Apoptosis Signal-regulating Kinase 1 Inhibitor Research Articles

Berberine attenuates CCN2-induced IL-1β expression and prevents cartilage degradation in a rat model of osteoarthritis

Connective tissue growth factor (CTGF; also known as CCN2) is an inflammatory mediator that is abundantly expressed in osteoarthritis (OA). Interleukin-1β (IL-1β) plays a pivotal role in OA pathogenesis. Berberine exhibits an anti-inflammatory effect, but the mechanisms by which it modulates CCN2-induced IL-1β expression in OA synovial fibroblasts (OASFs) remain unknown. We showed that CCN2-induced IL-1β expression is mediated by the activation of αvβ3/αvβ5 integrin-dependent reactive oxygen species (ROS) generation, and subsequent activation of apoptosis signal-regulating kinase 1 (ASK1), p38/JNK, and nuclear factor-κB (NF-κB) signaling pathways. This IL-1β expression in OASFs is attenuated by N-acetylcysteine (NAC), inhibitors of ASK1, p38, or JNK, or treatment with berberine. Furthermore, berberine also reverses cartilage damage in an experimental model of collagenase-induced OA (CIOA). We observed that CCN2 increased IL-1β expression via αvβ3/αvβ5 integrins, ROS, and ASK1, p38/JNK, and NF-κB signaling pathways. Berberine was found to inhibit these signaling components in OASFs in vitro and prevent cartilage degradation in vivo. We suggest a novel therapeutic strategy of using berberine for managing OA.

Identification of apoptosis signal-regulating kinase 1 (ASK1) inhibitors among the derivatives of benzothiazol-2-yl-3-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one

Apoptosis signal-regulating kinase 1 (ASK1) plays important roles in the pathogenesis of type 1 and type 2 diabetes, autoimmune disorders, cancer and neurodegenerative diseases suggesting that small compounds inhibiting ASK1 could be used for the treatment of these pathologies. We have identified novel chemical class of ASK1 inhibitors, namely benzothiazol-2-yl-3-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one, using molecular modeling techniques. It was found that the most active compound 1-(6-fluoro-benzothiazol-2-yl)-3-hydroxy-5-[3-(3-methyl-butoxy)-phenyl]-4-(2-methyl-2,3-dihydro-benzofuran-5-carbonyl)-1,5-dihydro-pyrrol-2-one (BPyO-34) inhibits ASK1 with IC50 of 0.52μM in vitro in kinase assay. The structure–activity relationships of 34 derivatives of benzothiazol-2-yl-3-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one have been studied and binding mode of this chemical class has been proposed.

Inhibitor of apoptosis signal-regulating kinase 1 protects against acetaminophen-induced liver injury

Metabolic activation and oxidant stress are key events in the pathophysiology of acetaminophen (APAP) hepatotoxicity. The initial mitochondrial oxidative stress triggered by protein adduct formation is amplified by c-jun-N-terminal kinase (JNK), resulting in mitochondrial dysfunction and ultimately cell necrosis. Apoptosis signal-regulating kinase 1 (ASK1) is considered the link between oxidant stress and JNK activation. The objective of the current study was to assess the efficacy and mechanism of action of the small-molecule ASK1 inhibitor GS-459679 in a murine model of APAP hepatotoxicity. APAP (300mg/kg) caused extensive glutathione depletion, JNK activation and translocation to the mitochondria, oxidant stress and liver injury as indicated by plasma ALT activities and area of necrosis over a 24h observation period. Pretreatment with 30mg/kg of GS-459679 almost completely prevented JNK activation, oxidant stress and injury without affecting the metabolic activation of APAP. To evaluate the therapeutic potential of GS-459679, mice were treated with APAP and then with the inhibitor. Given 1.5h after APAP, GS-459679 was still protective, which was paralleled by reduced JNK activation and p-JNK translocation to mitochondria. However, GS-459679 treatment was not more effective than N-acetylcysteine, and the combination of GS-459679 and N-acetylcysteine exhibited similar efficacy as N-acetylcysteine monotherapy, suggesting that GS-459769 and N-acetylcysteine affect the same pathway. Importantly, inhibition of ASK1 did not impair liver regeneration as indicated by PCNA staining. In conclusion, the ASK1 inhibitor GS-459679 protected against APAP toxicity by attenuating JNK activation and oxidant stress in mice and may have therapeutic potential for APAP overdose patients.

Design of a Phase 2 Clinical Trial of an ASK1 Inhibitor, GS-4997, in Patients with Diabetic Kidney Disease

Background: Most patients with diabetic kidney disease (DKD) experience disease progression despite receiving standard care therapy. Oxidative stress is associated with DKD severity and risk of progression, but currently approved therapies do not directly attenuate the pathologic consequences of oxidative stress. GS-4997 is a once daily, oral molecule that inhibits Apoptosis Signal-regulating Kinase 1 (ASK1), which is a key mediator of the deleterious effects of oxidative stress. Methods: We describe the rationale and design of a Phase 2 placebo-controlled clinical trial investigating the effects of GS-4997 in patients with T2DM and stage 3/4 DKD receiving standard of care therapy. Approximately, 300 subjects will be randomized in a stratified manner, based on the estimated glomerular filtration rate (eGFR) and urine albumin to creatinine ratio, to one of four arms in this dose-ranging study. The primary endpoint is change in eGFR at 48 weeks, and the key secondary endpoint is change in albuminuria. Conclusion: Guided by the biology of oxidative stress signaling through ASK1, the biology of DKD pathogenesis, and solid statistical methods, the decisions made for this Phase 2 study regarding delineating study population, efficacy outcomes, treatment period and statistical methods represent innovative attempts to resolve challenges specific to DKD study design.

Inhibition of apoptosis signal-regulating kinase 1 enhances endochondral bone formation by increasing chondrocyte survival.

Endochondral ossification is the result of chondrocyte differentiation, hypertrophy, death and replacement by bone. The careful timing and progression of this process is important for normal skeletal bone growth and development, as well as fracture repair. Apoptosis Signal-Regulating Kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK), which is activated by reactive oxygen species and other cellular stress events. Activation of ASK1 initiates a signaling cascade known to regulate diverse cellular events including cytokine and growth factor signaling, cell cycle regulation, cellular differentiation, hypertrophy, survival and apoptosis. ASK1 is highly expressed in hypertrophic chondrocytes, but the role of ASK1 in skeletal tissues has not been investigated. Herein, we report that ASK1 knockout (KO) mice display alterations in normal growth plate morphology, which include a shorter proliferative zone and a lengthened hypertrophic zone. These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death. Interestingly, under normal cell culture conditions, mouse embryonic fibroblasts (MEFs) derived from ASK1 KO mice show no differences in either MAPK signaling or osteogenic or chondrogenic differentiation when compared with wild-type (WT) MEFs. However, when cultured with stress activators, H2O2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways and a reduction in markers of terminal differentiation. Furthermore, in both WT mice treated with the ASK1 inhibitor, NQDI-1, and ASK1 KO mice endochondral bone formation was increased in an ectopic ossification model. These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation. Inhibition of ASK1 has clinical potential to treat fractures or to slow osteoarthritic progression by enhancing chondrocyte survival and slowing hypertrophy.

ASK1 pharmacophore model derived from diverse classes of inhibitors

The three-dimensional pharmacophore model of apoptosis signal-regulating kinase 1 (ASK1) inhibitors has been developed with PharmaGist program. The positions of pharmacophore features in the model correspond to conformations of ASK1 highly active inhibitors in which they interact with ATP-binding site of ASK1. The generated pharmacophore model allows accurately predict active and inactive compounds and can be of great use for virtual screening aimed at discovering novel ASK1 inhibitors.

Development of an HTS-Compatible Assay for the Discovery of ASK1 Signalosome Inhibitors Using AlphaScreen Technology

Genetic target validation studies have demonstrated that the apoptosis signal-regulating kinase 1 (ASK1) represents an important target for the treatment of rheumatoid arthritis, cardiac diseases, and several neurodegenerative disorders. To identify small-molecule inhibitors of ASK1, we have developed a high-throughput screening-compatible, homogenous, biochemical assay using AlphaScreen technology. This novel assay design utilizes purified stress-activated ASK1 signalosome complex, and it monitors phosphorylation of its full-length native substrate, MKK6. The assay has been optimized in a 384-well format and validated by screening the Sigma LOPAC library. The results presented here demonstrate that the assay is sensitive and robust with a Z' factor value of 0.88±0.04 and a signal-to-background ratio of 11, indicating that this assay can be used to screen large chemical libraries to discover novel inhibitors of ASK1.

Inhibition of JNK and p38 MAPK phosphorylation by 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester and 4-phenyl-butenoic acid decreases substance P-induced TNF-α upregulation in macrophages.

The interactions between the immune and nervous systems play an important role in immune and inflammatory conditions. Substance P (SP), the undecapeptide RPKPQQFFGLM-NH2, is known to upregulate the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α. We report here that 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester (AOPHA-Me) and 4-phenyl-3-butenoic acid (PBA), two anti-inflammatory compounds developed in our laboratory, reduce SP-stimulated TNF-α expression in RAW 264.7 macrophages. We also show that AOPHA-Me and PBA both inhibit SP-stimulated phosphorylation of JNK and p38 MAPK. Furthermore, molecular modeling studies indicate that both AOPHA-Me and PBA dock at the ATP binding site of apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPKs upstream of both JNK and p38 MAPK, with predicted interaction energies of -7.0 kcal/mol and -5.9 kcal/mol, respectively; this binding overlaps with that of staurosporine, a known inhibitor of ASK1. Taken together, these findings suggest that AOPHA-Me and PBA inhibition of TNF-α expression in SP-stimulated RAW 264.7 macrophages is a consequence of the inhibition of JNK and p38 MAPK phosphorylation. We have previously shown that AOPHA-Me and PBA inhibit the amidative bioactivation of SP, which also would be expected to decrease formation of pro-inflammatory cytokines. It is conceivable that this dual action of inhibiting amidation and MAPK phosphorylation may be of some advantage in enhancing the anti-inflammatory activity of a therapeutic molecule.

NQDI 1, An Inhibitor of ASK1 Attenuates Acute Ischemic Renal Injury by Modulating Oxidative Stress and Cell Death

Apoptosis signal-regulating kinase 1 (ASK1) is among the signaling events that lead to postischemic cell death. Inhibition of ASK1 pathway protected hearts from ischemic damage. The present study evaluated the renal protective effects of NQDI 1, an inhibitor of ASK1, in an animal model of acute ischemic renal failure. Male Wistar rats were subjected to right nephrectomy and clamping of left renal pedicle for 45 min, or sham operation. The administration of NQDI 1 attenuated renal dysfunction and histological changes characteristic for renal ischemia/reperfusion injury (IRI). Apoptosis of renal tissues, as detected by TUNEL staining, was also reduced together with p53 protein expression, and renal levels of MDA and SOD with NQDI 1 administration and BCL2 was up regulated. In conclusion, inhibition of ASK1 is of therapeutic potential against acute ischemic renal injury. Its protective effects are mediated via inhibition of apoptosis and oxidative stress.

Depletion of Apoptosis Signal-Regulating Kinase 1 Prevents Bile Duct Ligation–Induced Necroinflammation and Subsequent Peribiliary Fibrosis

Apoptosis signal-regulating kinase 1 (ASK1), also known as mitogen-activated protein kinase kinase kinase (MAP3K), is ubiquitously expressed and situated in an important upstream position of many signal transduction pathways. ASK1 plays a pivotal role in stressor-induced cell survival and inflammatory reactions. To ascertain the regulatory functions of ASK1 in bile duct ligation (BDL)-induced liver injury, we examined the net effects of ASK1 depletion on hepatic necroinflammation and/or fibrosis. We subjected C57BL/6 wild-type (WT) or ASK1-deficient (ASK1(-/-)) mice to sham or BDL surgery for 14 days. In day 3 BDL animals, ASK1(-/-) mice had significantly fewer bile infarcts along with more reduced interlobular or portal inflammatory infiltrate of various immune cells, including neutrophils, compared with WT mice in which ASK1 expression was markedly activated. Morphologically apoptotic hepatocytes or cholangiocytes were negligible in both the sham and BDL animals. In contrast, ASK1(-/-) mice had significantly less proliferating activity of not only hepatocytes but also large cholangiocytes than WT mice. Day 14 BDL ASK1(-/-) mice manifested potential antifibrogenic aspects of ASK1 deficiency, characterized by significantly fewer activated peribiliary fibrogenic cells and peribiliary fibrosis. These observations indicate that ASK1-mediated hepatic necroinflammation and proliferation, but not apoptosis, are closely linked to liver fibrosis and fibrogenesis. A specific ASK1 pathway blocker or inhibitor might offer a therapeutic strategy against human cholestatic diseases.

Interleukin-6 induces vascular endothelial growth factor expression and promotes angiogenesis through apoptosis signal-regulating kinase 1 in human osteosarcoma

Osteosarcoma is characterized by a high malignant and metastatic potential. Angiogenesis is essential for the caner metastasis. Interleukin-6 (IL-6) is a multifunctional cytokine that is associated with the disease status and outcomes of cancers. However, the relationship between IL-6 and vascular endothelial growth factor (VEGF) expression in human osteosarcoma is mostly unknown. Here we found that the IL-6 and VEGF expression was correlated with tumor stage and significantly higher than that in normal bone. Incubation of osteosarcoma cells with IL-6 increased VEGF mRNA and protein expression. Pretreatment of cells with IL-6R antibody reduced IL-6-mediated VEGF production. The apoptosis signal-regulating kinase 1 (ASK1)/p38/AP-1 pathway was activated after IL-6 treatment, and IL-6-induced VEGF expression was abolished by the specific inhibitor and siRNA of ASK1, p38, and AP-1 cascades. Importantly, knockdown IL-6 reduced VEGF expression and abolished osteosarcoma conditional medium-mediated angiogenesis. Taken together, these results indicate that IL-6 occurs through ASK1 and p38, which in turn activates AP-1, resulting in the activations of VEGF expression and contributing the angiogenesis of human osteosarcoma cells.

Apoptosis signal‐regulating kinase‐1 inhibitor as a potent therapeutic drug for the treatment of gastric cancer

Aside from the human epidermal growth factor receptor-2 (HER2)-targeting agent trastuzumab, molecular targeting therapy for gastric cancer (GC) has not been established. We previously reported that apoptosis signal-regulating kinase-1 (ASK1) was upregulated in human GC and that overexpression of ASK1 promoted GC cell proliferation. Here, we investigated the effect of ASK1 inhibitor K811 on GC cells. K811 efficiently prevented cell proliferation in cell lines with high ASK1 expression and in HER2-overexpressing GC cells. Treatment with K811 reduced sizes of xenograft tumors by downregulating proliferation markers. These results indicate that ASK1 inhibition prevents GC cell growth in vitro and in vivo, suggesting that ASK1 inhibitors can be potent therapeutic drugs for GC.

Design and biological evaluation of imidazo[1,2-a]pyridines as novel and potent ASK1 inhibitors

Imidazo[1,2-a]pyridine derivatives were designed, synthesized, and evaluated as inhibitors of the apoptosis signal-regulating kinase 1 (ASK1). These were based on a benzothiazole derivative that was discovered from high-throughput screening of our compound library. As a result, we identified potent, selective, and orally bioavailable ASK1 inhibitors for wide range of therapeutic targets.

Bortezomib/proteasome inhibitor triggers both apoptosis and autophagy-dependent pathways in melanoma cells

Generally, both endoplasmic reticulum (ER) stress and mitochondrial dysregulation are a potential therapeutic target of anticancer agents including bortezomib.The treatment of melanoma cells with bortezomib was found to induce apoptosis together with the upregulation of Noxa, Mcl-1, and HSP70 proteins, and the cleavage of LC3 and autophagic formation. Also, bortezomib induced ER-stress as evidenced by the increase of intracellular Ca2+ release. In addition, bortezomib enhanced the phosphorylation of inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α), apoptosis signal-regulating kinase 1 (ASK1), c-jun-N-terminal kinase (JNK) and p38, and the activation of the transcription factors AP-1, ATF-2, Ets-1, and HSF1. Bortezomib-induced mitochondrial dysregulation was associated with the accumulation of reactive oxygen species (ROS), the release of both apoptosis inducing factor (AIF) and cytochrome c, the activation of caspase-9 and caspase-3, and cleavage of Poly (ADP-ribose) polymerase (PARP). The pretreatment of melanoma cells with the inhibitor of caspase-3 (Ac-DEVD-CHO) was found to block bortezomib-induced apoptosis that subsequently led to the increase of autophagic formation. In contrast, the inhibition of ASK1 abrogated bortezomib-induced autophagic formation and increased apoptosis induction. Furthermore, the inhibition of JNK, of HSP70 also increased apoptosis induction without influence of bortezomib-induced autophagic formation. Based on the inhibitory experiments, the treatment with bortezomib triggers the activation of both ER-stress-associated pathways, namely IRE1α–ASK1–p38–ATF-2/ets-1–Mcl-1, and IRE1α–ASK1–JNK–AP-1/HSF1–HSP70 as well as mitochondrial dysregulation-associated pathways, namely ROS–ASK1–JNK–AP-1/HSF1–HS70, and AIF–caspase-3–PARP and Cyt.c, and caspase-9–caspase-3–PARP. Taken together, our data demonstrates for the first time the molecular mechanisms, whereby bortezomib triggers both apoptosis and autophagic formation in melanoma cells.

Rational design of apoptosis signal-regulating kinase 1 inhibitors: Discovering novel structural scaffold

Increased activity of apoptosis signal-regulating kinase 1 (ASK1) is associated with a number of human disorders and the inhibitors of ASK1 may become important compounds for pharmaceutical application. Here we report novel ASK1 inhibitor scaffold, namely 5-(5-Phenyl-furan-2-ylmethylene)-2-thioxo-thiazolidin-4-one, that has been identified using virtual screening and biochemical tests. A series of derivatives has been synthesized and evaluated in vitro towards human protein kinase ASK1. It was revealed that the most active compounds 4-((5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-4-oxo-2-thioxo-1,3-thiazolidin-3-yl)butanoic acid and 6-((5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-4-oxo-2-thioxo-1,3-thiazolidin-3-yl)hexanoic acid inhibit ASK1 with IC50 of 0.2 μM. Structure–activity relationships of 33 derivatives of 5-(5-Phenyl-furan-2-ylmethylene)-2-thioxo-thiazolidin-4-one have been studied and binding mode of this chemical class has been predicted.

An apoptosis signal‐regulating kinase 1 inhibitor reduces cardiomyocyte apoptosis and infarct size in a rat ischemia‐reperfusion model

Apoptosis Signal‐Regulating Kinase 1 (ASK1) is a critical mediator of oxidative stress‐induced apoptosis. We hypothesized that a small molecule inhibitor of ASK1 (ASK1‐i; Gilead Sciences, Palo Alto, CA) would reduce myocardial infarct size in a rat ischemia/reperfusion model. Forty‐seven SD rats were randomized to 3 groups: ASK1‐i infusion (n=16), vehicle infusion (n=16), or ischemic preconditioning (IPC; n=15). Infusion of ASK1‐i (10 mg/kg) or vehicle commenced 45 minutes prior to myocardial ischemia. IPC consisted of 3 cycles of 3 minutes coronary occlusion and 5 minutes reperfusion immediately prior to index myocardial ischemia. Rats were then subjected to 30 minutes of coronary occlusion followed by 180 minutes of reperfusion. Pathologic analysis revealed no significant difference in the size of the ischemic risk zone between the 3 groups. Infarct size, expressed as a percentage of the risk zone averaged 27.7 +/− 3.3%, 16.5 +/− 3.4%, and 41.5 +/− 4.8% in the ASK1‐i group, the IPC group, and the vehicle group, respectively (p < 0.05 for ASK1‐i and IPC versus vehicle). The percentage of apoptotic nuclei as assessed by TUNEL positivity averaged 12.4 +/− 1.1%, 11.1 +/− 1.9%, and 19.2 +/− 2.2% in the ASK1‐i group, IPC group, and vehicle group respectively (p < 0.05 for ASK1‐i and IPC versus vehicle). Inhibition of the ASK1 was shown to reduce apoptosis and myocardial infarct size in a rat model of ischemia/reperfusion.

Endoplasmic reticulum stress contributes to CRH-induced hippocampal neuron apoptosis

The hypothalamic–pituitary–adrenal (HPA) axis is critical to mediating the body's response to stress. Corticotropin releasing hormone (CRH) plays a central role in controlling the stress response and regulating the HPA axis. Recent findings support CRH participates in the stress-induced hippocampal neuron apoptosis, but the underlying mechanisms are not fully understood. Our present study demonstrates that CRH can independently decrease hippocampal neuron cell viability in vitro in a concentration- and time-dependent manner. CRH receptor 1 (CRHR1) is involved in CRH-induced neuron apoptosis. Endoplasmic reticulum (ER) stress response marker, glucose-regulated protein 78 (GRP78), either protein or mRNA, is significantly elevated after treatment of CRH, and decreased when co-treated with salubrinal, ER stress inhibitor. The ER stress associated proapoptotic transcription factor C/EBP homologous protein (CHOP) and cleavage of caspase-12 protein expression are also increased following CRH treatment. Furthermore, we investigate which ER stress cascades are affected by CRH. CRH activates inositol-requiring enzyme 1 (IRE1), apoptosis signal regulating kinase 1 (ASK1), and c-jun kinase (JNK). Neuron apoptotic rate, examined by flow cytometry, is increased when CRH treatment and attenuated by salubrinal, thioredoxin (ASK1 inhibitor) and SP600125 (JNK inhibitor). Therefore, current data indicate that ER stress, through activating the IRE1/ASK1/JNK cascade, plays an important role in CRH-induced neuron apoptosis.

Molecular hydrogen inhibits lipopolysaccharide/interferon γ-induced nitric oxide production through modulation of signal transduction in macrophages

Molecular hydrogen has been reported to be effective for a variety of disorders and its effects have been ascribed to the reduction of oxidative stress. However, we have recently demonstrated that hydrogen inhibits type I allergy through modulating intracellular signal transduction. In the present study, we examined the hydrogen effects on lipopolysaccharide/interferon γ LPS/IFNγ-induced nitric oxide (NO) production in murine macrophage RAW264 cells. Treatment with hydrogen reduced LPS/IFNγ-induced NO release, which was associated with a diminished induction of inducible isoform of nitric oxide synthase (iNOS). Hydrogen treatment inhibited LPS/IFNγ-induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) and its downstream signaling molecules, p38 MAP kinase and JNK, as well as IκBα, but did not affect activation of NADPH oxidase and production of reactive oxygen species (ROS). As ROS is an upstream activator of ASK1, inhibition of ASK1 by hydrogen without suppressing ROS implies that a potential target molecule of hydrogen should be located at the receptor or immediately downstream of it. These results suggested a role for molecular hydrogen as a signal modulator. Finally, oral intake of hydrogen-rich water alleviated anti-type II collagen antibody-induced arthritis in mice, a model for human rheumatoid arthritis. Taken together, our studies indicate that hydrogen inhibits LPS/IFNγ-induced NO production through modulation of signal transduction in macrophages and ameliorates inflammatory arthritis in mice, providing the molecular basis for hydrogen effects on inflammation and a functional interaction between two gaseous signaling molecules, NO and molecular hydrogen.

Identification of 3H-Naphtho[1,2,3-de]quinoline-2,7-diones as Inhibitors of Apoptosis Signal-Regulating Kinase 1 (ASK1)

Apoptosis signal-regulating kinase 1 (ASK1) has recently emerged as an attractive therapeutic target for the treatment of cardiac and neurodegenerative disorders. The selective inhibitors of ASK1 may become important compounds for the development of clinical agents. We have identified the ASK1 inhibitor among 3H-naphtho[1,2,3-de]quinoline-2,7-diones using receptor-based virtual screening. In vitro kinase assay revealed that ethyl 2,7-dioxo-2,7-dihydro-3H-naphtho[1,2,3-de]quinoline-1-carboxylate (NQDI-1) inhibited ASK1 with a K(i) of 500 nM. The competitive character of inhibition is demonstrated in Lineweaver-Burk plots. In our preliminary selectivity study this compound exhibited strong specific inhibitory activity toward ASK1.

Regulation of the severity of neuroinflammation and demyelination by TLR‐ASK1‐p38 pathway

Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase which plays important roles in stress and immune responses. Here, we show that ASK1 deficiency attenuates neuroinflammation in experimental autoimmune encephalomyelitis (EAE), without affecting the proliferation capability of T cells. Moreover, we found that EAE upregulates expression of Toll-like receptors (TLRs) in activated astrocytes and microglia, and that TLRs can synergize with ASK1-p38 MAPK signalling in the release of key chemokines from astrocytes. Consequently, oral treatment with a specific small molecular weight inhibitor of ASK1 suppressed EAE-induced autoimmune inflammation in both spinal cords and optic nerves. These results suggest that the TLR-ASK1-p38 pathway in glial cells may serve as a valid therapeutic target for autoimmune demyelinating disorders including multiple sclerosis.