Identification of Selective Small Molecule Inhibitors of the Nucleotide-Binding Oligomerization Domain 1 (NOD1) Signaling Pathway

<div><p>NOD1 is an intracellular pattern recognition receptor that recognizes diaminopimelic acid (DAP), a peptidoglycan component in gram negative bacteria. Upon ligand binding, NOD1 assembles with receptor-interacting protein (RIP)-2 kinase and initiates a signaling cascade leading to the production of pro-inflammatory cytokines. Increased NOD1 signaling has been associated with a variety of inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. We utilized a cell-based screening approach with extensive selectivity profiling to search for small molecule inhibitors of the NOD1 signaling pathway. Via this process we identified three distinct chemical series, xanthines (SB711), quinazolininones (GSK223) and aminobenzothiazoles (GSK966) that selectively inhibited iE-DAP-stimulated IL-8 release via the NOD1 signaling pathway. All three of the newly identified compound series failed to block IL-8 secretion in cells following stimulation with ligands for TNF receptor, TLR2 or NOD2 and, in addition, none of the compound series directly inhibited RIP2 kinase activity. Our initial exploration of the structure-activity relationship and physicochemical properties of the three series directed our focus to the quinazolininone biarylsulfonamides (GSK223). Further investigation allowed for the identification of significantly more potent analogs with the largest boost in activity achieved by fluoro to chloro replacement on the central aryl ring. These results indicate that the NOD1 signaling pathway, similarly to activation of NOD2, is amenable to modulation by small molecules that do not target RIP2 kinase. These compounds should prove useful tools to investigate the importance of NOD1 activation in various inflammatory processes and have potential clinical utility in diseases driven by hyperactive NOD1 signaling.</p></div

Identification of Benzimidazole Diamides as Selective Inhibitors of the Nucleotide-Binding Oligomerization Domain 2 (NOD2) Signaling Pathway

<div><p>NOD2 is an intracellular pattern recognition receptor that assembles with receptor-interacting protein (RIP)-2 kinase in response to the presence of bacterial muramyl dipeptide (MDP) in the host cell cytoplasm, thereby inducing signals leading to the production of pro-inflammatory cytokines. The dysregulation of NOD2 signaling has been associated with various inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. To identify inhibitors of the NOD2 signaling pathway, we utilized a cell-based screening approach and identified a benzimidazole diamide compound designated GSK669 that selectively inhibited an MDP-stimulated, NOD2-mediated IL-8 response without directly inhibiting RIP2 kinase activity. Moreover, GSK669 failed to inhibit cytokine production in response to the activation of Toll-like receptor (TLR)-2, tumor necrosis factor receptor (TNFR)-1 and closely related NOD1, all of which share common downstream components with the NOD2 signaling pathway. While the inhibitors blocked MDP-induced NOD2 responses, they failed to block signaling induced by NOD2 over-expression or single stranded RNA, suggesting specificity for the MDP-induced signaling complex and activator-dependent differences in NOD2 signaling. Investigation of structure-activity relationship allowed the identification of more potent analogs that maintained NOD2 selectivity. The largest boost in activity was achieved by N-methylation of the C2-ethyl amide group. These findings demonstrate that the NOD2 signaling pathway is amenable to modulation by small molecules that do not target RIP2 kinase activity. The compounds we identified should prove useful tools to investigate the importance of NOD2 in various inflammatory processes and may have potential clinical utility.</p></div

Scheme used to identify selective inhibitors of NOD1 stimulated IL-8 release in cells.

<p>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.</p

Structure-activity relationship for xanthine analogs (SB711 and compounds 1–7) in the iE-DAP-stimulated IL-8 production assay in 293/hNOD1 cells.

<p>Structure-activity relationship for xanthine analogs (SB711 and compounds 1–7) in the iE-DAP-stimulated IL-8 production assay in 293/hNOD1 cells.</p

Activity of the three selective NOD1 pathway inhibitor compounds in cells with endogenous NOD1.

<p>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.</p

Activity of three structurally distinct selective NOD1 pathway inhibitors.

<p>(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.</p

NOD1 pathway inhibitors block NF-κB and MAPK pathways.

<p>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.</p

Structure-activity relationship for aminobenzothiazole analogs (GSK966 and compounds 15–21) in the iE-DAP stimulated IL-8 production assay in 293/hNOD1 cells.

<p>Structure-activity relationship for aminobenzothiazole analogs (GSK966 and compounds 15–21) in the iE-DAP stimulated IL-8 production assay in 293/hNOD1 cells.</p

Activity of NOD1 selective compounds and inhibitors of IKK and RIP2 in cell-based assays used for hit identification and triage.

<p>IC₅₀ values are given in micromolar. For each of the cell-based assays, the concentration of IL-8 released into medium was the end-point measured. For the RIPK2 biochemical assay, the level of RIPK2 autophosphorylation was measured. Cell-based assays included are:- Monocyte = human primary peripheral blood monocytes; 293/hNOD1 and 293/hNOD2 = HEK293 cell lines stably expressing full-length human NOD1 or NOD2, respectively; HCT116 = human colon carcinoma cells.</p

SAR for additional quinazolininone analogs (compounds 22–29) in the 293/hNOD1 assay of iE-DAP stimulated IL-8 production.

<p>SAR for additional quinazolininone analogs (compounds 22–29) in the 293/hNOD1 assay of iE-DAP stimulated IL-8 production.</p