ABSTRACT
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.
Subject(s)
Benzothiazoles/pharmacology , Phosphates/pharmacology , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinase 2/antagonists & inhibitors , Animals , Benzothiazoles/chemistry , Benzothiazoles/pharmacokinetics , Benzothiazoles/therapeutic use , Colitis/drug therapy , Dogs , Drug Discovery , Humans , Male , Mice , Molecular Docking Simulation , Phosphates/chemistry , Phosphates/pharmacokinetics , Phosphates/therapeutic use , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Quinazolines/chemistry , Quinazolines/pharmacokinetics , Quinazolines/therapeutic use , Rats, Sprague-Dawley , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Swine , Swine, MiniatureABSTRACT
RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including inflammatory and neurological diseases. Currently, RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis and neurological diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In this paper, we report on the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting from a high-throughput screen and the lead-optimization of this series from a lead with minimal rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles in multiple species. Additionally, we identified a potent murine RIP1 kinase inhibitor 76 as a valuable in vivo tool molecule suitable for evaluating the role of RIP1 kinase in chronic models of disease. DHP 76 showed efficacy in mouse models of both multiple sclerosis and human retinitis pigmentosa.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Nuclear Pore Complex Proteins/antagonists & inhibitors , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , RNA-Binding Proteins/antagonists & inhibitors , Animals , Biological Availability , Cell Line , Chronic Disease , Drug Design , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Enzyme Inhibitors/pharmacokinetics , Haplorhini , High-Throughput Screening Assays , Humans , Mice , Mice, Inbred C57BL , Models, Molecular , Multiple Sclerosis/drug therapy , Pyrazoles/pharmacokinetics , Rats , Retinitis Pigmentosa/drug therapy , Structure-Activity RelationshipABSTRACT
The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-ß-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.
Subject(s)
Caspase 8/metabolism , Caspases/metabolism , Escherichia coli Infections/enzymology , Escherichia coli Infections/physiopathology , Shock, Septic/enzymology , Shock, Septic/physiopathology , Animals , Apoptosis , Apoptosis Regulatory Proteins/metabolism , Caspase 8/genetics , Caspases/genetics , Caspases, Initiator , Cells, Cultured , Female , Inflammation/metabolism , Inflammation/pathology , Interferon Regulatory Factor-3/genetics , Interferon-beta/blood , Interferon-beta/metabolism , Intestine, Small/pathology , Intracellular Signaling Peptides and Proteins , Lipopolysaccharides/toxicity , Male , Mice, Inbred C57BL , Mice, Knockout , Phosphate-Binding Proteins , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Spleen/pathology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/blood , Tumor Necrosis Factor-alpha/metabolismABSTRACT
RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.
Subject(s)
Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Colitis, Ulcerative/drug therapy , Inflammation/drug therapy , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Animals , Benzazepines/chemistry , Benzazepines/pharmacology , Colitis, Ulcerative/immunology , Cytokines/immunology , Dogs , Haplorhini , Humans , Inflammation/immunology , Mice , Molecular Docking Simulation , Rabbits , Rats , Receptor-Interacting Protein Serine-Threonine Kinases/immunology , Swine , Swine, Miniature , Tumor Necrosis Factor-alpha/immunologyABSTRACT
The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.
Subject(s)
DNA/chemistry , Isoxazoles/pharmacology , Oxazepines/pharmacology , Protein Kinase Inhibitors/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Animals , Cell Line, Tumor , Crystallography, X-Ray , Dose-Response Relationship, Drug , HT29 Cells , Humans , Isoxazoles/chemical synthesis , Isoxazoles/chemistry , Mice , Models, Molecular , Molecular Structure , Oxazepines/chemical synthesis , Oxazepines/chemistry , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity Relationship , U937 CellsABSTRACT
RIP1 (RIPK1) kinase is a key regulator of TNF-induced NF-κB activation, apoptosis, and necroptosis through its kinase and scaffolding activities. Dissecting the balance of RIP1 kinase activity and scaffolding function in vivo during development and TNF-dependent inflammation has been hampered by the perinatal lethality of RIP1-deficient mice. In this study, we generated RIP1 kinase-dead (Ripk1(K45A)) mice and showed they are viable and healthy, indicating that the kinase activity of RIP1, but not its scaffolding function, is dispensable for viability and homeostasis. After validating that the Ripk1(K45A) mice were specifically protected against necroptotic stimuli in vitro and in vivo, we crossed them with SHARPIN-deficient cpdm mice, which develop severe skin and multiorgan inflammation that has been hypothesized to be mediated by TNF-dependent apoptosis and/or necroptosis. Remarkably, crossing Ripk1(K45A) mice with the cpdm strain protected against all cpdm-related pathology. Together, these data suggest that RIP1 kinase represents an attractive therapeutic target for TNF-driven inflammatory diseases.
Subject(s)
Carrier Proteins/immunology , Receptor-Interacting Protein Serine-Threonine Kinases/immunology , Animals , Apoptosis/genetics , Apoptosis/immunology , Carrier Proteins/genetics , Carrier Proteins/metabolism , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , Intracellular Signaling Peptides and Proteins , Mice , Mice, Mutant Strains , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
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.
ABSTRACT
The purpose was to aid in determining termination of instrumentation and obturation. A meta-analysis was conducted as to success/failure of different obturation lengths. Inclusion criteria were (a) minimum follow-up of 2 yr, (b) data on obturation length, (c) definition of success/failure, (d) available data on success/failure, (e) radiographic evaluation. Correlations were made as to success/failure as related to length of obturation from the apex. When comparing group A (obturated 0-1 mm from apex) versus group C (obturated past apex) using the DerSimonian and Laird estimates, group A showed a marginally better (p < 0.10) success rate than group C by 28.8%. Group A had better success than group B (obturated >1 mm short); the difference was insignificant. The results were similar after controlling for study quality using a single random effects regression model. In conclusion, the meta-analysis indicated that a better success rate is achieved when treatment includes obturation short of the apex.
