Gasdermins assemble; recent developments in bacteriology and pharmacology.

The discovery of gasdermin D (GSDMD) as the terminal executioner of pyroptosis provided a large piece of the cell death puzzle, whilst simultaneously and firmly putting the gasdermin family into the limelight. In its purest form, GSDMD provides a connection between the innate alarm systems to an explosive, inflammatory form of cell death to jolt the local environment into immunological action. However, the gasdermin field has moved rapidly and significantly since the original seminal work and novel functions and mechanisms have been recently uncovered, particularly in response to infection.

RIPK1 kinase-dependent inflammation and cell death contribute to the pathogenesis of COPD.

Background: Receptor-interacting protein kinase 1 (RIPK1) is a key mediator of regulated cell death (including apoptosis and necroptosis) and inflammation, both drivers of COPD pathogenesis. We aimed to define the contribution of RIPK1 kinase-dependent cell death and inflammation in the pathogenesis of COPD.

Methods: We assessed expression in single-cell RNA sequencing (RNA-seq) data from human and mouse lungs, and validated RIPK1 levels in lung tissue of COPD patients immunohistochemistry.

Ubiquitylation of MLKL at lysine 219 positively regulates necroptosis-induced tissue injury and pathogen clearance.

Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL.

Extended pharmacodynamic responses observed upon PROTAC-mediated degradation of RIPK2.

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.

Discovery of a First-in-Class Receptor Interacting Protein 2 (RIP2) Kinase Specific Clinical Candidate, 2-((4-(Benzo[]thiazol-5-ylamino)-6-(-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 , currently in phase 1 clinical studies.

Discovery and Lead-Optimization of 4,5-Dihydropyrazoles as Mono-Kinase Selective, Orally Bioavailable and Efficacious Inhibitors of Receptor Interacting Protein 1 (RIP1) Kinase.

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.

Use of RIP1 Kinase Small-Molecule Inhibitors in Studying Necroptosis.

RIP1 kinase plays a key role in regulating signaling pathways downstream of a number of innate immune receptors such as TNFRI and TLRs. The discovery of Necrostatin-1 (Nec-1) as a small-molecule inhibitor of RIP1 kinase has been very instrumental in defining the necroptotic and other signalling pathways regulated by RIP1, but certain characteristics of Nec-1 limits its utility in experimental systems. Next generation RIP1 kinase inhibitors have been identified and the use of these tool inhibitors along with Nec-1 has revealed that RIP1 is emerging as a key driver of inflammation and tissue injury in the pathogenesis of various diseases.

LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis.

The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1 . Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype.

Autophagy protein ATG16L1 prevents necroptosis in the intestinal epithelium.

A variant of the autophagy gene is associated with Crohn's disease, an inflammatory bowel disease (IBD), and poor survival in allogeneic hematopoietic stem cell transplant recipients. We demonstrate that ATG16L1 in the intestinal epithelium is essential for preventing loss of Paneth cells and exaggerated cell death in animal models of virally triggered IBD and allogeneic hematopoietic stem cell transplantation. Intestinal organoids lacking ATG16L1 reproduced this loss in Paneth cells and displayed TNFα-mediated necroptosis, a form of programmed necrosis.

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.

MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses.

CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties.

Ndfip1 enforces a requirement for CD28 costimulation by limiting IL-2 production.

Although the pathways that permit IL-2 production and the full activation of T cells upon Ag encounter are fairly well defined, the negative regulatory circuits that limit these pathways are poorly understood. In this study, we show that the E3 ubiquitin ligase adaptor Ndfip1 directs one such negative regulatory circuit. T cells lacking Ndfip1 produce IL-2, upregulate IL-2Rα, and proliferate, in the absence of CD28 costimulation.

The E3 ubiquitin ligase adaptor Ndfip1 regulates Th17 differentiation by limiting the production of proinflammatory cytokines.

Ndfip1 is an adaptor for the E3 ubiquitin ligase Itch. Both Ndfip1- and Itch-deficient T cells are biased toward Th2 cytokine production. In this study, we demonstrate that lungs from Ndfip1(-/-) mice showed increased numbers of neutrophils and Th17 cells.

TGF-β induces the expression of the adaptor Ndfip1 to silence IL-4 production during iTreg cell differentiation.

Mice deficient in the adaptor Ndfip1 develop inflammation at sites of environmental antigen exposure. We show here that such mice had fewer inducible regulatory T cells (iT(reg) cells). In vitro, Ndfip1-deficient T cells expressed normal amounts of the transcription factor Foxp3 during the first 48 h of iT(reg) cell differentiation; however, this expression was not sustained.

Kinetics of early T cell receptor signaling regulate the pathway of lytic granule delivery to the secretory domain.

Cytolytic granules mediate killing of virus-infected cells by cytotoxic T lymphocytes. We show here that the granules can take long or short paths to the secretory domain. Both paths utilized the same intracellular molecular events, which have different spatial and temporal arrangements and are regulated by the kinetics of Ca(2+)-mediated signaling.

Protein kinase C theta regulates stability of the peripheral adhesion ring junction and contributes to the sensitivity of target cell lysis by CTL.

Destruction of virus-infected cells by CTL is an extremely sensitive and efficient process. Our previous data suggest that LFA-1-ICAM-1 interactions in the peripheral supramolecular activation cluster (pSMAC) of the immunological synapse mediate formation of a tight adhesion junction that might contribute to the sensitivity of target cell lysis by CTL. Herein, we compared more (CD8(+)) and less (CD4(+)) effective CTL to understand the molecular events that promote efficient target cell lysis.