Identification of benzimidazole diamides as selective inhibitors of the nucleotide-binding oligomerization domain 2 (NOD2) signaling pathway.
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.
Pattern Recognition Receptor Discovery Performance Unit, Immuno-Inflammation Therapeutic Area, GlaxoSmithKline, Collegeville, Pennsylvania, USA. David.J.Rickard@gsk.com
David J
DJ
Rickard
Clark A
CA
Sehon
Viera
V
Kasparcova
Lorena A
LA
Kallal
Xin
X
Zeng
Monica N
MN
Montoute
Tushar
T
Chordia
Derek D
DD
Poore
Hu
H
Li
Zining
Z
Wu
Patrick M
PM
Eidam
Pamela A
PA
Haile
Jong
J
Yu
John G
JG
Emery
Robert W
RW
Marquis
Peter J
PJ
Gough
John
J
Bertin
D000577:Amides / Q000737:chemistry*; D001562:Benzimidazoles / Q000737:chemistry* / Q000494:pharmacology*; D016207:Cytokines / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D053473:Nod2 Signaling Adaptor Protein / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D013329:Structure-Activity Relationship; D051195:Toll-Like Receptor 2 / Q000378:metabolism
D000577:Amides; D001562:Benzimidazoles; D016207:Cytokines; D016328:NF-kappa B; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; C495342:TLR2 protein, human; D051195:Toll-Like Receptor 2; C031000:benzimidazole