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Introduction: Receptor-interacting protein kinase 1 (RIPK1), a key mediator of inflammation through necroptosis and proinflammatory cytokine production, may play a role in the pathogenesis of immune-mediated inflammatory diseases such as chronic plaque psoriasis. An experimental medicine study of RIPK1 inhibition with GSK2982772 immediate-release formulation at doses up to 60 mg three times daily in mild to moderate plaque psoriasis indicated that efficacy may be improved with higher trough concentrations of GSK2982772., Methods: This multicenter, randomized, double-blind, placebo-controlled, repeat-dose study (NCT04316585) assessed the efficacy, safety, pharmacokinetics, and pharmacodynamics of 960 mg GSK2982772 (once-daily modified-release formulation) in patients with moderate to severe plaque psoriasis. Twenty-nine patients were randomized 2:1 to GSK2982772 (N = 19) or placebo (N = 10) for 12 weeks., Results: GSK2982772 was well tolerated with trough concentrations greater than tenfold higher than the previous phase 1 study with immediate release. Despite near complete RIPK1 target engagement in blood and modest reduction in circulating inflammatory cytokines, the proportion of patients achieving 75% improvement from baseline in Psoriasis Area Severity Index score at week 12 was similar between GSK2982772 and placebo (posterior median 1.8% vs 4.9%, respectively), with an estimated median treatment difference of - 2.3%. This analysis incorporated historical placebo data through the use of an informative prior distribution on the placebo arm. Week 4 changes in skin biopsy gene expression suggested sufficient local drug exposure to elicit a pharmacodynamic response., Conclusion: Administration of the RIPK1 inhibitor GSK2982772 to patients with moderate to severe plaque psoriasis did not translate into meaningful clinical improvements., (© 2024. The Author(s).)

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is an important kinase of the innate immune system. Herein, we describe the optimization of a series of RIPK2 PROTACs which recruit members of the inhibitor of apoptosis (IAP) family of E3 ligases. Our PROTAC optimization strategy focused on reducing the lipophilicity of the early lead which resulted in the identification of analogues with improved solubility and increased human and rat microsomal stability. We identified a range of IAP binders that were successfully incorporated into potent RIPK2 PROTACs with attractive pharmacokinetic profiles. Compound 20 possessed the best overall profile with good solubility, potent degradation of RIPK2, and associated inhibition of TNFα release. A proof-of-concept study utilizing a slow release matrix demonstrated the feasibility of a long-acting parenteral formulation with >1 month duration. This represents an attractive alternative dosing paradigm to oral delivery, especially for chronic diseases where compliance can be challenging.

Receptor Interacting Protein 2 (RIP2) kinase inhibitors have been reported for therapeutic opportunities in inflammatory bowel diseases such as Ulcerative Colitis and Crohn's disease. During lead optimization, team identified 4-aminoquinoline series and several compounds from this series were investigated in rat and dog pharmacokinetic studies. While compounds such as GSKA and GSKB demonstrated acceptable pharmacokinetics in rat and dog, further progression of these compounds was halted due to adverse findings in advanced safety studies. Structurally similar analogues incorporating polarity at C-7 position of 4-aminoquinoline resulted in identification of GSKC - GSKF. Interestingly, following oral administration to rat at similar low dose, GSKC - GSKF demonstrated significantly low systemic drug exposure compared to GSKA and GSKB (3-17-fold difference). However, in dog, dose normalized oral systemic exposure for GSKC - GSKF was comparable to GSKA and GSKB (within 2-fold). A series of studies were conducted to understand the disconnect which highlighted that an intrinsic reduction in permeability and high P-glycoprotein (P-gp) efflux ratio for C-7 substituted analogues were driving pharmacokinetic disconnect between rat and dog. Oral absorption was minimally impacted in dog by P-gp mediated efflux compared to rat because the leakier gastrointestinal tract in dog likely overcomes this effect., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines.

Herein we report the discovery of pyrazolocarboxamides as novel, potent, and kinase selective inhibitors of receptor interacting protein 2 kinase (RIP2). Fragment based screening and design principles led to the identification of the inhibitor series, and X-ray crystallography was used to inform key structural changes. Through key substitutions about the N1 and C5 N positions on the pyrazole ring significant kinase selectivity and potency were achieved. Bridged bicyclic pyrazolocarboxamide 11 represents a selective and potent inhibitor of RIP2 and will allow for a more detailed investigation of RIP2 inhibition as a therapeutic target for autoinflammatory disorders., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)

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.

Epoxyeicosatrienoic acids (EETs) are signaling lipids produced by cytochrome P450 epoxygenation of arachidonic acid, which are metabolized by EPHX2 (epoxide hydrolase 2, alias soluble epoxide hydrolase or sEH). EETs have pleiotropic effects, including anti-inflammatory activity. Using a Connectivity Map (CMAP) approach, we identified an inverse-correlation between an exemplar EPHX2 inhibitor (EPHX2i) compound response and an inflammatory bowel disease patient-derived signature. To validate the gene-disease link, we tested a pre-clinical tool EPHX2i (GSK1910364) in a mouse disease model, where it showed improved outcomes comparable to or better than the positive control Cyclosporin A. Up-regulation of cytoprotective genes and down-regulation of proinflammatory cytokine production were observed in colon samples obtained from EPHX2i-treated mice. Follow-up immunohistochemistry analysis verified the presence of EPHX2 protein in infiltrated immune cells from Crohn's patient tissue biopsies. We further demonstrated that GSK2256294, a clinical EPHX2i, reduced the production of IL2, IL12p70, IL10 and TNFα in both ulcerative colitis and Crohn's disease patient-derived explant cultures. Interestingly, GSK2256294 reduced IL4 and IFNγ in ulcerative colitis, and IL1β in Crohn's disease specifically, suggesting potential differential effects of GSK2256294 in these two diseases. Taken together, these findings suggest a novel therapeutic use of EPHX2 inhibition for IBD., Competing Interests: The GSK authors commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.

RIP2 kinase was recently identified as a therapeutic target for a variety of autoimmune diseases. We have reported previously a selective 4-aminoquinoline-based RIP2 inhibitor GSK583 and demonstrated its effectiveness in blocking downstream NOD2 signaling in cellular models, rodent in vivo models, and human ex vivo disease models. While this tool compound was valuable in validating the biological pathway, it suffered from activity at the hERG ion channel and a poor PK/PD profile thereby limiting progression of this analog. Herein, we detail our efforts to improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity and strengthening hinge binding ability. These efforts have led to inhibitor 7 , which possesses high binding affinity for the ATP pocket of RIP2 (IC 50 = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC 50 = 10 nM) with reduced hERG activity (14 μM)., Competing Interests: The authors declare the following competing financial interest(s): All GSK authors are/were employees and stockholders of GlaxoSmithKline when this work was completed.

Objective: To identify biomarkers of articular and ocular disease activity in patients with Blau syndrome (BS)., Methods: Multiplex plasma protein arrays were performed in five BS patients and eight normal healthy volunteers (NHVs). Plasma S100A12 and S100A8/9 were subsequently measured by ELISA at baseline and 1-year follow-up in all patients from a prospective multicentre cohort study. CRP was measured using Meso Scale Discovery immunoassay. Active joint counts, standardization uveitis nomenclature for anterior uveitis cells and vitreous haze by Nussenblatt scale were the clinical parameters., Results: Multiplex Luminex arrays identified S100A12 as the most significantly elevated protein in five selected BS vs eight NHVs and this was confirmed by ELISA on additional samples from the same five BS patients. In the patient cohort, S100A12 (n = 39) and S100A8/9 (n = 33) were significantly higher compared with NHVs (n = 44 for S100A12, n = 40 for S100A8/9) (P = 0.0000004 and P = 0.0003, respectively). Positive correlations between active joint counts and S100 levels were significant for S100A12 (P = 0.0008) and S100A8/9 (P = 0.015). CRP levels did not correlate with active joint count. Subgroup analysis showed significant association of S100 proteins with active arthritis (S100A12 P = 0.01, S100A8/9 P = 0.008). Active uveitis was not associated with increased S100 levels., Conclusion: S100 proteins are biomarkers of articular disease activity in BS and potential outcome measures in future clinical trials. As secreted neutrophil and macrophage products, S100 proteins may reflect the burden of granulomatous tissue in BS., (© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Therapies that suppress RIPK1 kinase activity are emerging as promising therapeutic agents for the treatment of multiple inflammatory disorders. The ability to directly measure drug binding of a RIPK1 inhibitor to its target is critical for providing insight into pharmacokinetics, pharmacodynamics, safety and clinical efficacy, especially for a first-in-class small-molecule inhibitor where the mechanism has yet to be explored. Here, we report a novel method for measuring drug binding to RIPK1 protein in cells and tissues. This TEAR1 (Target Engagement Assessment for RIPK1) assay is a pair of immunoassays developed on the principle of competition, whereby a first molecule (ie, drug) prevents the binding of a second molecule (ie, antibody) to the target protein. Using the TEAR1 assay, we have validated the direct binding of specific RIPK1 inhibitors in cells, blood and tissues following treatment with benzoxazepinone (BOAz) RIPK1 inhibitors. The TEAR1 assay is a valuable tool for facilitating the clinical development of the lead RIPK1 clinical candidate compound, GSK2982772, as a first-in-class RIPK1 inhibitor for the treatment of inflammatory disease., (© 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

GSK2982772 is a highly selective inhibitor of receptor-interacting protein kinase 1 (RIPK1) being developed to treat chronic inflammatory diseases. This first-in-human study evaluated safety, tolerability, pharmacokinetics (PK), and exploratory pharmacodynamics (PD) of GSK2982772 administered orally to healthy male volunteers. This was a Phase I, randomized, placebo-controlled, double-blind study. In Part A, subjects received single ascending doses of GSK2982772 (0.1-120 mg) or placebo in a crossover design during each of 4 treatment periods. In Part B, subjects received repeat doses of GSK2982772 (20 mg once daily [QD] to up to 120 mg twice daily [BID]) or placebo for 14 days. Part C was an open-label relative bioavailability study comparing 20-mg tablets vs capsules. Safety, tolerability, pharmacokinetics (PK), RIPK1 target engagement (TE), and pharmacodynamics (PD) were assessed. The most common adverse events (AEs) were contact dermatitis and headache. Most AEs were mild in intensity, and there were no deaths or serious AEs. The PK of GSK2982772 was approximately linear over the dose range studied (up to 120 mg BID). There was no evidence of drug accumulation upon repeat dosing. Greater than 90% RIPK1 TE was achieved over a 24-hour period for the 60-mg and 120-mg BID dosing regimens. Single and repeat doses of GSK2982772 were safe and well tolerated. PK profiles showed dose linearity. The high levels of RIPK1 TE support progression into Phase II clinical trials for further clinical development., (© 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

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.

Receptor interacting protein 2 (RIP2) is an intracellular kinase and key signaling partner for the pattern recognition receptors NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins 1 and 2). As such, RIP2 represents an attractive target to probe the role of these pathways in disease. In an effort to design potent and selective inhibitors of RIP2 we established a crystallographic system and determined the structure of the RIP2 kinase domain in an apo form and also in complex with multiple inhibitors including AMP-PCP (β,γ-Methyleneadenosine 5'-triphosphate, a non-hydrolysable adenosine triphosphate mimic) and structurally diverse ATP competitive chemotypes identified via a high-throughput screening campaign. These structures represent the first set of diverse RIP2-inhibitor co-crystal structures and demonstrate that the protein possesses the ability to adopt multiple DFG-in as well as DFG-out and C-helix out conformations. These structures reveal key protein-inhibitor structural insights and serve as the foundation for establishing a robust structure-based drug design effort to identify both potent and highly selective inhibitors of RIP2 kinase., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.