Discovery of the c-Jun N-Terminal Kinase Inhibitor CC-90001.

As a result of emerging biological data suggesting that within the c-Jun N-terminal kinase (JNK) family, JNK1 and not JNK2 or JNK3 may be primarily responsible for fibrosis pathology, we sought to identify JNK inhibitors with an increased JNK1 bias relative to our previous clinical compound tanzisertib (CC-930). This manuscript reports the synthesis and structure-activity relationship (SAR) studies for a novel series of JNK inhibitors demonstrating an increased JNK1 bias. SAR optimization on a series of 2,4-dialkylamino-pyrimidine-5-carboxamides resulted in the identification of compounds possessing low nanomolar JNK inhibitory potency, overall kinome selectivity, and the ability to inhibit cellular phosphorylation of the direct JNK substrate c-Jun. Optimization of physicochemical properties in this series resulted in compounds that demonstrated excellent systemic exposure following oral dosing, enabling in vivo efficacy studies and the selection of a candidate for clinical development, CC-90001, which is currently in clinical trials (Phase II) in patients with idiopathic pulmonary fibrosis (NCT03142191).

Immunomodulatory drugs reorganize cytoskeleton by modulating Rho GTPases.

IMiDs immunomodulatory drugs, including lenalidomide and pomalidomide represent a novel class of small molecule anticancer and anti-inflammatory drugs with broad biologic activities. However, the molecular mechanism through which these drugs exert their effects is largely undefined. Using pomalidomide and primary human monocytes, we report that pomalidomide rapidly and selectively activated RhoA and Rac1, but not Cdc42 or Ras, in the absence of any costimulation. Consistent with the activation of Rho GTPases, we found that pomalidomide enhanced F-actin formation, stabilized microtubules, and increased cell migration, all of which were blocked by selective inhibitors of ROCK1 and Rac1. Further, we showed that in Swiss 3T3 cells, pomalidomide only activated RhoA, not Rac1 or Cdc42, and potently induced stress fiber formation. The pomalidomide effect on actin cytoskeleton was blocked by the ROCK1 inhibitor, but not Rac1 inhibitor. Finally, we demonstrated that pomalidomide was able to regulate the activity of Rho GTPases and the formation of F-actin in primary human T cells as it did in monocytes and showed that the activation of RhoA was essential for pomalidomide-induced interleukin-2 expression in T cells. These novel activities provide what we believe a critical mechanism by which IMiDs drugs function as therapeutic immunomodulatory agents.

Blockade of the c-Jun amino terminal kinase prevents crescent formation and halts established anti-GBM glomerulonephritis in the rat.

Macrophages induce acute renal injury in anti-glomerular basement membrane (GBM) glomerulonephritis. This operates, in part, via activation of the c-Jun amino terminal kinase (JNK) signaling pathway. However, it is unknown whether inhibition of JNK signaling is effective once the proinflammatory response is established in the injured kidney. This study examined whether blockade of JNK signaling could halt disease progression, including crescent formation, in a model of severe crescentic anti-GBM glomerulonephritis. WKY rats were immunized with sheep IgG and then injected with sheep anti-GBM serum (day 0). Animals were treated with the JNK inhibitor, CC-401, vehicle alone, or no treatment from day 7 until being killed on day 24 of disease. Untreated animals at day 7 showed significant proteinuria, focal glomerular lesions, marked glomerular macrophage and T-cell accumulation, and upregulation of proinflammatory mediators (TNF-alpha, iNOS, MMP-12). Untreated and vehicle-treated groups displayed severe glomerulonephritis at day 24 with renal impairment and worsening proteinuria. These animals had severe glomerular lesions, with 60% of glomeruli exhibiting fibrocellular crescents, in association with increased macrophage and T-cell accumulation (including macrophage giant cells) and a further increase in mRNA levels of TNF-alpha, iNOS, MMP-12, and TGF-beta1. In contrast, CC-401 treatment prevented renal impairment, suppressed proteinuria, and prevented severe glomerular and tubulointerstitial lesions, including crescent formation and granulomatous-like lesions. These protective effects were independent of glomerular macrophage and T-cell accumulation, and of the humoral immune response. CC-401 treatment inhibited expression of both pro- and antiinflammatory molecules (interleukin-10 and heme oxygenase-1). In addition, IL-1 induced MMP-12 and IL-10 production by cultured macrophages was found to be JNK dependent. In conclusion, blockade of JNK signaling provides substantial protection against the progression of crescentic anti-GBM glomerulonephritis, which may be, in part, due to inhibition of the macrophage proinflammatory response.

CC-4047 promotes Th1 cell differentiation and reprograms polarized human Th2 cells by enhancing transcription factor T-bet.

The IMiDs immunomodulatory drugs are an expanding family of compounds under investigation in a broad range of diseases because they exhibit immunomodulatory and anti-tumorigenic properties. Although the molecular targets remain unidentified, the broad activity of select IMiDs immunomodulatory drugs on cell signaling pathways and transcription regulation has been partly described. One characteristic of these compounds is their ability to act as a co-stimulus of TCR ligation leading to increased IL-2, TNF-alpha and IFN-gamma expression indicative of a Th1 phenotype. Because clinical evidence for this response has been observed in thalidomide and lenalidomide treated patients, we investigated the effect of CC-4047 on T cell activation and differentiation at the molecular level. We used primary human CD4(+) T cells as a model and found that CC-4047 enhances the expression of transcription factor T-bet in both naive and pre-polarized Th2 cells. This modulation leads to upregulation of Th1 markers and cytokine production. By increasing the expression of T-bet, CC-4047 promotes the differentiation of naive T-cells to Th1 as well as effectively reverting Th2 cells into Th1-like effector cells in vitro. These findings elucidate a novel mechanism of action of CC-4047 on T cell differentiation, suggesting that certain IMiDs immunomodulatory drugs may have expanded clinical application in treating both allergic diseases and certain T cell lymphomas where a predominant Th2 phenotype is displayed.

Attenuation of ozone-induced airway inflammation and hyper-responsiveness by c-Jun NH2 terminal kinase inhibitor SP600125.

Ozone has potent oxidizing properties, and exposure to ozone causes airway hyper-responsiveness (AHR) and lung inflammation. We determined the importance of c-Jun NH(2) terminal kinase (JNK), a member of the mitogen-activated protein kinase pathway, in ozone-induced AHR and inflammation. SP600125 [anthra[1,9-cd] pyrazol-6 (2H)-one], a specific JNK inhibitor (30 mg/kg) or vehicle, was administered by intraperitoneal injection before and after ozone exposure (3 ppm for 3 h). SP600125 significantly reduced total cells, and neutrophils in bronchoalveolar fluid recovered at 20 to 24 h after exposure and inhibited ozone-induced AHR. Ozone exposure induced activation of JNK in the lung as measured by the expression of phosphorylated-c-Jun, an effect abolished by SP600125. Gene-microarray analysis revealed that ozone increased the expression of over 400 genes by more than 2-fold, including interleukin-6 (IL-6), CXCL1 (keratinocyte cytokine), and CCL2 (monocyte chemoattractant protein-1). SP600125 modulated the expression of a subset of 29 ozone-induced genes; IL-6 and CCL2 expression were further increased, whereas the expression of metallothionein 1, hemopexin, and mitogen-activated 3 kinase 6 was decreased in SP600125-treated ozone-exposed mice. Changes in mRNA for IL-6, CXCL1, and CCL2 were confirmed by real-time polymerase chain reaction. Ozone also decreased the expression of over 500 genes, with the most potent effect on angiopoietin-1. SP600125 modulated the expression of 15 of these genes, and in particular, SP600125 reversed ozone-induced decrease in expression of the redox-sensitive transcription factor, hypoxia-induced factor-1alpha. This study highlights an important role for JNK in response to oxidative stress through modulation of specific inflammatory and redox mediators. Inhibition of JNK with small molecule kinase inhibitors may be a means of reducing ozone-induced inflammation and AHR.

Effect of an inhibitor of Jun N-terminal protein kinase, SP600125, in single allergen challenge in sensitized rats.

Jun N-terminal kinase (JNK) has been implicated in the pathogenesis of inflammatory diseases including asthma. We examined the effect of SP600125 (anthra [1,9-cd] pyrazol-6 (2H)-one), a novel inhibitor of JNK in a model of asthma. Brown-Norway rats were sensitized to ovalbumin and treated with SP600125 intraperitoneally (90 mg/kg in total). SP600125 inhibited allergen-induced, increased activity of phosphorylated c-jun but not of phosphorylated-MAPKAPK2, indicative of activation of p38 MAPK, in the lung. SP600125 inhibited macrophage (P < 0.04), lymphocyte (P < 0.05), eosinophil (P < 0.04) and neutrophil (P < 0.005) numbers in bronchoalveolar lavage. Eosinophil and T-cell accumulation in the airways, mRNA expression for interleukin-1beta, tumour necrosis factor-beta, interleukin-3, interleukin-4 and interleukin-5, serum levels of allergen-specific immunoglobulin E and bronchial hyperresponsiveness were not affected by SP600125. Selective inhibition of JNK reduced inflammatory cell egress into the airway lumen after single allergen exposure. The role of JNK mitogen-activated protein kinase activation may be limited in the pathogenesis of bronchial hyperresponsiveness after single allergen exposure.

Differential requirement for c-Jun NH2-terminal kinase in TNFalpha- and Fas-mediated apoptosis in hepatocytes.

The c-Jun NH2-terminal kinase (JNK) is involved in the regulation of cell death, but its role in tumor necrosis factor (TNF)-alpha- and Fas-mediated apoptosis in primary cells is not well defined. In primary rat hepatocytes expressing an IkappaB superrepressor, the JNK inhibitor SP600125 strongly decreased TNF-alpha-induced cell death, caspase 3 activation, and DNA laddering. In contrast, SP600125 did not rescue mouse hepatocytes from Fas-induced apoptosis. Apoptosis in mouse hepatocytes, induced by human TNF-alpha, was blocked by SP600125, indicating that TNF-receptor (TNF-R) 1-mediated JNK activation is important for TNF-alpha-induced death. However, mouse TNF-alpha was more efficient than human TNF-alpha in activating JNK and killing mouse hepatocytes, suggesting that TNF-R1 and TNF-R2 cooperate in JNK activation and apoptosis. SP600125 rescued actinomycin D-pretreated hepatocytes and hepatocytes expressing a dominant negative c-Jun from TNF-alpha, indicating that JNK exerts its proapoptotic effect independently of transcription and c-Jun. SP600125 delayed the mitochondrial permeability transition, inhibited cytochrome c release and prevented bid degradation after TNF-alpha, suggesting that JNK-regulated proapoptotic factors act upstream of the mitochondria. Moreover, overexpression of JNK1 activated a mitochondrial death pathway in hepatocytes, albeit less efficiently than TNF-alpha. This study demonstrates that JNK augments TNF-alpha-induced apoptosis in hepatocytes through a signaling pathway that is distinct from the pathway by which it regulates proliferation.

Inhibition of cell proliferation and cell cycle progression by specific inhibition of basal JNK activity: evidence that mitotic Bcl-2 phosphorylation is JNK-independent.

The c-Jun NH(2)-terminal kinase (JNK) subgroup of mitogen-activated protein kinases has been implicated largely in stress responses, but an increasing body of evidence has suggested that JNK also plays a role in cell proliferation and survival. We examined the effect of JNK inhibition, using either SP600125 or specific antisense oligonucleotides, on cell proliferation and cell cycle progression. SP600125 was selective for JNK in vitro and in vivo versus other kinases tested including ERK, p38, cyclin-dependent protein kinase 1 (CDK1), and CDK2. SP600125 inhibited JNK activity and KB-3 cell proliferation with the same dose dependence, suggesting that inhibition of proliferation was a direct consequence of JNK inhibition. Inhibition of proliferation by SP600125 was associated with an increase in the G(2)-M and apoptotic fractions of cells but was not associated with p53 or p21 induction. Antisense oligonucleotides to JNK2 but not JNK1 caused highly significant inhibition of cell proliferation. Wild-type mouse fibroblasts responded similarly with proliferation inhibition and apoptosis induction, whereas c-jun(-/-) fibroblasts were refractory to the effects of SP600125, suggesting that JNK signaling to c-Jun is required for cell proliferation. Studies in synchronized KB-3 cells indicated that SP600125 delayed transit time through S and G(2)-M phases. Correspondingly, JNK activity increased in late S phase and peaked in late G(2) phase. During synchronous mitotic progression, cyclin B levels increased concomitant with phosphorylation of c-Jun, H1 histone, and Bcl-2. In the presence of SP600125, mitotic progression was prolonged, and c-Jun phosphorylation was inhibited, but neither H1 nor Bcl-2 phosphorylation was inhibited. However, the CDK inhibitor roscovitine inhibited mitotic Bcl-2 phosphorylation. These results indicate that JNK, and more specifically the JNK2 isoform, plays a key role in cell proliferation and cell cycle progression. In addition, conclusive evidence is presented that a kinase other than JNK, most likely CDK1 or a CDK1-regulated kinase, is responsible for mitotic Bcl-2 phosphorylation.

JNK: a new therapeutic target for diabetes.

Jun N-terminal kinase (JNK) regulates the transcription factor AP-1, which is implicated in the controlled expression of many genes involved in the immune response. For this reason, drug discovery efforts have focused on the development of JNK inhibitors for chronic inflammatory diseases. However, recent genetic evidence and emerging pharmacological data indicate that activated JNK could be critical in causing diabetes, insulin resistance and obesity. Indeed, if JNK is considered as a stress-activated protein kinase, there appear to be multiple mechanisms through which it might promote diabetes.

c-Jun-N-terminal kinase drives cyclin D1 expression and proliferation during liver regeneration.

The c-Jun-N-terminal kinase (JNK) pathway is strongly activated after partial hepatectomy (PH), but its role in hepatocyte proliferation is not known. In this study, JNK activity was blocked with the small molecule inhibitor JNK SP600125 in vivo and in vitro as shown by a reduction of c-Jun phosphorylation, AP-1 DNA binding activity, and c-jun messenger RNA (mRNA) expression. SP600125 inhibited proliferating cell nuclear antigen (PCNA) expression, cyclin D1 mRNA and protein expression and reduced mitotic figures after PH. Survival was reduced significantly 3 days after PH in SP600125-treated versus vehicle-treated rats (3 of 11 vs. 8 of 9, P <.01). In epidermal growth factor (EGF)-treated primary cultures of rat hepatocytes, SP600125 decreased (3)H-thymidine uptake, cyclin D1 mRNA and protein expression, and inhibited the EGF-induced transcription of a cyclin D1 promoter-driven reporter gene. The defective regeneration and the decreased survival in SP600125-treated rats did not result from a major increase in apoptosis as shown by normal levels of caspase 3 activity and only slight increases in apoptotic figures. In conclusion, our data show that JNK drives G0 to G1 transition in hepatocytes and that cyclin D1 is a downstream target of the JNK pathway during liver regeneration.

Potential new therapeutics for Waldenstrom's macroglobulinemia.

Thalidomide the first commercially available immune modulatory drug (IMiD), has activity in the treatment of Waldenstrom's macroglobulinemia (WM), as well as multiple myeloma, myelodysplastic syndrome, myelofibrosis with myeloid metaplasia, chronic lymphocytic leukemia (CLL), and B-cell lymphomas. Although its molecular mechanisms of action have not yet been elucidated, thalidomide and the IMiDs affect a variety of cytokines and inflammatory mediators including tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-1beta, interferon gamma (IFNgamma), IL-6, IL-10, IL-12, and COX-2 and angiogenesis factors such as vascular endothelial growth factor (VEGF) and its receptor. The IMiDs also affect adhesion molecules such as ICAM-1, ICAM-2, and L-CAM, in addition to preferentially stimulating CD8 cells and expanding natural killer (NK) cell populations. Since most IMiDs share these properties, it would be expected that the second-generation IMiDs (REVIMID, ACTIMID) would have activity similar to thalidomide in WM with an improved safety profile. TNFalpha and angiogenesis most likely play a role in promoting the growth and development of WM. The selective cytokine inhibitory drugs (SelCIDs) are potent phosphodiesterase 4 (PDE-4) inhibitors that inhibit TNFalpha production and are highly antiangiogenic. In addition, inhibition of PDE-4 induces apoptosis in human CLL lymphocytes. It is therefore expected that the SelCIDs might have activity in Waldenstrom's tumors. Jun N-terminal kinase (JNK) is a component of signaling cascades that modulate apoptosis, the induction of an inflammatory response via the AP-1 pathway, and modulation of cellular proliferation. In a variety of tumors, including multiple myeloma, JNK is induced as part of a protective mechanism. It is hypothesized that inhibition of JNK activity might allow other chemotherapeutic agents to be more effective in a similar manner to corticosteroids. Work is in progress to evaluate this. Inhibitors of the E3 subunit of ubiquitin ligase may also selectively modulate the expression of receptors, growth factors, and transcription factors essential to the growth, survival, and spread of tumors. We hypothesize that the IMiDs, SelCIDs, JNK inhibitors, and ligase inhibitors will be the basis for a new nonchemotherapeutic approach to the treatment of WM and other related diseases.

Differential requirement for NF-kappaB-inducing kinase in the induction of NF-kappaB by IL-1beta, TNF-alpha, and Fas.

In this study, we examined the role of the nuclear factor-kappaB (NF-kappaB)-inducing kinase (NIK) in distinct signaling pathways leading to NF-kappaB activation. We show that a dominant-negative form of NIK (dnNIK) delivered by adenoviral (Ad5dnNIK) vector inhibits Fas-induced IkappaBalpha phosphorylation and NF-kappaB-dependent gene expression in HT-29 and HeLa cells. Interleukin (IL)-1beta- and tumor necrosis factor-alpha (TNF-alpha)-induced NF-kappaB activation and kappaB-dependent gene expression are inhibited in HeLa cells but not in Ad5dnNIK-infected HT-29 cells. Moreover, Ad5dnNIK failed to sensitize HT-29 cells to TNF-alpha-induced apoptosis at an early time point. However, cytokine- and Fas-induced signals to NF-kappaB are finally integrated by the IkappaB kinase (IKK) complex, since IkappaBalpha phosphorylation, NF-kappaB DNA binding activity, and IL-8 gene expression were strongly inhibited in HT-29 and HeLa cells overexpressing dominant-negative IKKbeta (Ad5dnIKKbeta). Our findings support the concept that cytokine signaling to NF-kappaB is redundant at the level of NIK. In addition, this study demonstrates for the first time the critical role of NIK and IKKbeta in Fas-induced NF-kappaB signaling cascade.