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Background Janus kinase (JAK) inhibitors have shown encouraging results in the treatment of alopecia areata (AA), an autoimmune form of hair loss, in small, uncontrolled studies and case reports. Objective To conduct a biopsy sub-study during the randomized, double-blind, placebo-controlled first 24 weeks of a phase 2a clinical trial that evaluated the efficacy and safety of ritlecitinib, an inhibitor of JAK3 and the tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase family, and brepocitinib, an inhibitor of tyrosine kinase 2 (TYK2)/JAK1 in the treatment of AA. Methods Change in biomarkers in lesional scalp biopsies between baseline and weeks 12 and 24 was an exploratory endpoint, and 46 patients participated from the ritlecitinib (n = 18), brepocitinib (n = 16), and placebo (n = 12) groups. Correlations of biomarkers with hair regrowth, measured using the Severity of Alopecia Tool (SALT) score, were also evaluated. Results At week 24, both ritlecitinib and brepocitinib demonstrated improvement exceeding 100% in the lesional scalp transcriptome toward a nonlesional profile. At week 12, the improvements in scalp tissue were greater with brepocitinib than ritlecitinib; however, at week 24, the improvements were greater with ritlecitinib. Conclusions For both ritlecitinib and brepocitinib, improvement in the SALT scores was positively associated with expression of Th1 markers and negatively associated with expression of hair keratins. Larger, long-term clinical trials are warranted.

The Janus kinase-signal transducer and activator of transcription (JAK-STAT) intracellular signaling pathway is implicated in the pathogenesis of a number of inflammatory dermatoses. Clinical trials and other studies have demonstrated the efficacy of JAK inhibitors in the treatment of a variety of dermatologic conditions. Here we review JAK inhibitors currently under investigation for the treatment of alopecia areata, vitiligo, sarcoidosis, necrobiosis lipoidica, granuloma annulare, and systemic lupus erythematosus with a special emphasis on safety and the implications of JAK inhibitors during the novel coronavirus 2019 pandemic.

Activating TYK2-rearrangements have recently been identified and implicated in the leukemogenesis of high-risk acute lymphoblastic leukemia (HR-ALL) cases. Pre-clinical studies indicated the JAK/TYK2 inhibitor (JAKi), cerdulatinib, as a promising therapeutic against TYK2-rearranged ALL, attenuating the constitutive JAK/STAT signaling resulting from the TYK2 fusion protein. However, following a period of clinical efficacy, JAKi resistance often occurs resulting in relapse. In this study, we modeled potential mechanisms of JAKi resistance in TYK2-rearranged ALL cells in vitro in order to recapitulate possible clinical scenarios and provide a rationale for alternative therapies. Cerdulatinib resistant B-cells, driven by the MYB-TYK2 fusion oncogene, were generated by long-term exposure to the drug. Sustained treatment of MYB-TYK2-rearranged ALL cells with cerdulatinib led to enhanced and persistent JAK/STAT signaling, co-occurring with JAK1 overexpression. Hyperactivation of JAK/STAT signaling and JAK1 overexpression was reversible as cerdulatinib withdrawal resulted in re-sensitization to the drug. Importantly, histone deacetylase inhibitor (HDACi) therapies were efficacious against cerdulatinib-resistant cells demonstrating a potential alternative therapy for use in TYK2-rearranged B-ALL patients who have lost response to JAKi treatment regimens.

Pseudouridine is the most abundant yet unexplored RNA modification in glioblastoma. Cui and coworkers find that PUS7, a pseudouridine depositing enzyme, promotes tumor growth and can be targeted by small molecule inhibitors. Mechanistically, PUS7 modifies tRNAs, reduces TYK2 translation, and downregulates a proliferation-restricting interferon-STAT1 pathway in glioblastoma.

Lemur tyrosine kinase 2 (LMTK2) was recently identified as a novel cancer-related gene in several human cancers. However, little is known of its function in hepatocellular carcinoma (HCC). Here we aim to investigate the expression pattern, biological function, and regulatory mechanism of LMTK2 in HCC. We found that LMTK2 was highly expressed in HCC tissues, and patients with high expression of LMTK2 in tumor tissues had shorter survival times. LMTK2 expression was also elevated in HCC cell lines, and LMTK2 silencing markedly repressed the proliferation and invasion of HCC cells. By contrast, LMTK2 overexpression exerted promotion effects on HCC cell proliferation and invasion. Our results demonstrate that LMTK2 silencing decreases the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and the expression of an active β-catenin protein, leading to inhibition of Wnt/β-catenin signaling. Notably, GSK-3β inhibition significantly reversed the LMTK2 silencing-mediated antitumor effect on proliferation, invasion, and Wnt/β-catenin signaling in HCC cells. LMTK2 silencing retarded the tumor growth of HCC cells in an in vivo xenograft tumor model, associated with downregulation of Wnt/β-catenin signaling. In conclusion, our findings suggest that silencing of LMTK2 suppresses the proliferation and invasion of HCC cells through the inhibition of Wnt/β-catenin signaling, via GSK-3β, highlighting the importance of LMTK2/GSK-3β/Wnt/β-catenin signaling in HCC progression.

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations were important for the leukemogenesis of acute leukemia (AL). The JAK2 R867Q somatic mutation is detected in a subset of AL patients. However, roles of JAK2 R867Q mutation in the pathogenesis of AL remain unclear. In this study, homology modeling analysis showed that loss of interaction between R867 and Y613 disrupted the JAK2 JH1/JH2 domain's interactions was responsible for its activation. JAK2 R867Q and mutations (R867A and R867G) abolished this interaction caused JAK2 constitutive activation. While, mutations (R867K, Y613E, R867K/Y613E) repairing this interaction reduced JAK2 R867Q mutation's activity. Furthermore, our studies showed that abolished R867 and Y613 interaction disrupted JH1/JH2 domains' interactions and led to JAK2 constitutive activation. More importantly, mutations (R867Q, R867A and R867G) disrupted this interaction enhanced the activity of JAK2-STAT5 pathway and the proliferation of Ba/F3 and MV4-11 cells. Further study showed that JAK2 R867Q mutation promoted the expression of proliferation marker and inhibited the differentiation marker of Ba/F3 and MV4-11 cells. Thus our studies provide clues in understanding the pathogenesis of JAK2 R867Q mutation in AL.

Background Janus kinase (JAK) 2 plays pivotal roles in signaling by several cytokine receptors. The mutant JAK2 V617F is the most common molecular event associated with myeloproliferative neoplasms. Selective targeting of the mutant would be ideal for treating these pathologies by sparing essential JAK2 functions. Objective We characterize inhibitory strategies for JAK2 V617F and assess their effect on physiologic signaling by distinct cytokine receptors. Methods Through structure-guided mutagenesis, we assessed the role of key residues around F617 and used a combination of cellular and biochemical assays to measure the activity of JAKs in reconstituted cells. We also assessed the effect of several specific JAK2 V617F inhibitory mutations on receptor dimerization using the NanoBiT protein complementation approach. Results We identified a novel Janus kinase homology 2 (JH2) αC mutation, A598F, which is suggested to inhibit the aromatic stacking between F617 with F594 and F595. Like other JAK2 V617F inhibitory mutations, A598F decreased oncogenic activation and spared cytokine activation while preventing JAK2 V617F–promoted erythropoietin receptor dimerization. Surprisingly, A598F and other V617F-inhibiting mutations (F595A, E596R, and F537A) significantly impaired IFN-γ signaling. This was specific for IFN-γ because the inhibitory mutations preserved responses to ligands of a series of receptor complexes. Similarly, homologous mutations in JAK1 prevented signaling by IFN-γ. Conclusions The JH2 αC region, which is required for JAK2 V617F hyperactivation, is crucial for relaying cytokine-induced signaling of the IFN-γ receptor. We discuss how strategies aiming to inhibit JAK2 V617F could be used for identifying inhibitors of IFN-γ signaling.

Disruption of interleukin-13 (IL-13) signaling with large molecule antibody therapies has shown promise in diseases of allergic inflammation. Given that IL-13 recruits several members of the Janus Kinase family (JAK1, JAK2, and TYK2) to its receptor complex, JAK inhibition may offer an alternate small molecule approach to disrupting IL-13 signaling. Herein we demonstrate that JAK1 is likely the isoform most important to IL-13 signaling. Structure-based design was then used to improve the JAK1 potency of a series of previously reported JAK2 inhibitors. The ability to impede IL-13 signaling was thereby significantly improved, with the best compounds exhibiting single digit nM IC50's in cell-based assays dependent upon IL-13 signaling. Appropriate substitution was further found to influence inhibition of a key off-target, LRRK2. Finally, the most potent compounds were found to be metabolically labile, which makes them ideal scaffolds for further development as topical agents for IL-13 mediated diseases of the lungs and skin (for example asthma and atopic dermatitis, respectively).

BACKGROUND: The incidence of eosinophilic esophagitis (EoE) is greater in male than female subjects, and the underlying molecular basis for this sex bias remains unclear. OBJECTIVE: We sought to delineate the contribution of the sex hormone estrogen to the EoE phenotype and esophageal epithelial barrier function and remodeling. METHODS: We performed demographic and incidence analyses of EoE in male and female subjects from a single-center pediatric cohort. Estrogen-responsive gene expression analyses and estrogen receptor (ESR) immunofluorescence staining of esophageal biopsy specimens from patients with EoE and control subjects were performed. The effect of 17β-estradiol (E2) on IL-13–induced signaling pathways, gene expression, and esophageal epithelial architecture and barrier function in a primary human esophageal keratinocyte cell (EPC2) culture system (EPC2–air-liquid interface) was examined. RESULTS: We observed a male predominance in patients with EoE. Analyses of RNA sequencing data sets revealed a significant dysregulation of the estrogen-responsive gene network and expression of ESR1 and ESR2 in esophageal biopsy specimens from patients with EoE compared with control subjects. IL-13 stimulation of EPC2–air-liquid interface cells led to altered cellular architecture with induced dilation of intercellular spaces and barrier dysfunction. Pretreatment of EPC2s with E2 prior to IL-13 exposure abrogated IL-13– induced architectural changes and esophageal barrier dysfunction. Mechanistically, E2-protective effects were dependent on ESR2 and associated with diminishing of IL-13– induced tyrosine kinase 2 and signal transducer and activator of transcription 6 phosphorylation and EoE-dysregulated gene expression. CONCLUSIONS: Estrogen-responsive genes are modified in patients with EoE compared with control subjects. E2 attenuated IL-13– induced architectural changes and esophageal epithelial barrier dysfunction through inhibition of the IL-13/tyrosine kinase 2/ signal transducer and activator of transcription 6 pathway via ESR2-dependent process. Estrogen hormone signaling may protect against development of EoE in female subjects.

Emerging evidence indicates that cancer stem cells (CSCs) are involved in tumorigenesis, tumor recurrence, and therapeutic resistance in hepatocellular carcinoma (HCC). However, the mechanisms underlying HCC CSC regulation remain largely unknown. Here we report our analysis of 97 paraffin-embedded HCC tumor specimens. We found that protein tyrosine kinase 2 (PTK2) expression correlated with liver CSC marker expression, overall survival, and recurrence-free survival in HCC patients. Our results further showed that PTK2 activated Wnt/β-catenin signaling by promoting nuclear accumulation of β-catenin in HCC cells. In this manner, PTK2 activates CSC traits and drives tumorigenicity in HCC cells, leading to HCC recurrence and sorafenib resistance. Moreover, PTK2 expression was negatively correlated with its level of promoter methylation. PTK2 apparently acts as an oncogene by increasing CSC traits and tumorigenicity in HCC. The present data suggest that PTK2 may be a novel prognostic biomarker for HCC recurrence, and a therapeutic target for HCC treatment.

Recently, two studies demonstrated that a relevant percentage of primary antiphospholipid syndrome (PAPS) patients had an upregulation of interferon (IFN) genes. However, 20%-28% of these patients had anti-dsDNA, a highly specific systemic lupus erythematosus (SLE) autoantibody. This study aimed to determine the prevalence of the type I IFN signature in the peripheral blood mononuclear cells of PAPS patients without specific SLE autoantibodies and search for its clinical associations.Fifty-three PAPS patients (Sydney's criteria) were consecutively selected and age-matched with 50 healthy controls. A third group of nonimmune-mediated thrombophilia patients was also included. The expression of 41 IFN-induced genes was analyzed using real time quantitative PCR. A principal component analysis determined which genes composed the IFN signature, and the z-score was calculated. An ROC curve defined the signature cut-off.Six genes remained in the IFN signature DNAJA1, IFIT5, IFI27, MX1, IFI6, and TYK2. The ROC cutoff was 3.9-fold (AUC = 0.706, S = 0.49, E = 0.86, PPV = 0.79, NPV = 0.61). The type I IFN signature was present in 49% of the patients with PAPS compared with 14.0% of the healthy controls and 17% of the nonimmune-mediated thrombophilia patients (p .0001). The IFN signature was associated with a younger age at the first antiphospholipid syndrome event (p = .023) and with preeclampsia (p = .032).Our results indicate that PAPS patients without lupus-specific antibodies have an enhanced type I IFN gene signature that is not observed in nonimmune-mediated thrombophilia. Also, this overexpression of type I IFN-regulated genes associated with an earlier onset of antiphospholipid syndrome event and preeclampsia.

The structure and function of spines and excitatory synapses are under the dynamic control of multiple signalling networks. Although tyrosine phosphorylation is involved, its regulation and importance are not well understood. Here we study the role of Pyk2, a non-receptor calcium-dependent protein-tyrosine kinase highly expressed in the hippocampus. Hippocampal-related learning and CA1 long-term potentiation are severely impaired in Pyk2-deficient mice and are associated with alterations in NMDA receptors, PSD-95 and dendritic spines. In cultured hippocampal neurons, Pyk2 has autophosphorylation-dependent and -independent roles in determining PSD-95 enrichment and spines density. Pyk2 levels are decreased in the hippocampus of individuals with Huntington and in the R6/1 mouse model of the disease. Normalizing Pyk2 levels in the hippocampus of R6/1 mice rescues memory deficits, spines pathology and PSD-95 localization. Our results reveal a role for Pyk2 in spine structure and synaptic function, and suggest that its deficit contributes to Huntington's disease cognitive impairments., Several kinases regulate spine morphology and plasticity. Here, the authors show that the tyrosine kinase Pyk2 regulates hippocampal plasticity and contributes to cognitive and hippocampal plasticity deficits in a mouse model of Huntington's disease.

Background & Aims: Identification and validation of new functionally relevant and pharmacologically actionable targets for pancreatic ductal adenocarcinoma (PDAC) remains a great challenge. Premalignant acinar cell reprogramming (acinar-to-ductal metaplasia [ADM]) is a precursor of pancreatic intraepithelial neoplasia (PanIN) lesions that can progress to PDAC. This study investigated the role of proline-rich tyrosine kinase 2 (PYK2) in mutant Kras-induced and pancreatitis-associated ADM and PanIN formation, as well as in PDAC maintenance. Methods: Genetically engineered mouse models of mutant Kras (glycine 12 to aspartic acid) and Pyk2 deletion were used for investigating the role of PYK2 in PDAC genesis in mice. In vitro ADM assays were conducted using primary pancreatic acinar cells isolated from mice. Immunohistochemistry, immunofluorescence, and a series of biochemical experiments were used to investigate upstream regulators/downstream targets of PYK2 in pancreatic carcinogenesis. PDAC cell line xenograft experiments were performed to study the role of PYK2 and its downstream target in PDAC maintenance. Results: PYK2 was increased substantially in ADM lesions induced by mutant Kras or inflammatory injury. Pyk2 deletion remarkably suppressed ADM and PanIN formation in a mutant Kras-driven and pancreatitis-associated PDAC model, whereas PYK2 knockdown substantially inhibited PDAC cell growth in vitro and in nude mice. This study uncovered a novel yes-associated protein 1/transcriptional co-activator with PDZ binding motif/signal transducer and activator of transcription 3/PYK2/β-catenin regulation axis in PDAC. Our results suggest that PYK2 contributes to PDAC genesis and maintenance by activating the Wnt/β-catenin pathway through directly phosphorylating β-cateninY654. Conclusions: The current study uncovers PYK2 as a novel downstream effector of mutant KRAS signaling, a previously unrecognized mediator of pancreatitis-induced ADM and a novel intervention target for PDAC. Keywords: Acinar-to-Ductal Metaplasia, Inflammation, KRAS, Wnt/β-Catenin Signaling

There is increasing evidence of a significant correlation between prolonged drug-target residence time and increased drug efficacy. Here, we report a structural rational for kinetic selectivity between two closely related kinases: Focal Adhesion Kinase (FAK) and Proline-rich Tyrosine Kinase 2 (PYK2). We found that slow off-rate FAK inhibitors induce helical structure at the DFG motif of FAK but not PYK2. Binding kinetic data, high resolution structures and mutagenesis data support the role of hydrophobic interactions of inhibitors with the DFG helical region providing a structural rational for slow dissociation rates from FAK and kinetic selectivity over PYK2. Our experimental data correlated well with computed relative residence times from molecular simulations providing a feasible strategy for rationally optimizing ligand residence times. We suggest that the interplay between the protein structural mobility and ligand-induced effects is a key regulator of the kinetic selectivity of inhibitors of FAK versus PYK2.

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations play key roles in the pathology of myeloproliferative neoplasms (MPNs). Recently, germline JAK2 mutations are also associated with triple-negative MPNs. A novel germline mutation JAK2 V625F is reported to be involved in a subset of MPNs patients. However, the pathogenesis of this mutation caused MPN is still unclear. In this study, the homology models of JAK2 V625F showed that the newly formed interaction between F625 and Y613 disrupted the JAK2 JH1-JH2 domain interactions was responsible for its activation, when F625 and Y613 interaction was disrupted, its activity significantly decreased. While, when this interaction was repaired whether by forming hydrogen bond or salt bond, it would cause JAK2 activation. Biochemical studies also demonstrated that JAK2 V625F mutation led to JAK2-STAT5 pathway activation and promoted the proliferation of BaF3 cells. Thus, our results herein provide clues to understand the mechanism JAK2 V625F mutation caused MPNs and give information for the development of JAK2 mutation specific inhibitors.

Mitochondrial dysfunction caused by Ca2+ overload plays an important role in ischemia-induced brain damage. Mitochondrial calcium uniporter (MCU), located on the mitochondrial inner membrane, is the major channel responsible for mitochondrial Ca2+ uptake. Activated proline-rich tyrosine kinase 2 (Pyk2) can directly phosphorylate MCU, which enhances mitochondrial Ca2+ uptake in cardiomyocytes. It has been suggested that the Pyk2/MCU pathway may be a novel therapeutic target in stress-induced cellular apoptosis. In this study, we explored the role of the Pyk2/MCU pathway in the ischemic brain following a stroke injury. We found that the Pyk2/MCU pathway is activated in a rat cerebral ischemia model, and is responsible for mitochondrial dysfunction and neuronal apoptosis. Inhibiting the Pyk2/MCU pathway with a Pyk2 inhibitor (PF-431396) prevented mitochondrial Ca2+ overload, mitochondrial injury, proapoptotic protein release, and cell death. Interestingly, human urinary kallidinogenase (HUK) alleviated neuronal ischemic injury by inhibiting the Pyk2/MCU pathway, suggesting that the Pyk2/MCU pathway may be a protective target for ischemic stroke treatment.

Background/aim Increasing evidence show microRNAs (miRNAs) are associated with hepatopulmonary syndrome (HPS). The aim of this study was to investigate the role of miR-144 in the angiogenesis of HPS, as well as to identify its underlying mechanism. Methods The expression levels of miR-144-3p were assessed in pulmonary micro-vascular endothelial cells (PMVECs), as well as in lung tissues from rats with HPS. We predicted the potential target of miR-144-3p. Tyrosine kinase 2(Tie2) was identified as a target gene of miR144-3p, which has an essential role in the angiogenesis of lung vessel. In addition, the effects of miR-144-3p regulated on Tie2 was examined. The upregulation and down-regulation of miR-144-3p can affect the proliferation of PMVECs. Results We found that the levels of miR-144-3p were frequently downregulated in HPS tissues and cell lines, and overexpression of miR-144-3p dramatically inhibited PMVECs proliferation and cell cycle. We further verified the Tie2 as a novel and direct target of miR-144-3p in HPS. Conclusion miR-144-3p can negatively regulate PMVECs proliferation by Tie2 expression. In addition, overexpression of miR-144-3p may prove beneficial as a therapeutic strategy for HPS treatment.

Tofacitinib, an oral Janus kinase (JAK) inhibitor for treatment of rheumatoid arthritis, targets JAK1, JAK3, and to a lesser extent JAK2 and TYK2. JAK1/3 inhibition impairs gamma common chain cytokine receptor signaling, important in lymphocyte development, homeostasis and function. Adult and juvenile cynomolgus monkey and rat studies were conducted and the impact of tofacitinib on immune parameters (lymphoid tissues and lymphocyte subsets) and function (T-dependent antibody response (TDAR), mitogen-induced T cell proliferation) assessed. Tofacitinib administration decreased circulating T cells and NK cells in juvenile and adult animals of both species. B cell decreases were observed only in rats. These changes and decreased lymphoid tissue cellularity are consistent with the expected pharmacology of tofacitinib. No differences were observed between juvenile and adult animals, either in terms of doses at which effects were observed or differential effects on immune endpoints. Lymphomas were observed in three adult monkeys. Tofacitinib impaired the primary TDAR in juvenile monkeys, although a recall response was generated. Complete or partial reversal of the effects on the immune system was observed.

Background Janus kinase (JAK) inhibitors are thought to be promising candidates to aid renal transplantation. However, the effectiveness of JAK inhibitors against features of chronic rejection, including interstitial fibrosis/tubular atrophy (IF/TA) and glomerulosclerosis, has not been elucidated. Here, we investigated the effect of AS2553627, a novel JAK inhibitor, on the development of chronic rejection in rat renal transplantation. Methods Lewis (LEW) to Brown Norway (BN) rat renal transplantation was performed. Tacrolimus (TAC) at 0.1 mg/kg was administered intramuscularly once a day for 10 consecutive days starting on the day of transplantation (days 0 to 9) to prevent initial acute rejection. After discontinuation of TAC treatment from days 10 to 28, AS2553627 (1 and 10 mg/kg) was orally administered with TAC. At 13 weeks after renal transplantation, grafts were harvested for histopathological and mRNA analysis. Creatinine and donor-specific antibodies were measured from plasma samples. Urinary protein and kidney injury markers were also evaluated. Results AS2553627 in combination with TAC exhibited low plasma creatinine and a marked decrease in urinary protein and kidney injury markers, such as tissue inhibitor of metalloproteinase-1 and kidney injury molecule-1. At 13 weeks, histopathological analysis revealed that AS2553627 treatment inhibited glomerulosclerosis and IF/TA. In addition, upregulation of cell surface markers, fibrosis/epithelial–mesenchymal transition and inflammation-related genes were reduced by the combination of AS2553672 and TAC, particularly CD8 and IL-6 mRNAs, indicating that AS2553627 prevented cell infiltration and inflammation in renal allografts. Conclusions These results indicate the therapeutic potential of JAK inhibitors in chronic rejection progression, and suggest that AS2553627 is a promising agent to improve long-term graft survival after renal transplantation.

This study aimed to examine the expression of TYK2, CBLB and LMP7 genes at both mRNA and protein levels in relapsing-remitting MS (RRMS) patients in compare with healthy controls. Seventy-eight RRMS patients treated with IFNβ-1a and 79 age- and ethnic-matched healthy subjects were studied. The mRNA expression levels of TYK2, CBLB and LMP7 in PBMCs were quantified by real-time PCR and plasma concentrations of three molecules were measured by ELISA. Results were compared between patients and controls, IFNβ-responders and non-responders. Forty-nine of 78 patients were classified as IFNβ-responders and 29 cases were non-responders. Significantly down-regulated expression of TYK2, CBLB and LMP7 genes was found in the patients group versus controls (P0.001). Decreased plasma levels of three molecules were observed in patients compared to controls (P0.001). IFNβ-responders had significantly higher expressions for CBLB (P=0.001) and LMP7 (P=0.02) than non-responders. Also, we observed increased expressions of LMP7 (P=0.39) and CBLB (P=0.02) genes in patients under 30y and increased expression of TYK2 in patients40years (P=0.002). Our results suggest that expression analysis of TYK2, CBLB and LMP7 genes could be useful for evaluation of T cells immunity and clinical response to IFNβ-therapy in RRMS patients.

Significant advances in the understanding of the molecular basis of innate immunity have led to the identification of interferons (IFNs), particularly IFN-α, as central mediators in the pathogenesis of Systemic Lupus Erythematosus. Therefore, targeting of IFNs and of their downstream pathways has emerged as important developments for novel drug research in SLE. Based on this, several specific interferon blocking strategies using anti-IFN-α antibodies, anti-type I interferon receptor antibodies, Interferon-α-kinoid, or anti-IFN-γ antibodies have all been assessed in recent clinical trials. Alternative strategies targeting the plasmacytoid dendritic cells (pDCs), Toll-Like Receptors (TLRs)-7/9 or their downstream pathways such as the myeloid differentiation primary-response protein 88 (MYD88), spleen tyrosine kinase (Syk), Janus-kinases (JAKs), interleukin-1 receptor-associated kinase 4 (IRAK4), or the Tyrosine Kinase 2 (TYK2) are also investigated actively in SLE, at more preliminary clinical development stages, except for JAK inhibitors which have reached phase 2 studies. In a near future, in-depth and personalized functional characterization of IFN pathways may provide further guidance for the selection of the most relevant therapeutic strategy in SLE, tailored at the patient-level.

Regulatory elements present in the promoter of a gene drive the expression of the gene in response to various stimuli. Lemur Tyrosine Kinase 2 (LMTK2) is a membrane-anchored Serine/Threonine kinase involved in endosomal protein trafficking and androgen signaling amongst other processes. Previous studies have shown this protein to be of therapeutic importance in cystic fibrosis and prostate cancer. However, nothing is known about the endogenous expression of this protein and its regulation. In this study, we analyzed the gene encoding human LMTK2, to look for possible regulatory elements that could affect its expression. Interestingly, the human lmtk2 gene contains a consensus TPA (12- O-Tetradecanoylphorbol-13-acetate)-responsive element (TRE) in the region preceding its start codon. The element with the sequence TGAGTCA modulates LMTK2 expression in response to treatment with TPA, a synthetic Protein Kinase C (PKC) activator. It serves as the binding site for c-Fos, a member of the Activator Protein −1 (AP-1) transcription factor complex, which is transactivated by PKC. We observed that TPA, at low concentrations, increases the promoter activity of LMTK2, which leads to a subsequent increase in the mRNA transcript and protein levels. This modulation occurs through binding of the AP-1 transcription factor complex to the lmtk2 promoter. Thus, our current study has established LMTK2 as a TPA-responsive element-containing gene, which is upregulated downstream of PKC activation. Considering the involvement of LMTK2 in intracellular processes as well as pathological conditions, our findings demonstrate a way to modulate intracellular LMTK2 levels pharmacologically for potentially therapeutic purposes.

The JAK signal transducer and STAT signaling pathway is an important regulator of cell proliferation, differentiation, survival, motility, apoptosis, immune response, and development. In this study, we used RNA-Sequencing, qRT-PCR, and bioinformatics tools to investigate the differential expression of JAK-STAT pathway genes, their interactions, and regulators in the spleen of two genetically disparate chicken lines (Marek's disease-resistant line 6.3 and MD-susceptible line 7.2) induced necrotic enteritis (NE) disease by co-infection with Eimeria maxima and Clostridium perfringens. Using RNA-Seq analysis, we identified a total of 116 JAK-STAT pathway genes that were differentially expressed in the spleen of these chickens. All of the identified genes were analyzed through clustering, mapping to the KEGG chicken JAK-STAT pathway, and the Pathway Studio program. Of the 116 JAK-STAT pathway genes, 20 were further verified by qRT-PCR. According to the RNA-Seq results, several key genes, including STAT1-6, JAK1-3, TYK2, AKT1, AKT3, SOCS1-5, PIAS1, PIAS2, PIAS4, SHP1, SHP2, and PIK3, showed marked differential expression in the two lines, relative to their respective controls. Moreover, the RNA-Seq results of many key genes were highly correlated with the qRT-PCR results. Finally, we predicted 63 mature miRNAs that variably target JAK-STAT pathway genes and are differentially expressed in the spleen of chickens of both lines. To the best of our knowledge, this study is the first to analyze most of the genes, interactions, and regulators of the JAK-STAT pathway in the innate immune response to NE disease in chickens.

Herein, we describe the design, synthesis, and structure−activity relationships of a series of imidazopyrrolopyridines derivatives that selectively inhibit Janus kinase 2 (JAK2). These screening cascades revealed that 6k was a preferred compound, with IC50 values of 10 nM for JAK2. Moreover, 6k was a selective JAK2 inhibitor with 19-fold, >30-fold and >30-fold selectivity over JAK1, JAK3 and TYK2 respectively. In cytokine-stimulated cell-based assays, 6k exhibited a higher JAK2 selectivity over JAK1 isoforms. Indeed, at a dose of 20 mg/kg compound 6k, pSTAT3 and pSTAT5 expression was reduced to levels comparable to those of control animals untreated with GM-CSF. Additionally, 6k showed a relatively good bioavailability (F = 38%), a suitable half-life time (T1/2 = 1.9 h), a satisfactory metabolic stability, suggesting that 6k might be a promising inhibitor of JAK2 for further development research for the treatment of MPNs.

Kawasaki disease (KD) is a paediatric vasculitis whose pathogenesis remains unclear. Based on experimental studies using a mouse model for KD, we report here that proline-rich protein tyrosine kinase 2 (Pyk2) plays a critical role in the onset of KD-like murine vasculitis. The mouse model for KD was prepared by administrating a Candida albicans water-soluble fraction (CAWS). Unlike CAWS-treated WT mice, CAWS-treated Pyk2-Knockout (Pyk2-KO) mice did not develop apparent vasculitis. A sustained increase in MIG/CXCL9 and IP-10/CXCL10, both of which have potent angiostatic activity, was observed in CAWS-treated Pyk2-KO mice. CAWS-induced activation of STAT3, which negatively regulates the expression of these chemokines, was also attenuated in macrophages derived from Pyk2-KO mice. The present study suggests that defects in Pyk2 suppress KD-like experimental vasculitis, presumably through CXCL9- and CXCL10-dependent interference with neo-angiogenesis. Since Pyk2-KO mice show no life-threatening phenotype, Pyk2 may be a promising therapeutic molecular target for KD.

The mammalian Janus Kinases (JAK1, JAK2, JAK3 and TYK2) are intracellular, non-receptor tyrosine kinases whose activities have been associated in the literature and the clinic with a variety of hyperproliferative diseases and immunological disorders. At the onset of the program, it was hypothesized that a JAK1 selective compound over JAK2 could lead to an improved therapeutic index relative to marketed non-selective JAK inhibitors by avoiding the clinical AEs, such as anemia, presumably associated with JAK2 inhibition. During the course of the JAK1 program, a number of diverse chemical scaffolds were identified from both uHTS campaigns and de novo scaffold design. As part of this effort, a (benz)imidazole scaffold evolved via a scaffold-hopping exercise from a mature chemical series. Concurrent crystallography-driven exploration of the ribose pocket and the solvent front led to analogs with optimized kinome and JAK1 selectivities over the JAK2 isoform by targeting several residues unique to JAK1, such as Arg-879 and Glu-966.

Tyrosine kinase 2 (TYK2), Janus kinase 1 (JAK1), and signal transducer and activator of transcription 3 (STAT3) all play critical roles in innate and acquired immunity. In this study, we investigated the association between the TYK2, JAK1, and STAT3 genes and genetic susceptibility to nonspecific digestive disorder (NSDD) in rabbits. Five coding single-nucleotide polymorphisms (cSNPs) in TYK2 were detected in New Zealand White (NZW) rabbits with direct sequencing, and two of them, c.1477 (C>T) and c.2013 (C>T), from two different linkage disequilibrium blocks, were further genotyped. A case–control association analysis revealed that allele C (c.1477) in TYK2 increases the risk of NSDD (odds ratio [OR]: 1.21; 95% confidence interval [CI] 1.036–2.145; P=0.021), whereas allele C (c.2013) plays a potentially protective role against NSDD (OR: 0.41; 95% CI 0.2996–0.5705; P

The IL-23/T helper type 17 cell axis is a target for psoriasis. The TYK2/Janus kinase 1 inhibitor PF-06700841 will directly suppress TYK2-dependent IL-12 and IL-23 signaling and Janus kinase 1–dependent signaling in cells expressing these signaling molecules, including T cells and keratinocytes. This clinical study sought to define the inflammatory gene and cellular pathways through which PF-06700841 improves the clinical manifestations of psoriasis. Patients (n = 30) with moderate-to-severe psoriasis were randomized to once-daily 30 mg (n = 14) or 100 mg (n = 7) PF-06700841 or placebo (n = 9) for 28 days. Biopsies were taken from nonlesional and lesional skin at baseline and weeks 2 and 4. Changes in the psoriasis transcriptome and genes induced by IL-17 in keratinocytes were evaluated with microarray profiling and reverse transcriptase–PCR. Reductions in IL-17A, IL-17F, and IL-12B mRNA were observed as early as 2 weeks and approximately 70% normalization of lesional gene expression after 4 weeks. Immunohistochemistry showed significant decreases in markers of keratinocyte activation, epidermal thickness, KRT16 and Ki-67 expression, and immune cell infiltrates CD3+/CD8+ (T cells) and CD11c (dendritic cells) after 2 weeks of treatment, corresponding with improvement in histologic score. PF-06700841 improves clinical symptoms of chronic plaque psoriasis by inhibition of proinflammatory cytokines that require TYK2 and Janus kinase 1 for signal transduction.

Janus kinases (JAKs) including JAK1, JAK2, JAK3, and TYK2 are members of a family of intracellular nonreceptor tyrosine kinases, which have been demonstrated to be critical in the cell signaling pathway and involved in inflammatory diseases and cancer. V617F mutation in JAK2 has been implicated in polycythaemia vera (PV), essential thrombocythaemia (ET) and myelofibrosis (MF). Here, we described the design, synthesis, and biological evaluation of a series of 2-aminopyridine derivatives. The results of enzymatic activity assays supported compound 16m-(R) as a potential and selective JAK2 inhibitor, which exhibited high inhibitory activity with an IC50 of 3 nM against JAK2, and 85- and 76-fold selectivity over JAK1 and JAK3, respectively. Structure-activity relationships (SAR) and mechanistic analysis demonstrated that 16m-(R) might be a promising selective JAK2 inhibitor for further study.

PROTACs have recently emerged as a novel paradigm in drug discovery. They can hijack existing biological machinery to selectively degrade proteins of interest, in a catalytic fashion. Here we describe the design, optimisation and biological activity of a set of novel PROTACs targeting the Janus kinase family (JAK1, JAK2, JAK3 and TYK2) of proximal membrane-bound proteins. The JAK family proteins display membrane localisation by virtue of their association with cytoplasmic tails of cytokine receptors, and there are no reports of a successful PROTAC strategy being deployed against this class of proteins. JAK PROTACs from two distinct JAK chemotypes were designed, optimising the physicochemical properties for each template to enhance cell permeation. These PROTACs are capable of inducing JAK1 and JAK2 degradation, demonstrating an extension of the PROTAC methodology to an unprecedented class of protein targets. A number of known ligase binders were explored, and it was found that PROTACs bearing an inhibitor of apoptosis protein (IAP) ligand induced significantly more JAK degradation over Von Hippel–Lindau (VHL) and Cereblon (CRBN) PROTACs. In addition, the mechanism of action of the JAK PROTACs was elucidated, and it was confirmed that JAK degradation was both IAP- and proteasome-dependent.

Emerging evidence has demonstrated that cancer stem cells (CSCs) are involved in tumorigenesis, tumor recurrence and therapy resistance in hepatocellular carcinoma (HCC). However, the mechanisms underlying the regulation of CSCs in HCC remain largely unknown. Here, we report that protein tyrosine kinase 2 (PTK2) expression correlates with liver CSC marker expression, overall survival and recurrence-free survival in HCC patients. PTK2 has an activating effect on Wnt/β-catenin signaling caused by PTK2 promotion of nuclear accumulation of β-catenin protein in HCC cells. PTK2 activates CSC traits and tumorigenicity of HCC cells, leading to HCC recurrence and sorafenib resistance. Moreover, PTK2 expression negatively correlates with the methylation level of its promoter. PTK2 acts as an oncogene by increasing CSC traits and tumorigenicity in HCC. Our findings suggest PTK2 as a novel prognostic biomarker for HCC recurrence and a therapeutic target for HCC treatment. Funding Statement: This work was supported by the National Natural Science Foundation of China (81702936, 81472589, 81672602, 81402345), the Natural Science Foundation of Beijing (7172199) and a General Financial Grant from the Postdoctoral Science Foundation of China (2017M613389) and Logistics Scientific Research project (BWS16J010). Declaration of Interests: The authors declare no competing financial interests. Ethics Approval Statement: The study was conducted with informed consent of the patients and with the approval of the Institutional Review Committees of the Chinese PLA General Hospital.

Background Recently, natural mutation of Tyrosine kinase 2 (Tyk2) gene has been shown to determine susceptibility to murine virus-induced diabetes. In addition, a previous human genome-wide study suggested the type 1 diabetes (T1D) susceptibility region to be 19p13, where the human TYK2 gene is located (19p13.2). Methods Polymorphisms of TYK2 gene at the promoter region and exons were studied among 331 healthy controls, and 302 patients with T1D and 314 with type 2 diabetes (T2D) in the Japanese. Findings A TYK2 promoter haplotype with multiple genetic polymorphisms, which are in complete linkage disequilibrium, named TYK2 promoter variant, presenting decreased promoter activity, is associated with an increased risk of not only T1D (odds ratio (OR), 2.4; 95% confidence interval (CI), 1.2 to 4.6; P = 0.01), but also T2D (OR, 2.1; 95% CI, 1.1 to 4.1; P = 0.03). The risk is high in patients with T1D associated with flu-like syndrome at diabetes onset and also those without anti-glutamic acid decarboxylase autoantibody. Interpretation The TYK2 promoter variant is associated with an overall risk for diabetes, serving a good candidate as a virus-induced diabetes susceptibility gene in humans. Funding Ministry of Education, Culture, Sports, Science and Technology and of Health, Labor and Welfare of Japan., Highlights • TYK2 promoter variant with multiple polymorphisms was identified in the Japanese. • TYK2 promoter variant is associated with an increased risk of both type 1 and type 2 diabetes. • TYK2 promoter variant serves a good candidate as a virus-induced diabetes susceptibility gene in humans.

Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified that bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor but not Tyk2-independent signaling and transcriptional cellular assays, including stimulation through the receptors for IL-2 (JAK1- and JAK3-dependent) and thrombopoietin (JAK2-dependent), demonstrating the high functional selectivity of this approach. A crystal structure of the pseudokinase domain liganded with a representative example showed the compound bound to a site analogous to the ATP-binding site in catalytic kinases with features consistent with high ligand selectivity. The results support a model where the pseudokinase domain regulates activation of the catalytic domain by forming receptor-regulated inhibitory interactions. Tyk2 pseudokinase stabilizers, therefore, represent a novel approach to the design of potent and selective agents for the treatment of autoimmunity.

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.

A new class of Janus kinase (JAK) inhibitors was discovered using a rationally designed pyrrolo[1,2-b]pyridazine-3-carboxamide scaffold. Preliminary studies identified (R)-(2,2-dimethylcyclopentyl)amine as a preferred C4 substituent on the pyrrolopyridazine core (3b). Incorporation of amino group to 3-position of the cyclopentane ring resulted in a series of JAK3 inhibitors (4g-4j) that potently inhibited IFNγ production in an IL2-induced whole blood assay and displayed high functional selectivity for JAK3-JAK1 pathway relative to JAK2. Further modifications led to the discovery of an orally bioavailable (2-fluoro-2-methylcyclopentyl)amino analogue 5g which is a nanomolar inhibitor of both JAK3 and TYK2, functionally selective for the JAK3-JAK1 pathway versus JAK2, and active in a human whole blood assay.

EGFR signaling pathway and microRNAs (miRNAs) are two important factors for development and treatment in non-small cell lung cancer (NSCLC). Microarray analysis enables the genome-wide expression profiling. However, the information from microarray data may not be fully deciphered through the existing approaches. In this study we present an mRNA:miRNA stepwise regression model supported by miRNA target prediction databases. This model is applied to explore the roles of miRNAs in the EGFR signaling pathway. The results show that miR-145 is positively associated with epidermal growth factor (EGF) in the pre-surgery NSCLC group and miR-199a-5p is positively associated with EGF in the post-surgery NSCLC group. Surprisingly, miR-495 is positively associated with protein tyrosine kinase 2 (PTK2) in both groups. The coefficient of determination (R(2)) and leave-one-out cross-validation (LOOCV) demonstrate good performance of our regression model, indicating that it can identify the miRNA roles as oncomirs and tumor suppressor mirs in NSCLC.

IL-6 a multi-functional cytokine with important effects in both inflammation and haematopoiesis. SOCS3 is the primary inhibitor of IL-6 signalling, interacting with gp130, the common shared chain of the IL-6 family of cytokines, and JAK1, JAK2 and TYK2 to control both the duration of signalling and the biological response. Recent biochemical and structural studies have shown SOCS3 binds to only these three JAKs, all of which are associated with IL-6 signalling, and not JAK3. This specificity is determined by a three residue "GQM" motif in the kinase domain of JAK1, JAK2 and TYK2. SOCS3 binds to JAK and gp130 simultaneously, and inhibits JAK activity in an ATP-independent manner by partially occluding the kinase's substrate binding groove with its kinase inhibitory region. We therefore propose a model in which each of gp130, JAK and SOCS3 are directly bound to the other two, allowing SOCS3 to inhibit IL6 signalling with high potency and specificity.

Microglia are immune cells that maintain brain homeostasis at a resting state by surveying the environment and engulfing debris. However, in some pathological conditions, microglia can produce neurotoxic factors such as pro-inflammatory cytokines and nitric oxide (NO) that lead to neuronal degeneration. Inflammation-induced calcium (Ca(2+)) signaling is thought to underlie this abnormal activation of microglia, but the mechanisms are still obscure. We previously showed that combined application of lipopolysaccharide and interferon γ (LPS/IFNγ) induced-production of NO in microglia from wild-type (WT) mice is significantly reduced in microglia from transient receptor potential melastatin 2 (TRPM2)-knockout (KO) mice. Here, we found that LPS/IFNγ produced a late-onset Ca(2+) signaling in WT microglia, which was abolished by application of the NADPH oxidase inhibitor diphenylene iodonium (DPI) and ML-171. In addition, pharmacological blockade or gene deletion of TRPM2 channel in microglia did not show this Ca(2+) signaling. Furthermore, pharmacological manipulation and Western blotting revealed that Ca(2+) mobilization, the proline-rich tyrosine kinase 2 (Pyk2), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH2-terminal kinase (JNK) contributed to TRPM2-mediated LPS/IFNγ-induced activation, while the extracellular signal-regulated protein kinase (ERK) did not. These results suggest that LPS/IFNγ activates TRPM2-mediated Ca(2+) signaling, which in turn increases downstream p38 MAPK and JNK signaling and results in increased NO production in microglia.

Tyk2, a member of the Janus Kinase (JAK) family of protein tyrosine kinases, is required for interferon-α/β binding and signaling in higher vertebrates. Currently, little is known about the role of the different JAKs in signaling responses to interferon (IFN) in lower vertebrates including fish. In this paper we report the identification and characterization of Atlantic salmon (Salmo salar) Tyk2. Four cDNA sequences, two containing an open reading frame encoding full-length Tyk protein and two with an up-stream in frame stop codon, were identified. The deduced amino acid sequences of the salmon full-length Tyk2 proteins showed highest identity with Tyk2 from other species and their transcripts were ubiquitously expressed. Like in mammals the presented data suggests that salmon Tyk2 is auto-phosporylated when ectopically expressed in cells. In our experiments, full-length salmon Tyk2 overexpressed in CHSE-cells phosphorylated itself, while both a kinase-deficient mutant and the truncated Tyk2 (Tyk-short) were inactive. Interestingly, the overexpression of full length Tyk2 was shown to up-regulate the transcript levels of the IFN induced gene Mx, thus indicating the involvement of salmon Tyk2 in the salmon IFN I pathway.

Natural product-inspired discovery of new drug leads plays a key role in drug development. Recently, small-molecule JAK inhibitors have been pursued for the development of anticancer therapeutics. However, most of these inhibitors reported up to now are multi-nitrogen polycyclic aromatic heterocycles. Undoubtedly, the discovery of new types of promising JAK-inhibitory leads is pivotal for JAK inhibitor-based anticancer drug development. Herein we report an unprecedented sesquiterpenoid-alkaloid named thespesilactam, containing a benzo[cd]indole scaffold, from the heartwood of the Portia tree, Thespesia populnea. Its 3-O-Me product, i.e. 8-hydroxy-5-isopropyl-3-methoxy-7-methylbenzo[cd]indol-2(1H)-one, named 3-O-methylthespesilactam, of which the structure was identified by NMR investigations and single-crystal X-ray diffraction analysis, was discovered as a new type of small-molecule anticancer pan-JAK inhibitor against A2058 human melanoma cells, and selective and potent inhibitor of JAK1 and TYK2.

Interleukin-10 (IL-10) is an important immunomodulatory cytokine that plays an obligate role in regulating inflammatory responses. Here we demonstrated the role of IL-10 in regulating crypts length and breadth as well as maintaining the survival of epithelial cells using rhesus colon explant cultures. Anti-IL-10 antibody treatment of colon explant cultures induced increased production of inflammatory cytokines/molecules like IFNγ, TNFα, CD107a and perforin as well as increased epithelial cell apoptosis compared to media controls tested. Our results suggest that IL-10 plays a crucial role in maintaining mucosal homeostasis by regulating mucosal IFNγ and TNFα cytokine production.

Pegylated interferon lambda-1a (Lambda) is currently in clinical development for the treatment of chronic hepatitis C virus (HCV) infection. To gain insight into the potential mechanisms of non-responsiveness that may occur in patients treated with Lambda, HCV subgenomic replicon cell-lines with impaired susceptibility to the unpegylated recombinant (r) form of interferon (IFN) lambda-1 (rIFNλ) were isolated and characterized. The selected replicon cell populations showed a defect in the activation of the IFN-dependent JAK-STAT signaling pathway. Reduced phosphorylation of STAT proteins and lower expression levels of the cellular janus kinases Jak1 and Tyk2 were observed in these cell populations, which may account for the impaired JAK-STAT signaling and reduced antiviral responses to rIFNλ. Overall, this in vitro study provides molecular insights into the possible mechanism of viral evasion to rIFNλ in the HCV replicon cell system.

Several studies in the last decade have highlighted the role of the type I interferon (IFN-I) pathway, and particularly interferon alpha (IFNα) in SLE pathogenesis. As a result, a multitude of potential treatments targeting IFNα have emerged in the last few years, a few of which have already completed phase II clinical trials. Some of the treatment strategies have focused on blocking IFNα or its receptor and others the plasmacytoid dendritic cell (pDC), which is the principal IFNα producing cell. In this review, we will discuss the evidence supporting a pathogenic role of IFNα and pDC in SLE, provide an update on the current status of these therapeutic strategies, and discuss the potential advantages and disadvantages of each therapeutic approach.

A therapeutic rationale is proposed for the treatment of inflammatory diseases, such as psoriasis and inflammatory bowel diseases (IBD), by selective targeting of TYK2. Hit triage, following a high-throughput screen for TYK2 inhibitors, revealed pyridine 1 as a promising starting point for lead identification. Initial expansion of 3 separate regions of the molecule led to eventual identification of cyclopropyl amide 46, a potent lead analog with good kinase selectivity, physicochemical properties, and pharmacokinetic profile. Analysis of the binding modes of the series in TYK2 and JAK2 crystal structures revealed key interactions leading to good TYK2 potency and design options for future optimization of selectivity.

Human bone marrow-derived mesenchymal stromal cells (MSCs) express Toll-like receptors (TLRs) and produce cytokines and chemokines, all of which contribute to these cells' immunomodulatory and proangiogenic properties. Among the secreted cytokines, colony-stimulating factors (CSFs) regulate angiogenesis through activation of endothelial cell proliferation and migration. Since MSC are recruited within hypoxic tumors where they signal paracrine-regulated angiogenesis, the aim of this study was to evaluate which CSF members are expressed and are inducible in activated MSC. Furthermore, we investigated the JAK/STAT signal transducing pathway that may impact on CSF transcription. MSC were activated with Concanavalin-A (ConA), a TLR-2/6 agonist as well as a membrane type-1 matrix metalloproteinase (MT1-MMP) inducer, and we found increased transcription of granulocyte macrophage-CSF (GM-CSF, CSF-2), granulocyte CSF (G-CSF, CSF-3), and MT1-MMP. Gene silencing of either STAT3 or MT1-MMP prevented ConA-induced phosphorylation of STAT3, and reversed ConA effects on CSF-2 and CSF-3. Treatment with the Janus Kinase (JAK)2 inhibitor AG490 antagonized the ConA induction of MT1-MMP and CSF-2, while the pan-JAK inhibitor Tofacitinib reversed ConA-induced CSF-2 and -3 gene expression. Silencing of JAK2 prevented the ConA-mediated increase of CSF-2, while silencing of JAK1, JAK3 and TYK2 prevented the increase in CSF-3. Given that combined TLR-activation and locally-produced CSF-2 and CSF-3 could regulate immunomodulation and neovascularization, pharmacological targeting of TLR-2/6-induced MT1-MMP/JAK/STAT3 signalling pathway may prevent MSC contribution to tumor development.

The signalling pathway of Janus tyrosine Kinases-Signal Transducers and Activators of Transcription (JAK-STAT) is activated by a number of cytokines, hormones (GH, erythropoietin and prolactin), and growth factors. JAK-STAT signalling is involved in regulation of cell proliferation, differentiation and apoptosis. These activities are due to different members of JAK-STAT family consisting of: JAK1, JAK2, JAK3, Tyk2 and STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6. Recent studies suggest a key role for STAT family proteins, in particular for STAT3, in selectively inducing and maintaining a pro-carcinogenic inflammatory microenvironment, that promote tumour cells transformation. Moreover, a striking correlation between cancer development/progression and STAT3 persistent activation exists, probably due to STAT3 promoting of the pro-oncogenic inflammatory pathways, like NF-kB, IL-6 and JAK family kinases. Recent study demonstrated that carbazoles can inhibit STAT3 mediated transcription. From these evidences, STAT3 represents a therapeutic target, so we have synthesized a new set of N-alkylcarbazole derivatives substituted in positions 2, 4 and 6, to evaluate their activity on STAT3. Some of these compounds showed an interesting activity as STAT3 selective inhibitors; in particular, compounds 9a 9b and 9c revealed to inhibit the STAT3 activation for the 50%, 90% and 95%, respectively.