ABSTRACT
Apoptosis signal regulated kinase 1 (ASK1, MAP3K5) is a member of the mitogen activated protein kinase (MAPK) signaling pathway, involved in cell survival, differentiation, stress response, and apoptosis. ASK1 kinase inhibition has become a promising strategy for the treatment of Non-alcoholic steatohepatitis (NASH) disease. A series of novel ASK1 inhibitors with indazole scaffolds were designed and synthesized, and their ASK1 kinase activities were evaluated. The System Structure Activity Relationship (SAR) study discovered a promising compound 33c, which has a strong inhibitory effect on ASK1. Noteworthy observations included a discernible reduction in lipid droplets within LO2 cells stained with Oil Red O, coupled with a decrease in LDL, CHO, and TG content within the NASH model cell group. Mechanistic inquiries revealed that compound 33c could inhibit the protein expression levels of the upregulated ASK1-p38/JNK signaling pathway in TNF-α treated HGC-27 cells and regulate apoptotic proteins. In summary, these findings suggest that compound 33c may be valuable for further research as a potential candidate compound against NASH.
Subject(s)
Drug Design , Indazoles , MAP Kinase Kinase Kinase 5 , Molecular Docking Simulation , Protein Kinase Inhibitors , Humans , Apoptosis/drug effects , Dose-Response Relationship, Drug , Indazoles/pharmacology , Indazoles/chemical synthesis , Indazoles/chemistry , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , MAP Kinase Kinase Kinase 5/metabolism , Molecular Structure , Non-alcoholic Fatty Liver Disease/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity Relationship , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolismABSTRACT
BACKGROUND AND AIMS: Apoptosis Signal-regulating Kinase 1 (ASK1) is activated by various pathological stimuli and induces cell apoptosis through downstream p38 activation. We studied the effect of pharmacological ASK1 inhibition on cirrhosis and its sequelae using comprehensive preclinical in vivo and in vitro systems. APPROACH AND RESULTS: Short-term (4-6 wk) and long-term (24-44 wk) ASK1 inhibition using small molecule GS-444217 was tested in thioacetamide-induced and BALB/c. Mdr2-/- murine models of cirrhosis and HCC, and in vitro using primary hepatocyte cell death assays. Short-term GS-444217 therapy in both models strongly reduced phosphorylated p38, hepatocyte death, and fibrosis by up to 50%. Profibrogenic release of mitochondrial DAMP mitochondrial deoxyribonucleic acid from dying hepatocytes was blocked by ASK1 or p38 inhibition. Long-term (24 wk) therapy in BALBc.Mdr2 - / - model resulted in a moderate 25% reduction in bridging fibrosis, but not in net collagen deposition. Despite this, the development of cirrhosis was effectively prevented, with strongly reduced p21 + hepatocyte staining (by 72%), serum ammonia levels (by 46%), and portal pressure (average 6.07 vs. 8.53 mm Hg in controls). Extended ASK1 inhibition for 44 wk in aged BALB/c. Mdr2-/- mice resulted in markedly reduced tumor number and size by ~50% compared to the control group. CONCLUSIONS: ASK1 inhibition suppresses the profibrogenic release of mitochondrial deoxyribonucleic acid from dying hepatocytes in a p38-dependent manner and protects from liver fibrosis. Long-term ASK1 targeting resulted in diminished net antifibrotic effect, but the progression to liver cirrhosis and cancer in BALBc/ Mdr2- / - mice was effectively inhibited. These data support the clinical evaluation of ASK1 inhibitors in fibrotic liver diseases.
Subject(s)
Disease Progression , Hepatocytes , Liver Cirrhosis , Liver Neoplasms , MAP Kinase Kinase Kinase 5 , Mice, Inbred BALB C , p38 Mitogen-Activated Protein Kinases , Animals , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , MAP Kinase Kinase Kinase 5/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , Mice , Liver Cirrhosis/drug therapy , Liver Cirrhosis/pathology , Liver Cirrhosis/metabolism , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Liver Neoplasms/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Male , Thioacetamide/toxicity , Mitochondria/drug effects , Mitochondria/metabolism , Disease Models, AnimalABSTRACT
Oxidative stress and neuroapoptosis are key pathological processes after subarachnoid hemorrhage (SAH). The present study evaluated the antioxidation and antiapoptotic neuroprotective effects of the apoptosis signalregulating kinase 1 (ASK1) inhibitor ethyl2,7dioxo2,7dihydro3Hnaphtho(1,2,3de)quinoline1carboxylate (NQDI1) in early brain injury (EBI) following SAH in a rat model. A total of 191 rats were used and the SAH model was induced using monofilament perforation. Western blotting was subsequently used to detect the endogenous expression levels of proteins. Immunofluorescence was then used to confirm the nerve cellular localization of ASK1. Shortterm neurological function was assessed using the modified Garcia scores and the beam balance test 24 h after SAH, whereas longterm neurological function was assessed using the rotarod test and the Morris water maze test. Apoptosis of neurons was assessed by TUNEL staining and oxidative stress was assessed by dihydroethidium staining 24 h after SAH. The protein expression levels of phosphorylated (p)ASK1 and ASK1 rose following SAH. NQDI1 was intracerebroventricularly injected 1 h after SAH and demonstrated significant improvements in both short and longterm neurological function and significantly reduced oxidative stress and neuronal apoptosis. Injection of NQDI1 caused a significant decrease in protein expression levels of pASK1, pp38, pJNK, 4 hydroxynonenal, and Bax and significantly increased the protein expression levels of heme oxygenase 1 and Bcl2. The use of the p38 inhibitor BMS582949 or the JNK inhibitor SP600125 led to significant decreases in the protein expression levels of pp38 or pJNK, respectively, and a significant reduction in oxidative stress and neuronal apoptosis; however, these inhibitors did not demonstrate an effect on pASK1 or ASK1 protein expression levels. In conclusion, treatment with NQDI1 improved neurological function and decreased oxidative stress and neuronal apoptosis in EBI following SAH in rats, possibly via inhibition of ASK1 phosphorylation and the ASK1/p38 and JNK signaling pathway. NQDI1 may be considered a potential agent for the treatment of patients with SAH.
Subject(s)
Apoptosis , Brain Injuries , MAP Kinase Kinase Kinase 5 , MAP Kinase Signaling System , Neuroprotective Agents , Subarachnoid Hemorrhage , Animals , Rats , Apoptosis/drug effects , Brain Injuries/drug therapy , Brain Injuries/etiology , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , MAP Kinase Signaling System/drug effects , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Oxidative Stress/drug effects , p38 Mitogen-Activated Protein Kinases , Rats, Sprague-Dawley , Subarachnoid Hemorrhage/complications , Subarachnoid Hemorrhage/drug therapyABSTRACT
BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. The advanced stage of NAFLD, non-alcoholic steatohepatitis (NASH), has been recognized as a leading cause of end-stage liver injury for which there are no FDA-approved therapeutic options. Glutathione S-transferase Mu 2 (GSTM2) is a phase II detoxification enzyme. However, the roles of GSTM2 in NASH have not been elucidated. METHODS: Multiple RNA-seq analyses were used to identify hepatic GSTM2 expression in NASH. In vitro and in vivo gain- or loss-of-function approaches were used to investigate the role and molecular mechanism of GSTM2 in NASH. RESULTS: We identified GSTM2 as a sensitive responder and effective suppressor of NASH progression. GSTM2 was significantly downregulated during NASH progression. Hepatocyte GSTM2 deficiency markedly aggravated insulin resistance, hepatic steatosis, inflammation and fibrosis induced by a high-fat diet and a high-fat/high-cholesterol diet. Mechanistically, GSTM2 sustained MAPK pathway signaling by directly interacting with apoptosis signal-regulating kinase 1 (ASK1). GSTM2 directly bound to the N-terminal region of ASK1 and inhibited ASK1 N-terminal dimerization to subsequently repress ASK1 phosphorylation and the activation of its downstream JNK/p38 signaling pathway under conditions of metabolic dysfunction. CONCLUSIONS: These data demonstrated that hepatocyte GSTM2 is an endogenous suppressor that protects against NASH progression by blocking ASK1 N-terminal dimerization and phosphorylation. Activating GSTM2 holds promise as a therapeutic strategy for NASH. CLINICAL TRIAL NUMBER: IIT-2021-277. LAY SUMMARY: New therapeutic strategies for non-alcoholic steatohepatitis are urgently needed. We identified that the protein GSTM2 exerts a protective effect in response to metabolic stress. Therapies that aim to increase the activity of GSTM2 could hold promise for the treatment of non-alcoholic steatohepatitis.
Subject(s)
Glutathione Transferase/pharmacology , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , Non-alcoholic Fatty Liver Disease/prevention & control , Animals , Biopsy/methods , Biopsy/statistics & numerical data , Disease Models, Animal , Gene Targeting/methods , Gene Targeting/standards , Gene Targeting/statistics & numerical data , Glutathione Transferase/metabolism , Hepatocytes/metabolism , Hepatocytes/physiology , Liver/pathology , MAP Kinase Kinase Kinase 5/therapeutic use , Mice , Non-alcoholic Fatty Liver Disease/drug therapy , Sequence Analysis, RNA/methods , Sequence Analysis, RNA/statistics & numerical dataABSTRACT
Oxidative stress-induced dopaminergic neuronal loss and apoptosis play a crucial role in the pathogenesis of Parkinson's disease (PD), and as a vital antioxidant protein, thioredoxin (Trx) exerts neuroprotection against PD. In this study, we investigated the effect of Schisanhenol (Sal), an active component from a traditional Chinese herb Schisandra rubriflora (Franch.), on MPP+-induced apoptosis and its association with thioredoxin-1 (Trx1) in SH-SY5Y cells. The protein levels of Trx1 and apoptosis-related proteins were detected by Western blot, the expression of Trx1 mRNA by real time qPCR, and apoptosis was detected by fluorescence microscopy and flow cytometry. Pretreatment with Sal (1 µM, 10 µM, and 50 µM) dose-dependently ameliorated MPP+-induced neuronal injury, confirmed by the improvement of the viability and morphological changes. Sal decreased the apoptosis rate of cells, suppressed the production of DNA ladder and sub-G1 peak, inhibited the Caspase-3 activity and the expression of apoptosis-related proteins. Sal enhanced the expression of Trx1 both in the protein and mRNA levels. However, the Trx1 inhibitor PX-12 suppressed the protective effects of Sal. In addition, Sal inhibited NF-κB translocation and activation. These results suggest that Sal has a protective effect against MPP+-induced apoptosis in SH-SY5Y cells via up-regulation of Trx1 expression and suppression of ASK1-P38-NF-κB pathway.
Subject(s)
1-Methyl-4-phenylpyridinium/adverse effects , Cyclooctanes/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , NF-kappa B/antagonists & inhibitors , Neuroblastoma/pathology , Polycyclic Compounds/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Proliferation , Herbicides/adverse effects , Humans , MAP Kinase Kinase Kinase 5/genetics , MAP Kinase Kinase Kinase 5/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Neuroblastoma/genetics , Neuroblastoma/metabolism , Neuroprotection , Tumor Cells, Cultured , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Apoptosis signal-regulating kinase 1 (ASK1) is one of the key mediators of the cellular stress response that regulates inflammation and apoptosis. To probe the therapeutic value of modulating this pathway in preclinical models of neurological disease, we further optimized the profile of our previously reported inhibitor 3. This effort led to the discovery of 32, a potent (cell IC50 = 25 nM) and selective ASK1 inhibitor with suitable pharmacokinetic and brain penetration (rat Cl/Clu = 1.6/56 L/h/kg and Kp,uu = 0.46) for proof-of-pharmacology studies. Specifically, the ability of 32 to inhibit ASK1 in the central nervous system (CNS) was evaluated in a human tau transgenic (Tg4510) mouse model exhibiting elevated brain inflammation. In this study, transgenic animals treated with 32 (at 3, 10, and 30 mg/kg, BID/PO for 4 days) showed a robust reduction of inflammatory markers (e.g., IL-1ß) in the cortex, thus confirming inhibition of ASK1 in the CNS.
Subject(s)
Brain/drug effects , Drug Discovery , Inflammation/drug therapy , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Animals , Brain/metabolism , Dose-Response Relationship, Drug , Humans , Inflammation/metabolism , MAP Kinase Kinase Kinase 5/metabolism , Mice , Mice, Transgenic , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Rats , Structure-Activity RelationshipABSTRACT
Diabetic cardiomyopathy (DCM) significantly increased the morbidity of heart failure in diabetic patients. Long-time oxidative stress is an indisputable contributor for DCM development. Apocynin (APO) has been suggested to be a potential drug against oxidative stress. The study aims to find out the effects of APO on DCM and the related mechanisms. Mice were randomly divided into four groups: control (CON), APO, DCM and DCM + APO. Echocardiography analyses, histological analyses, Western blot and RT-PCR were used to explore the roles and mechanisms of APO in DCM. Isolated neonatal rat cardiomyocytes (NRCMs) and cardiac fibroblasts (CFs) were used for further confirming the APO treatment effects in vitro. Deteriorated cardiac function, enlarged cardiomyocytes, excess cardiac fibrosis and significant cardiac oxidative stress were observed in DCM group. However, APO treatment successfully improved cardiac function, decreased cardiac hypertrophy and fibrosis, and depressed oxidative stress. Mechanistically, APO treatment markedly suppressed apoptosis signal regulating kinase 1(ASK1)-p38/c-jun N-terminal kinase (JNK) signaling and reduced apoptosis. It also inhibited NRCM apoptosis and CF activation via depressing ASK1-p38/JNK signaling in vitro. Moreover, adenovirus-mediated ASK1 overexpression completely removed the protection of APO in vitro. In conclusion, APO treatment could effectively attenuate DCM-associated injuries in vivo and protect against high glucose-induced NRCM and CF injuries in vitro via suppressing ASK1-p38/JNK signaling. APO might be a potential ASK1 inhibitor for treating DCM.
Subject(s)
Acetophenones/pharmacology , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Type 2/complications , Diabetic Cardiomyopathies/drug therapy , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , Acetophenones/therapeutic use , Animals , Animals, Newborn , Cells, Cultured , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Type 2/chemically induced , Diabetes Mellitus, Type 2/drug therapy , Diabetic Cardiomyopathies/etiology , Diabetic Cardiomyopathies/pathology , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Kinase Kinase 5/metabolism , MAP Kinase Signaling System/drug effects , Male , Mice , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Oxidative Stress/drug effects , Primary Cell Culture , Rats , Streptozocin/administration & dosage , Streptozocin/toxicity , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The functions of dual-specificity phosphatase 9 (DUSP9) in hepatic steatosis and metabolic disturbance during nonalcoholic fatty liver disease were discussed in our prior study. However, its roles in the pathophysiology of pressure overload-induced cardiac hypertrophy remain to be illustrated. This study attempted to uncover the potential contributions and underpinning mechanisms of DUSP9 in cardiac hypertrophy. Utilizing the gain-and-loss-of-functional approaches of DUSP9 the cardiac phenotypes arising from the pathological, echocardiographic, and molecular analysis were quantified. The results showed increased levels of DUSP9 in hypertrophic mice heart and angiotensin II treated cardiomyocytes. In accordance with the results of cellular hypertrophy in response to angiotensin II, cardiac hypertrophy exaggeration, fibrosis, and malfunction triggered by pressure overload was evident in the case of cardiac-specific conditional knockout of DUSP9. In contrast, transgenic mice hearts with DUSP9 overexpression portrayed restoration of the hypertrophic phenotypes. Further explorations of molecular mechanisms indicated the direct interaction of DUSP9 with ASK1, which further repressed p38 and JNK signaling pathways. Moreover, blocking ASK1 with ASK1-specific inhibitor compensated the pro-hypertrophic effects induced by DUSP9 deficiency in cardiomyocytes. The main findings of this study suggest the potential of DUSP9 in alleviating cardiac hypertrophy at least partially by repressing ASK1, thereby looks promising as a prospective target against cardiac hypertrophy.
Subject(s)
Cardiomegaly/metabolism , Dual-Specificity Phosphatases/metabolism , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , Myocardium/pathology , Pressure/adverse effects , Animals , Animals, Newborn , Cardiomegaly/prevention & control , Dual-Specificity Phosphatases/genetics , HEK293 Cells , Humans , MAP Kinase Kinase Kinase 5/metabolism , MAP Kinase Signaling System/physiology , Male , Mice , Mice, Knockout , Mice, Transgenic , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Cytokines/biosynthesis , Endothelial Cells/metabolism , MAP Kinase Kinase Kinase 5/physiology , Toll-Like Receptor 4/physiology , Cells, Cultured , Humans , JNK Mitogen-Activated Protein Kinases/physiology , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , MAP Kinase Signaling System/physiology , Nitric Oxide Synthase Type III/physiology , Permeability , p38 Mitogen-Activated Protein Kinases/physiologyABSTRACT
Apoptosis signal-regulating kinase 1 (ASK1, MAP3K5), a member of the mitogen-activated protein kinase (MAPK) signaling pathway, is involved in cell survival, differentiation, stress response, and apoptosis. ASK1 kinase inhibition has emerged as a promising therapeutic strategy for inflammatory disease. A series of novel ASK1 inhibitors with 1H-indazole scaffold were designed, synthesized and evaluated for their ASK1 kinase activity and AP1-HEK293 cell inhibitory effect. Systematic structure-activity relationship (SAR) efforts led to the discovery of promising compound 15, which showed excellent in vitro ASK1 kinase activity and potent inhibitory effects on ASK1 in AP1-HEK293 cells. In a tumor necrosis factor-α (TNF-α)-induced HT-29 intestinal epithelial cell model, compound 15 exhibited a significantly protective effect on cell viability comparable to that of GS-4997; moreover, compound 15 exhibited no obvious cytotoxicity against HT-29 cells at concentrations up to 25 µM. Mechanistic research demonstrated that compound 15 suppresses phosphorylation in the ASK1-p38/JNK signaling pathway in HT-29 cells, and regulates the expression levels of apoptosis-related proteins. Altogether, these results show that compound 15 may serve as a potential candidate compound for the treatment of inflammatory bowel disease (IBD).
Subject(s)
Antineoplastic Agents/pharmacology , Drug Design , Indazoles/pharmacology , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Indazoles/chemical synthesis , Indazoles/chemistry , MAP Kinase Kinase Kinase 5/metabolism , Molecular Docking Simulation , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
AIMS: Indoxyl sulfate (IS), a protein-bound uremic toxin, is implicated in endothelial dysfunction, which contributes to adverse cardiovascular events in chronic kidney disease. Apoptosis signal regulating kinase 1 (ASK1) is a reactive oxygen species-driven kinase involved in IS-mediated adverse effects. This study assessed the therapeutic potential of ASK1 inhibition in alleviating endothelial effects induced by IS. MAIN METHODS: IS, in the presence and absence of a selective ASK1 inhibitor (GSK2261818A), was assessed for its effect on vascular reactivity in rat aortic rings, and cultured human aortic endothelial cells where we evaluated phenotypic and mechanistic changes. KEY FINDINGS: IS directly impairs endothelium-dependent vasorelaxation and endothelial cell migration. Mechanistic studies revealed increased production of reactive oxygen species-related markers, reduction of endothelial nitric oxide synthase and increased protein expression of tissue inhibitor of matrix metalloproteinase 1 (TIMP1). IS also increases angiopoietin-2 and tumour necrosis factor α gene expression and promotes transforming growth factor ß receptor abundance. Inhibition of ASK1 ameliorated the increase in oxidative stress markers, promoted autocrine interleukin 8 pro-angiogenic signalling and decreased anti-angiogenic responses at least in part via reducing TIMP1 protein expression. SIGNIFICANCE: ASK1 inhibition attenuated vasorelaxation and endothelial cell migration impaired by IS. Therefore, ASK1 is a viable intracellular target to alleviate uremic toxin-induced impairment in the vasculature.
Subject(s)
Endothelium/metabolism , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , MAP Kinase Kinase Kinase 5/metabolism , Animals , Apoptosis/drug effects , Cells, Cultured , Endothelial Cells/metabolism , Endothelium/drug effects , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Indican/adverse effects , Indican/pharmacology , MAP Kinase Kinase Kinase 5/physiology , Male , NADPH Oxidases/metabolism , Nitric Oxide Synthase Type III/metabolism , Oxidative Stress/drug effects , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Vasodilation/drug effectsABSTRACT
Ischemia-reperfusion injury (IRI) consequent to major liver surgery is a still unmet clinical problem. The activation of endogenous systems of hepatoprotection can prevent the damaging effects of ischemia-reperfusion (IR) as shown by the phenomenon known as 'ischemic preconditioning'. The identification of endogenous signal mediators of hepatoprotection is of main interest since they could be targeted in future therapeutic interventions. Qiu et al. recently reported in Clin. Sci. (Lond.) (2020) 134(17), 2279-2294, the discovery of a novel protective molecule against hepatic IR damage: dual-specificity phosphatase 12 (DUSP12). IR significantly decreased DUSP12 expression in liver whereas DUSP12 overexpression in hepatocytes protected IRI and DUSP12 deletion in DUSP12 KO mice exacerbated IRI. The protective effects of DUSP12 depended on apoptosis signal-regulating kinase 1 (ASK1) and acted through the inhibition of the ASK1-dependent kinases c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). These results enlighten DUSP12 as a novel intermediate negative regulator of the pro-inflammatory and pro-apoptotic ASK1/JNK-p38 MAPK pathway activated during hepatic IR and identify DUSP12 as potential therapeutic target for IRI.
Subject(s)
Dual-Specificity Phosphatases/metabolism , Liver/pathology , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , Reperfusion Injury/prevention & control , Signal Transduction , Animals , Endothelial Cells/metabolism , Endothelial Cells/pathology , MAP Kinase Kinase Kinase 5/metabolism , MiceABSTRACT
Inflammation is a complex biological process which alters the normal physiological function of the immune system resulting in an abnormal microenvironment that leads to several clinical complications. The process of inflammation is mediated through various intracellular signaling factors inside the cells. Apoptosis signal-regulating kinase 1 (ASK1) is an inflammation-derived kinase that controls the activation of other family of kinases such as p38 mitogen-activated protein kinases (p38 MAPKs), which mediates various the inflammatory processes. In this study, we cultured THP-1 macrophage cells to undergo inflammatory proliferation with LPS (1 µg/ml) and TNFα (10 ng/ml) stimulation. Initial in silico analysis was utilized to predict novel microRNAs (miRNAs) that target ASK1 signaling and its expression levels in LPS and TNFα stimulated THP-1 cells were estimated. Among the miRNAs, miR-532-3p showcased the highest binding affinity towards ASK1 kinase. We witnessed that transient transfection of miR-532-3p diminished the levels of ASK1 and downstream phosphorylation/translocation of p38 MAPK. Furthermore, direct targeting of ASK1 resulted in regulation of uncontrolled release of cytokines (TNFα, IL-6, and IL-23) and chemokines (GM-CSF and MIP-2α). Overall, we suggest that miR-532-3p attenuates the pro-inflammatory nature of macrophages by targeting ASK1/p38 MAPK signaling pathway and can be used as a molecular intervention for treating inflammatory diseases.
Subject(s)
Inflammation Mediators/metabolism , MAP Kinase Kinase Kinase 5/biosynthesis , MAP Kinase Signaling System/physiology , Macrophages/metabolism , MicroRNAs/biosynthesis , Humans , Inflammation Mediators/antagonists & inhibitors , Lipopolysaccharides/toxicity , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , MAP Kinase Signaling System/drug effects , Macrophages/drug effects , THP-1 CellsABSTRACT
BACKGROUND: Chronic kidney disease (CKD) is a common cause of morbidity and mortality in human immunodeficiency virus (HIV)-positive individuals. Among the HIV-related kidney diseases, HIV-associated nephropathy (HIVAN) is a rapidly progressive renal disease characterized by collapsing focal glomerulosclerosis (GS), microcystic tubular dilation, interstitial inflammation and fibrosis. Although the incidence of end-stage renal disease due to HIVAN has dramatically decreased with the widespread use of antiretroviral therapy, the prevalence of CKD continues to increase in HIV-positive individuals. Recent studies have highlighted the role of apoptosis signal-regulating kinase 1 (ASK1) in driving kidney disease progression through the activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase and selective ASK-1 inhibitor GS-444217 was recently shown to reduce kidney injury and disease progression in various experimental models. Therefore we examined the efficacy of ASK1 antagonism by GS-444217 in the attenuation of HIVAN in Tg26 mice. METHODS: GS-444217-supplemented rodent chow was administered in Tg26 mice at 4 weeks of age when mild GS and proteinuria were already established. After 6 weeks of treatment, the kidney function assessment and histological analyses were performed and compared between age- and gender-matched control Tg26 and GS-444217-treated Tg26 mice. RESULTS: GS-444217 attenuated the development of GS, podocyte loss, tubular injury, interstitial inflammation and renal fibrosis in Tg26 mice. These improvements were accompanied by a marked reduction in albuminuria and improved renal function. Taken together, GS-4442217 attenuated the full spectrum of HIVAN pathology in Tg26 mice. CONCLUSIONS: ASK1 signaling cascade is central to the development of HIVAN in Tg26 mice. Our results suggest that the select inhibition of ASK1 could be a potential adjunctive therapy for the treatment of HIVAN.
Subject(s)
AIDS-Associated Nephropathy/drug therapy , Disease Models, Animal , Fibrosis/prevention & control , Inflammation/prevention & control , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Proteinuria/prevention & control , AIDS-Associated Nephropathy/metabolism , AIDS-Associated Nephropathy/pathology , Animals , Mice , Mice, TransgenicABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Tiao Geng (TG) decoction is a Chinese herbal medicine extract that has been utilized for the treatment of menopausal symptoms for a history of over 30 years. In our previous study, we suggest that TG decoction possibly exerts an anti-apoptotic effect on hypothalamic neurons of ovariectomized rats via the ASK1/MKK7/JNK pathway. Tributyltin chloride (TBTC) causes oxidative damage and induces apoptosis of primary hypothalamic neurons in rats. AIM OF THE STUDY: The present work aimed to explore the inhibition of TG decoction on TBTC-induced GT1-7 cell apoptosis and its possible molecular mechanism. MATERIALS AND METHODS: The GT1-7 cell line was exposed to TG decoction at diverse doses (31.25, 62.5, 125 µg/mL) for 24 h and later with TBTC (1 mg/L) for 1 h, with 17ß-E2 (100 nM) treatment being the positive control. Then, CCK8 assay was conducted to evaluate cell viability, while flow cytometric analysis was conducted to examine the apoptosis level. Related pathways and differentially expressed proteins were identified by tandem mass tag (TMT)-based quantitative phosphoproteomics. qRT-PCR was carried out to examine mRNA levels of Bax and B-cell lymphoma-2 (Bcl-2). Western blotting was performed to detect the levels of Bax, Bcl-2, c-Jun, c-Jun N-terminal kinase (JNK), Caspase-3 (Casp3), Mitogen-activated protein kinase kinase 7 (MKK7), and apoptosis signal-regulating kinase 1 (ASK1) . Finally, cells were pretreated with SP600125, an inhibitor of JNK, later the expression of JNK and Casp3 was measured. RESULTS: Application of TG decoction mitigated the GT1-7 cell apoptosis and injury caused by TBTC; besides, it inhibited the activation of the ASK1/MKK7/JNK pathway. Moreover, Bcl-2/Bax ratio became higher, and the MKK7, ASK1, Casp3 and c-Jun levels were inhibited. Besides, TG decoction combined with SP600125 (the JNK inhibitor) more significantly inhibited GT1-7 cell apoptosis caused by TBTC. CONCLUSION: As discovered from the experiment in this study, TG decoction has a neuroprotective effect, which is achieved through inhibiting the ASK1/MKK7/JNK signal transduction pathway to reduce GT1-7 cell apoptosis.
Subject(s)
Apoptosis/drug effects , Drugs, Chinese Herbal/pharmacology , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Trialkyltin Compounds/toxicity , Animals , Apoptosis/physiology , Cell Line , Cell Survival/drug effects , Cell Survival/physiology , Dose-Response Relationship, Drug , MAP Kinase Kinase Kinase 5/metabolism , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinases/metabolism , Neurons/drug effects , Neurons/metabolism , Rats , Trialkyltin Compounds/antagonists & inhibitorsABSTRACT
Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase (MAPK) family, is implicated in many human diseases. Here, we describe the structural optimization of hit compound 7 and conduct further structure-activity relationship (SAR) studies that result in the development of compound 19 with a novel indole-2-carboxamide hinge scaffold. Compound 19 displays potent anti-ASK1 kinase activity and stronger inhibitory effect on ASK1 in AP1-HEK293 cells than previously described ASK1 inhibitor GS-4997. Besides improved in vitro activity, compound 19 also exhibits an appropriate in vivo PK profile. In a dextran sulfate sodium (DSS)-induced mouse model of ulcerative colitis (UC), compound 19 shows significant anti-UC efficacy and markedly attenuates DSS-induced body weight loss, colonic shortening, elevation in disease activity index (DAI) and inflammatory cell infiltration in colon tissues. Mechanistically, compound 19 represses the phosphorylation of ASK1-p38/JNK signaling pathways and suppresses the overexpression of inflammatory cytokines. Together, these findings suggest that ASK1 inhibitors can potentially be used as a therapeutic strategy for UC.
Subject(s)
Colitis, Ulcerative/drug therapy , Indoles/therapeutic use , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , Humans , Indoles/pharmacology , Molecular StructureABSTRACT
Apoptosis Signal-Regulating Kinase-1 (ASK1) is a known member of the Mitogen-Activated Protein Kinase Kinase Kinase (MAP3K) family and upon stimulation will activate the p38- and JNK-pathways leading to cardiac apoptosis, fibrosis, and hypertrophy. Using Structure-Based Drug Design (SBDD) in parallel with deconstruction of a published compound, a novel series of ASK1 inhibitors was optimized, which incorporated a saturated heterocycle proximal to the hinge-binding motif. This yielded a unique chemical series with excellent selectivity across the broader kinome, and desirable drug-like properties. The lead compound (10) is highly soluble and permeable, and exhibits a cellular EC50 = 24 nM and Kd < 1 nM. Of the 350 kinases tested, 10 has an IC50 ≤ 500 nM for only eight of them. This paper will describe the design hypotheses behind this series, key data points during the optimization phase, as well as a possible structural rationale for the kinome selectivity. Based on crystallographic data, the presence of an aliphatic cycle adjacent to the hinge-binder in the active site of the protein kinase showed up in <1% of the >5000 structures in the Protein Data Bank, potentially conferring the selectivity seen in this series.
Subject(s)
MAP Kinase Kinase Kinase 5/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Animals , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Drug Design , Humans , Imidazoles/chemistry , Imidazoles/metabolism , Imidazoles/therapeutic use , Inhibitory Concentration 50 , MAP Kinase Kinase Kinase 5/metabolism , Mice , Molecular Dynamics Simulation , Myocardial Reperfusion Injury/drug therapy , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/therapeutic useABSTRACT
Type 2 diabetes (T2D) is associated with diabetic nephropathy as well as nonalcoholic steatohepatitis (NASH), which can be called "diabetic hepatopathy or diabetic liver disease". NASH, a severe form of nonalcoholic fatty disease (NAFLD), can sometimes progress to cirrhosis, hepatocellular carcinoma and hepatic failure. T2D patients are at higher risk for liver-related mortality compared with the nondiabetic population. NAFLD is closely associated with chronic kidney disease (CKD) or diabetic nephropathy according to cross-sectional and longitudinal studies. Simultaneous kidney liver transplantation (SKLT) is dramatically increasing in the United States, because NASH-related cirrhosis often complicates end-stage renal disease. Growing evidence suggests that NAFLD and CKD share common pathogenetic mechanisms and potential therapeutic targets. Glucagon-like peptide 1 (GLP-1) receptor agonists and sodium-glucose cotransporter 2 (SGLT2) inhibitors are expected to ameliorate NASH and diabetic nephropathy/CKD. There are no approved therapies for NASH, but a variety of drug pipelines are now under development. Several agents of them can also ameliorate diabetic nephropathy/CKD, including peroxisome proliferator-activated receptors agonists, apoptosis signaling kinase 1 inhibitor, nuclear factor-erythroid-2-related factor 2 activator, C-C chemokine receptor types 2/5 antagonist and nonsteroidal mineral corticoid receptor antagonist. This review focuses on common drug pipelines in the treatment of diabetic nephropathy and hepatopathy.
Subject(s)
Diabetic Nephropathies/drug therapy , Non-alcoholic Fatty Liver Disease/drug therapy , Anti-Inflammatory Agents/therapeutic use , Antihypertensive Agents/therapeutic use , Antioxidants/therapeutic use , Apoptosis/drug effects , Clinical Trials as Topic , Diabetes Mellitus, Type 2/complications , Diabetic Nephropathies/physiopathology , Dysbiosis/complications , Dysbiosis/therapy , Gastrointestinal Microbiome , Humans , Hypoglycemic Agents/therapeutic use , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , Models, Biological , Non-alcoholic Fatty Liver Disease/physiopathology , Peroxisome Proliferator-Activated Receptors/agonists , Prebiotics , Probiotics/therapeutic use , Renal Insufficiency, Chronic/drug therapyABSTRACT
Aberrant activation of mitogen-activated protein kinases (MAPKs) like c-Jun N-terminal kinase (JNK) and p38 is an event involved in the pathophysiology of numerous human diseases. The apoptosis signal-regulating kinase 1 (ASK1) is an upstream target that gets activated only under pathological conditions and as such is a promising target for therapeutic intervention. In the first part of this review the molecular mechanisms leading to ASK1 activation and regulation will be described as well as the evidences supporting a pathogenic role for ASK1 in human disease. In the second part, an update on drug discovery efforts towards the discovery and development of ASK1-targeting therapies will be provided.