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1.  Discovery of Small Molecule RIP1 Kinase Inhibitors for the Treatment of Pathologies Associated with Necroptosis 
ACS Medicinal Chemistry Letters  2013;4(12):1238-1243.
Potent inhibitors of RIP1 kinase from three distinct series, 1-aminoisoquinolines, pyrrolo[2,3-b]pyridines, and furo[2,3-d]pyrimidines, all of the type II class recognizing a DLG-out inactive conformation, were identified from screening of our in-house kinase focused sets. An exemplar from the furo[2,3-d]pyrimidine series showed a dose proportional response in protection from hypothermia in a mouse model of TNFα induced lethal shock.
PMCID: PMC4027519  PMID: 24900635
RIP1; type II kinase inhibitors; necroptosis
2.  Blau Syndrome, the prototypic auto-inflammatory granulomatous disease 
Blau syndrome is a monogenic disease resulting from mutations in the pattern recognition receptor NOD2, and is phenotypically characterized by the triad of granulomatous polyarthritis, dermatitis and uveitis. This paper reviews briefly the classical clinical features of the disease, as well as more recently described extra-triad symptoms. From an ongoing prospective multicenter study, we provide new data on the natural history of Blau syndrome, focusing on functional status and visual outcome. We also present an update of the range of different NOD2 mutations found in Blau syndrome as well as recent data on morphologic and immunohistochemical characteristics of the Blau granuloma. Finally, emerging insights into pathogenic mechanisms including activation of NOD2 signal transduction, and potential biomarkers of disease activity are discussed.
PMCID: PMC4136643  PMID: 25136265
Blau syndrome; Granulomatous diseases; NOD2; RIP2 kinase; Sarcoidosis; Uveitis
3.  Identification of Selective Small Molecule Inhibitors of the Nucleotide-Binding Oligomerization Domain 1 (NOD1) Signaling Pathway 
PLoS ONE  2014;9(5):e96737.
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.
PMCID: PMC4013053  PMID: 24806487
4.  Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors 
PLoS ONE  2014;9(4):e91119.
Human embryonic stem cell-derived endothelial cells (hESC-EC), as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR) Toll-like receptor (TLR)-4 and nucleotide-binding oligomerisation domain-containing protein (NOD)-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC). HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC), and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.
PMCID: PMC3972153  PMID: 24690886
5.  The NOD-like receptor NLRP12 attenuates colon inflammation and tumorigenesis 
Cancer cell  2011;20(5):649-660.
NLRP12 is a member of the intracellular Nod-like receptor (NLR) family that has been suggested to down-regulate the production of inflammatory cytokines, but its physiological role in regulating inflammation has not been characterized. We generated mice deficient in Nlrp12 and studied its role in inflammatory diseases such as colitis and colorectal tumorigenesis. We show that Nlrp12-deficient mice are highly susceptible to colon inflammation and tumorigenesis, which is associated with increased production of inflammatory cytokines, chemokines and tumorigenic factors. Enhanced colon inflammation and colorectal tumor development in Nlrp12-deficient mice are due to a failure to dampen NF-κB and ERK activation in macrophages. These results reveal a critical role for NLRP12 in maintaining intestinal homeostasis and providing protection against colorectal tumorigenesis.
PMCID: PMC3761879  PMID: 22094258
NLRP12; NLR; colon; inflammation; tumorigenesis; colitis; NF-κB; ERK
6.  The NLRP12 inflammasome recognizes Yersinia pestis 
Immunity  2012;37(1):96-107.
Yersinia pestis, the causative agent of plague, is able to suppress production of inflammatory cytokines IL-18 and IL-1β, which are generated through caspase-1–activating nucleotide-binding domain and leucine-rich repeat (NLR)-containing inflammasomes. Here, we sought to elucidate the role of NLRs and IL-18 during plague. Lack of IL-18 signaling led to increased susceptibility to Y. pestis, producing tetra-acylated lipid A,and an attenuated strain producing a Y. pseudotuberculosis-like hexa-acylated lipid A. We found that the NLRP12 inflammasome was an important regulator controlling IL-18 and IL-1β production after Y. pestis infection, and NLRP12-deficient mice were more susceptible to bacterial challenge. NLRP12 also directed interferon-γ production via induction of IL-18, but had minimal effect on signaling to the transcription factor NF-κB._ These studies reveal a role for NLRP12 in host resistance against pathogens. Minimizing NLRP12 inflammasome activation may have been a central factor in evolution of the high virulence of Y. pestis.
PMCID: PMC3753114  PMID: 22840842
7.  Identification of Benzimidazole Diamides as Selective Inhibitors of the Nucleotide-Binding Oligomerization Domain 2 (NOD2) Signaling Pathway 
PLoS ONE  2013;8(8):e69619.
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.
PMCID: PMC3731320  PMID: 23936340
8.  NLRP6 Negatively Regulates Innate Immunity and Host Defense Against Bacterial Pathogens 
Nature  2012;488(7411):389-393.
Members of the intracellular nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family contribute to immune responses through activation of NF-κB, type I interferon and inflammasome signaling1. Mice lacking the NLR family member NLRP6 were recently shown to be susceptible to colitis and colorectal tumorigenesis2-4, but the role of NLRP6 in microbial infections and the nature of the inflammatory signaling pathways regulated by NLRP6 remain unclear. Here, we show that Nlrp6-deficient mice were highly resistant to infection with the bacterial pathogens Listeria monocytogenes, Salmonella typhimurium and Escherichia coli. Infected Nlrp6-deficient mice had increased numbers of monocytes and neutrophils in circulation, and NLRP6 signaling in both hematopoietic and radio-resistant cells contributed to increased susceptibility. Nlrp6-deficiency enhanced activation of MAPK and canonical NF-κB upon TLR, but not cytosolic NOD1/2 ligation in vitro. Consequently, infected Nlrp6-deficient cells produced elevated levels of NF-κB- and MAPK-dependent cytokines and chemokines. Thus, our results reveal NLRP6 as a negative regulator of inflammatory signaling, and demonstrate a role for this NLR in impeding clearance of both Gram-positive and –negative bacterial pathogens.
PMCID: PMC3422416  PMID: 22763455
NLRP6; NLR; Listeria; Salmonella; NF-kB; ERK
9.  A Key Role for the Endothelium in NOD1 Mediated Vascular Inflammation: Comparison to TLR4 Responses 
PLoS ONE  2012;7(8):e42386.
Understanding the mechanisms by which pathogens induce vascular inflammation and dysfunction may reveal novel therapeutic targets in sepsis and related conditions. The intracellular receptor NOD1 recognises peptidoglycan which features in the cell wall of Gram negative and some Gram positive bacteria. NOD1 engagement generates an inflammatory response via activation of NFκB and MAPK pathways. We have previously shown that stimulation of NOD1 directly activates blood vessels and causes experimental shock in vivo. In this study we have used an ex vivo vessel-organ culture model to characterise the relative contribution of the endothelium in the response of blood vessels to NOD1 agonists. In addition we present the novel finding that NOD1 directly activates human blood vessels. Using human cultured cells we confirm that endothelial cells respond more avidly to NOD1 agonists than vascular smooth muscle cells. Accordingly we have sought to pharmacologically differentiate NOD1 and TLR4 mediated signalling pathways in human endothelial cells, focussing on TAK1, NFκB and p38 MAPK. In addition we profile novel inhibitors of RIP2 and NOD1 itself, which specifically inhibit NOD1 ligand induced inflammatory signalling in the vasculature. This paper is the first to demonstrate activation of whole human artery by NOD1 stimulation and the relative importance of the endothelium in the sensing of NOD1 ligands by vessels. This data supports the potential utility of NOD1 and RIP2 as therapeutic targets in human disease where vascular inflammation is a clinical feature, such as in sepsis and septic shock.
PMCID: PMC3411636  PMID: 22870324
10.  A Functional Role for Nlrp6 in Intestinal Inflammation and Tumorigenesis* 
The Nod-like receptor (NLR) family member, Nlrp6, has been implicated in inflammasome signaling to activate caspase-1, which is essential for the production of mature IL-1β and IL-18. However, a function for Nlrp6 in vivo has never been demonstrated. Due to the relative high expression of Nlrp6 in intestinal tissue, we hypothesized that Nlrp6 has a role in intestinal homeostasis. Indeed, Nlrp6-deficient mice are more susceptible to chemically-induced colitis as well as colitis-induced tumorigenesis than wildtype mice. Nlrp6-deficient mice exhibited significantly more inflammation within the colon than wildtype mice after dextran sulfate sodium treatment. Their inability to resolve inflammation and repair damaged epithelium as efficiently as wildtype mice resulted in prolonged increases in epithelial proliferative activity that likely underlie the increased propensity for tumors in these mice during chronic inflammation. We further show that that the activity of Nlrp6 in hematopoietic cells is critical for protection against inflammation-related colon tumorigenesis. This study highlights the importance of NLR function in maintaining intestinal homeostasis to prevent the development of aberrant inflammation and tumor development within the colon.
PMCID: PMC3133458  PMID: 21543645
11.  NLRP6 inflammasome is a regulator of colonic microbial ecology and risk for colitis 
Cell  2011;145(5):745-757.
Inflammasomes are multi-protein complexes that function as sensors of endogenous or exogenous damage-associated molecular patterns. Here we show that deficiency of NLRP6 in mouse colonic epithelial cells results in reduced IL-18 levels and altered fecal microbiota characterized by expanded representation of the bacterial phyla Bacteroidetes (Prevotellaceae) and TM7. NLRP6 inflammasome-deficient mice were characterized by spontaneous intestinal hyperplasia, inflammatory cell recruitment, and exacerbation of chemical colitis induced by exposure to dextran sodium sulfate (DSS). Cross-fostering and cohousing experiments revealed that the colitogenic activity of this microbiota is transferable to neonatal or adult wild-type mice, leading to exacerbation of DSS colitis via induction of CCL5. Antibiotic treatment and electron microscopy studies further supported the role of Prevotellaceae as a key representative of this microbiota-associated phenotype. Altogether, perturbations in this inflammasome pathway, including NLRP6, ASC, caspase-1 and IL-18 may constitute a predisposing or initiating event in some cases of human IBD.
PMCID: PMC3140910  PMID: 21565393
Inflammasome; NLRP6; ASC; IL-18; microbiota; colitis
12.  The NLR gene family: An official nomenclature 
Immunity  2008;28(3):285-287.
Iimmune regulatory proteins such as CIITA, NAIP, IPAF, NOD1, NOD2, NALP1, cryopyrin/NALP3 are members of a family characterized by the presence of a nucleotide-binding domain (NBD) and leucine-rich repeats (LRR). Members of this gene family encode a protein structure similar to the NB-LRR subgroup of disease-resistance genes in plants and are involved in the sensing of pathogenic products and the regulation of cell signaling and apoptosis. Several members of this family have been associated with immunologic disorders. NOD2 for instance is associated with both Crohn's disease and Blau syndrome.
A variety of different names are currently used to describe this gene family, its subfamilies and individual genes, including CATERPILLER (CLR), NOD-LRR, NACHT-LRR, CARD, NALP, NOD, PAN and PYPAF, and this lack of consistency has led to a pressing need to unify the nomenclature. Consequently, we collectively propose the family designation NLR (nucleotide-binding domain and leucine-rich repeat containing) and provide unique and standardized gene designations for all family members.
PMCID: PMC2630772  PMID: 18341998
13.  Role of the caspase-1 inflammasome in Salmonella typhimurium pathogenesis 
The Journal of Experimental Medicine  2006;203(6):1407-1412.
Caspase-1 is activated by a variety of stimuli after the assembly of the “inflammasome,” an activating platform made up of a complex of the NOD-LRR family of proteins. Caspase-1 is required for the secretion of proinflammatory cytokines, such as interleukin (IL)-1β and IL-18, and is involved in the control of many bacterial infections. Paradoxically, however, its absence has been reported to confer resistance to oral infection by Salmonella typhimurium. We show here that absence of caspase-1 or components of the inflammasome does not result in resistance to oral infection by S. typhimurium, but rather, leads to increased susceptibility to infection.
PMCID: PMC2118315  PMID: 16717117
15.  Binding of FADD and Caspase-8 to Molluscum Contagiosum Virus MC159 v-FLIP Is Not Sufficient for Its Antiapoptotic Function 
Journal of Virology  2002;76(2):697-706.
Molluscum contagiosum virus (MCV), a member of the human poxvirus family, encodes the MC159 protein that inhibits Fas-, tumor necrosis factor (TNF)-, and TNF-related apoptosis-inducing ligant (TRAIL)-induced apoptosis. We used site-directed mutagenesis to change charged or hydrophobic amino acid residues to alanines to identify regions of MC159 that are critical for protection from apoptosis and for protein-protein interactions. Surprisingly, while MC159 is thought to block apoptosis by binding to Fas-associated death domain (FADD) or caspase-8, several mutants that lost apoptosis blocking activity still bound to both FADD and caspase-8. Mutations in the predicted hydrophobic patch 1 and α2 regions of both death effector domains (DEDs) within MC159 resulted in loss of the ability to bind to FADD or caspase-8 and to block apoptosis. Amino acid substitutions in the RXDL motif located in the α6 region of either DED resulted in loss of protection from apoptosis induced by Fas, TNF, and TRAIL and abolished the ability of MC159 to block death effector filament formation. Thus, charged or hydrophobic amino acids in three regions of the MC159 DEDs (hydrophobic patch 1, α2, and α6) are critical for the protein’s ability to interact with cellular proteins and to block apoptosis.
PMCID: PMC136828  PMID: 11752160
16.  Death-effector Filaments: Novel Cytoplasmic Structures that Recruit Caspases and Trigger Apoptosis  
The Journal of Cell Biology  1998;141(5):1243-1253.
The death-effector domain (DED) is a critical protein interaction domain that recruits caspases into complexes with members of the TNF-receptor superfamily. Apoptosis can also be induced by expressing certain DED-containing proteins without surface receptor cross-linking. Using Green Fluorescent Protein to examine DED-containing proteins in living cells, we show that these proteins cause apoptosis by forming novel cytoplasmic filaments that recruit and activate pro-caspase zymogens. Formation of these filaments, which we term death-effector filaments, was blocked by coexpression of viral antiapoptotic DED-containing proteins, but not by bcl-2 family proteins. Thus, formation of death-effector filaments allows a regulated intracellular assembly of apoptosis-signaling complexes that can initiate or amplify apoptotic stimuli independently of receptors at the plasma membrane.
PMCID: PMC2137190  PMID: 9606215

Results 1-16 (16)