Identification of novel ASK1 inhibitors using virtual screening

Apoptosis signal-regulating kinase 1 (ASK1, also called MAP3K5) is a mitogen-activated protein kinase kinase kinase (MAP3K) that plays important roles in stress-induced cell death and inflammation, and is expected as a new therapeutic target for cancer, cardiovascular diseases, and neurodegenerative diseases. We identified novel ASK1 inhibitors by virtual screening from the public chemical library collected by Chemical Biology Research Initiative (CBRI) at the University of Tokyo.     

ASK1 and its role in cardiovascular and other disorders: available treatments and future prospects

ABSTRACT

Introduction: Apoptosis signal-regulating kinase 1 (ASK1), also known as MAP3K5, is a member of mitogen-activated protein kinase kinase kinase (MAP3K) family and is well reported as crucial in the regulation of the JNK and P38 pathways. ASK1 is activated in response to a diverse array of stresses such as endoplasmic reticulum stress, lipopolysaccharides, tumor necrosis factor alpha, and reactive oxygen species. The activation of ASK1 induces various stress responses.

Areas covered: Considering ASK1 as an important therapeutic drug target, here we have discussed the role of ASK1 in the progression of various diseases. We have also provided an overview of the available inhibitors for ASK1. The success of computational-based approaches toward ASK1 inhibitor design has also been discussed.

Expert opinion: A number of reports have outlined the prominent role of ASK1 in the pathogenesis of several diseases. The discovery of novel ASK1 inhibitors would have a wide range of applications in medical science. In-silico techniques have been successfully used in the design of some novel ASK1 inhibitors. The use of machine learning-based approaches in combination with structure-based virtual screening (SBVS) and ligand-based virtual screening (LBVS) will be helpful toward the development of potent ASK1 inhibitors.     

Apoptosis signal-regulating kinase 1 is involved not only in apoptosis but also in non-apoptotic cardiomyocyte death

The molecular basis of myocardial cell death in the ischemia-reperfused heart still remains to be clarified. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in stress-induced apoptosis. We studied ASK1(-/-) mice to examine the role of ASK1 in ischemia-reperfusion injury. In the wild-type heart, ischemia-reperfusion resulted in necrotic injury, whereas infarct size was drastically reduced in the ASK1(-/-) heart. The necrotic injury was not accompanied with any evidence of apoptosis such as an increase in TUNEL-positive cells, DNA fragmentation or the activation of caspase-3. ASK1(-/-) cardiomyocytes were more resistant to H(2)O(2)- or Ca(2+)-induced apoptotic and non-apoptotic cell death compared with wild-type cells. These data suggest that ASK1 is involved in necrosis as well as apoptosis and that ASK1-dependent necrosis is likely to contribute to myocardial cell death in the ischemia-reperfused heart.     

Interaction of apoptosis signal-regulating kinase 1 with isoforms of 14-3-3 proteins

Apoptosis signal-regulating kinase 1 (ASK1) is a critical mediator of apoptotic signaling pathways initiated by a variety of death stimuli. Its activity is tightly controlled by various mechanisms such as covalent modification and protein-protein interaction. One of the proteins that control ASK1 function is 14-3-3zeta, a member of the 14-3-3 protein family. Here, we report that ASK1 is capable of binding to other isoforms of 14-3-3, suggesting that binding ASK1 is a general property of the 14-3-3 family. In support of this notion, mutational analysis revealed that the ASK1/14-3-3 interaction was mediated by the conserved amphipathic groove of 14-3-3 with some residue selectivity. Functionally, expression of various isoforms of 14-3-3 suppressed ASK1-induced apoptosis. To understand how 14-3-3 controls the ASK1 activity, we examined intracellular localization of ASK1 upon 14-3-3 co-expression. We found that 14-3-3 co-expression is correlated with the translocation of ASK1 from the cytoplasm to a perinuclear localization, likely the ER compartment. Consistent with this notion, ASK1(S967A), a 14-3-3 binding defective mutant of ASK, showed no change in intracellular distribution upon 14-3-3 co-expression. These data support a model that 14-3-3 proteins regulate the proapoptotic function of ASK1 in part by controlling its subcellular distribution.     

Heteromeric complex formation of ASK2 and ASK1 regulates stress-induced signaling

Apoptosis signal-regulating kinase 2 (ASK2) is an interaction partner of the highly related ASK1. Here, we describe a regulatory function of ASK2 in stress signaling-induced cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Increased cleavage of caspase-3 and PARP was demonstrated by overexpression as well as knockdown of ASK2 after stress-induction by serum-starvation. We show that ectopically expressed ASK2 homo-oligomerized while endogenous ASK2 and ASK1 formed hetero-oligomers, which decreased upon serum-starvation. Co-expression of ASK2 and ASK1 stabilized these two proteins and reduced starvation-induced caspase-3 activation and degradation of PARP. Analysis of the intracellular localization of ASK2 exhibited a similar localization compared with ASK1 in the nucleus, cytoplasm, and in mitochondria. We propose that ASK2 regulates stress-induced caspase-3 and PARP cleavage in a dose-dependent manner by heteromeric complex formation with ASK1.     

Involvement of ASK1 in Ca2+-induced p38 MAP kinase activation

The mammalian mitogen-activated protein (MAP) kinase kinase kinase apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component in cytokine- and stress-induced apoptosis. It also regulates cell differentiation and survival through p38 MAP kinase activation. Here we show that Ca²⁺ signalling regulates the ASK1–p38 MAP kinase cascade. Ca²⁺ influx evoked by membrane depolarization in primary neurons and synaptosomes induced activation of p38, which was impaired in those derived from ASK1-deficient mice. Ca²⁺/calmodulin-dependent protein kinase type II (CaMKII) activated ASK1 by phosphorylation. Moreover, p38 activation induced by the expression of constitutively active CaMKII required endogenous ASK1. Thus, ASK1 is a critical intermediate of Ca²⁺ signalling between CaMKII and p38 MAP kinase.     

Apoptosis signal-regulating kinase (ASK) 2 functions as a mitogen-activated protein kinase kinase kinase in a heteromeric complex with ASK1

Apoptosis signal-regulating kinase (ASK) 1 is a mitogen-activated protein kinase kinase kinase (MAP3K) in the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways that play multiple important roles in cytokine and stress responses. Here we show that ASK2, a highly related serine/threonine kinase to ASK1, also functions as a MAP3K only in a heteromeric complex with ASK1. We found that endogenous ASK2 was constitutively degraded in ASK1-deficient cells, suggesting that ASK1 is required for the stability of ASK2. ASK2 in a heteromeric complex with a kinase-negative mutant of ASK1 (ASK1-KN) effectively activated MAP2K and was more competent to respond to oxidative stress than ASK2 alone. Knockdown of ASK2 revealed that ASK2 was required for oxidative stress-induced JNK activation. These results suggest that ASK2 forms a functional MAP3K complex with ASK1, in which ASK1 supports the stability and the active configuration of ASK2. Moreover, ASK2 was found to activate ASK1 by direct phosphorylation, suggesting that ASK1 and ASK2 in a heteromeric complex facilitate their activities to each other by distinct mechanisms. Such a formation of functional heteromeric complex between different MAP3Ks may be advantageous for cells to cope with a wide variety of stimuli by fine regulation of cellular responses.     

Roles of MAPKKK ASK1 in stress-induced cell death

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein (MAP) kinase kinase kinase that activates the c-Jun N-terminal kinase (JNK) and p38 MAP kinase signaling cascades. Recent findings from analyses of ASK1-deficient mice have revealed that ASK1 is required for apoptosis induced by oxidative stress, TNF and endoplasmic reticulum (ER) stress. In addition, several lines of evidence have suggested that ASK1 has diverse functions in the decision of cell fate beyond its pro-apoptotic activity. Thus, ASK1 appears to be a pivotal component not only in stress-induced cell death but also in a broad range of biological activities in order for cells to adapt to or oppose various stresses.     

GSK-3β-induced ASK1 stabilization is crucial in LPS-induced endotoxin shock

Glycogen synthase kinase-3β (GSK-3β), a multifunctional kinase, is a regulator of lipopolysaccharide (LPS)-mediated septic shock. Apoptosis signal-regulating kinase 1 (ASK1) is also required for LPS-induced activation of p38, which is a crucial determinant for the production of pro-inflammatory cytokines via Toll-like receptor 4 (TLR4) in endotoxemia. Here, we show that attenuation of endotoxemia induced by GSK-3 inhibition is caused by the ASK1 reduction-mediated inhibition of p38, a representative downstream kinase of ASK1. LPS-stimulated activation of p38 was blocked by the reduction of ASK1 via the knockdown of GSK-3β. In addition, compared with L929 control cells, ASK1 protein was reduced in L929 cells stably expressing Wnt-3a and in which β-catenin was active, due to the inhibition of GSK-3β activity. GSK-3β inhibition-mediated ASK1 reduction was also confirmed by reduced ASK1 in GSK-3β-deficient mouse embryo fibroblasts (MEFs) and MCF7 GSK-3β siRNA cells. Furthermore, ASK1 protein stability was also attenuated in MCF7 GSK-3β siRNA cells compared with GFP control cells. Consistent with stability data, a much stronger ubiquitination of ASK1 was observed in cells in which GSK-3β was knocked down. These findings suggest that GSK-3β crosstalks with p38 kinase via the regulation of ASK1 protein stability in endotoxemia.     

Knockdown of apoptosis signal-regulating kinase 1 modulates basal glycogen synthase kinase-3β kinase activity and regulates cell migration

GSK-3β is a basally active kinase. Axin forms a complex with GSK-3β and β-catenin; this complex promotes the GSK-3β-dependent phosphorylation of β-catenin, thereby inducing its degradation. However, the inhibition of GSK-3β provokes cell migration via the dysregulation of β-catenin. In this study, we determined that the level of apoptosis signal-regulating kinase 1 (ASK1) was lower in a metastatic breast cancer cell line, compared to that of non-metastatic cancer cell lines and the knockdown of ASK1 not only induces β-catenin activation via the inhibition of GSK-3β and collapsing the subsequent protein complex by regulating Axin dynamics, but also stimulates cell migration. Together, the blockage of the GSK-3β-β-catenin pathway resulting from the knockdown of ASK1 modulates the migration of breast cancer cells.     

Apoptosis signal-regulating kinase 1 (ASK1) is an intracellular inducer of keratinocyte differentiation

Cells differentiate in response to various extracellular stimuli. This cellular response requires intracellular signaling pathways. The mitogen-activated protein (MAP) kinase cascade is a core signal transduction pathway that determines the fate of many kinds of cell. MAP kinase kinase kinase activates MAP kinase kinase, which in turn activates MAP kinase. Apoptosis signal-regulating kinase (ASK1) was identified as a MAP kinase kinase kinase involved in the stress-induced apoptosis-signaling cascade that activates the SEK1-JNK and MKK3/MKK6-p38 MAP kinase cascades. Expression of the constitutively active form of ASK1 (ASK1-DeltaN) in keratinocytes induced significant morphological changes and differentiation markers, transglutaminase-1, loricrin, and involucrin. A transient increase in p21(Cip1/WAF1) reduced DNA synthesis, and cell cycle analysis verified the differentiation. p38 MAP kinase inhibitors, SB202190 and SB203580, abolished the induction of differentiation markers, transglutaminase-1, loricrin, and involucrin. In turn, the induction of differentiation with ceramide in keratinocytes caused an increase in ASK1 expression and activity. Furthermore, normal human skin expresses ASK1 protein in the upper epidermis, implicating ASK1 in in vivo keratinocyte differentiation. We propose that the ASK1-p38 MAP kinase cascade is a new intracellular regulator of keratinocyte differentiation.     

S-nitrosylation of thioredoxin mediates activation of apoptosis signal-regulating kinase 1

Apoptosis signal-regulating kinase 1 (ASK1) was recently discovered as a typical member of the mitogen-activated protein (MAP) kinase kinase kinase family, which induces apoptosis by activation of c-Jun-N-terminal kinase/p38 MAP kinase pathways. In normal cells ASK1 is directly inhibited by thioredoxin (Trx), a 12-kDa protein ubiquitously expressed in all living cells, which has a variety of biological functions related to cell proliferation and apoptosis. Here we found that purified Trx is sensitive to S-nitrosylation. Stimulation of HEK-293 cells with S-nitrosoglutathione (GSNO) for 2, 4, 8, and 16h also caused Trx S-nitrosylation, which showed straight correlation with ASK1 activation based on Western blot detection of the enzyme, immunoprecipitation assay, and measurement of its catalytic activity. These results suggest that S-nitrosylation of Trx induces ASK1 activation. Treatment of cells with N-acetyl-cysteine for 2h after 8h of pretreatment with GSNO caused an increase in glutathione and nullified ASK1 activation.     

ASK Family Proteins in Stress Response and Disease

Cells are continuously exposed to reactive oxygen species (ROS) generated by aerobic metabolism. Excessively generated ROS causes severe dysfunctions to cells as oxidative stress. On the other hand, there is increasing evidence that ROS plays important roles as a signaling intermediate that induces a wide variety of cellular responses such as proliferation, differentiation, senescence, and apoptosis. To transmit physiological ROS-mediated signals and to adapt to oxidative stress, cells are equipped with various intracellular signal transduction systems, represented by mitogen-activated protein kinase (MAPK) cascades. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream regulator of the stress-activated MAPK cascades and has been shown to play critical roles in ROS-mediated cellular responses. Here, we highlight the roles of members of the ASK family, which consists of ASK1 and newly characterized ASK2, in ROS signaling with their possible involvement in human diseases.     

Ghrelin inhibits apoptosis signal-regulating kinase 1 activity via upregulating heat-shock protein 70

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), which has been originally isolated from rat stomach. It has been reported that ghrelin inhibited apoptosis in several cells, such as cardiomyocytes, endothelial cells, adipocyte, adrenal zona glomerulosa cells, pancreatic beta-cells, osteoblastic MC3T3-E1 cells, intestinal epithelial cells and hypothalamic neurons. However, it is unknown whether heat-shock protein 70 (HSP70) or apoptosis signal-regulating kinase 1 (ASK1) is the important target molecule which mediates the anti-apoptotic effects of ghrelin. We show that ghrelin inhibited ASK1 activity induced by sodium nitroprusside (SNP), inhibited ASK1-mediated caspase 3 activation and apoptosis in PC12 cells. Ghrelin promoted expression of HSP70. Quercetin, an inhibitor of HSP70, blocked the effects of ghrelin on ASK1 activity. Thus, ghrelin inhibits ASK1-mediated apoptosis and ASK1 activation by a mechanism involving induction of HSP70 expression. The results of the present study suggest the therapeutic potential of ghrelin for some pathological processes or disorders.     

Diacylglycerol kinase η colocalizes and interacts with apoptosis signal-regulating kinase 3 in response to osmotic shock

Diacylglycerol kinase (DGK) η translocates from the cytoplasm to punctate vehicles via osmotic shock. Apoptosis signal-regulating kinase (ASK) 3 (MAP kinase kinase kinase (MAPKKK) 15) is also reported to respond to osmotic shock. Therefore, in the present study, we examined the subcellular localization of DGKη and ASK3 expressed in COS-7 cells under osmotic stress. We found that DGKη was almost completely colocalized with ASK3 in punctate structures in response to osmotic shock. In contrast, DGKδ, which is closely related to DGKη structurally, was not colocalized with ASK3, and DGKη failed to colocalize with another MAPKKK, C-Raf, even under osmotic stress. The structures in which DGKη and ASK3 localized were not stained with stress granule makers. Notably, DGKη strongly interacted with ASK3 in an osmotic shock-dependent manner. These results indicate that DGKη and ASK3 undergo osmotic shock-dependent colocalization and associate with each other in specialized structures.     

Type 1 insulin-like growth factor receptor (IGF-IR) signaling inhibits apoptosis signal-regulating kinase 1 (ASK1)

The type 1 insulin-like growth factor receptor (IGF-IR) is a receptor-tyrosine kinase that plays a critical role in signaling cell survival and proliferation. IGF-IR binding to its ligand, insulin-like growth factor (IGF-I) activates phosphoinositide 3-kinase (PI3K), promotes cell proliferation by activating the mitogen-activated protein kinase (MAPK) cascade, and blocks apoptosis by inducing the phosphorylation and inhibition of proapoptotic proteins such as BAD. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that is required for c-Jun N-terminal kinase (JNK) and p38 activation in response to Fas and tumor necrosis factor (TNF) receptor stimulation, and for oxidative stress- and TNFalpha-induced apoptosis. The results presented here indicate that ASK1 forms a complex with the IGF-IR and becomes phosphorylated on tyrosine residue(s) in a manner dependent on IGF-IR activity. IGF-IR signaling inhibited ASK1 irrespective of TNFalpha-induced ASK1 activation and resulted in decreased ASK1-dependent JNK1 stimulation. Signaling through IGF-IR rescued cells from ASK1-induced apoptotic cell death in a manner independent of PI3K activity. These results indicate that IGF-IR signaling suppresses the ASK-1-mediated stimulation of JNK/p38 and the induction of programmed cell death. The simultaneous activation of MAP kinases and the inhibition of the stress-activated arm of the cascade by IGF-IR may constitute a potent proliferative signaling system and is possibly a mechanism by which IGF-I can stimulate growth and inhibit cell death in a wide variety of cell types and biological settings.     

Identification of a novel antiapoptotic protein that antagonizes ASK1 and CAD activities

Diverse stimuli initiate the activation of apoptotic signaling pathways that often causes nuclear DNA fragmentation. Here, we report a new antiapoptotic protein, a caspase-activated DNase (CAD) inhibitor that interacts with ASK1 (CIIA). CIIA, by binding to apoptosis signal-regulating kinase 1 (ASK1), inhibits oligomerization-induced ASK1 activation. CIIA also associates with CAD and inhibits the nuclease activity of CAD without affecting caspase-3-mediated ICAD cleavage. Overexpressed CIIA reduces H2O2- and tumor necrosis factor-alpha-induced apoptosis. CIIA antisense oligonucleotides, which abolish expression of endogenous CIIA in murine L929 cells, block the inhibitory effect of CIIA on ASK1 activation, deoxyribonucleic acid fragmentation, and apoptosis. These findings suggest that CIIA is an endogenous antagonist of both ASK1- and CAD-mediated signaling.     

Thioredoxin and HSP90α modulate ASK1–JNK1/2 signaling in stressed hepatocytes of Mugil cephalus

Induction of antioxidant proteins like thioredoxin (Trx) and heat shock protein 90α (HSP90α) is a crucial step in the cellular response to oxidative stress. Here, we report the impact of environmental stress on Trx and HSP90α expressions in freshly isolated hepatocytes of Mugil cephalus living in either a contaminated (Test; Ennore) or uncontaminated (Control; Kovalam) estuary. Modulation in the activities of signal transduction molecules like apoptosis signal-regulating kinase 1 (ASK1) and c-Jun NH₂-terminal kinase 1/2 (JNK1/2) were also investigated to understand their functional role under natural stressed condition. The expression pattern of the proteins was determined by immunoblotting and the relationship between the proteins was identified by regression analysis. Test fish hepatocytes demonstrated significant upregulation (P < 0.05) in the levels of Trx and HSP90α and insignificant inductions in the expression pattern of ASK1 and JNK1/2 than control fish hepatocytes. These findings provide direct evidence that Trx and HSP90α induction in fish hepatocytes under stress may aid cell survival by negatively regulating ASK1 expression and thereby functionally antagonizing the apoptotic role of JNK1/2 in natural aquatic systems.     

Phosphorylation-dependent scaffolding role of JSAP1/JIP3 in the ASK1-JNK signaling pathway. A new mode of regulation of the MAP kinase cascade

JSAP1 (also termed JIP3) is a scaffold protein that interacts with specific components of the JNK signaling pathway. Apoptosis signal-regulating kinase (ASK) 1 is a MAP kinase kinase kinase that activates the JNK and p38 mitogen-activated protein (MAP) kinase cascades in response to environmental stresses such as reactive oxygen species. Here we show that JSAP1 bound ASK1 and enhanced ASK1- and H(2)O(2)-induced JNK activity. ASK1 phosphorylated JSAP1 in vitro and in vivo, and the phosphorylation facilitated interactions of JSAP1 with SEK1/MKK4, MKK7 and JNK3. Furthermore, ASK1-dependent phosphorylation was required for JSAP1 to recruit and thereby activate JNK in response to H(2)O(2). We thus conclude that JSAP1 functions not only as a simple scaffold, but it dynamically participates in signal transduction by forming a phosphorylation-dependent signaling complex in the ASK1-JNK signaling module.