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

<i>In vitro</i> and <i>in vivo</i> induction of fetal hemoglobin with a reversible and selective DNMT1 inhibitor

Pharmacological induction of fetal hemoglobin (HbF) expression is an effective therapeutic strategy for the management of beta-hemoglobinopathies such as sickle cell disease. DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-aza) and 5-aza-2′-deoxycytidine (decitabine) have been shown to induce fetal hemoglobin expression in both preclinical models and clinical studies, but are not currently approved for the management of hemoglobinopathies. We report here the discovery of a novel class of orally bioavailable DNMT1-selective inhibitors as exemplified by GSK3482364. This molecule potently inhibits the methyltransferase activity of DNMT1, but not DNMT family members DNMT3A or DNMT3B. In contrast with cytidine analog DNMT inhibitors, the DNMT1 inhibitory mechanism of GSK3482364 does not require DNA incorporation and is reversible. In cultured human erythroid progenitor cells (EPCs), GSK3482364 decreased overall DNA methylation resulting in de-repression of the gamma globin genes HBG1 and HBG2 and increased HbF expression. In a transgenic mouse model of sickle cell disease, orally administered GSK3482364 caused significant increases in both HbF levels and in the percentage HbF-expressing erythrocytes, with good overall tolerability. We conclude that in these preclinical models, selective, reversible inhibition of DNMT1 is sufficient for the induction of HbF, and is well-tolerated. We anticipate that GSK3482364 will be a useful tool molecule for the further study of selective DNMT1 inhibition both in vitro and in vivo

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

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 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

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

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