Receptor-interacting Protein Research Articles

Immunomodulatory and clinical effects of receptor-interacting protein kinase 1 (RIPK1) inhibitor eclitasertib (SAR443122) in patients with severe COVID-19: a phase 1b, randomized, double-blinded, placebo-controlled study

BackgroundTargeting receptor-interacting serine/threonine protein kinase 1 could mitigate the devastating sequelae of the hyperinflammatory state observed in severe cases of COVID-19. This study explored the immunomodulatory and clinical effects of the receptor-interacting serine/threonine protein kinase 1 inhibitor SAR443122 (eclitasertib) in patients with severe COVID-19.MethodsIn this Phase 1b, double-blinded, placebo-controlled study (NCT04469621) a total of 82 patients were screened, of whom 68 patients were eligible and randomized (2:1) to receive eclitasertib 600 mg (300 mg twice daily) or placebo up to 14 days. Primary outcome was relative change in C-reactive protein from baseline to Day 7. Time to clinical improvement using 7-point ordinal scale, ventilator/respiratory failure-free days, change in SpO2/FiO2 ratio, and biomarkers of severe COVID-19 were explored.ResultsGeometric mean ratio (point estimate [90% confidence interval]) of the relative change from baseline in C-reactive protein with eclitasertib vs. placebo on Day 7 was 0.85 (0.49–1.45; p = 0.30). Median time to 50% decrease in C-reactive protein from baseline was 3 days vs. 5 days (p = 0.056) with eclitasertib vs. placebo. Median time to ≥ 2-point improvement on 7-point clinical symptoms scale was 8 days vs. 10 days with eclitasertib vs. placebo (p = 0.38). Mean ventilator/respiratory failure-free days, change in baseline-adjusted SpO2/FiO2 ratio, and clinical biomarkers showed consistent numerical improvements with eclitasertib vs. placebo. The most frequently reported treatment-emergent adverse events were gastrointestinal disorders and condition aggravated/worsened COVID-19 pneumonia.ConclusionsEclitasertib was well tolerated with consistent trends toward more rapid resolution of inflammatory biomarkers and clinical improvement in severe COVID-19 patients than placebo.ClinicalTrials.gov identifierNCT04469621, first posted on clinicaltrials.gov on July 14, 2020.

TRIP6 promotes neural stem cell maintenance through YAP-mediated Sonic Hedgehog activation.

In the adult mammalian brain, new neurons are continuously generated from neural stem cells (NSCs) in the subventricular zone (SVZ)-olfactory bulb (OB) pathway. YAP, a transcriptional co-activator of the Hippo pathway, promotes cell proliferation and inhibits differentiation in embryonic neural progenitors. However, the role of YAP in postnatal NSCs remains unclear. Here, we showed that YAP was present in NSCs of the postnatal mouse SVZ. Forced expression of Yap promoted NSC maintenance and inhibited differentiation, whereas depletion of Yap by RNA interference or conditional knockout led to the decline of NSC maintenance, premature neuronal differentiation, and collapse of neurogenesis. For the molecular mechanism, thyroid hormone receptor-interacting protein 6 (TRIP6) recruited protein phosphatase PP1A to dephosphorylate LATS1/2, therefore inducing YAP nuclear localization and activation. Moreover, TRIP6 promoted NSC maintenance, cell proliferation, and inhibited differentiation through YAP. In addition, YAP regulated the expression of the Sonic Hedgehog (SHH) pathway effector Gli2 and Gli1/2 mediated the effect of YAP on NSC maintenance. Together, our findings demonstrate a novel TRIP6-YAP-SHH axis, which is critical for regulating postnatal neurogenesis in the SVZ-OB pathway.

Effect of electroacupuncture on neuronal programmed necrosis by regulating RIP1/RIP3/MLKL pathway in rats with cerebral ischemia reperfusion injury.

To investigate the neuroprotective effect of electroacupuncture (EA) at "Quchi"(LI11) and "Zusanli"(ST36) in the rats with cerebral ischemia reperfusion injury and its influence on programmed necrosis of cerebral cortical neurons. Sixty male SD rats were randomly divided into sham-operation group, model group, EA group and inhibitor group, with 15 rats in each group. Left middle cerebral artery occlusion model was established using the modified thread embolism method. In the sham-operation group, the carotid artery was exposed and dissociated in each rat. EA was applied to "Quchi"(LI11) and "Zusanli"(ST36) on the right side for 30 min each time, once daily for 7 days in the rats of the EA group. The rats in the inhibitor group were intraperitoneally injected with norstatin-1 (0.6 mg/kg) for consecutive 7 days. The neurological deficit score of rats in each group was observed. HE staining was adopted to detect the degree of pathological damage of the cerebral cortex in the infarction area. Using TUNEL staining, the apoptosis of cortical neurons in the infarction area was determined；the contents of tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 were detected by ELISA；the mRNA and protein expression of the receptor interacting protein-1 (RIP1), the receptor interacting protein-3 (RIP3) and the substrate mixed lineage kinase like protein (MLKL) were detected by fluorescence quantitative PCR and Western blot, respectively. In comparison with the sham-operation group, the neurological deficit score in the model group was higher(P<0.01)；HE staining showed that there was the pathological damage in the infarction area；the neuron apoptosis rate, the contents of TNF-α, IL-1β and IL-6, and the mRNA and protein expressions of RIP1, RIP3 and MLKL increased(P<0.01) in the model group. In the EA group, the neurological deficit score was reduced(P<0.01)；HE staining showed that the pathological damage was ameliorated in the infarction area；the neuron apoptosis rate, the contents of TNF-α, IL-1β and IL-6, and the mRNA and protein expressions of RIP1, RIP3, MLKL decreased(P<0.05, P<0.01) when compared with those in the model group. EA can attenuate cerebral ischemia reperfusion injury and display its neuroprotective effect probably through inhibiting programmed necrosis of cerebral cortical neurons in the rats.

Understanding a point mutation signature D54K in the caspase activation recruitment domain of NOD1 capitulating concerted immunity via atomistic simulation

Point mutation D54K in the human N-terminal caspase recruitment domain (CARD) of nucleotide-binding oligomerization domain −1 (NOD1) abrogates an imperative downstream interaction with receptor-interacting protein kinase (RIPK2) that entails combating bacterial infections and inflammatory dysfunction. Here, we addressed the molecular details concerning conformational changes and interaction patterns (monomeric-dimeric states) of D54K by signature-based molecular dynamics simulation. Initially, the sequence analysis prioritized D54K as a pathogenic mutation, among other variants, based on a sequence signature. Since the mutation is highly conserved, we derived the distant ortholog to predict the sequence and structural similarity between native and mutant. This analysis showed the utility of 33 communal core residues associated with structural-functional preservation and variations, concurrently served to infer the cryptic hotspots Cys39, Glu53, Asp54, Glu56, Ile57, Leu74, and Lys78 determining the inter helical fold forming homodimers for putative receptor interaction. Subsequently, the atomistic simulations with free energy (MM/PB(GB)SA) calculations predicted structural alteration that takes place in the N-terminal mutant CARD where coils changed to helices (45 α3- L4-α4-L6- α683) in contrast to native (45T2-L4-α4-L6-T483). Likewise, the C-terminal helices 93T1-α7105 connected to the loops distorted compared to native 93α6-L7105 may result in conformational misfolding that promotes functional regulation and activation. These structural perturbations of D54K possibly destabilize the flexible adaptation of critical homotypic NOD1CARD-CARDRIPK2 interactions (α4Asp42-Arg488α5 and α6Phe86-Lys471α4) is consistent with earlier experimental reports. Altogether, our findings unveil the conformational plasticity of mutation-dependent immunomodulatory response and may aid in functional validation exploring clinical investigation on CARD-regulated immunotherapies to prevent systemic infection and inflammation. Communicated by Ramaswamy H. Sarma

Limited WKY chromosomal regions confer increases in anxiety and fear memory in a F344 congenic rat strain.

This study investigated the interaction between genetic differences in stress reactivity/coping and environmental challenges, such as acute stress during adolescence on adult contextual fear memory and anxiety-like behaviors. Fischer 344 (F344) and the inbred F344;WKY-Stresp3/Eer congenic strain (congenic), in which chromosomal regions from the Wistar-Kyoto (WKY) strain were introgressed into the F344 background, were exposed to a modified forced swim test during adolescence, while controls were undisturbed. In adulthood, fear learning and memory, assessed by contextual fear conditioning, were significantly greater in congenic animals compared with F344 animals, and stress during adolescence increased them even further in males of both strains. Anxiety-like behavior, measured by the open field test, was also greater in congenic than F344 animals, and stress during adolescence increased it further in both strains of adult males. Whole genome sequencing of the F344;WKY-Stresp3/Eer strain revealed an enrichment of WKY genotypes in chromosomes 9, 14, and 15. An example of functional WKY sequence variations in the congenic strain, cannabinoid receptor interacting protein 1 (Cnrip1) had a Cnrip1 transcript isoform that lacked two exons. Although the original hypothesis that the genetic predisposition to increased anxiety of the WKY donor strain would exaggerate fear memory relative to the background strain was confirmed, the consequences of adolescent stress were strain independent but sex dependent in adulthood. Molecular genomic approaches combined with genetic mapping of WKY sequence variations in chromosomes 9, 14, and 15 could aid in finding quantitative trait genes contributing to the variation in fear memory.NEW & NOTEWORTHY This study found that 1) whole genome sequencing of congenic strains should be a criterion for their recognition; 2) sequence variations between Wistar-Kyoto and Fischer 344 strains at regions of chromosomes 9, 14, and 15 contribute to differences in contextual fear memory and anxiety-like behaviors; and 3) stress during adolescence affects these behaviors in males, but not females, and is independent of strain.

Different concentrations of betaxolol switch cell fate between necroptosis, apoptosis, and senescence in human corneal stromal cells

Betaxolol is commonly used to manage glaucoma in clinical practice. However, its long-term use may damage the cornea. Thus, the cytotoxicity and mechanisms of betaxolol in human corneal stromal cells (HCSCs) warrant further study. In this study, we used in vitro HCSCs and in vivo rabbit corneal models to investigate betaxolol cytotoxic effects and mechanism of action. At near-clinical concentrations (0.28% and 0.14%), betaxolol inhibited caspase-8 activity, activated receptor-interacting protein kinase (RIPK)1, RIPK3, and mixed-spectrum kinase-like domain (MLKL), and phosphorylated MLKL to induce necroptosis in HCSCs. Similarly, moderate concentrations of betaxolol (0.07%–0.0175%) activated caspase-8 to trigger the exogenous apoptotic pathway. Through the intrinsic apoptotic pathway, betaxolol upregulated the expression of Bcl-2 family apoptotic proteins Bax and Bad and downregulated that of anti-apoptotic proteins Bcl-2 and Bcl-xL. This subsequently disrupted the mitochondrial membrane potential and cytoplasmic transfer of cytochrome c and apoptosis-inducing factor, activated caspase-9, and induced apoptosis in HCSCs. Furthermore, continuous treatment with low betaxolol concentrations (0.00875%) for three generations of HCSCs prevented apoptosis by promoting the expression of Bcl-xL and suppressing that of Bax. However, its toxic effects initiated cellular senescence by increasing reactive oxygen species, leading to the disruption of energy metabolism and DNA damage. Finally, clinical concentrations of betaxolol had a pro-apoptotic effect on rabbit corneal stromal cells in vivo. These results suggest that betaxolol induces cytotoxicity in a concentration-dependent manner in HCSCs, and that caspase-8 and Bcl-2 family proteins may be critical switches in the conversion of different HCSC death mechanisms.

5-Hydroxytryptamine 4 Receptor Agonist Attenuates Diabetic Enteric Neuropathy through Inhibition of the Receptor-Interacting Protein Kinase 3 Pathway

Necroptosis, considered as a form of programmed cell death, contributes to the neural loss. The 5-hydroxytryptamine 4 receptor (5-HT4R) is involved in neurogenesis in the enteric nervous system (ENS). However, it is unclear whether the activation of 5-HT4R can alleviate diabetic enteric neuropathy by inhibiting receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis. This study aimed to explore the beneficial effects of 5-HT4R agonist on enteric neuropathy in a mouse model of diabetes and the mechanisms underlying these effects. Diabetes developed neural loss in the colon of mice. 5-HT4Rs localized in submucosal and myenteric plexuses were confirmed. Administration of 5-HT4R agonist attenuated diabetes-induced colonic hypomotility and neural loss of the colon in mice. Remarkably, RIPK3, phosphorylated RIPK3 (pRIPK3) and its downstream target mixed lineage kinase domain-like protein (MLKL), two key proteins regulating necroptosis, were significantly upregulated in the colon of diabetic mice. Treatment with 5-HT4R agonist appeared to inhibit diabetes-induced elevation of RIPK3, pRIPK3 and MLKL in the colon of mice. Diabetes-induced upregulation of MLKL in both the mucosa and muscularis of the colon was prevented by RIPK3 deletion. Moreover, Diabetes-evoked neural loss and delayed colonic transit were significantly inhibited by RIPK3 removal. These findings suggest that activation of 5-HT4R receptors could potentially provide a protective effect against diabetic enteric neuropathy by suppressing RIPK3-mediated necroptosis.

Autophagy inhibitors 3-MA and BAF may attenuate hippocampal neuronal necroptosis after global cerebral ischemia-reperfusion injury in male rats by inhibiting the interaction of the RIP3/AIF/CypA complex.

Our previous study found that receptor interacting protein 3 (RIP3) and apoptosis-inducing factor (AIF) were involved in neuronal programmed necrosis during global cerebral ischemia-reperfusion (I/R) injury. Here, we further studied its downstream mechanisms and the role of the autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin A1 (BAF). A 20-min global cerebral I/R injury model was constructed using the 4-vessel occlusion (4-VO) method in male rats. 3-MA and BAF were injected into the lateral ventricle 1 h before ischemia. Spatial and activation changes of proteins were detected by immunofluorescence (IF), and protein interaction was determined by immunoprecipitation (IP). The phosphorylation of H2AX (γ-H2AX) and activation of mixed lineage kinase domain-like protein (p-MLKL) occurred as early as 6 h after reperfusion. RIP3, AIF, and cyclophilin A (CypA) in the neurons after I/R injury were spatially overlapped around and within the nucleus and combined with each other after reperfusion. The survival rate of CA1 neurons in the 3-MA and BAF groups was significantly higher than that in the I/R group. Autophagy was activated significantly after I/R injury, which was partially inhibited by 3-MA and BAF. Pretreatment with both 3-MA and BAF almost completely inhibited nuclear translocation, spatial overlap, and combination of RIP3, AIF, and CypA proteins. These findings suggest that after global cerebral I/R injury, RIP3, AIF, and CypA translocated into the nuclei and formed the DNA degradation complex RIP3/AIF/CypA in hippocampal CA1 neurons. Pretreatment with autophagy inhibitors could reduce neuronal necroptosis by preventing the formation of the RIP3/AIF/CypA complex and its nuclear translocation.

Abstract TP26: Hypothermic and Non-Hypothermic Neuroprotection by Phenothiazine in Acute Ischemic Stroke: Modulation of NLRP3 Inflammasome-Related-Inflammation via Different Pathways of RIPK1/RIPK3-DRP1 or HIF-1α

Background: Inflammation after successful revascularization worsened outcomes in ischemic stroke. NLRPyrin domain containing 3 (NLRP3) inflammasome mediated inflammatory responses, in which receptor-interacting protein kinase (RIPK1) activated dynamin-related protein 1 (DRP1) in a RIPK3-dependent fashion. Hypoxia-inducible factor 1α (HIF-1α) regulated ischemic inflammation through NLRP3 inflammasome complex, thus apoptosis after stroke. Phenothiazine, including chlorpromazine and promethazine (C+P), induced hypothermic and non-hypothermic neuroprotection. We determined effects of RIPK1/RIPK3-DRP1 or HIF-1α pathways underlying the dual neuroprotection by C+P. Methods: 144 adult male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO), followed by 24 h reperfusion. 8mg/kg of C+P was injected at the onset of reperfusion. Infarct volumes, mRNA and protein expressions of HIF-1α, RIPK1, RIPK3, DRP-1, NLRP3-inflammation (NLRP3, ASC, Caspase-1, IL-1β and IL-18) and cytochrome C-apoptosis (cytochrome C, APAF-1, Caspase-9 and Caspase-3) were assessed. Apoptotic cell death, and the infiltration of neutrophils and macrophages were evaluated. Co-localization of RIPK1/RIPK3 and HIF-1α/NLRP3 were determined. Results: C+P significantly reduced brain inflammation, neuronal apoptosis and immune cell infiltration, with or without hypothermia. C+P with induced-hypothermia and RIPK1 inhibitor significantly reduced infarct volumes, the expression of RIPK1, RIPK3, DRP-1, NLRP3-inflammation process and cytochrome C-apoptosis, as well as the co-localization of RIPK1/RIPK3, suggesting the hypothermic effect underlying the RIPK1/RIPK3-DRP1 pathway. In non-hypothermia effect, HIF-1α expression was reduced by C+P or HIF-1α inhibitor, in concomitant with reduced NLRP3 inflammasome, cytochrome C-apoptosis, and decreased interaction of HIF-1α and NLRP3. Conclusion: Hypothermic and non-hypothermic neuroprotection of C+P involve different pathways. The hypothermic effect was via RIPK1/RIPK3-DRP1 signaling, while non-hypothermic effect was mediated by HIF-1α. This provides a theoretical basis for future precise exploration of hypothermic and non-hypothermic neuroprotection.

Inhibition of Receptor-Interacting Protein Kinase1 in Chronic Plaque Psoriasis: A Multicenter, Randomized, Double-Blind, Placebo-Controlled Study.

Receptor-interacting protein kinase1 (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 60mg three times daily in mild to moderate plaque psoriasis indicated that efficacy may be improved with higher trough concentrations of GSK2982772. This multicenter, randomized, double-blind, placebo-controlled, repeat-dose study (NCT04316585) assessed the efficacy, safety, pharmacokinetics, and pharmacodynamics of 960mg 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 12weeks. GSK2982772 was well tolerated with trough concentrations greater than tenfold higher than the previous phase1 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 week12 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. Week4 changes in skin biopsy gene expression suggested sufficient local drug exposure to elicit a pharmacodynamic response. Administration of the RIPK1 inhibitor GSK2982772 to patients with moderate to severe plaque psoriasis did not translate into meaningful clinical improvements.

P043 RIPK3 – a new marker for assessment of activity in Crohn’s disease

Abstract Background RIPK3 (Receptor Interacting Protein Kinase-3) is a key molecule involved in the process of necroptosis. Necroptosis is a regulated form of cell death that involves the activation of specific signaling pathways leading to cell lysis. The released endogenous molecules - DAMPs (Danger Associated Molecular Patterns) activate the immune system and lead to inflammation. This relationship suggests a role for necroptosis in the pathogenesis of IBD (Inflammatory Bowel Disease). Methods The aim of the study was to evaluate RIPK3 expression in patients with Crohn's disease (CD) according to disease activity and some clinical parameters. Tissue expression of RIPK3 in the inflamed areas of 85 patients with Crohn's disease was determined immunohistochemically. The CDAI (Crohn’s Disease Activity Index) was used to assess activity, and the Montreal Classification to determine disease location and bahavior. Results A correlation was found between RIPK3 expression and CDAI (p=0.038), with patients in remission having the lowest expression (154.67), while patients with severe activity had overexpression of the marker (180.0) (Fig.1). According to the localization of CD, a significant difference was found in the expression of RIPK3 (p&amp;lt;0.05), with the highest expression of the marker in the ileum (L1) and colon (L2) with involvement of the upper GIT (L4) – L1+L4 (225.50) and L2 + L4 (218.00). Isolated involvement of terminal ileum (L1) was characterized by the lowest expression of the marker, while patients with colonic localization (L2) had high expression of RIPK3 (184.87). Regarding disease behavior, it was found that the highest expression of the marker is in the patients with phenotype B2+B3 – both stricturing and penetrating disease, followed by inflammatory disease (B1) (Fig.2). Perianal disease further increased RIPK3 expression, with patients with concurrent perianal disease showing high expression of the marker (181.85 vs. 167.86 for those without perianal disease; p&amp;lt;0.05). Conclusion High RIPK3 expression in CD patients correlated with severe disease activity as measured by CDAI, involvement of upper GIT - L1+L4 and L2+L4, stricturing with penetrating disease - B2+B3 and concurrent perianal disease.

Creatine supplementation with exercise reduces α-synuclein oligomerization and necroptosis in Parkinson's disease mouse model

Parkinson's disease (PD) is an incurable neurological disorder that causes typical motor deficits. In this study, we investigated the effects of creatine supplementation and exercise in the subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. We found that 2% creatine supplementation and/or exercise intervention for 4 weeks elicited neurobehavioral recovery and neuroprotective effects regarding dopaminergic cell loss in MPTP-treated mice; this effect implies functional preservation of dopaminergic cells in the substantia nigra, as reflected by tyrosine hydroxylase expression recovery. Creatine and exercise reduced necroptotic activity in dopaminergic cells by lowering mixed lineage kinase domain-like protein (MLKL) modification to active phenotypes (phosphorylation at Ser345 and oligomerization) and phosphorylated receptor-interacting protein kinase 1 (RIPK1) (Ser166-p) and RIPK3 (Ser232-p) levels. In addition, creatine and exercise reduced the MPTP-induced increase in pathogenic α-synuclein forms, such as Ser129 phosphorylation and oligomerization. Furthermore, creatine and exercise had anti-inflammatory and antioxidative effects in MPTP mice, as evidenced by a decrease in microglia activation, NF-κB-dependent pro-inflammatory molecule expression, and increase in antioxidant enzyme expression. These phenotypic changes were associated with the exercise/creatine-induced AMP-activated protein kinase (AMPK)/nuclear factor erythroid 2-related factor 2 (Nrf2) and sirtuin 3 (SIRT3)/forkhead box O3 (FoxO3a) signaling pathways. In all experiments, combining creatine with exercise resulted in considerable improvement over either treatment alone. Consequently, these findings suggest that creatine supplementation with exercise has anti-inflammatory, antioxidative, and anti-α-synucleinopathy effects, thereby reducing necroptotic cell death in a PD mouse model.

Clinical Significance and Expression Pattern of RIP5 and VGLL4 in Clear Cell Renal Cell Carcinoma Patients Treated with Sunitinib.

While clear cell renal cell carcinoma (ccRCC) is curable, advanced metastatic (mRCC) remains a clinical challenge. We analyzed clinical, pathohistological, and molecular data (Receptor Interacting Protein 5-RIP5 and Vestigial Like Family Member 4-VGLL4 expression) of 55 mRCC patients treated with first-line treatment with sunitinib. The trend of linear increase in the protein expression of RIP5 was observed with the progression of tumor grade. Overall, 80% of RIP5-positive cells were in the control kidneys and high-grade mRCC. On the contrary, RIP5 displayed low expression in grade 2 mRCC (5.63%). The trend of linear decrease in the expression of VGLL4 was observed with the progression of tumor grade. The highest protein expression of VGLL4 was observed in grade 2 (87.82%) in comparison to grade 3 and 4 and control. High expression of RIP5 mRNA was associated with longer first-line overall survival and longer progression-free survival in mRCC. In addition, a high VGLL4 mRNA expression showed better overall survival in patients with ccRCC. In conclusion, high mRNA expression of RIP5 and VGLL4 are important markers of better survival rates in mRCC patients.

The Role of Sgt1 in Methamphetamine/Hyperthermia-induced Necroptosis.

Methamphetamine (METH) is a synthetic drug widely abused globally and can result in hyperthermia (HT) and psychiatric symptoms. Our previous studies showed that heat shock protein 90 alpha (HSP90α) plays a vital role in METH/HT-elicited neuronal necroptosis; however, the detailed mechanism of HSP90α regulation remained obscure. Herein, we demonstrated a function of the suppressor of G-two allele of SKP1 (Sgt1) in METH/HT-induced necroptosis. Sgt1 was mainly expressed in neurons, co-located with HSP90α, and increased in rat striatum after METH treatment. METH/HT injury triggered necroptosis and increased Sgt1 expression in PC-12 cells. Data from computer simulations indicated that Sgt1 might interact with HSP90α. Geldanamycin (GA), the specific inhibitor of HSP90α, attenuated the interaction between Sgt1 and HSP90α. Knockdown of Sgt1 expression did not affect the expression level of HSP90α. Still, it inhibited the expression of receptor-interacting protein 3 (RIP3), mixed lineage kinase domain-like protein (MLKL), p-RIP3, and p-MLKL, as well as necroptosis induced by METH/HT injury. In conclusion, Sgt1 may regulate the expression of RIP3, p-RIP3, MLKL, and p-MLKL by assisting HSP90α in affecting the METH/HT-induced necroptotic cell death.

Circ_0079471 Regulates the Proliferation, Migration, Invasion and Apoptosis of Osteosarcoma Cells by Mediating miR-485-3p and TRIP6.

Circular RNAs (circRNAs) are a special class of non-coding RNA molecules that show a closed circular structure and have been implicated in both tumour formation and oncogenesis. This study aimed to learn more about how circ_0079471 functions in osteosarcomas (OSs). Quantitative real-time polymerase chain reaction was used to detect the expression levels of thyroid hormone receptor-interacting protein 6 (TRIP6), miR-485-3p and circ_0079471. Methyl-thiazolyl-tetrazolium and flow cytometry were used to track cell growth and cell-cycle progression, and the research explored wound healing (migration) and invasion using Transwell plates. Western blotting was used to determine the protein expression of TRIP6, proliferating cell nuclear antigen and cyclin D1, and a dual-luciferase assay revealed the target relationship. A xenograft experiment evaluated the in vivo effects of circ_0079471 on OS, and the results revealed the high expression of circ_0079471 in OS tissue and cells. The proliferation, cell-cycle migration and invasion of cells were reduced after circ_0079471 knockdown in OS cells; however, the effects of this knockdown were reversed when TRIP6 was overexpressed in the OS cells. The function of circ_0079471 was also achieved by in vivo miR-485-3p sponging. The upregulation of miR-485-3p and the downregulation of TRIP6 partly resulted in circ_0079471 downregulation, which subsequently inhibited OS progression. According to these results, circ_0079471 influences the development of OS by regulating miR-485-3p and TRIP6.

Evaluation of specific binding of [11C]TZ7774 to the receptor-interacting protein kinase 1 (RIPK1) in the brain

Evaluation of specific binding of [11C]TZ7774 to the receptor-interacting protein kinase 1 (RIPK1) in the brain

Receptor-interacting protein kinase 2 is associated with tumor immune infiltration, immunotherapy-related biomarkers, and affects gastric cancer cells growth in vivo.

Background: The objective of this study was to analyze the research trend of four RIPK genes (RIPK1, RIPK2, RIPK3, and RIPK4), their expression variations in tumors, and the correlation between RIPK2 expression and immune-related biomarkers in gastric cancer (GC). Methods: The PubMed database was utilized to investigate the research trend surrounding four RIPKs genes in tumors. The ULCAN database was employed to analyze the differential expression of these four RIPKs genes. TCGA data were utilized to examine the association between RIPK2 expression and various factors including tumor immune infiltration and immune-related biomarkers. Lastly, the impact of targeting RIPK2 on the growth of GC cells was confirmed through tumor formation assay, immunohistochemistry, and Tunnel assays. Results: In the field of tumor biology, there has been a sustained increase in research focused on the four RIPKs genes over the past decade. Four RIPKs genes are differentially expressed in a majority of tumors. Furthermore, this investigation has unveiled a connection between the expression of RIPK2 and the infiltration of four immune cells, as well as the presence of RNA methylation modifying enzymes, specifically m1A, m6A, and m5C, in GC. Additionally, RIPK2 expression was associated with the genes related to immune checkpoint regulation, as well as genes associated with immunoinhibitors and immunostimulators. It was also revealed that RIPK2 expression was correlated to immunotherapy response biomarkers, namely MSI and TMB, and tumor stemness. Ultimately, it was demonstrated that targeting the RIPK2 effectively regulated GC cells growth through the suppression of PCNA expression and the induction of apoptosis. Conclusion: The expression of RIPK2 is correlated with immune cell infiltration, RNA methyltransferase activity, tumor stemness, checkpoint-related genes, and immunotherapy-related biomarkers. Suppression of RIPK2 impedes the growth of GC cells in vivo. Consequently, RIPK2 holds promise as a viable immunotherapy target for various types of cancer.

Design, synthesis, and structure-activity relationship studies of 6,7-dihydro-5H-pyrrolo[1,2-b][1,2,4]triazole derivatives as necroptosis inhibitors.

The development of necroptosis inhibitors has emerged as a promising strategy to effectively mitigate necroptosis-related inflammatory diseases, neurodegenerative diseases, and cancers. In this paper, we reported a series of 6,7-dihydro-5H-pyrrolo[1,2-b][1,2,4]triazole derivatives as potent necroptosis inhibitors. The representative compound 26 displayed potent anti-necroptotic activity in both human and mouse cellular assays and exhibited potent inhibitory activity against receptor-interacting protein kinase 1 (RIPK1). In vivo pharmacokinetic studies were performed to determine the oral exposure of compound 26. Finally, molecular docking elucidated that compound 26 could effectively bind to the allosteric pocket of RIPK1 and serve as a type III inhibitor. Taken together, our findings highlighted that compound 26 represented a promising lead compound for future necroptosis inhibitor development.

Inhibition of the RIP3/MLKL/TRPM7 necroptotic pathway ameliorates diabetes mellitus-induced erectile dysfunction by reducing cell death, fibrosis, and inflammation.

Diabetes mellitus-induced erectile dysfunction (DMED) is a common complication in patients with diabetes mellitus. Necroptosis is regarded as a form of cell death that is intimately associated with the inflammatory response, which is not only initiated by inflammatory factors such as TNF-α, but also triggers the inflammatory cascade through the rupture of the dying cell. There is no definitive study on the role of necroptosis in the pathological process of DMED. In light of the pathological features of high inflammation levels in DMED patients, we assessed whether the necroptosis plays an important role in the course of DMED. Our study revealed that penile tissues of DMED rats showed high levels of key necroptosis factors such as receptor-interacting protein kinase 3 (RIP3), mixed-lineage kinase domain-like protein (MLKL), and transient receptor potential melatonin 7 (TRPM7). Furthermore, the inhibition of necroptosis with a receptor-interacting protein kinase 3 (RIP3) inhibitor or Yimusake (a common herbal remedy for ED) effectively rescued damage to corpus cavernosum smooth muscle cells (CCSMC) under high glucose conditions. Our findings suggest that inhibition of the RIP3/MLKL/TRPM7 necroptotic pathway could effectively ameliorate CCSMCs fibrosis and death induced by high glucose and inhibited the inflammatory response.

RIPK4 downregulation impairs Wnt3A-stimulated invasiveness via Wnt/β-catenin signaling in melanoma cells and tumor growth in vivo.

The role of Wnt signaling in oncogenesis and drug resistance is well known. Receptor-interacting protein kinase (RIPK4) contributing to the increased activity of many signaling pathways, including Wnt/β-catenin, may be an important target for designing new drugs for metastatic melanoma, but its role in melanoma is not fully understood. We tested the effect of genetic manipulation of RIPK4 (CRISPR/Cas9) on xenograft growth. In addition, immunohistochemistry was used to detect active β-catenin, Ki67 and necrosis in xenografts. Wnt signaling pathway activity was examined using Western blot and Top-Flash. The effect of RIPK4 knockout on melanoma cells in vitro stimulated Wnt3A on wound overgrowth, migration and invasion ability was then evaluated. Our study showed that CRISPR/Cas9-mediated RIPK4 knockout (KO) significantly reduced tumor growth in a mouse model of melanoma, particularly of WM266.4 cells. RIPK4 KO tumors exhibited lower percentages of Ki67+ cells as well as reduced necrotic area and decreased levels of active β-catenin. In addition, we observed that RIPK4 knockout impaired Wnt3A-induced activation of LRP6 and β-catenin, as manifested by a decrease in the transcriptional activity of β-catenin in Top-Flash in both tested melanoma cell lines, A375 and WM266.4. Prolonged incubation (48h) with Wnt3A showed reduced level of MMP9, C-myc, and increased SOX10, proteins whose transcription is also dependent on β-catenin activity. Moreover, RIPK4 knockout led to the inhibition of scratch overgrowth, migration and invasion of these cells compared to their controls. RIPK4 knockdown inhibits melanoma tumor growth and Wnt3A stimulated migration and invasion indicating that RIPK4 might be a potential target for melanoma therapy.