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.

Summary Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo . sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence.

Background and aimThymus-derived regulatory T cells (Tregs) mediate dominant peripheral tolerance and treat experimental colitis. Tregs can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. Treg cell therapy is also conceptually attractive for Crohn's disease (CD). However, barriers exist to this approach. The stability of Tregs expanded from Crohn's blood is unknown. The potential for adoptively transferred Tregs to express interleukin-17 and exacerbate Crohn's lesions is of concern. Mucosal T cells are resistant to Treg-mediated suppression in active CD. The capacity for expanded Tregs to home to gut and lymphoid tissue is unknown.MethodsTo define the optimum population for Treg cell therapy in CD, CD4+CD25+CD127loCD45RA+ and CD4+CD25+CD127loCD45RA− Treg subsets were isolated from patients’ blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background.ResultsTregs can be expanded from the blood of patients with CD to potential target dose within 22–24 days. Expanded CD45RA+ Tregs have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA− Tregs. CD45RA+ Tregs highly express α4β7 integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA+ Tregs also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA+ Tregs. These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohn's mucosa.ConclusionsCD4+CD25+CD127loCD45RA+ Tregs may be the most appropriate population from which to expand Tregs for autologous Treg therapy for CD, paving the way for future clinical trials.

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.

Arginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasing cAMP, which promotes the redistribution of AQP2 from intracellular vesicles into the plasma membrane. cAMP also increases AQP2 transcription, but whether altered degradation also modulates AQP2 protein levels is not well understood. Here, elevation of cAMP increased AQP2 protein levels within 30 minutes in primary inner medullary collecting duct (IMCD) cells, in human embryonic kidney (HEK) 293 cells ectopically expressing AQP2, and in mouse kidneys. Accelerated transcription or translation did not explain this increase in AQP2 abundance. In IMCD cells, cAMP inhibited p38-mitogen-activated protein kinase (p38-MAPK) via activation of protein kinase A (PKA). Inhibition of p38-MAPK associated with decreased phosphorylation (serine 261) and polyubiquitination of AQP2, preventing proteasomal degradation. Our results demonstrate that AVP enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA-and p38-MAPK-dependent pathway. Aquaporin-2 (AQP2) is the water channel mediating arginine-vasopressin (AVP)-increases in water re-absorption in renal collecting duct principal cells. 1-4 AVP binds to plasma membrane-located vasopressin V2 receptors, thereby stimulating adenylyl cyclase and elevating cAMP. cAMP activates protein kinase A (PKA), which phosphorylates AQP2 at serine 256 (S256), inducing its redistribution from intracellular vesicles into the plasma membrane. 3,4 This short-term regulation of AQP2 occurs within seconds to minutes. In the case of long-term regulation, cAMP enhances AQP2 mRNA expression, followed by a rise in the AQP2 protein level within hours. 5,6 AQP2 can be degraded in proteasomes and lysosomes. 7,8 Ubiquitination directs proteins for degradation to both compartments. Monoubiquitination (mUb) is a signal for degradation in lysosomes, whereas polyubiquitination (pUb) is mainly linked to proteasomal degradation. 9 mUb of AQP2 is induced by FSK stimulation and occurs at the apical plasma membrane. 10 In WT5 cells, a model for AQP2 regulation, increased mUb of AQP2 persists