Discovery of CNS-Penetrant Apoptosis Signal-Regulating Kinase 1 (ASK1) Inhibitors.
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ABSTRACT:
SUBMITTER: Xin Z
PROVIDER: S-EPMC7153032 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Apoptosis signal-regulating kinase 1 (ASK1) is a critical cellular stress sensor that senses diverse reactive chemotypes and integrates these chemical signals into a single biological pathway response. It is unknown whether ASK1 senses all stressors in the same way or if unique stress-specific mechanisms detect distinct chemotypes. In order to answer this question, we treated ASK1-expressing cells with two distinct stress activators, H2O2 and 4-hydroxy-2-nonenal (HNE), and monitored the phosphorylation state of ASK1. Phosphorylation is an important regulator for the activity of ASK1, and we hypothesized that these two chemically distinct molecules may produce differences in the phosphorylation state of ASK1. Shotgun mass spectrometry and manual validation identified 12 distinct ASK1 phosphosites. Targeted parallel reaction monitoring assays were used to track the phosphorylation dynamics of each confirmed site in response to treatment. Eleven phosphosites exhibited dynamic response to one or both treatments. Six of these sites were identified in both H2O2- and HNE-treated cells, and four of these exhibited a consistent response between the two molecules. The results confirm that different chemotypes produce distinct phosphorylation patterns in concert with activation of a common MAPK pathway.
| S-EPMC5937698 | biostudies-literature
Project description:Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, regulates diverse physiological processes. The activity of ASK1 is triggered by various stress stimuli and is involved in the pathogenesis of cancer, neurodegeneration, inflammation, and diabetes. ASK1 forms a high molecular mass complex whose activity is, under non-stress conditions, suppressed through interaction with thioredoxin and the scaffolding protein 14-3-3. The 14-3-3 protein binds to the phosphorylated Ser-966 motif downstream of the ASK1 kinase domain. The role of 14-3-3 in the inhibition of ASK1 has yet to be elucidated. In this study we performed structural analysis of the complex between the ASK1 kinase domain phosphorylated at Ser-966 (pASK1-CD) and the 14-3-3ζ protein. Small angle x-ray scattering (SAXS) measurements and chemical cross-linking revealed that the pASK1-CD·14-3-3ζ complex is dynamic and conformationally heterogeneous. In addition, structural analysis coupled with the results of phosphorus NMR and time-resolved tryptophan fluorescence measurements suggest that 14-3-3ζ interacts with the kinase domain of ASK1 in close proximity to its active site, thus indicating this interaction might block its accessibility and/or affect its conformation.
| S-EPMC5034064 | biostudies-literature
Project description:Most animal cell types regulate their cell volume after an osmotic volume change. The regulatory volume increase (RVI) occurs through uptake of NaCl and osmotically obliged water after osmotic shrinkage. However, apoptotic cells undergo persistent cell shrinkage without showing signs of RVI. Persistence of the apoptotic volume decrease is a prerequisite to apoptosis induction. We previously demonstrated that volume regulation is inhibited in human epithelial HeLa cells stimulated with the apoptosis inducer. Here, we studied signaling mechanisms underlying the apoptotic inhibition of RVI in HeLa cells. Hypertonic stimulation was found to induce phosphorylation of a Ser/Thr protein kinase Akt (protein kinase B). Shrinkage-induced Akt activation was essential for RVI induction because RVI was suppressed by an Akt inhibitor, expression of a dominant negative form of Akt, or small interfering RNA-mediated knockdown of Akt1 (but not Akt2). Staurosporine, tumor necrosis factor-alpha, or a Fas ligand inhibited both RVI and hypertonicity-induced Akt activation in a manner sensitive to a scavenger for reactive oxygen species (ROS). Any of apoptosis inducers also induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) in a ROS-dependent manner. Suppression of (ASK1) expression blocked the effects of apoptosis, in hypertonic conditions, on both RVI induction and Akt activation. Thus, it is concluded that in human epithelial cells, shrinkage-induced activation of Akt1 is involved in the RVI process and that apoptotic inhibition of RVI is caused by inhibition of Akt activation, which results from ROS-mediated activation of ASK1.
| S-EPMC2825405 | biostudies-literature
Project description:A novel series of biaryl amides was identified as RORγt inhibitors through core replacement of a starting hit 1. Structure-activity relationship exploration on the biaryl moiety led to discovery of potent RORγt inhibitors with good oral bioavailability and CNS penetration. Compounds 9a and 9g demonstrated excellent in vivo efficacy in EAE mice dose dependently with once daily oral administration.
| S-EPMC4499822 | biostudies-literature
Project description:Systemic hypertension increases cardiac workload causing cardiomyocyte hypertrophy and increased cardiac fibrosis. An underlying feature is increased production of reactive oxygen species. Redox-sensitive ASK1 (apoptosis signal-regulating kinase 1) activates stress-regulated protein kinases (p38-MAPK [mitogen-activated protein kinases] and JNKs [c-Jun N-terminal kinases]) and promotes fibrosis in various tissues. Here, we determined the specificity of ASK1 signaling in the heart, with the hypothesis that ASK1 inhibitors may be used to manage fibrosis in hypertensive heart disease. Using immunoblotting, we established that moderate levels of H2O2 activate ASK1 in neonatal rat cardiomyocytes and perfused rat hearts. ASK1 was activated during ischemia in adult rat hearts, but not on reperfusion, consistent with activation by moderate (not high) reactive oxygen species levels. In contrast, IL (interleukin)-1β activated an alternative kinase, TAK1 (transforming growth factor-activated kinase 1). ASK1 was not activated by IL1β in cardiomyocytes and activation in perfused hearts was due to increased reactive oxygen species. Selonsertib (ASK1 inhibitor) prevented activation of p38-MAPKs (but not JNKs) by oxidative stresses in cultured cardiomyocytes and perfused hearts. In vivo (C57Bl/6J mice with osmotic minipumps for drug delivery), selonsertib (4 mg/[kg·d]) alone did not affect cardiac function/dimensions (assessed by echocardiography). However, it suppressed hypertension-induced cardiac hypertrophy resulting from angiotensin II (0.8 mg/[kg·d], 7d), with inhibition of Nppa/Nppb mRNA upregulation, reduced cardiomyocyte hypertrophy and, notably, significant reductions in interstitial and perivascular fibrosis. Our data identify a specific reactive oxygen species→ASK1→p38-MAPK pathway in the heart and establish that ASK1 inhibitors protect the heart from hypertension-induced cardiac remodeling. Thus, targeting the ASK1→p38-MAPK nexus has potential therapeutic viability as a treatment for hypertensive heart disease.
| S-EPMC7480944 | biostudies-literature
Project description:Stem cell therapy using adult stem cells, such as mesenchymal stem cells (MSCs) has produced some promising results in treating the damaged heart. However, the low survival rate of MSCs after transplantation is still one of the crucial factors that limit the therapeutic effect of stem cells. In the damaged heart, oxidative stress due to reactive oxygen species (ROS) production can cause the death of transplanted MSCs. Apoptosis signal-regulating kinase 1 (ASK1) has been implicated in the development of oxidative stress-related pathologic conditions. Thus, we hypothesized that down-regulation of ASK1 in human MSCs (hMSCs) might attenuate the post-transplantation death of MSCs. To test this hypothesis, we screened microRNAs (miRNAs) based on a miRNA-target prediction database and empirical data and investigated the anti-apoptotic effect of selected miRNAs on human adipose-derived stem cells (hASCs) and on rat myocardial infarction (MI) models. Our data indicated that miRNA-301a most significantly suppressed ASK1 expression in hASCs. Apoptosis-related genes were significantly down-regulated in miRNA-301a-enriched hASCs exposed to hypoxic conditions. Taken together, these data show that miRNA-mediated down-regulation of ASK1 protects MSCs during post-transplantation, leading to an increase in the efficacy of MSC-based cell therapy.
| S-EPMC5085777 | biostudies-literature
Project description:Sepsis represents a life-threatening event often mediated by the host's response to pathogens such as gram-negative organisms, which release the proinflammatory lipopolysaccharide (LPS). Within the endothelium, the mitogen-activated protein kinase (MAPK) pathway is an important driver of endothelial injury during sepsis, of which oxidant-sensitive apoptosis signal-regulating kinase 1 (ASK1) is postulated to be a critical upstream regulator. We hypothesized that ASK1 would play a key role in endothelial inflammation during bacterial challenge. Utilizing RNA sequencing data from patients and cultured human microvascular endothelial cells (HMVECs), ASK1 expression was increased in sepsis and after LPS challenge. Two ASK1 inhibitors, GS444217 and MSC2023964A, reduced cytokine production in HMVECs following LPS stimulation, but had no effect on permeability as measured by transendothelial electrical resistance and intercellular space. MAPKs are known to interact with endothelial nitric oxide synthase (eNOS) and ASK1 expression levels correlated with eNOS expression in patients with septic shock. In addition, eNOS physically interacted with ASK1, though this interaction was not altered by ASK1 inhibition, nor did inhibition alter MAPK p38 activity. Instead, among MAPKs, ASK1 inhibition only impaired LPS-induced JNK phosphorylation. The reduction in JNK activation caused by ASK1 inhibition impaired JNK-mediated cytokine production without affecting permeability. Thus, LPS triggers JNK-dependent cytokine production that requires ASK1 activation, but both its effects on permeability and activation of p38 are ASK1-independent. These data demonstrate how distinct MAPK signaling pathways regulate endothelial inflammatory outputs during acute infectious challenge.
| S-EPMC8328918 | biostudies-literature
Project description:Cell transformation by growth-promoting oncoproteins renders cells extremely sensitive to apoptosis through an unknown mechanism affecting the mitochondrial pathway of apoptosis. We have shown previously that sensitization to apoptosis also correlated with the activation of the stress-activated protein kinase p38. In the present study, we investigated the role of p38 in c-Myc-dependent apoptosis induced by the anticancer agent cisplatin. Cisplatin treatment of Rat1 cells with deregulated expression of c-Myc resulted in nuclear fragmentation that was accompanied in all cells by the activation of Bax and the translocation of cytochrome c from the mitochondria to the cytoplasm. None of these features of apoptosis was induced in control Rat-1 cells. p38 was also activated by cisplatin only in cells with deregulated expression of c-Myc, but, in contrast with all features of apoptosis, this activation was not affected by Bcl-2. Remarkably, overexpression of an interfering mutant of the p38alpha isoform, but not p38beta, blocked cisplatin-induced Bax activation or cytochrome c release and nuclear fragmentation. Analysis of the kinase cascade upstream of p38 revealed a c-Myc-dependent activation by cisplatin of mitogen-activated protein kinase kinase (MKK) 3/6 and apoptosis signal-regulating kinase 1 (Ask1). Inhibition of Ask1 blocked p38 activation by cisplatin and all features of apoptosis. Several of these data were confirmed using other DNA-damaging agents. The findings indicated that c-Myc potentiation of the mitochondrial pathway of apoptosis results, at least in part, from a sensitization of Ask1 activation, allowing DNA-damaging agents to induce in cascade Ask1, p38alpha and Bax.
| S-EPMC1223429 | biostudies-other
Project description:The peroxiredoxin (PRX) family of antioxidant enzymes helps maintain the intracellular reducing milieu and suppresses apoptosis in non-neuronal cells. However, whether PRX can inhibit neuronal apoptosis through specific signaling mechanisms remains poorly understood. Induction of PRX2, the most abundant neuronal PRX, occurs in Parkinson's disease (PD) patient brains, but its functional impact is unclear. In the present study, we used the dopaminergic (DA) toxin 6-hydroxydopamine (6-OHDA) to model PD and explore the protective effect and mechanisms of PRX on DA neurons. Of the 2-cysteine PRXs that were tested in MN9D DA neurons, endogenous PRX2 was most beneficial to cell survival. Lentivirus-mediated PRX2 overexpression conferred marked in vitro and in vivo neuroprotection against 6-OHDA toxicity in DA neurons, and preserved motor functions involving the dopamine system in mouse. In addition to its role as an antioxidant enzyme, PRX2 exhibited anti-apoptotic effects in DA neurons via suppression of apoptosis signal-regulating kinase (ASK1)-dependent activation of the c-Jun N-terminal kinase/c-Jun and p38 pro-death pathways, which are also activated in DA neurons of postmortem PD brains. PRX2 inhibited 6-OHDA-induced ASK1 activation by modulating the redox status of the endogenous ASK1 inhibitor thioredoxin (Trx). PRX2 overexpression maintained Trx in a reduced state by inhibiting the cysteine thiol-disulfide exchange, thereby preventing its dissociation from ASK1. This study describes a previously undefined mechanism by which redox-sensitive molecules signal via apoptotic pathways in response to PD-relevant toxic stress in DA neurons. Our results also suggest that PRX2 and ASK1 may be potential targets for neuroprotective intervention in PD.
| S-EPMC3078571 | biostudies-other
Project description:The dopaminergic system is susceptible to dysfunction in numerous neurological diseases, including Parkinson's disease (PD). In addition to motor symptoms, some PD patients may experience non-motor symptoms, including cognitive and memory deficits. A possible explanation for their manifestation is a disturbed pattern of dopamine release in brain regions involved in learning and memory, such as the hippocampus. Therefore, investigating neuropathological alterations in dopamine release prior to neurodegeneration is imperative. This study aimed to characterize evoked hippocampal dopamine release and assess the impact of the neurotoxin MPP+ using a genetically encoded dopamine sensor and gene expression analysis. Additionally, considering the potential neuroprotective attributes demonstrated by apoptosis signal-regulating kinase 1 (Ask1) in various animal-disease-like models, the study also aimed to determine whether Ask1 knockdown restores MPP+-altered dopamine release in acute hippocampal slices. We applied variations of low- and high-frequency stimulation to evoke dopamine release within different hippocampal regions and discovered that acute application of MPP+ reduced the amount of dopamine released and hindered the recovery of dopamine release after repeated stimulation. In addition, we observed that Ask1 deficiency attenuated the detrimental effects of MPP+ on the recovery of dopamine release after repeated stimulation. RNA sequencing analysis indicated that genes associated with the synaptic pathways are involved in response to MPP+ exposure. Notably, Ask1 deficiency was found to downregulate the expression of Slc5a7, a gene encoding a sodium-dependent high-affinity choline transporter that regulates acetylcholine levels. Respective follow-up experiments indicated that Slc5a7 plays a role in Ask1 deficiency-mediated protection against MPP+ neurotoxicity. In addition, increasing acetylcholine levels using an acetylcholinesterase inhibitor could exacerbate the toxicity of MPP+. In conclusion, our data imply that the modulation of the dopamine-acetylcholine balance may be a crucial mechanism of action underlying the neuroprotective effects of Ask1 deficiency in PD.
| S-EPMC10896914 | biostudies-literature