[This corrects the article DOI: 10.1021/acsmedchemlett.8b00344.].

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.

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.

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.

NOD1 and NOD2 are cytoplasmic PRRs, which bind peptides derived from bacterial peptidoglycans. Signaling via these receptors involves recruitment and activation of RIP2 kinase, which results in NF-κB- and MAPK-dependent pro-inflammatory cytokine production. To enable a chemical biology approach to evaluate the role of RIP2 in inflammation we screened an in-house compound collection. Optimization of an aminoquinoline template led to the discovery of GSK’214 and related analogs. GSK’214 potently and reversibly inhibited human, rat and mouse RIP2 enzymatic activity (IC50 3-10 nM). Selectivity was demonstrated against a panel of 273 commercially-available kinases. NOD2-mediated signaling induced by muramyl dipeptide (MDP) was used to characterize functional activity, in vitro and in vivo. GSK’214 inhibited cytokine (TNFα, IL1β, IL-6 and IL-8) production (IC50 20 nM) by human monocytes stimulated by MDP, but not by ligands specific for TLR2, TLR4, TLR7, TLR9, IL1R and TNFR. Using an acute mouse peritonitis assay, orally administered GSK’214 inhibited neutrophil influx and cytokine release induced by MDP, but not LPS. Next, we used these inhibitors to probe the role of RIP2 in a murine TNBS-colitis model of intestinal inflammation. Selective inhibition of RIP2 kinase activity reduced disease severity to a degree comparable to prednisolone. These results demonstrate the potential of these inhibitors to elucidate the contribution of RIP2 kinase-dependent signaling in inflammation.

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.