Fig. 1: Identification of NVS-STG2 as a potent allosteric small molecule STING agonist.
From: Activation of human STING by a molecular glue-like compound
a, High-throughput screen of a 250,000 small-molecule compound library with the THP1-Dual reporter cell line identified NVS-STG1 (red circle). One representative from biological duplicates is shown. b, Structures of NVS-STG1 and its more potent analog NVS-STG2. AC50, half-maximal activity concentration; Amax, maximum activity. c, NVS-STG2 induces STING-dependent IRF3 phosphorylation in THP1 cells. One representative from three biological replicates is shown. d, Structures of NVS-STG3 and PAL probe, NVS-STG4. e, Identification of proteins that are enriched by NVS-STG4 and where this labeling can be competed by NVS-STG3. The x axis denotes the enrichment observed in the presence of 1 µM NVS-STG4 relative to 0 µM NVS-STG4 (log2(0 µM NVS-STG4/1 µM NVS-STG4)). STING(TMEM173) was enriched over twofold with PAL probe NVS-STG4 over no probe control in chemoproteomics pull-down. Proteins below the dashed horizontal line show reduction in the enrichment in the presence of 20 µM NVS-STG3. STING(TMEM173) was also competed by adding NVS-STG3. f, NVS-STG2 selectively activates hSTING (solid circle), but not mSTING (solid square). A fusion STING with hSTING N-terminal TMD (1–153) and mouse C-terminal LBD (153–378) responds to NVS-STG2 (empty circle). g, hSTING mutants (S27V, V31M, L93I, R95C, I103S, P115I) were generated according to key amino acid differences between human and mouse STING N-terminal TMD. NVS-STG2 activity is sensitive to hSTING R95C mutation. h, Comparison of residues in human and mouse STING tested by mutation in g. In f and g, the axis represents concentrations of STG2 in log scale, and the symbols and error bars represent mean and s.e.m., respectively, from three biological replicates. KO, knockout; MW, molecular weight.
