Covalent Targeting As a Common Mechanism for Inhibiting NLRP3 Inflammasome Assembly.

The NLRP3 inflammasome is a cytosolic protein complex important for the regulation and secretion of inflammatory cytokines, including IL-1ß and IL-18. Aberrant overactivation of NLRP3 is implicated in numerous inflammatory disorders. However, the activation and regulation of NLRP3 inflammasome signaling remain poorly understood, limiting our ability to develop pharmacologic approaches to target this important inflammatory complex. Here, we developed and implemented a high-throughput screen to identify compounds that inhibit the inflammasome assembly and activity. From this screen, we identify and profile inflammasome inhibition of 20 new covalent compounds across nine different chemical scaffolds, as well as many known inflammasome covalent inhibitors. Intriguingly, our results indicate that NLRP3 possesses numerous reactive cysteines on multiple domains whose covalent targeting blocks the activation of this inflammatory complex. Specifically, focusing on compound VLX1570, which possesses multiple electrophilic moieties, we demonstrate that this compound allows covalent, intermolecular cross-linking of NLRP3 cysteines to inhibit inflammasome assembly. Our results, along with the recent identification of numerous covalent molecules that inhibit NLRP3 inflammasome activation, further support the continued development of electrophilic compounds that target reactive cysteine residues on NLRP3 to regulate its activation and activity.

Pharmacological inhibition of CLK2 activates YAP by promoting alternative splicing of AMOTL2.

Yes-associated protein (YAP), the downstream effector of the evolutionarily conserved Hippo pathway, promotes cellular proliferation and coordinates certain regenerative responses in mammals. Small molecule activators of YAP may, therefore, display therapeutic utility in treating disease states involving insufficient proliferative repair. From a high-throughput chemical screen of the comprehensive drug repurposing library ReFRAME, here we report the identification of SM04690, a clinical stage inhibitor of CLK2, as a potent activator of YAP-driven transcriptional activity in cells. CLK2 inhibition promotes alternative splicing of the Hippo pathway protein AMOTL2, producing an exon-skipped gene product that can no longer associate with membrane-bound proteins, resulting in decreased phosphorylation and membrane localization of YAP. This study reveals a novel mechanism by which pharmacological perturbation of alternative splicing inactivates the Hippo pathway and promotes YAP-dependent cellular growth.

Succinylation of a KEAP1 sensor lysine promotes NRF2 activation.

Cross talk between metabolism and stress-responsive signaling is essential for maintaining cellular homeostasis. This cross talk is often achieved through covalent modification of proteins by endogenous, reactive metabolites that regulate key stress-responsive transcription factors like NRF2. Metabolites including methylglyoxal, glyceraldehyde 3-phosphate, fumarate, and itaconate covalently modify sensor cysteines of the NRF2 repressor KEAP1, resulting in stabilization of NRF2 and activation of its cytoprotective transcriptional program. Here, we employed a shRNA-based screen targeting the enzymes of central carbon metabolism to identify additional regulatory nodes bridging metabolism to NRF2 activation. Succinic anhydride, increased by genetic depletion of the TCA cycle enzyme succinyl-CoA synthetase or by direct administration, results in N-succinylation of lysine 131 of KEAP1 to activate NRF2 signaling. This study identifies KEAP1 as capable of sensing reactive metabolites not only by several cysteine residues but also by a conserved lysine residue, indicating its potential to sense an expanded repertoire of reactive metabolic messengers.

Covalent targeting as a common mechanism for inhibiting NLRP3 inflammasome assembly.

The NLRP3 inflammasome is a cytosolic protein complex important for the regulation and secretion of inflammatory cytokines including IL-1ß and IL-18. Aberrant overactivation of NLRP3 is implicated in numerous inflammatory disorders. However, the activation and regulation of NLRP3 inflammasome signaling remains poorly understood, limiting our ability to develop pharmacologic approaches to target this important inflammatory complex. Here, we developed and implemented a high-throughput screen to identify compounds that inhibit inflammasome assembly and activity. From this screen we identify and profile inflammasome inhibition of 20 new covalent compounds across 9 different chemical scaffolds, as well as many known inflammasome covalent inhibitors. Intriguingly, our results indicate that NLRP3 possesses numerous reactive cysteines on multiple domains whose covalent targeting blocks activation of this inflammatory complex. Specifically, focusing on compound VLX1570, which possesses multiple electrophilic moieties, we demonstrate that this compound allows covalent, intermolecular crosslinking of NLRP3 cysteines to inhibit inflammasome assembly. Our results, along with the recent identification of numerous covalent molecules that inhibit NLRP3 inflammasome activation, suggests that NLRP3 serves as a cellular electrophile sensor important for coordinating inflammatory signaling in response to redox stress. Further, our results support the potential for covalent cysteine modification of NLRP3 for regulating inflammasome activation and activity.

Succinylation of a KEAP1 sensor lysine promotes NRF2 activation.

Crosstalk between metabolism and stress-responsive signaling is essential to maintaining cellular homeostasis. One way this crosstalk is achieved is through the covalent modification of proteins by endogenous, reactive metabolites that regulate the activity of key stress-responsive transcription factors such as NRF2. Several metabolites including methylglyoxal, glyceraldehyde 3-phosphate, fumarate, and itaconate covalently modify sensor cysteines of the NRF2 regulatory protein KEAP1, resulting in stabilization of NRF2 and activation of its cytoprotective transcriptional program. Here, we employed a shRNA-based screen targeting the enzymes of central carbon metabolism to identify additional regulatory nodes bridging metabolic pathways to NRF2 activation. We found that succinic anhydride, increased by genetic depletion of the TCA cycle enzyme succinyl-CoA synthetase or by direct administration, results in N-succinylation of lysine 131 of KEAP1 to activate NRF2 transcriptional signaling. This study identifies KEAP1 as capable of sensing reactive metabolites not only by several cysteine residues but also by a conserved lysine residue, indicating its potential to sense an expanded repertoire of reactive metabolic messengers.

Pharmacological inhibition of CLK2 activates YAP by promoting alternative splicing of AMOTL2.

Yes-associated protein (YAP), the downstream effector of the evolutionarily conserved Hippo pathway, promotes cellular proliferation and coordinates certain regenerative responses in mammals. Small molecule activators of YAP may therefore display therapeutic utility in treating disease states involving insufficient proliferative repair. From a high-throughput chemical screen of the comprehensive drug repurposing library ReFRAME, here we report the identification of SM04690, a clinical stage inhibitor of CLK2, as a potent activator of YAP driven transcriptional activity in cells. CLK2 inhibition promotes alternative splicing of the Hippo pathway protein AMOTL2, producing an exon-skipped gene product that can no longer associate with membrane-bound proteins, resulting in decreased phosphorylation and membrane localization of YAP. This study reveals a novel mechanism by which pharmacological perturbation of alternative splicing inactivates the Hippo pathway and promotes YAP dependent cellular growth.

Protective effect of Renqing Changjue on acute respiratory distress syndrome in rabbits / 中国药理学与毒理学杂志

OBJECTIVE To investigate whether Renqing Changjue has a protective effect on acute respiratory dis?tress syndrome (ARDS) induced by endotoxin lipopolysaccharide (LPS) in rabbits. METHODS Thirty-six healthy male New Zealand white rabbits were randomly divided into six groups: normal control group, model group, dexamethasone group, Renqing Changjue high, middle and low dose group, with six rabbits in each group. LPS was used to replicate the ARDS model after five consecutive days of gavage. Arterial pressure, respiratory rate and anal temperature blood were recorded for arterial blood gas analysis at 0, 0.5, 1, 2 and 4 h, respectively. At the end of the four-hour experiment, rab?bits were killed by bloodletting, and the lung tissue was quickly removed to determine the cytokines, SOD, MDA and pathological examination of rabbit lung. RESULTS Renqing Changjue can significantly reduce the pathological changes of lung in ARDS model group. The expression of AQP1 and MPO in rabbit lung was significantly decreased by immuno?histochemistry (P<0.05) ,reduce the lung wet/dry weight ratio, increase the ratio of PaO2/FiO2, inhibit the release of inflammatory factors and scavenge free radicals and antioxidant effects. CONCLUSION Renqing Changjue can effec?tively protect rabbits with acute respiratory distress syndrome induced by LPS, and may protect the lung by inhibiting the release of cytokines and anti-oxidation.

Huntington disease and Huntington disease-like in a case series from Brazil.

The aim of this study was to identify the relative frequency of Huntington's disease (HD) and HD-like (HDL) disorders HDL1, HDL2, spinocerebellar ataxia type 2 (SCA2), SCA17, dentatorubral-pallidoluysian degeneration (DRPLA), benign hereditary chorea, neuroferritinopathy and chorea-acanthocytosis (CHAC), in a series of Brazilian families. Patients were recruited in seven centers if they or their relatives presented at least chorea, besides other findings. Molecular studies of HTT, ATXN2, TBP, ATN1, JPH3, FTL, NKX2-1/TITF1 and VPS13A genes were performed. A total of 104 families were ascertained from 2001 to 2012: 71 families from South, 25 from Southeast and 8 from Northeast Brazil. There were 93 HD, 4 HDL2 and 1 SCA2 families. Eleven of 104 index cases did not have a family history: 10 with HD. Clinical characteristics were similar between HD and non-HD cases. In HD, the median expanded (CAG)n (range) was 44 (40-81) units; R(2) between expanded HTT and age-at-onset (AO) was 0.55 (p=0.0001, Pearson). HDL2 was found in Rio de Janeiro (2 of 9 families) and Rio Grande do Sul states (2 of 68 families). We detected HD in 89.4%, HDL2 in 3.8% and SCA2 in 1% of 104 Brazilian families. There were no cases of HDL1, SCA17, DRPLA, neuroferritinopathy, benign hereditary chorea or CHAC. Only six families (5.8%) remained without diagnosis.

Middle-preserving pancreatectomy: report of two cases and review of the literature.

BACKGROUND: Middle-preserving pancreatectomy (MPP) is a parenchyma-sparing surgical procedure which has recently been sporadically reported for the treatment of multicentric periampullary-pancreatic lesions. However, a comprehensive recognition of this procedure has not been clearly elucidated. CASE PRESENTATION: We herein report two patients undergoing MPP due to synchronous multicentric pancreatic neoplasm. Patient one was a 24-year-old woman with a multicentric solid pseudopapillary neoplasm (SPN) and patient two was a 36-year-old woman with a multicentric serous cystic neoplasm (SCN). Simultaneous atypical pancreaticoduodenectomy and atypical left pancreatectomy were performed in patient one; simultaneous standard pancreaticoduodenectomy and atypical left pancreatectomy with spleen preservation were performed in patient two. Approximately 6 cm and 5 cm segments of the middle portion of the pancreas were preserved, respectively. At follow-up at 36 months and 6 months respectively, patient one had developed diabetes and malabsorption requiring dietary control, exercise and pancreatic enzyme supplement whereas patient two showed normal fasting blood glucose without diarrhea. Both patients were disease-free and in good nutritional condition. We reviewed twenty cases of MPP that were previously reported in the literature. Patient characteristics, surgical techniques and short- and long-term outcomes were analyzed. CONCLUSION: MPP is mainly beneficial for multicentric noninvasive periampullary-pancreatic lesions. However, for multicentric periampullary-pancreatic lesions involving even primary invasive cancers, as long as the invasive cancers affect only one side of the pancreas (proximal or distal), MPP could serve as a rational choice in well-selected patients.

Evaluation of the cost-effectiveness of home monitoring of uterine contractions.

In 1988, the major health insurance carrier in the state of Hawaii initiated a prospective study to determine the cost-effectiveness of ambulatory uterine activity monitoring. At the end of the study, 79 patients had completed 3189 days on the monitor. Thirty-six patients (45.6%) did not have preterm labor; the daily monitoring resulted in an average loss to the insurance carrier of $3300 per patient, or $118,800. Forty-three patients (54.4%) experienced preterm labor and were found to have benefited from the monitoring system. Earlier detection of preterm labor and better management of oral tocolysis in this group resulted in earlier initiation of maternal tocolytic therapy, which ultimately decreased the preterm birth rate and hospitalization days in the neonatal intensive care unit. Cost analysis of this group demonstrated an average savings of $24,000 per patient, or an overall savings of $1,032,000. The total group of high-risk patients (79) resulted in a net savings to the insurance carrier of $913,200, or $11,500 per patient, suggesting that use of the ambulatory uterine activity monitoring system significantly reduced the cost associated with preterm labor and early delivery.