected cells were used as control