Design, synthesis and activity screening of cedrol derivatives as small molecule JAK3 inhibitors.

The JAK-STAT signalling pathway is considered to be a significant role involved in the regulation of inflammatory diseases and immune responses, which indicate that specific inhibition of JAK-STAT pathway would be a potential key strategy for RA (Rheumatoid arthritis) treatment. Cedrol (CE), found from ginger by our group earlier, has been proven to play an excellent role in ameliorating RA via acting on JAK3. In this study, 27 new (1, 3-28), along with one known (2) derivatives of CE were synthesized by using chloroacetic acid and acryloyl chloride as intermediate ligands. In vitro, the inhibition effect on JAK kinases were performed using HTRF (Homogenous Time-Resolved Fluorescence) detection technology, which is more convenient and stable than traditional methods. The results compared with the secretion of LPS-induced p-JAK3 can better reflect the true kinase-selective effect of the compounds. Compound 22 was identified as a potent inhibitor to reduce the secretion of LPS-induced p-JAK3 with a dose-dependent manner. Given these results, compound 22 could serve as a favourable inhibitor of JAK3 for further research.

The Pharmacokinetics, Metabolism, and Clearance Mechanisms of Ritlecitinib, a Janus Kinase 3 and Tyrosine-Protein Kinase Family Inhibitor, in Humans.

Ritlecitinib is an oral once-daily irreversible inhibitor of Janus kinase 3 and tyrosine-protein kinase family being developed for the treatment of moderate-to-severe alopecia areata. This study examined the disposition of ritlecitinib in male participants following oral and intravenous administration using accelerator mass spectroscopy methodology to estimate pharmacokinetic parameters and characterize metabolite profiles. The results indicated ritlecitinib had a systemic clearance of 43.7 L/h, a steady state volume of distribution of 73.8 L, extent of absorption of 89%, time to maximum plasma concentration of ∼0.5 hours, and absolute oral bioavailability of 64%. An observed long terminal half-life of total radioactivity was primarily attributed to ritlecitinib binding to plasma albumin. Ritlecitinib was the main circulating drug species in plasma (∼30%), with one major pharmacologically inactive cysteine conjugated metabolite (M2) at >10%. Oxidative metabolism (fractional clearance 0.47) and glutathione-related conjugation (fractional clearance 0.24) were the primary routes of elimination for ritlecitinib with the greatest disposition of radioactivity shown in the urine (∼71%). In vitro phenotyping indicated ritlecitinib cytochrome P450 (CYP) fraction of metabolism assignments of 0.29 for CYP3A, 0.09 for CYP2C8, 0.07 for CYP1A2, and 0.02 for CYP2C9. In vitro phenotyping in recombinant human glutathione S-transferases indicated ritlecitinib was turned over by a number of cytosolic and microsomal enzyme isoforms. SIGNIFICANCE STATEMENT: This study provides a detailed understanding of the disposition and metabolism of ritlecitinib, a JAK3 and TEC family kinase inhibitor for alopecia areata in humans, as well as characterization of clearance pathways and pharmacokinetics of ritlecitinib and its metabolites. As an AMS-based ADME study design, we have expanded on reporting the standard ADME endpoints, providing key pharmacokinetic parameters, such as clearance, volume of distribution, and bioavailability, allowing for a more comprehensive understanding of drug disposition.

Synthesis of Thiazolidin-4-Ones Derivatives, Evaluation of Conformation in Solution, Theoretical Isomerization Reaction Paths and Discovery of Potential Biological Targets.

Thiazolin-4-ones and their derivatives represent important heterocyclic scaffolds with various applications in medicinal chemistry. For that reason, the synthesis of two 5-substituted thiazolidin-4-one derivatives was performed. Their structure assignment was conducted by NMR experiments (2D-COSY, 2D-NOESY, 2D-HSQC and 2D-HMBC) and conformational analysis was conducted through Density Functional Theory calculations and 2D-NOESY. Conformational analysis showed that these two molecules adopt exo conformation. Their global minimum structures have two double bonds (C=N, C=C) in Z conformation and the third double (C=N) in E. Our DFT results are in agreement with the 2D-NMR measurements. Furthermore, the reaction isomerization paths were studied via DFT to check the stability of the conformers. Finally, some potential targets were found through the SwissADME platform and docking experiments were performed. Both compounds bind strongly to five macromolecules (triazoloquinazolines, mglur3, Jak3, Danio rerio HDAC6 CD2, acetylcholinesterase) and via SwissADME it was found that these two molecules obey Lipinski's Rule of Five.

CS12192: A novel selective and potent JAK3 inhibitor mitigates acute graft-versus-host disease in bone marrow transplantation.

BACKGROUND: Despite the significant role of JAK3 in various autoimmune diseases, including graft-versus-host disease (GVHD), there has been a lack of potent and selective JAK3 inhibitors specifically studied for GVHD. In our preclinical investigations, we evaluated a novel JAK3 inhibitor called CS12192, which is already undergoing clinical investigation in autoimmune diseases. METHODS: We evaluated the efficacy of CS12192 in GVHD through mixed lymphocyte reaction (MLR) in both mouse and human cells, as well as allogeneic bone marrow transplantation (BMT) in a murine model. RESULTS: CS12192, starting at a concentration of 0.5 µM, dose-dependently reduced the intracellular positivity for cytokines TNF-α and IFN-γ in CD4+ T cells (p < 0.05 to p < 0.0001) and CD8+ T cells (p < 0.01 to p < 0.0001) during mouse allogeneic MLR assays. This effect was observed for both single and double positivity of the cytokines. Moreover, In MLR assays with three different human donors, CS12192 also demonstrated a dose-dependent reduction in the proportion of IFN-γ positive CD4+ T cells (p < 0.0001) and CD8+ T cells (p < 0.01 to p < 0.0001). Additionally, it suppressed T cell proliferation in the mouse MLR (p < 0.05 to p < 0.0001), but this effect was observed in only one human donor (p < 0.001 to p < 0.0001). Furthermore, the administration of CS12192 at 40 and 80 mg/kg BID significantly improved the survival rate in the BMT model, resulting in cumulative 62-day survival rates of 88.89% (p < 0.01) and 100% (p < 0.001), respectively, compared with prednisolone (p < 0.05). CONCLUSIONS: CS12192 is a novel, potent and selective JAK3 inhibitor demonstrating great potential to mitigate acute GVHD.

The JAK-STAT signaling-related signature serves as a prognostic and predictive biomarker for renal cell carcinoma immunotherapy.

Renal cell carcinoma (RCC) represents a significant portion of genitourinary cancers, marked by challenging prognosis and high metastasis rates. Immunotherapy has been applied in managing advanced renal cell carcinoma, but the therapeutic outcomes are unsatisfactory. In this study, we order to construct a Janus kinase/signal transduction and activator transcriptional (JAK/STAT)-related signature linked to kidney patient outcomes for better predicting the efficacy to immune checkpoint inhibitors (ICIs) and to provide guidance for effective combination therapy. We screened 25 differentially expressed genes (DEGs) that exhibited high expression in RCC samples and were enriched in the JAK-STAT signaling pathway. Among these genes, 11 key genes were identified and correlated with the expectation of Kidney Clear Cell Carcinoma (KIRC) patients and all these genes was significantly elevated in RCC tumor tissues and cancer cells compared to para-cancer tissues and normal renal cells. Utilizing these 11 genes, we divided RCC patients into high-risk and low-risk groups. We found a clear correlation between the clinicopathologic factors of KIRC patients and the JAK-STAT-related risk score. And the IHC results shown that the JAK3 and STAT4 expression of tumor was significantly higher than normal tissue in RCC patients, the level of JAK3 and STAT4 was positively related to the T stage of RCC patients. In addition, high-risk patients had a poorer prognosis and greater protumor immune cell infiltration, and benefitted less from immunotherapy than did low-risk patients. Furthermore, the JAK-STAT-related risk score can predict disease-free survival (DFS) in RCC patients according to the nomogram, which constructed in combination with other clinical features such as age, TNM-staging and stage. Our study demonstrated the JAK-STAT signaling pathway's important regulatory function in RCC tumor immunity. This insight not only enhances our ability to accurately predict the survival rate of RCC patients, but also underscores a potential therapeutic alternative for RCC, involving the combined targeting of the JAK-STAT pathway and immune checkpoints.

Structural optimizations on the 7H-pyrrolo[2,3-d]pyrimidine scaffold to develop highly selective, safe and potent JAK3 inhibitors for the treatment of Rheumatoid arthritis.

Janus Kinase 3 (JAK3) is important for the signaling transduction of cytokines in immune cells and is identified as potential target for treatment of rheumatoid arthritis (RA). Recently, we designed and synthesized two JAK3 inhibitors J1b and J1f, which featured with high selectivity but mild bioactivity. Therefore, in present study the structure was optimized to increase the potency. As shown in the results, most of the compounds synthesized showed stronger inhibitory activities against JAK3 in contrast to the lead compounds, among which 9a was the most promising candidate because it had the most potent effect in ameliorating carrageenan-induced inflammation of mice and exhibited low acute in vivo toxicity (MTD > 2 g/kg). Further analysis revealed that 9a was highly selective to JAK3 (IC50 = 0.29 nM) with only minimal effect on other JAK members (>3300-fold) and those kinases bearing a thiol in a position analogous to that of Cys909 in JAK3 (>150-fold). Meanwhile, the selectivity of JAK3 was also confirmed by PBMC stimulation assay, in which 9a irreversibly bound to JAK3 and robustly inhibited the signaling transduction with mild suppression on other JAKs. Moreover, it was showed that 9a could remarkably inhibited the proliferation of lymphocytes in response to concanavalin A and significantly mitigate disease severity in collagen induced arthritis. Therefore, present data indicate that compound 9a is a selective JAK3 inhibitor and could be a promising candidate for clinical treatment of RA.

IRG1/Itaconate inhibits proliferation and promotes apoptosis of CD69+CD103+CD8+ tissue-resident memory T cells in autoimmune hepatitis by regulating the JAK3/STAT3/P53 signalling pathway.

To investigate the protective role of immune response gene 1 (IRG1) and exogenous itaconate in autoimmune hepatitis (AIH) and elucidate the underlying mechanisms. Wild-type and IRG1-/- AIH mouse models were established, and samples of liver tissue and ocular blood were collected from each group of mice to assess the effects of IRG1/itaconate on the expression of pro- and anti-inflammatory cytokines. The levels of liver enzymes and related inflammatory factors were determined using enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction (PCR). Liver histomorphology was detected through hematoxylin and eosin staining and then scored for liver injury, and the infiltration levels of tissue-resident memory T (TRM) cells and related molecules in the liver tissue were detected through immunofluorescence staining in vitro. RNA sequencing and gene enrichment analysis were conducted to identify the corresponding molecules and pathways, and lentiviral transfection was used to generate TRM cell lines with IRG1, Jak3, Stat3, and p53 knockdown. Real-time quantitative PCR and western blot were performed to detect the expression levels of relevant mRNAs and proteins in the liver tissue and cells. The percentage of apoptotic cells was determined using flow cytometry. IRG1/itaconate effectively reduced the release of pro-inflammatory cytokines and the pathological damage to liver tissue, thereby maintaining normal liver function. At the same time, IRG1/itaconate inhibited the JAK3/STAT3 signaling pathway, regulated the expression of related downstream proteins, and inhibited the proliferation and promoted the apoptosis of CD69+CD103+CD8+ TRM cells. For the first time, P53 was found to act as a downstream molecule of the JAK3/STAT3 pathway and was regulated by IRG1/itaconate to promote the apoptosis of CD8+ TRM cells. IRG1/itaconate can alleviate concanavalin A-induced autoimmune hepatitis in mice by inhibiting the proliferation and promoting the apoptosis of CD69+CD103+CD8+ TRM cells via the JAK3/STAT3/P53 pathway.

Janus Kinase 3 (JAK3): A Critical Conserved Node in Immunity Disrupted in Immune Cell Cancer and Immunodeficiency.

The Janus kinase (JAK) family is a small group of protein tyrosine kinases that represent a central component of intracellular signaling downstream from a myriad of cytokine receptors. The JAK3 family member performs a particularly important role in facilitating signal transduction for a key set of cytokine receptors that are essential for immune cell development and function. Mutations that impact JAK3 activity have been identified in a number of human diseases, including somatic gain-of-function (GOF) mutations associated with immune cell malignancies and germline loss-of-function (LOF) mutations associated with immunodeficiency. The structure, function and impacts of both GOF and LOF mutations of JAK3 are highly conserved, making animal models highly informative. This review details the biology of JAK3 and the impact of its perturbation in immune cell-related diseases, including relevant animal studies.

Peficitinib alleviated acute lung injury by blocking glycolysis through JAK3/STAT3 pathway.

Peficitinib is a selective Janus kinase (JAK3) inhibitor recently developed and approved for the treatment of rheumatoid arthritis in Japan. Glycolysis in macrophages could induce NOD-like receptor (NLR) family and pyrin domain-containing protein 3 (NLRP3) inflammasome activation, thus resulting in pyroptosis and acute lung injury (ALI). The aim of our study was to investigate whether Peficitinib could alleviate lipopolysaccharide (LPS)-induced ALI by inhibiting NLRP3 inflammasome activation. Wild type C57BL/6J mice were intraperitoneally injected with Peficitinib (5 or 10 mg·kg-1·day-1) for 7 consecutive days before LPS injection. The results showed that Peficitinib pretreatment significantly relieved LPS-induced pulmonary edema, inflammation, and apoptosis. NLRP3 inflammasome and glycolysis in murine lung tissues challenged with LPS were also blocked by Peficitinib. Furthermore, we found that the activation of JAK3/signal transducer and activator of transcription 3 (STAT3) was also suppressed by Peficitinib in mice with ALI. However, in Jak3 knockout mice, Peficitinib did not show obvious protective effects after LPS injection. In vitro experiments further showed that Jak3 overexpression completely abolished Peficitinib-elicited inhibitory effects on pyroptosis and glycolysis in LPS-induced RAW264.7 macrophages. Finally, we unveiled that LPS-induced activation of JAK3/STAT3 was mediated by toll-like receptor 4 (TLR4) in RAW264.7 macrophages. Collectively, our study proved that Peficitinib could protect against ALI by blocking JAK3-mediated glycolysis and pyroptosis in macrophages, which may serve as a promising candidate against ALI in the future.

Design and synthesis of highly selective Janus kinase 3 covalent inhibitors for the treatment of rheumatoid arthritis.

Selective inhibition of Janus kinase 3 (JAK3) is a promising strategy for the treatment of autoimmune diseases. Based on the discovery of a hydrophobic pocket unutilized between the lead compound RB1 and the JAK3 protein, a series of covalent JAK3 inhibitors were prepared by introducing various aromatic fragments to RB1. Among them, J1b (JAK3 IC50 = 7.2 nM, other JAKs IC50 > 1000 nM) stood out because of its low toxicity (MTD > 2 g/kg) and superior anti-inflammatory activity in Institute of Cancer Research mice. Moreover, the acceptable bioavailability (F% = 31.69%) ensured that J1b displayed excellent immune regulation in collagen-induced arthritis mice, whose joints in the high-dose group were almost recovered to a normal state. Given its clear kinase selectivity (Bmx IC50 = 539.9 nM, other Cys909 kinases IC50 > 1000 nM), J1b was nominated as a highly selective JAK3 covalent inhibitor, which could be used to safely treat arthritis and other autoimmune diseases.

N-Acetylcysteine Alters Disease Progression and Increases Janus Kinase Mutation Frequency in a Mouse Model of Precursor B-Cell Acute Lymphoblastic Leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) is the most prevalent type of cancer in young children and is associated with high levels of reactive oxygen species (ROS). The antioxidant N-acetylcysteine (NAC) was tested for its ability to alter disease progression in a mouse model of B-ALL. Mb1-CreΔPB mice have deletions in genes encoding PU.1 and Spi-B in B cells and develop B-ALL at 100% incidence. Treatment of Mb1-CreΔPB mice with NAC in drinking water significantly reduced the frequency of CD19+ pre-B-ALL cells infiltrating the thymus at 11 weeks of age. However, treatment with NAC did not reduce leukemia progression or increase survival by a median 16 weeks of age. NAC significantly altered gene expression in leukemias in treated mice. Mice treated with NAC had increased frequencies of activating mutations in genes encoding Janus kinases 1 and 3. In particular, frequencies of Jak3 R653H mutations were increased in mice treated with NAC compared with control drinking water. NAC opposed oxidization of PTEN protein ROS in cultured leukemia cells. These results show that NAC alters leukemia progression in this mouse model, ultimately selecting for leukemias with high Jak3 R653H mutation frequencies. SIGNIFICANCE STATEMENT: In a mouse model of precursor B-cell acute lymphoblastic leukemia associated with high levels of reactive oxygen species, treatment with N-acetylcysteine did not delay disease progression but instead selected for leukemic clones with activating R653H mutations in Janus kinase 3.

The disruptor of telomeric silencing 1-like (DOT1L) promotes peritoneal fibrosis through the upregulation and activation of protein tyrosine kinases.

The disruptor of telomeric silencing 1-like (DOT1L), a specific histone methyltransferase that catalyzed methylation of histone H3 on lysine 79, was associated with the pathogenesis of many diseases, but its role in peritoneal fibrosis remained unexplored. Here, we examined the role of DOT1L in the expression and activation of protein tyrosine kinases and development of peritoneal fibrosis. We found that a significant rise of DOT1L expression in the fibrotic peritoneum tissues from long-term PD patients and mice. Inhibition of DOT1L significantly attenuated the profibrotic phenotypic differentiation of mesothelial cells and macrophages, and alleviated peritoneal fibrosis. Mechanistically, RNA sequencing and proteomic analysis indicated that DOT1L was mainly involved in the processes of protein tyrosine kinase binding and extracellular matrix structural constituent in the peritoneum. Chromatin immunoprecipitation (ChIP) showed that intranuclear DOT1L guided H3K79me2 to upregulate EGFR in mesothelial cells and JAK3 in macrophages. Immunoprecipitation and immunofluorescence showed that extranuclear DOT1L could interact with EGFR and JAK3, and maintain the activated signaling pathways. In summary, DOT1L promoted the expression and activation of tyrosine kinases (EGFR in mesothelial cells and JAK3 in macrophages), promoting cells differentiate into profibrotic phenotype and thus peritoneal fibrosis. We provide the novel mechanism of dialysis-related peritoneal fibrosis (PF) and the new targets for clinical drug development. DOT1L inhibitor had the PF therapeutic potential.

New JAK3-INSL3 Fusion Transcript-An Oncogenic Event in Cutaneous T-Cell Lymphoma.

Constitutively activated tyrosine kinase JAK3 is implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCL). The mechanisms of constitutive JAK3 activation are unknown although a JAK3 mutation was reported in a small portion of CTCL patients. In this study, we assessed the oncogenic roles of a newly identified JAK3-INSL3 fusion transcript in CTCL. Total RNA from malignant T-cells in 33 patients with Sézary syndrome (SS), a leukemic form of CTCL, was examined for the new JAK3-INSL3 fusion transcript by RT-PCR followed by Sanger sequencing. The expression levels were assessed by qPCR and correlated with patient survivals. Knockdown and/or knockout assays were conducted in two CTCL cell lines (MJ cells and HH cells) by RNA interference and/or CRISPR/Cas9 gene editing. SS patients expressed heterogeneous levels of a new JAK3-INSL3 fusion transcript. Patients with high-level expression of JAK3-INSL3 showed poorer 5-year survival (n = 19, 42.1%) than patients with low-level expression (n = 14, 78.6%). CTCL cells transduced with specific shRNAs or sgRNAs had decreased new JAK3-INSL3 fusion transcript expression, reduced cell proliferation, and decreased colony formation. In NSG xenograft mice, smaller tumor sizes were observed in MJ cells transduced with specific shRNAs than cells transduced with controls. Our results suggest that the newly identified JAK3-INSL3 fusion transcript confers an oncogenic event in CTCL.

Janus Kinase 3 phosphorylation and the JAK/STAT pathway are positively modulated by follicle-stimulating hormone (FSH) in bovine granulosa cells.

Janus kinase 3 (JAK3) is a member of the JAK family of tyrosine kinase proteins involved in cytokine receptor-mediated intracellular signal transduction through the JAK/STAT signaling pathway. JAK3 was previously shown as differentially expressed in granulosa cells (GC) of bovine pre-ovulatory follicles suggesting that JAK3 could modulate GC function and activation/inhibition of downstream targets. We used JANEX-1, a JAK3 inhibitor, and FSH treatments and analyzed proliferation markers, steroidogenic enzymes and phosphorylation of target proteins including STAT3, CDKN1B/p27Kip1 and MAPK8IP3/JIP3. Cultured GC were treated with or without FSH in the presence or not of JANEX-1. Expression of steroidogenic enzyme CYP11A1, but not CYP19A1, was upregulated in GC treated with FSH and both were significantly decreased when JAK3 was inhibited. Proliferation markers CCND2 and PCNA were reduced in JANEX-1-treated GC and upregulated by FSH. Western blots analyses showed that JANEX-1 treatment reduced pSTAT3 amounts while JAK3 overexpression increased pSTAT3. Similarly, FSH treatment increased pSTAT3 even in JANEX-1-treated GC. UHPLC-MS/MS analyses revealed phosphorylation of specific amino acid residues within JAK3 as well as CDKN1B and MAPK8IP3 suggesting possible activation or inhibition post-FSH or JANEX-1 treatments. We show that FSH activates JAK3 in GC, which could phosphorylate target proteins and likely modulate other signaling pathways involving CDKN1B and MAPK8IP3, therefore controlling GC proliferation and steroidogenic activity.

JAK3/STAT5b/PPARγ Pathway Mediates the Association between Di(2-ethylhexyl) Phthalate Exposure and Lipid Metabolic Disorder in Chinese Adolescent Students.

Our previous studies found that di (2-ethylhexyl) phthalate (DEHP) could disorder lipid metabolism in adolescents but the mechanisms underlying this association remained unclear. This study was undertaken to clarify the mediating effect of JAK3/STAT5/PPARγ on disorder lipid levels induced by DEHP in adolescents. We recruited 478 adolescent students (median age 18.1 years). The mRNA expression and DNA methylation levels of JAK3/STAT5/PPARγ were detected by real-time PCR and the MethylTarget, respectively. We used multiple linear regression to analyze the association between DEHP metabolites (MEHP, MEOHP, MEHHP, MECPP, MCMHP, and ΣDEHP) levels, mRNA expression, and DNA methylation levels. The mediating effect of JAK3/STAT5/PPARγ mRNA expression levels was examined by mediation analysis. We found that all DEHP metabolite levels were positively correlated with TC/HDL-C and LDL-C/HDL-C (P < 0.05). The MEOHP level was negatively associated with DNA methylation levels and positively associated with mRNA levels of PPARγ and STAT5b (P < 0.05). The MEHP level was negatively associated with the DNA methylation level and positively associated with the mRNA level of JAK3 (P < 0.05). Higher MEOHP was associated with a higher level of TC/HDL-C, the mediation analysis showed the mediation effect was 17.18% for the JAK3 level, 10.76% for the STAT5b level, and 11% for the PPARγ level. Higher MEHP was associated with a higher level of LDL-C/HDL-C, the mediation effect was 14.49% for the JAK3 level. In conclusion, DEHP metabolites decreased the DNA methylation levels, inducing the increase of the mRNA levels of JAK3/STAT5/PPARγ. In addition, the mRNA levels mediated the association between DEHP exposure and disorder lipid levels.

JAK1 Pseudokinase V666G Mutant Dominantly Impairs JAK3 Phosphorylation and IL-2 Signaling.

Overactive Janus kinases (JAKs) are known to drive leukemia, making them well-suited targets for treatment. We sought to identify new JAK-activating mutations and instead found a JAK1-inactivating pseudokinase mutation, V666G. In contrast to other pseudokinase mutations that canonically lead to an active kinase, the JAK1 V666G mutation led to under-activation seen by reduced phosphorylation. To understand the functional role of JAK1 V666G in modifying kinase activity we investigated its influence on other JAK kinases and within the Interleukin-2 pathway. JAK1 V666G not only inhibited its own activity, but its presence could inhibit other JAK kinases. These findings provide new insights into the potential of JAK1 pseudokinase to modulate its own activity, as well as of other JAK kinases. Thus, the features of the JAK1 V666 region in modifying JAK kinases can be exploited to allosterically inhibit overactive JAKs.

Structural basis of Janus kinase trans-activation.

Janus kinases (JAKs) mediate signal transduction downstream of cytokine receptors. Cytokine-dependent dimerization is conveyed across the cell membrane to drive JAK dimerization, trans-phosphorylation, and activation. Activated JAKs in turn phosphorylate receptor intracellular domains (ICDs), resulting in the recruitment, phosphorylation, and activation of signal transducer and activator of transcription (STAT)-family transcription factors. The structural arrangement of a JAK1 dimer complex with IFNλR1 ICD was recently elucidated while bound by stabilizing nanobodies. While this revealed insights into the dimerization-dependent activation of JAKs and the role of oncogenic mutations in this process, the tyrosine kinase (TK) domains were separated by a distance not compatible with the trans-phosphorylation events between the TK domains. Here, we report the cryoelectron microscopy structure of a mouse JAK1 complex in a putative trans-activation state and expand these insights to other physiologically relevant JAK complexes, providing mechanistic insight into the crucial trans-activation step of JAK signaling and allosteric mechanisms of JAK inhibition.

Novel Potent EGFR-JAK3 Dual-Target Inhibitor that Overcomes KRAS Mutation Resistance in Colorectal Cancer.

BACKGROUND: In-depth and clear mechanistic study is a prerequisite for new drugs to enter clinical research. METHODS: New chemical entity BY4008 was identified by our lab as a novel and highly potent EGFR and JAK3 dualtarget inhibitor. A cell-based test exhibited strong antiproliferative activities against SW620 and HCT116 colon cancer cells harboring KRAS mutation with IC50 of nanomolar potency. Furthermore, acridine orange/ethidium bromide (AO/EB), Hematoxylin-Eosin (H&E) and DAPI staining assays and flow cytometry analyses indicated that BY4008 has the function of pro-apoptosis and arresting the cell cycle. In addition, BY4008 inhibited the autophosphorylation of EGFR and blocked the activation of downstream signaling and the JAK-STAT3 pathway. RESULTS: Meanwhile, a decreased level of reactive oxygen species (ROS) and an increased level of malondialdehyde (MDA) in SW620 and HCT116 cells were observed after exposure to BY4008. CONCLUSION: In summary, this study provides an important structural basis and mechanistic study for future effective treatment of colorectal cancer.

A novel cDNA-uPA/SCID/Rag2-/- /Jak3-/- mouse model for hepatitis virus infection and reconstruction of human immune system.

Although human hepatocyte-transplanted immunodeficient mice support infection with hepatitis viruses, these mice fail to develop viral hepatitis due to the lack of an adaptive immune system. In this study, we generated new immunodeficiency cDNA-urokinase-type plasminogen activator (uPA)/SCID/Rag2-/- /Jak3-/- mice and established a mouse model with both a humanized liver and immune system. Transplantation of human hepatocytes with human leukocyte antigen (HLA)-A24 resulted in establishment of a highly replaced liver in cDNA-uPA/SCID/Rag2-/- /Jak3-/- mice. These mice were successfully infected with hepatitis B virus (HBV) and hepatitis C virus (HCV) for a prolonged period and facilitate analysis of the effect of anti-HCV drugs. Administration of peripheral blood mononuclear cells (PBMCs) obtained from an HLA-A24 donor resulted in establishment of 22.6%-81.3% human CD45-positive mononuclear cell chimerism in liver-infiltrating cells without causing graft-versus-host disease in cDNA-uPA/SCID/Rag2-/- /Jak3-/- mice without human hepatocyte transplantation. When mice were transplanted with human hepatocytes and then administered HLA-A24-positive human PBMCs, an alloimmune response between transplanted human hepatocytes and PBMCs occurred, with production of transplanted hepatocyte-specific anti-HLA antibody. In conclusion, we succeeded in establishing a humanized liver/immune system characterized by an allo-reaction between transplanted human immune cells and human liver using a novel cDNA-uPA/SCID/Rag2-/- /Jak3-/- mouse. This mouse model can be used to generate a chronic hepatitis mouse model with a human immune system with application not only to hepatitis virus virology but also to investigation of the pathology of post-transplantation liver rejection.

The mRNA Expression of PTEN, LEF1, JAK3, LC3 and p62/SQSTM1 Genes in Patients with Chronic Myeloid Leukemia.

INTRODUCTION: Chronic myeloid leukemia (CML) is a progressive myeloproliferative disorder resulting from forming a chimeric BCR-ABL gene. The proteins derived from this gene can affect some genes from various signaling pathways such as PI3K/AKT/Wnt/catenin/JAK/Stat involved in proliferation, differentiation, cell death, and genes related to autophagy. Imatinib is the first-line treatment for CML patients, with durable and proper responses in Iranian children and adult CML patients. Hence, we aimed to evaluate the mRNA expression of some selected key genes from those pathways in patients with CML before and under treatment. METHODS: In the case-control study, the mRNA expression of PTEN, LEF1, JAK3, LC3 and p62 genes were measured in 51 CML patients (6 patients before treatment and 45 patients under treatment with imatinib mesylate) and 40 healthy controls using the Real-time PCR method. RESULTS: The mRNA expression of PTEN and P62 were significantly higher in newly diagnosed patients than in controls (P<0.0001 and P = 0.0183, respectively), while the expression of the LC3 gene was significantly lower in the untreated newly diagnosed group than in control subjects (P = 0.0191). The expression level of PTEN, LEF1, JAK3 and P62 genes were significantly decreased in patients under treatment than in the group before treatment (P = 0.0172, P = 0.0002, P = 0.0047 and P = 0.0038, respectively). A positive correlation was seen between the gene expression of P62 and BCR-ABL in the patients under treatment (r 0529, P = 0.016). CONCLUSION: Our findings showed that the changes in expression of these genes were related to the patient's treatment. Due to the key role of these genes in proliferation, differentiation and tumor suppression, it is proposed that these genes may be helpful for follow-up of treatment in CML patients.