Figure 1.
Scheme used to identify selective inhibitors of NOD1 stimulated IL-8 release in cells.
In the primary HTS compounds that prevented iE-DAP induced cytokine in 293/hNOD1 cells were determined, followed by counter-screens to eliminate those compounds that also inhibited IL-8 induced via activation of NOD2, TNFR1 or TLR2, as well as compounds which directly inhibited RIP2 kinase activity. The activity of selective NOD1 inhibitors was then confirmed in HCT116 intestinal epithelial cells which express NOD1/2 endogenously, stimulated with either Tri-DAP or MDP.
Figure 2.
Activity of three structurally distinct selective NOD1 pathway inhibitors.
(A) Chemical structure of the original hits in each series. (B–D) Concentration response curves for inhibition of IL-8 release by 293/hNOD1 and 293/hNOD2 stable cell lines stimulated with iE-DAP or MDP, respectively, and pre-incubated with the xanthine SB711 (B), quinazolininone GSK223 (C) or aminobenzothiazole GSK966 (D) over the concentration range 2.5 nM –50 µM. Data are the mean ± SD from at least 5 (293/hNOD1– iE-DAP) and from 2 (293/hNOD2– MDP) separate assays for each compound.
Table 1.
Activity of NOD1 selective compounds and inhibitors of IKK and RIP2 in cell-based assays used for hit identification and triage.
Figure 3.
Activity of the three selective NOD1 pathway inhibitor compounds in cells with endogenous NOD1.
Concentration response curves for the aminobenzothiazole GSK966, quinazolininone GSK223 and xanthine SB711 for inhibition of IL-8 secretion by HCT116 colon carcinoma cells pre-incubated for 1 hour with compounds (16 nM–16 µM) and then stimulated with 25 µg/mL Tri-DAP (A) or 1 µg/mL MDP (B). The concentration of IL-8 in medium was determined by HTRF assay after 24 hour treatment. Data are mean ± SD from a single experiment, repeated at least 3 times in Tri-DAP stimulated cells with similar results.
Figure 4.
NOD1 pathway inhibitors block NF-κB and MAPK pathways.
Serum-starved 293/hNOD1 (A) or 293/hNOD2 (B) stable cells were pre-incubated with compounds at the indicated concentrations prior to stimulation for 1 hour with either Tri-DAP (50 µg/mL) or MDP (25 µg/mL), respectively. Compounds included the three selective NOD1 pathway inhibitors or previously identified inhibitors of NOD2 signaling (GSK669 and GSK400). Levels of total IκBα, as well as total and phosphorylated MAPKs (p38, JNK and ERK1/2) were determined by immunoblotting of whole cell lysates. A RIP2 inhibitor was used as a positive control with 293/hNOD2 cells in which the NOD1 pathway inhibitors had no effect on MDP responses. Results shown are representative of two separate experiments performed in both cell lines.
Figure 5.
Structure-activity relationship for xanthine analogs (SB711 and compounds 1–7) in the iE-DAP-stimulated IL-8 production assay in 293/hNOD1 cells.
Figure 6.
Structure-activity relationship for quinazolininone analogs (GSK223 and compounds 8–14) in the iE-DAP stimulated IL-8 production assay in 293/hNOD1 cells.
Figure 7.
Structure-activity relationship for aminobenzothiazole analogs (GSK966 and compounds 15–21) in the iE-DAP stimulated IL-8 production assay in 293/hNOD1 cells.
Figure 8.
SAR for additional quinazolininone analogs (compounds 22–29) in the 293/hNOD1 assay of iE-DAP stimulated IL-8 production.
Figure 9.
NOD1 inhibitors antagonize Tri-DAP induced type I IFN response.
HT-29 colon carcinoma cells were pre-incubated with more potent analogues from the quinazolininone (compound 22) and aminobenzothiazole (compound 16) series (5 nM –16 µM), or with a RIP2 kinase inhibitor (0.5 nM –1.6 µM) followed by stimulation with 50 µg/mL Tri-DAP for 24 hours. IL-8 and IP-10/CXCL10 secreted into conditioned medium were measured by HTRF assay and ELISA, respectively. Results are shown as the mean ± SD of chemokine concentrations determined from triplicate treatment wells. Data are representative of two experiments.
Figure 10.
Suppression of human monocyte IL-8 secretion by selective NOD1 inhibitors.
(A) Comparison of activity between original hit compounds and more potent analogues 16 and 22. Monocytes freshly isolated from human whole blood were pre-treated with NOD1 compounds (0.05, 0.5 and 5 µM) prior to stimulation with 25 µg/mL Tri-DAP. An inhibitor of RIP2 kinase was included as a positive control. The amount of IL-8 present in the medium after 24-hour incubation was determined by HTRF assay. (B) The more potent quinazolininone compound 22 selectively inhibits Tri-DAP but not MDP stimulated IL-8 secretion. Monocytes were pre-treated with compound at the indicated concentrations for 1 hour prior to stimulation with either 25 µg/mL Tri-DAP or 0.1 µg/mL MDP for 24 hours. IL-8 release was determined by HTRF assay. The RIP2 inhibitor blocked responses to both NOD agonists, as expected, and was included as a positive control. Results are expressed as percent inhibition of stimulated IL-8 secretion. The data shown in (A) is the mean inhibition (± SD) observed across multiple experiments (n = 2 or 4) for each compound and data in (B) is the mean ± SD of triplicate treatment wells from an individual experiment repeated once with similar results.