Development of an oral small molecule protein-protein inhibitor of NKG2D with a novel SNAP-Tag cell-based assay

Abstract ID 128111 Poster Board 364 Natural killer group 2D (NKG2D) is a homodimeric transmembrane activating immunoreceptor whose function is to detect and eliminate infected, stressed, or transformed cells that are targeted due to upregulation of NKG2D ligands (NKG2DL). NKG2D associates with a specific adaptor molecule, the DNAX-activating protein of 10kDa (DAP10), which contains the signaling sequence needed for activation. NKG2D signaling has also been associated with chronic inflammatory and autoimmune diseases and is therefore a considerable target for immune intervention. The discovery of small-molecule protein–protein interaction (PPI) inhibitors remain challenging due to the complex nature of most PPI interfaces. We developed an extensive small-molecule hit identification strategy, including a high-throughput screen that utilized a ligand binding time-resolved fluorescence resonance energy transfer (TR-FRET) cellular-based assay. This assay helped to identify a series of PPI inhibitors, that directly blocked the interaction between NKG2D and NKG2DLs. We created a HEK293 cell line stably coexpressing the full-length NKG2D/DAP10 receptors, wherein the TR-FRET SNAP-tag technology was used as a primary assay to drive structure–activity relationship (SAR) to study PPI and identify binding pocket formations. This assay underwent two orthogonal screenings with our internal library of about half a million compounds and one-billion-member DEL screen to find small-molecule inhibitors with potent binding affinity to the NKG2D protein. These efforts resulted in the identification of two structurally distinct NKG2D binders/inhibitors and several notable analogs, that share a unique allosteric mechanism of action that differ from any known antibody based NKG2D PPIs.