Pharmacological inhibition of receptor-interacting protein kinase 2 (RIPK2) elicits neuroprotective effects following experimental ischemic stroke.

Ischemic stroke induces a debilitating neurological insult, where inflammatory processes contribute greatly to the expansion and growth of the injury. Receptor-interacting protein kinase 2 (RIPK2) is most well-known for its role as the obligate kinase for NOD1/2 pattern recognition receptor signaling and is implicated in the pathology of various inflammatory conditions. Compared to a sham-operated control, ischemic stroke resulted in a dramatic increase in the active, phosphorylated form of RIPK2, indicating that RIPK2 may be implicated in the response to stroke injury. Here, we assessed the effects of pharmacological inhibition of RIPK2 to improve post-stroke outcomes in mice subjected to experimental ischemic stroke. We found that treatment at the onset of reperfusion with a RIPK2 inhibitor, which inhibits the phosphorylation and activation of RIPK2, resulted in marked improvements in post-stroke behavioral outcomes compared to the vehicle-administered group assessed 24 h after stroke. RIPK2 inhibitor-treated mice exhibited dramatic reductions in infarct volume, concurrent with reduced damage to the blood-brain barrier, as evidenced by reduced levels of active matrix metalloproteinase-9 (MMP-9) and leakage of blood-borne albumin in the ipsilateral cortex. To explore the protective mechanism of RIPK2 inhibition, we next pretreated mice with RIPK2 inhibitor or vehicle and examined transcriptomic alterations occurring in the ischemic brain 6 h after stroke. We observed a dramatic reduction in neuroinflammatory markers in the ipsilateral cortex of the inhibitor-treated group while also attaining a comprehensive view of the vast transcriptomic alterations occurring in the brain with inhibitor treatment through bulk RNA-sequencing of the injured cortex. Overall, we provide significant novel evidence that RIPK2 may represent a viable target for post-stroke pharmacotherapy and potentially other neuroinflammatory conditions.

Design, synthesis and evaluation of novel thieno[2,3d]pyrimidine derivatives as potent and specific RIPK2 inhibitors.

In human cells, receptor-interacting protein kinase 2 (RIPK2) is mainly known to mediate downstream enzymatic cascades from the nucleotide-binding oligomerization domain-containing receptors 1 and 2 (NOD1/2), which are regulators of pro-inflammatory signaling. Thus, the targeted inhibition of RIPK2 has been proposed as a pharmacological strategy for the treatment of a variety of pathologies, in particular inflammatory and autoimmune diseases. In this work, we designed and developed novel thieno[2,3d]pyrimidine derivatives, in order to explore their activity and selectivity as RIPK2 inhibitors. Primary in vitro evaluations of the new molecules against purified RIPKs (RIPK1-4) demonstrated outstanding inhibitory potency and selectivity for the enzyme RIPK2. Moreover, investigations for efficacy against the RIPK2-NOD1/2 signaling pathways, conducted in living cells, showed their potency could be tuned towards a low nanomolar range. This could be achieved by solely varying the substitutions at position 6 of the thieno[2,3d]pyrimidine scaffold. A subset of lead inhibitors were ultimately evaluated for selectivity against 58 human kinases other than RIPKs, displaying great specificities. We therefore obtained new inhibitors that might serve as starting point for the preparation of targeted tools, which could be useful to gain a better understanding of biological roles and clinical potential of RIPK2.

RIPK2 inhibitors for disease therapy: Current status and perspectives.

Receptor-interacting protein kinase 2 (RIPK2) belongs to the receptor-interacting protein family (RIPs), which is mainly distributed in the cytoplasm. RIPK2 is widely expressed in human tissues, and its mRNA level is highly expressed in the spleen, leukocytes, placenta, testis, and heart. RIPK2 is a dual-specificity kinase with multiple domains, which can interact with tumor necrosis factor receptor (TNFR), and participate in the Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) signaling pathways. It is considered as a vital adapter molecule involved in the innate immunity, adaptive immunity, and apoptosis. Functionally, RIPK2 and its targeted small molecules are of great significance in inflammatory responses, autoimmune diseases and tumors. The present study reviews the molecule structure and biological functions of RIPK2, and its correlation between human diseases. In addition, we focus on the structure-activity relationship of small molecule inhibitors of RIPK2 and their therapeutic potential in human diseases.

Receptor-interacting protein kinase 2 (RIPK2) stabilizes c-Myc and is a therapeutic target in prostate cancer metastasis.

Despite progress in prostate cancer (PC) therapeutics, distant metastasis remains a major cause of morbidity and mortality from PC. Thus, there is growing recognition that preventing or delaying PC metastasis holds great potential for substantially improving patient outcomes. Here we show receptor-interacting protein kinase 2 (RIPK2) is a clinically actionable target for inhibiting PC metastasis. RIPK2 is amplified/gained in ~65% of lethal metastatic castration-resistant PC. Its overexpression is associated with disease progression and poor prognosis, and its genetic knockout substantially reduces PC metastasis. Multi-level proteomics analyses reveal that RIPK2 strongly regulates the stability and activity of c-Myc (a driver of metastasis), largely via binding to and activating mitogen-activated protein kinase kinase 7 (MKK7), which we identify as a direct c-Myc-S62 kinase. RIPK2 inhibition by preclinical and clinical drugs inactivates the noncanonical RIPK2/MKK7/c-Myc pathway and effectively impairs PC metastatic outgrowth. These results support targeting RIPK2 signaling to extend metastasis-free and overall survival.

The potent and selective RIPK2 inhibitor BI 706039 improves intestinal inflammation in the TRUC mouse model of inflammatory bowel disease.

Mouse and human data implicate the NOD1 and NOD2 sensors of the intestinal microbiome and the associated signal transduction via the receptor interacting protein kinase 2 (RIPK2) as a potential key signaling node for the development of inflammatory bowel disease (IBD) and an attractive target for pharmacological intervention. The TRUC mouse model of IBD was strongly indicated for evaluating RIPK2 antagonism for its effect on intestinal inflammation based on previous knockout studies with NOD1, NOD2, and RIPK2. We identified and profiled the BI 706039 molecule as a potent and specific functional inhibitor of both human and mouse RIPK2 and with favorable pharmacokinetic properties. We dosed BI 706039 in the spontaneous TRUC mouse model from age 28 to 56 days. Oral, daily administration of BI 706039 caused dose-responsive and significant improvement in colonic histopathological inflammation, colon weight, and terminal levels of protein-normalized fecal lipocalin (all P values <0.001). These observations correlated with dose responsively increasing systemic levels of the BI 706039 compound, splenic molecular target engagement of RIPK2, and modulation of inflammatory genes in the colon. This demonstrates that a relatively low oral dose of a potent and selective RIPK2 inhibitor can modulate signaling in the intestinal immune system and significantly improve disease associated intestinal inflammation.NEW & NOTEWORTHY The RIPK2 kinase at the apex of microbiome immunosensing is an attractive target for pharmacological intervention. A low oral dose of a RIPK2 inhibitor leads to significantly improved intestinal inflammation in the murine TRUC model of colitis. A selective and potent inhibitor of the RIPK2 kinase may represent a new class of therapeutics that target microbiome-driven signaling for the treatment of IBD.

Design of pyrido[2,3-d]pyrimidin-7-one inhibitors of receptor interacting protein kinase-2 (RIPK2) and nucleotide-binding oligomerization domain (NOD) cell signaling.

Receptor interacting protein kinase-2 (RIPK2) is an enzyme involved in the transduction of pro-inflammatory nucleotide-binding oligomerization domain (NOD) cell signaling, a pathway implicated in numerous chronic inflammatory conditions. Herein, a pyrido[2,3-d]pyrimidin-7-one based class of RIPK2 kinase and NOD2 cell signaling inhibitors is described. For example, 33 (e.g. UH15-15) inhibited RIPK2 kinase (IC50 = 8 ± 4 nM) and displayed > 300-fold selectivity versus structurally related activin receptor-like kinase 2 (ALK2). This molecule blocked NOD2-dependent HEKBlue NF-κB activation (IC50 = 20 ± 5 nM) and CXCL8 production (at concentrations > 10 nM). Molecular docking suggests that engagement of Ser25 in the glycine-rich loop may provide increased selectivity versus ALK2 and optimal occupancy of the region between the gatekeeper and the αC-helix may contribute to potent NOD2 cell signaling inhibition. Finally, this compound also demonstrated favorable in vitro ADME and pharmacokinetic properties (e.g. Cmax = 5.7 µM, Tmax = 15 min, t1/2 = 3.4 h and Cl = 45 mL/min/kg following single 10 mg/kg intraperitoneal administration) further supporting the use of pyrido[2,3-d]pyrimidin-7-ones as a new structure class of RIPK2 kinase and NOD cell signaling inhibitors.

Renoprotective Effects of a Novel Receptor-Interacting Protein Kinase 2 Inhibitor, AS3334034, in Uninephrectomized Adriamycin-Induced Chronic Kidney Disease Rats.

Renal inflammation is a final common pathway of chronic kidney disease (CKD), and its progression can be used to effectively gauge the degree of renal dysfunction. Inflammatory mechanisms contribute to glomerulosclerosis and tubulointerstitial fibrosis, which are hallmarks of CKD leading to end-stage renal disease. Receptor-interacting protein kinase 2 (RIP2) is largely committed to nucleotide-binding oligomerization domain signaling as a direct effector and transmits nuclear factor-κB (NF-κB)-mediated proinflammatory cytokine production. In the present study, we hypothesized that if inflammation via RIP2 and NF-κB signaling plays an important role in renal failure, then the anti-inflammatory effect of RIP2 inhibitors should be effective in improving CKD. To determine its pharmacologic potency, we investigated the renoprotective properties of the novel RIP2 inhibitor AS3334034 [7-methoxy-6-(2-methylpropane-2-sulfonyl)-N-(4-methyl-1H-pyrazol-3-yl)quinolin-4-amine] in uninephrectomized adriamycin-induced CKD rats. Six weeks' repeated administration of AS3334034 (10 mg/kg, once daily) significantly reduced urinary protein excretion and prevented the development of glomerulosclerosis and tubulointerstitial fibrosis. In addition, AS3334034 showed beneficial effects on renal function, as demonstrated by a decrease in levels of plasma creatinine and blood urea nitrogen and attenuation of a decline in creatinine clearance. Furthermore, AS3334034 significantly attenuated inflammation, renal apoptosis, and glomerular podocyte loss. These results suggest that the RIP2 inhibitor AS3334034 suppresses the progression of chronic renal failure via an anti-inflammatory effect and is therefore potentially useful in treating patients with CKD. SIGNIFICANCE STATEMENT: The receptor-interacting protein kinase 2 (RIP2) inhibitor AS3334034 suppresses the progression of chronic renal failure via an anti-inflammatory effect, suggesting that the nucleotide-binding oligomerization domain-RIP2 axis might play a crucial role in the pathogenesis of inflammatory kidney diseases. AS3334034 is expected to be potentially useful in the treatment of patients with chronic kidney disease.

Gefitinib Inhibits Invasion and Metastasis of Osteosarcoma via Inhibition of Macrophage Receptor Interacting Serine-Threonine Kinase 2.

Most patients with osteosarcoma have subclinical pulmonary micrometastases at diagnosis. Mounting evidence suggests that macrophages facilitate metastasis. As the EGFR has been implicated in carcinoma-macrophage cross-talk, in this study, we asked whether gefitinib, an EGFR inhibitor, reduces osteosarcoma invasion and metastatic outgrowth using the K7M2-Balb/c syngeneic murine model. Macrophages enhanced osteosarcoma invasion in vitro, which was suppressed by gefitinib. Oral gefitinib inhibited tumor extravasation in the lung and reduced the size of metastatic foci, resulting in reduced metastatic burden. Gefitinib also altered pulmonary macrophage phenotype, increasing MHCII and decreasing CD206 expression compared with controls. Surprisingly, these effects are mediated through inhibition of macrophage receptor interacting protein kinase 2 (RIPK2), rather than EGFR. Supporting this, lapatinib, a highly specific EGFR inhibitor that does not inhibit RIPK2, had no effect on macrophage-promoted invasion, and RIPK2-/- macrophages failed to promote invasion. The selective RIPK2 inhibitor WEHI-345 blocked tumor cell invasion in vitro and reduced metastatic burden in vivo In conclusion, our results indicate that gefitinib blocks macrophage-promoted invasion and metastatic extravasation by reprogramming macrophages through inhibition of RIPK2.

Immune Modulation of Allergic Asthma by Early Pharmacological Inhibition of RIP2.

Exposure to house dust mite (HDM) is highly associated with the development of allergic asthma. The adaptive immune response to HDM is largely Th2 and Th17 dominant, and a number of innate immune receptors have been identified that recognize HDM to initiate these responses. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is a cytosolic sensor of peptidoglycan, which is important for Th2 and Th17 polarization. NOD2 mediates its signaling through its downstream effector kinase, receptor-interacting serine/threonine protein kinase 2 (RIP2). We have previously shown that RIP2 promotes HDM-associated allergic airway inflammation and Th2 and Th17 immunity, acting early in the HDM response and likely within airway epithelial cells. However, the consequences of inhibiting RIP2 during this critical period has not yet been examined. In this study, we pharmacologically inhibited RIP2 activity during the initial exposure to allergen in an acute HDM model of asthma and determined the effect on the subsequent development of allergic airway disease. We show that early inhibition of RIP2 was sufficient to reduce lung histopathology and local airway inflammation while reducing the Th2 immune response. Using a chronic HDM asthma model, we demonstrate that inhibition of RIP2, despite attenuating airway inflammation and airway remodeling, was insufficient to reduce airway hyperresponsiveness. These data demonstrate the potential of pharmacological targeting of this kinase in asthma and support further development and optimization of RIP2-targeted therapies.

Discovery of a First-in-Class Receptor Interacting Protein 2 (RIP2) Kinase Specific Clinical Candidate, 2-((4-(Benzo[d]thiazol-5-ylamino)-6-(tert-butylsulfonyl)quinazolin-7-yl)oxy)ethyl Dihydrogen Phosphate, for the Treatment of Inflammatory Diseases.

RIP2 kinase has been identified as a key signal transduction partner in the NOD2 pathway contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP2 kinase or its signaling partners on the NOD2 pathway that are suitable for advancement into the clinic have yet to be described. Herein, we report our discovery and profile of the prodrug clinical compound, inhibitor 3, currently in phase 1 clinical studies. Compound 3 potently binds to RIP2 kinase with good kinase specificity and has excellent activity in blocking many proinflammatory cytokine responses in vivo and in human IBD explant samples. The highly favorable physicochemical and ADMET properties of 3 combined with high potency led to a predicted low oral dose in humans.

Identification of a novel class of RIP1/RIP3 dual inhibitors that impede cell death and inflammation in mouse abdominal aortic aneurysm models.

Receptor interacting protein kinase-1 and -3 (RIP1 and RIP3) are essential mediators of cell death processes and participate in inflammatory responses. Our group recently demonstrated that gene deletion of Rip3 or pharmacological inhibition of RIP1 attenuated pathogenesis of abdominal aortic aneurysm (AAA), a life-threatening degenerative vascular disease characterized by depletion of smooth muscle cells (SMCs), inflammation, negative extracellular matrix remodeling, and progressive expansion of aorta. The goal of this study was to develop drug candidates for AAA and other disease conditions involving cell death and inflammation. We screened 1141 kinase inhibitors for their ability to block necroptosis using the RIP1 inhibitor Necrostatin-1s (Nec-1s) as a selection baseline. Positive compounds were further screened for cytotoxicity and virtual binding to RIP3. A cluster of top hits, represented by GSK2593074A (GSK'074), displayed structural similarity to the established RIP3 inhibitor GSK'843. In multiple cell types including mouse SMCs, fibroblasts (L929), bone marrow derived macrophages (BMDM), and human colon epithelial cells (HT29), GSK'074 inhibited necroptosis with an IC50 of ~3 nM. Furthermore, GSK'074, but not Nec-1s, blocked cytokine production by SMCs. Biochemical analyses identified both RIP1 and RIP3 as the biological targets of GSK'074. Unlike GSK'843 which causes profound apoptosis at high doses (>3 µM), GSK'074 showed no detectable cytotoxicity even at 20 µM. Daily intraperitoneal injection of GSK'074 at 0.93 mg/kg significantly attenuated aortic expansion in two mouse models of AAA (calcium phosphate: DMSO 66.06 ± 9.17% vs GSK'074 27.36 ± 8.25%, P < 0.05; Angiotensin II: DMSO 85.39 ± 15.76% vs GSK'074 36.28 ± 5.76%, P < 0.05). Histologically, GSK'074 treatment diminished cell death and macrophage infiltration in aneurysm-prone aortae. Together, our data suggest that GSK'074 represents a new class of necroptosis inhibitors with dual targeting ability to both RIP1 and RIP3. The high potency and minimum cytotoxicity make GSK'074 a desirable drug candidate of pharmacological therapies to attenuate AAA progression and other necroptosis related diseases.

Development of a virtual reality platform for effective communication of structural data in drug discovery.

In drug discovery, structural knowledge of a target enables structure-based design approaches and thereby reduces the time and labor required to develop a therapy. Whilst molecular graphics frameworks coupled with computational analysis are now ubiquitous tools for the structural and computational biologist, sharing the detailed visualization and derived structural information with non-expert users still presents a challenge. Here we describe an intuitive virtual world for viewing, manipulating, and modifying chemical and macromolecular structures in a fully immersive and collaborative 3D environment. By reducing the barriers to viewing and interacting with structural data, structural analysis can be democratized to a general scientist, which in turn fosters novel collaboration, ideas, and findings in structural biology and structure-based drug discovery.

Computational design of new protein kinase 2 inhibitors for the treatment of inflammatory diseases using QSAR, pharmacophore-structure-based virtual screening, and molecular dynamics.

Receptor-interacting protein kinase 2 (RIPK2) plays an essential role in autoimmune response and is suggested as a target for inflammatory diseases. A pharmacophore model was built from a dataset with ponatinib (template) and 18 RIPK2 inhibitors selected from BindingDB database. The pharmacophore model validation was performed by multiple linear regression (MLR). The statistical quality of the model was evaluated by the correlation coefficient (R), squared correlation coefficient (R2), explanatory variance (adjusted R2), standard error of estimate (SEE), and variance ratio (F). The best pharmacophore model has one aromatic group (LEU24 residue interaction) and two hydrogen bonding acceptor groups (MET98 and TYR97 residues interaction), having a score of 24.739 with 14 aligned inhibitors, which were used in virtual screening via ZincPharmer server and the ZINC database (selected in function of the RMSD value). We determined theoretical values of biological activity (logRA) by MLR, pharmacokinetic and toxicology properties, and made molecular docking studies comparing binding affinity (kcal/mol) results with the most active compound of the study (ponatinib) and WEHI-345. Nine compounds from the ZINC database show satisfactory results, yielding among those selected, the compound ZINC01540228, as the most promising RIPK2 inhibitor. After binding free energy calculations, the following molecular dynamics simulations showed that the receptor protein's backbone remained stable after the introduction of ligands.

Small molecule inhibitors reveal an indispensable scaffolding role of RIPK2 in NOD2 signaling.

RIPK2 mediates inflammatory signaling by the bacteria-sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD-mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP-binding and XIAP-mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between ß2 and ß3 of the N-lobe of the kinase, which is in close proximity to the ATP-binding pocket. Through characterization of a new series of ATP pocket-binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2-XIAP interaction, and of cellular and in vivoNOD2 signaling. Our study exemplifies how targeting of the ATP-binding pocket in RIPK2 can be exploited to interfere with the RIPK2-XIAP interaction for modulation of NOD signaling.

Identification and Characterization of Novel Receptor-Interacting Serine/Threonine-Protein Kinase 2 Inhibitors Using Structural Similarity Analysis.

Receptor-interacting protein kinase 2 (RIP2 or RICK, herein referred to as RIPK2) is linked to the pathogen pathway that activates nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) and autophagic activation. Using molecular modeling (docking) and chemoinformatics analyses, we used the RIPK2/ponatinib crystal structure and searched in chemical databases for small molecules exerting binding interactions similar to those exerted by ponatinib. The identified RIPK2 inhibitors potently inhibited the proliferation of cancer cells by > 70% and also inhibited NFκB activity. More importantly, in vivo inhibition of intestinal and lung inflammation rodent models suggests effectiveness to resolve inflammation with low toxicity to the animals. Thus, our identified RIPK2 inhibitor may offer possible therapeutic control of inflammation in diseases such as inflammatory bowel disease, asthma, cystic fibrosis, primary sclerosing cholangitis, and pancreatitis.

Identification of Novel Protein Kinase Receptor Type 2 Inhibitors Using Pharmacophore and Structure-Based Virtual Screening.

The Protein Kinase Receptor type 2 (RIPK2) plays an important role in the pathogenesis of inflammatory diseases; it signals downstream of the NOD1 and NOD2 intracellular sensors and promotes a productive inflammatory response. However, excessive NOD2 signaling has been associated with various diseases, including sarcoidosis and inflammatory arthritis; the pharmacological inhibition of RIPK2 is an affinity strategy that demonstrates an increased expression of pro-inflammatory secretion activity. In this study, a pharmacophoric model based on the crystallographic pose of ponatinib, a potent RIPK2 inhibitor, and 30 other ones selected from the BindingDB repository database, was built. Compounds were selected based on the available ZINC compounds database and in silico predictions of their pharmacokinetic, toxicity and potential biological activity. Molecular docking was performed to identify the probable interactions of the compounds as well as their binding affinity with RIPK2. The compounds were analyzed to ponatinib and WEHI-345, which also used as a control. At least one of the compounds exhibited suitable pharmacokinetic properties, low toxicity and an interesting binding affinity and high fitness compared with the crystallographic pose of WEHI-345 in complex with RIPK2. This compound also possessed suitable synthetic accessibility, rendering it a potential and very promising RIPK2 inhibitor to be further investigated in regards to different diseases, particularly inflammatory ones.

Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors.

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity.

Disruption of XIAP-RIP2 Association Blocks NOD2-Mediated Inflammatory Signaling.

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.

Impaired antibacterial autophagy links granulomatous intestinal inflammation in Niemann-Pick disease type C1 and XIAP deficiency with NOD2 variants in Crohn's disease.

OBJECTIVE: Patients with Niemann-Pick disease type C1 (NPC1), a lysosomal lipid storage disorder that causes neurodegeneration and liver damage, can present with IBD, but neither the significance nor the functional mechanism of this association is clear. We studied bacterial handling and antibacterial autophagy in patients with NPC1. DESIGN: We characterised intestinal inflammation in 14 patients with NPC1 who developed IBD. We investigated bacterial handling and cytokine production of NPC1 monocytes or macrophages in vitro and compared NPC1-associated functional defects to those caused by IBD-associated nucleotide-binding oligomerization domain-containing protein 2 (NOD2) variants or mutations in X-linked inhibitor of apoptosis (XIAP). RESULTS: Patients with the lysosomal lipid storage disorder NPC1 have increased susceptibility to early-onset fistulising colitis with granuloma formation, reminiscent of Crohn's disease (CD). Mutations in NPC1 cause impaired autophagy due to defective autophagosome function that abolishes NOD2-mediated bacterial handling in vitro similar to variants in NOD2 or XIAP deficiency. In contrast to genetic NOD2 and XIAP variants, NPC1 mutations do not impair NOD2-receptor-interacting kinase 2 (RIPK2)-XIAP-dependent cytokine production. Pharmacological activation of autophagy can rescue bacterial clearance in macrophages in vitro by increasing the autophagic flux and bypassing defects in NPC1. CONCLUSIONS: NPC1 confers increased risk of early-onset severe CD. Our data support the concept that genetic defects at different checkpoints of selective autophagy cause a shared outcome of CD-like immunopathology linking monogenic and polygenic forms of IBD. Muramyl dipeptide-driven cytokine responses and antibacterial autophagy induction are parallel and independent signalling cascades downstream of the NOD2-RIPK2-XIAP complex.

The Identification and Pharmacological Characterization of 6-(tert-Butylsulfonyl)-N-(5-fluoro-1H-indazol-3-yl)quinolin-4-amine (GSK583), a Highly Potent and Selective Inhibitor of RIP2 Kinase.

RIP2 kinase is a central component of the innate immune system and enables downstream signaling following activation of the pattern recognition receptors NOD1 and NOD2, leading to the production of inflammatory cytokines. Recently, several inhibitors of RIP2 kinase have been disclosed that have contributed to the fundamental understanding of the role of RIP2 in this pathway. However, because they lack either broad kinase selectivity or strong affinity for RIP2, these tools have only limited utility to assess the role of RIP2 in complex environments. We present, herein, the discovery and pharmacological characterization of GSK583, a next-generation RIP2 inhibitor possessing exquisite selectivity and potency. Having demonstrated the pharmacological precision of this tool compound, we report its use in elucidating the role of RIP2 kinase in a variety of in vitro, in vivo, and ex vivo experiments, further clarifying our understanding of the role of RIP2 in NOD1 and NOD2 mediated disease pathogenesis.