RESUMO
Our previous study identified round scad neuroprotective peptides with different characteristics. However, the intrinsic relationship between their structure and bioactivity, as well as their bioavailability, remains unclear. The aim of this study is to elucidate the bioavailability of these peptides and their structure-activity relationship against neuroinflammation. Results showed that the SR and WCP peptides were resistant to gastrointestinal digestion. Additionally, peptides SR, WCP, and WCPF could transport Caco-2 monolayers as intact peptides. The permeability coefficients (Papp) of SR, WCP, and WCPF in Caco-2 monolayer were (1.53 ± 0.01) × 10-5, (2.12 ± 0.01) × 10-5, and (8.86 ± 0.03) × 10-7 cm/s, respectively. Peptides SR, WCP, and WCPF, as promising inhibitors of JAK2 and STAT3, could attenuate the levels of pro-inflammatory cytokines and regulate the NFκB and JAK2/STAT3 signaling pathway in LPS-treated BV-2 cells. WCPF exerted the highest anti-inflammatory activity. Moreover, bioinformatics, molecular docking, and quantum chemistry studies indicated that the bioactivity of SR was attributed to Arg, whereas those of WCP and WCPF were attributed to Trp. This study supports the application of round-scad peptides and deepens the understanding of the structure-activity relationship of neuroprotective peptides.
Assuntos
Anti-Inflamatórios , Janus Quinase 2 , Peptídeos , Humanos , Relação Estrutura-Atividade , Peptídeos/química , Peptídeos/farmacologia , Células CACO-2 , Janus Quinase 2/metabolismo , Janus Quinase 2/química , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Animais , Camundongos , Proteínas de Peixes/química , Proteínas de Peixes/farmacologia , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/genética , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , NF-kappa B/genética , NF-kappa B/químicaRESUMO
Ruxolitinib {RUX; systematic name: (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile, C17H18N6} is an orally bioavailable JAK1/2 inhibitor approved for treating intermediate- or high-risk myelofibrosis (MF) and high-risk polycythemia vera (PV). Recent patents claim that RUX can exist in many different forms, information for which is important for the clinical utilization of RUX, especially for the formulation and bioavailability of the drug. But there has been no detailed study on its forms so far. Herein crystals of RUX and its dihydrate (RUX-2H; C17H18N6·2H2O) and phosphate (RUX-P; systematic name: 4-{1-[(1R)-2-cyano-1-cyclopentylethyl]-1H-pyrazol-4-yl}-7H-pyrrolo[2,3-d]pyrimidin-3-ium dihydrogen phosphate, C17H19N6+·H2PO4-) were prepared successfully and their structures studied in detail for the first time. Our study shows that the three crystals of RUX differ in the orientation of the pyrimidine ring relative to the pyrazole ring of the RUX molecule, and in their hydrogen-bond interactions. The water molecules in RUX-2H and the dihydrogen phosphate anion in RUX-P enrich the hydrogen-bond networks in these forms. The expected proton transfer occurs in RUX phosphate and the protonated N atom is engaged in a charge-assisted hydrogen bond with the counter-anion. Hydrogen-bonding interactions dominate in the crystal packing of the three forms. The detailed conformations and packing of the three forms were compared through the calculation of both Hirshfeld surfaces and fingerprint plots.
Assuntos
Ligação de Hidrogênio , Janus Quinase 1 , Janus Quinase 2 , Nitrilas , Fosfatos , Pirazóis , Pirimidinas , Pirimidinas/química , Pirimidinas/farmacologia , Pirazóis/química , Pirazóis/farmacologia , Nitrilas/química , Cristalografia por Raios X , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/química , Janus Quinase 2/metabolismo , Fosfatos/química , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/química , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , HumanosRESUMO
Janus kinase 2 (JAK2) plays a critical role in orchestrating hematopoiesis, and its deregulation leads to various blood disorders, most importantly myeloproliferative neoplasms (MPNs). Ruxolitinib, fedratinib, momelotinib, and pacritinib are FDA-/EMA-approved JAK inhibitors effective in relieving symptoms in MPN patients but show variable clinical profiles due to poor JAK selectivity. The development of next-generation JAK2 inhibitors is hampered by the lack of comparative functional analysis and knowledge of the molecular basis of their selectivity. Here, we provide mechanistic profiling of the four approved and six clinical-stage JAK2 inhibitors and connect selectivity data with high-resolution structural and thermodynamic analyses. All of the JAK inhibitors potently inhibited JAK2 activity. Inhibitors differed in their JAK isoform selectivity and potency for erythropoietin signaling, but their general cytokine inhibition signatures in blood cells were comparable. Structural data indicate that high potency and moderate JAK2 selectivity can be obtained by targeting the front pocket of the adenosine 5'-triphosphate-binding site.
Assuntos
Janus Quinase 2 , Inibidores de Proteínas Quinases , Humanos , Sítios de Ligação , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Janus Quinase 2/química , Modelos Moleculares , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Pirazóis/química , Pirazóis/farmacologia , Pirazóis/síntese química , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinas/síntese química , Relação Estrutura-Atividade , Termodinâmica , Trifosfato de Adenosina/química , Trifosfato de Adenosina/farmacologiaRESUMO
Janus kinase 2 (JAK2) inhibitors are potential anticancer drugs in the treatment of lymphoma, leukemia, thrombocytosis and particularly myeloproliferative diseases. However, the resemblance among JAK family members has challenged the identification of highly selective inhibitors for JAK2 to reduce undesired side effects. As a result, a robust search for promising JAK2 inhibitors using a computational approach that can effectively nominate new potential candidates to be further analyzed through laborious experimental operations has become necessary. In this study, the binding affinities of JAK2 inhibitors were rapidly and precisely estimated using the fast pulling of ligand (FPL) simulations combined with a modified linear interaction energy (LIE) method. The approach correlates with the experimental binding affinities of JAK2 inhibitors with a correlation coefficient of R = 0.82 and a root-mean-square error of 0.67 kcalâ¢mol-1. The data reveal that the FPL/LIE method is highly approximate in anticipating the relative binding free energies of known JAK2 inhibitors with an affordable consumption of computational resources, and thus, it is very promising to be applied in in silico screening for new potential JAK2 inhibitors from a large number of molecules available.
Assuntos
Antineoplásicos , Inibidores de Janus Quinases , Janus Quinase 2/química , Janus Quinase 2/metabolismo , Janus Quinases , Ligantes , Inibidores de Proteínas Quinases/químicaRESUMO
Myeloproliferative neoplasms (MPN) are a group of blood cancers in which the bone marrow (BM) produces an overabundance of erythrocyte, white blood cells, or platelets. Philadelphia chromosome-negative MPN has three subtypes, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The over proliferation of blood cells is often associated with somatic mutations, such as JAK2, CALR, and MPL. JAK2V617F is present in 95% of PV and 50-60% of ET and PMF. Based on current molecular dynamics simulations of full JAK2 and the crystal structure of individual domains, it suggests that JAK2 maintains basal activity through self-inhibition, whereas other domains and linkers directly/indirectly enhance this self-inhibited state. Nevertheless, the JAK2V617F mutation is not the only determinant of MPN phenotype, as many normal individuals carry the JAK2V617F mutation without a disease phenotype. Here we review the major MPN phenotypes, JAK-STAT pathways, and mechanisms of development based on structural biology, while also describing the impact of other contributing factors such as gene mutation allele burden, JAK-STAT-related signaling pathways, epigenetic modifications, immune responses, and lifestyle on different MPN phenotypes. The cross-linking of these elements constitutes a complex network of interactions and generates differences in individual and cellular contexts that determine the phenotypic development of MPN.
Assuntos
Substituição de Aminoácidos , Janus Quinase 2/metabolismo , Transtornos Mieloproliferativos/patologia , Epigênese Genética , Humanos , Janus Quinase 2/química , Janus Quinase 2/genética , Sistema de Sinalização das MAP Quinases , Modelos Moleculares , Transtornos Mieloproliferativos/genética , Domínios ProteicosRESUMO
JAK2 is cytokine-activated non-receptor tyrosine kinase. Although JAK2 is mainly localized at the plasma membrane, it is also present on the centrosome. In this study, we demonstrated that JAK2 localization to the centrosome depends on the SH2 domain and intact kinase activity. We created JAK2 mutants deficient in centrosomal localization ΔSH2, K882E and (ΔSH2, K882E). We showed that JAK2 WT clone strongly enhances cell proliferation as compared to control cells while JAK2 clones ΔSH2, K882E and (ΔSH2, K882E) proliferate slower than JAK2 WT cells. These mutant clones also progress much slower through the cell cycle as compared to JAK2 WT clone and the enhanced proliferation of JAK2 WT cells is accompanied by increased S -> G2 progression. Both the SH2 domain and the kinase activity of JAK2 play a role in prolactin-dependent activation of JAK2 substrate STAT5. We showed that JAK2 is an important regulator of centrosome function as the SH2 domain of JAK2 regulates centrosome amplification. The cells overexpressing ΔSH2 and (ΔSH2, K-E) JAK2 have almost three-fold the amplified centrosomes of WT cells. In contrast, the kinase activity of JAK2 is dispensable for centrosome amplification. Our observations provide novel insight into the role of SH2 domain and kinase activity of JAK2 in centrosome localization of JAK2 and in the regulation of cell growth and centrosome biogenesis.
Assuntos
Proliferação de Células , Centrossomo/metabolismo , Janus Quinase 2/metabolismo , Domínios de Homologia de src/genética , Animais , Células COS , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Chlorocebus aethiops , Humanos , Interferon gama/farmacologia , Janus Quinase 2/química , Janus Quinase 2/genética , Mutagênese Sítio-Dirigida , Transporte Proteico/efeitos dos fármacos , Fator de Transcrição STAT5/metabolismoRESUMO
The SH2B family of adaptor proteins, SH2-B, APS, and LNK are key modulators of cellular signalling pathways. Whilst SH2-B and APS have been partially structurally and biochemically characterised, to date there has been no such characterisation of LNK. Here we present two crystal structures of the LNK substrate recognition domain, the SH2 domain, bound to phosphorylated motifs from JAK2 and EPOR, and biochemically define the basis for target recognition. The LNK SH2 domain adopts a canonical SH2 domain fold with an additional N-terminal helix. Targeted analysis of binding to phosphosites in signalling pathways indicated that specificity is conferred by amino acids one- and three-residues downstream of the phosphotyrosine. Several mutations in LNK showed impaired target binding in vitro and a reduced ability to inhibit signalling, allowing an understanding of the molecular basis of LNK dysfunction in variants identified in patients with myeloproliferative disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Janus Quinase 2/química , Janus Quinase 2/metabolismo , Janus Quinase 3/química , Janus Quinase 3/metabolismo , Camundongos , Mutação , Transtornos Mieloproliferativos/genética , Fosfotirosina , Ligação Proteica , Proteínas Proto-Oncogênicas c-kit/química , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores da Eritropoetina/química , Receptores da Eritropoetina/metabolismo , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/química , Tirosina Quinase 3 Semelhante a fms/metabolismo , Domínios de Homologia de srcRESUMO
Sepsis-induced myocardial dysfunction (SIMD) represents one of the serious complications secondary to sepsis, which is a leading cause of the high mortality rate among septic cases. Subsequent cardiomyocyte apoptosis, together with the uncontrolled inflammatory response, has been suggested to be closely related to SIMD. Piceatannol (PIC) is verified with potent anti-apoptotic and anti-inflammatory effects, but its function and molecular mechanism in SIMD remain unknown so far. This study aimed to explore the potential role and mechanism of action of PIC in resisting SIMD. The interaction of PIC with JAK2 proteins was evaluated by molecular docking, molecular dynamics (MD) simulation and surface plasmon resonance imaging (SPRi). The cecal ligation and puncture-induced septicemia mice and the LPS-stimulated H9C2 cardiomyocytes were prepared as the models in vivo and in vitro, separately. Molecular docking showed that JAK2-PIC complex had the -8.279 kcal/mol binding energy. MD simulations showed that JAK2-PIC binding was stable. SPRi analysis also showed that PIC has a strong binding affinity to JAK2. PIC treatment significantly ameliorated the cardiac function, attenuated the sepsis-induced myocardial loss, and suppressed the myocardial inflammatory responses both in vivo and in vitro. Further detection revealed that PIC inhibited the activation of the JAK2/STAT3 signaling, which was tightly associated with apoptosis and inflammation. Importantly, pre-incubation with a JAK2 inhibitor (AG490) partially blocked the cardioprotective effects of PIC. Collectively, the findings demonstrated that PIC restored the impaired cardiac function by attenuating the sepsis-induced apoptosis and inflammation via suppressing the JAK2/STAT3 pathway both in septic mice and H9C2 cardiomyocytes.
Assuntos
Cardiomiopatias/prevenção & controle , Cardiotônicos/farmacologia , Janus Quinase 2/antagonistas & inibidores , Sepse/complicações , Estilbenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cardiomiopatias/etiologia , Cardiomiopatias/patologia , Cardiotônicos/química , Cardiotônicos/uso terapêutico , Linhagem Celular , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/etiologia , Inflamação/metabolismo , Janus Quinase 2/química , Janus Quinase 2/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Sepse/tratamento farmacológico , Sepse/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estilbenos/química , Estilbenos/uso terapêutico , Tirfostinas/farmacologiaRESUMO
Extracellular cytokines are enriched in the tumor microenvironment and regulate various important properties of cancers, including autophagy. However, the precise molecular mechanisms underlying the link between autophagy and extracellular cytokines remain to be elucidated. In the present study, we demonstrate that IL-6 activates autophagy through the IL-6/JAK2/BECN1 pathway and promotes chemotherapy resistance in colorectal cancer (CRC). Mechanistically, IL-6 triggers the interaction between JAK2 and BECN1, where JAK2 phosphorylates BECN1 at Y333. We demonstrate that BECN1 Y333 phosphorylation is crucial for BECN1 activation and IL-6-induced autophagy by regulating PI3KC3 complex formation. Furthermore, we investigate BECN1 Y333 phosphorylation as a predictive marker for poor CRC prognosis and chemotherapy resistance. Combination treatment with autophagy inhibitors or pharmacological agents targeting the IL-6/JAK2/BECN1 signaling pathway may represent a potential strategy for CRC cancer therapy.
Assuntos
Autofagia/fisiologia , Proteína Beclina-1/metabolismo , Tratamento Farmacológico , Interleucina-6/metabolismo , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/metabolismo , Proteína Beclina-1/química , Proteína Beclina-1/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-6/farmacologia , Janus Quinase 2/química , Janus Quinase 2/metabolismo , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease in which T-helper type 2 (Th2) immune responses are dominant. SH3 and multiple ankyrin repeat domains (SHANK)-associated RH domain-interacting protein (SHARPIN) is expressed at low levels in AD, resulting in the upregulation of the signal transducer and activator of transcription (STAT)3 protein and the Th2 cytokine, interleukin (IL)-33. However, the roles of SHARPIN in AD are not yet fully elucidated. AIM: To evaluate the signalling interactions of SHARPIN and IL-33 in order to improve understanding of AD pathogenesis. METHODS: Western blotting was used to detect the Janus kinase (JAK)/STAT signalling proteins and IL-33 protein in HaCaT cells to determine the key proteins mediating the interaction between SHARPIN and IL-33. The findings were validated by immunofluorescence and immunohistochemical staining. Chromatin immunoprecipitation assays were used to evaluate the activity of STAT3 at the IL-33 promoter. RESULTS: We found that phosphorylated (p)JAK2 and pSTAT3 were upregulated in SHARPIN-knockdown HaCaT cells. Subsequent chromatin immunoprecipitation assays revealed that STAT3 binds to the IL-33 promoter to mediate IL-33 expression. Moreover, SHARPIN-mediated expression of IL-33 was reduced after treatment of HaCaT cells with the JAK/STAT inhibitor ruxolitinib. STAT3 and IL-33 expression levels were higher in AD skin lesion tissues than in normal skin tissues. CONCLUSION: These findings suggest that SHARPIN modulates inflammation in HaCaT cells by inhibiting JAK/STAT signalling, supporting the application of SHARPIN as a potential therapeutic target for AD.
Assuntos
Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/metabolismo , Interleucina-33/metabolismo , Fator de Transcrição STAT2/metabolismo , Ubiquitinas/farmacologia , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Imunoprecipitação da Cromatina/métodos , Dermatite Atópica/imunologia , Regulação para Baixo , Feminino , Imunofluorescência/métodos , Células HaCaT/metabolismo , Células HaCaT/patologia , Humanos , Imuno-Histoquímica/métodos , Inflamação/metabolismo , Janus Quinase 2/química , Janus Quinase 2/metabolismo , Masculino , Pessoa de Meia-Idade , Nitrilas/uso terapêutico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Células Th2/imunologia , Células Th2/metabolismo , Ubiquitinas/metabolismo , Adulto JovemRESUMO
Janus kinase 2 (JAK2) is the most important signal-transducing tyrosine kinase in erythropoietic precursor cells. Its malfunction drives several myeloproliferative disorders. Heme is a small metal-ion-carrying molecule that is incorporated into hemoglobin in erythroid precursor cells to transport oxygen. In addition, heme is a signaling molecule and regulator of various biochemical processes. Here, we show that heme exposure leads to hyperphosphorylation of JAK2 in a myeloid cancer cell line. Two peptides identified in JAK2 are heme-regulatory motifs and show low-micromolar affinities for heme. These peptides map to the kinase domain of JAK2, which is essential for downstream signaling. We suggest these motifs to be the interaction sites of heme with JAK2, which drive the heme-induced hyperphosphorylation. The results presented herein could facilitate the development of heme-related pharmacological tools to combat myeloproliferative disorders.
Assuntos
Heme/química , Heme/metabolismo , Janus Quinase 2/química , Janus Quinase 2/metabolismo , Leucemia Mieloide/patologia , Tirosina/química , Humanos , Leucemia Mieloide/metabolismo , Fosforilação , Conformação Proteica , Transdução de Sinais , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
Ethyl 5-arylpyridopyrimidine-6-carboxylates 3a-d were prepared as a one pot three component reaction via the condensation of different aromatic aldehydes and ethyl acetoacetate with 6-amino-1-benzyluracil 1a under reflux condition in ethanol. Additionally, condensation of ethyl 2-(2-hydroxybenzylidene) acetoacetate with 6-amino-1-benzyluracil in DMF afforded 6-acetylpyridopyrimidine-7-one 3e; a facile, operationally, simple and efficient one-pot synthesis of 8-arylxanthines 6a-f is reported by refluxing 5,6-diaminouracil 4 with aromatic aldehydes in DMF. Moreover, 6-aryllumazines 7a-d was obtained via the reaction of 5,6-diaminouracil with the appropriate aromatic aldehydes in triethyl orthoformate under reflux condition. The synthesized compounds were characterized by spectral (1H-NMR, 13C-NMR, IR and mass spectra) and elemental analyses. The newly synthesized compounds were screened for their anticancer activity against lung cancer A549 cell line. Furthermore, a molecular-docking study was employed to determine the possible mode of action of the synthesized compounds against a group of proteins highly implicated in cancer progression, especially lung cancer. Docking results showed that compounds 3b, 6c, 6d, 6e, 7c and 7d were the best potential docked compounds against most of the tested proteins, especially CDK2, Jak2, and DHFR proteins. These results are in agreement with cytotoxicity results, which shed a light on the promising activity of these novel six heterocyclic derivatives for further investigation as potential chemotherapeutics.
Assuntos
Antineoplásicos/síntese química , Neoplasias/tratamento farmacológico , Pteridinas/síntese química , Piridinas/síntese química , Pirimidinas/síntese química , Xantina/síntese química , Células A549 , Antineoplásicos/farmacologia , Sítios de Ligação , Técnicas de Química Sintética , Quinase 2 Dependente de Ciclina/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Ácido Fólico/química , Humanos , Concentração Inibidora 50 , Janus Quinase 2/química , Células MCF-7 , Simulação de Acoplamento Molecular , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-mdm2/química , Pteridinas/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Tetra-Hidrofolato Desidrogenase/química , Proteína Supressora de Tumor p53/química , Xantina/farmacologiaRESUMO
The binding energies for cation-π complexation are underestimated by traditional fixed-charge force fields owing to their lack of explicit treatment of ion-induced dipole interactions. To address this deficiency, an explicit treatment of cation-π interactions has been introduced into the OPLS-AA force field. Following prior work with atomic cations, it is found that cation-π interactions can be handled efficiently by augmenting the usual 12-6 Lennard-Jones potentials with 1/r4 terms. Results are provided for prototypical complexes as well as protein-ligand systems of relevance for drug design. Alkali cation, ammonium, guanidinium, and tetramethylammonium were chosen for the representative cations, while benzene and six heteroaromatic molecules were used as the π systems. The required nonbonded parameters were fit to reproduce structure and interaction energies for gas-phase complexes from density functional theory (DFT) calculations at the ωB97X-D/6-311++G(d,p) level. The impact of the solvent was then examined by computing potentials of mean force (pmfs) in both aqueous and tetrahydrofuran (THF) solutions using the free-energy perturbation (FEP) theory. Further testing was carried out for two cases of strong and one case of weak cation-π interactions between druglike molecules and their protein hosts, namely, the JH2 domain of JAK2 kinase and macrophage migration inhibitory factor. FEP results reveal greater binding by 1.5-4.4 kcal/mol from the addition of the explicit cation-π contributions. Thus, in the absence of such treatment of cation-π interactions, errors for computed binding or inhibition constants of 101-103 are expected.
Assuntos
Teoria da Densidade Funcional , Modelos Moleculares , Furanos/química , Janus Quinase 2/química , Domínios Proteicos , Termodinâmica , Água/químicaRESUMO
The traditional Chinese medicinal herb Notopterygium incisum Ting ex H.T. Chang has anti-rheumatism activity, and a mass spectrometry assay of patients' serum after administration of the herb revealed that notopterol is the most abundant component enriched. However, the functions of notopterol and its molecular target in rheumatoid arthritis (RA) treatment remain unknown. Here, we show in different RA mouse strains that both oral and intraperitoneal administration of notopterol result in significant therapeutic effects. Mechanistically, notopterol directly binds Janus kinase (JAK)2 and JAK3 kinase domains to inhibit JAK/signal transducers and activators of transcription (JAK-STAT) activation, leading to reduced production of inflammatory cytokines and chemokines. Critically, combination therapy using both notopterol and tumor necrosis factor (TNF) blocker results in enhanced therapeutic effects compared to using TNF blocker alone. We demonstrate that notopterol ameliorates RA pathology by targeting JAK-STAT signaling, raising the possibility that notopterol could be effective in treating other diseases characterized by aberrant JAK-STAT signaling pathway.
Assuntos
Artrite Reumatoide/patologia , Cumarínicos/farmacologia , Inflamação/patologia , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 3/antagonistas & inibidores , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/enzimologia , Artrite Experimental/patologia , Artrite Experimental/prevenção & controle , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/enzimologia , Produtos Biológicos/administração & dosagem , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Quimiocinas/metabolismo , Cumarínicos/administração & dosagem , Cumarínicos/química , Cumarínicos/uso terapêutico , Etanercepte/farmacologia , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Mediadores da Inflamação/metabolismo , Interferon gama/farmacologia , Janus Quinase 2/química , Janus Quinase 3/metabolismo , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Domínios Proteicos , Fatores de Transcrição STAT/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: Fraxetin, extracted from the bark of Fraxinus rhynchophylla, has been shown to exhibit antitumour and anti-inflammatory pharmacological properties. However, the mechanism underlying its anticancer activity towards colon adenocarcinoma (COAD) is not well understood. We aimed to determine the antitumour effect of fraxetin on COAD cell lines and elucidate its biochemical and molecular targets. METHODS: The cell lines HCT116 and DLD-1 were used to evaluate the in vitro antitumour efficacy of fraxetin. Cytotoxicity and viability were assessed by CCK-8 and plate colony formation assays. Flow cytometry was used to assess apoptosis and cell cycle progression in fraxetin-treated COAD cells. Western blot, RT-qPCR, molecular docking, immunohistochemical, and immunofluorescence analyses were used to gain insights into cellular and molecular mechanisms. Preclinical curative effects were evaluated in nude mouse xenograft models. RESULTS: Fraxetin significantly inhibited COAD cell proliferation in both dose- and time-dependent manners, specifically by inducing S-phase cell cycle arrest and triggering intrinsic apoptosis. Additionally, the level of p-JAK2 was decreased by fraxetin via the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signalling pathway. Interestingly, in COAD cells, fraxetin directly targeted the Y1007 and Y1008 residues of JAK2 to suppress its auto- or transphosphorylation, leading to decreased activation of its downstream effector STAT3 and blocking its nuclear translocation. Finally, fraxetin exhibited good tumour growth suppression activity and low toxicity. CONCLUSIONS: Fraxetin inhibits the proliferation of COAD cells by regulating the JAK2/STAT3 signalling pathway, providing evidence that targeting JAK2 with fraxetin may offer a novel potential auxiliary therapy for COAD treatment.
Assuntos
Adenocarcinoma/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Neoplasias do Colo/metabolismo , Cumarínicos/farmacologia , Janus Quinase 2/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Cumarínicos/química , Cumarínicos/uso terapêutico , Fraxinus/química , Humanos , Janus Quinase 2/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Fosforilação , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Colorectal cancer (CRC) is a global pressing healthcare priority. Dysregulation of the IL6/JAK2/STAT3 and p53/caspase downstreaming pathways are significantly involved in the progression of CRC, and mainly affecting apoptosis. Discovery of new anti-cancer agents is laborious, time consuming, and costly with obvious socioeconomic burden. In the present study, we are proposing new molecular insights on the anti-proliferative and apoptotic therapeutic effects of nitazoxanide (NTZ) on CRC. NTZ is FDA-approved thiazolide antiparasitic agent, which has excellent safety and pharmacokinetic profiles. The molecular docking study revealed that NTZ has better binding affinity and docking score against JAK2 and BCL2 proteins compared to 5-Fluorouracil, which is the standard drug for treatment of CRC. The current in vitro work on a human HCT116 cell line displayed that NTZ had lower IC50 value (11.20 µM) than 5-flurouracil (23.78 µM), and NTZ induced a statistically significant down-regulation of IL6/JAK2/STAT3. NTZ also modulated significantly the p53/caspases-dependent signaling pathways, leading to enhancement of apoptosis and an increase of DNA fragmentation. Moreover, NTZ regulated the Bcl-2 gene family and promoted the loss of mitochondrial function which was depicted by release of cytochrome c (Cyt c), and caspase activation in apoptotic HCT116 cells. Additionally, NTZ was able to reduce the expression of VEGF in CRC cell line, which needs future thorough molecular investigations. In conclusion, our findings provided a novel evidence that NTZ could be a dual potential IL6/JAK2/STAT3 signaling inhibitor and p53/caspases-dependent pathway activator in CRC cell line. These potentials support further exploratory molecular researches targeting the therapeutic roles of NTZ in CRC; individually and simultaneously with current approved chemotherapeutic regimens.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Interleucina-6/metabolismo , Janus Quinase 2/metabolismo , Fator de Transcrição STAT3/metabolismo , Tiazóis/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Antiprotozoários/farmacologia , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Citocromos c/metabolismo , Fluoruracila/química , Fluoruracila/farmacologia , Células HCT116 , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Janus Quinase 2/química , Simulação de Acoplamento Molecular , Nitrocompostos , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazóis/química , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Janus kinases (JAKs) are non-receptor tyrosine kinases that are essential components of the JAK-STAT signaling pathway. Associated aberrant signaling is responsible for many forms of cancer and disorders of the immune system. The present focus is on the discovery of molecules that may regulate the activity of JAK2 by selective binding to the JAK2 pseudokinase domain, JH2. Specifically, the Val617Phe mutation in JH2 stimulates the activity of the adjacent kinase domain (JH1) resulting in myeloproliferative disorders. Starting from a non-selective screening hit, we have achieved the goal of discovering molecules that preferentially bind to the ATP binding site in JH2 instead of JH1. We report the design and synthesis of the compounds and binding results for the JH1, JH2, and JH2 V617F domains, as well as five crystal structures for JH2 complexes. Testing with a selective and non-selective JH2 binder on the autophosphorylation of wild-type and V617F JAK2 is also contrasted.
Assuntos
Amitrol (Herbicida)/química , Amitrol (Herbicida)/metabolismo , Ativadores de Enzimas/química , Ativadores de Enzimas/metabolismo , Janus Quinase 2/química , Janus Quinase 2/metabolismo , Animais , Células HEK293 , Humanos , Ligantes , Ligação Proteica/fisiologia , Células Sf9 , Difração de Raios X/métodosRESUMO
Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.
Assuntos
Carcinogênese/genética , Membrana Celular/química , Janus Quinase 2/química , Janus Quinase 2/genética , Multimerização Proteica , Receptores da Eritropoetina/química , Receptores da Somatotropina/química , Receptores de Trombopoetina/química , Substituição de Aminoácidos/genética , Células HeLa , Humanos , Janus Quinase 2/antagonistas & inibidores , Ligantes , Microscopia de Fluorescência , Modelos Moleculares , Mutação , Nitrilas , Fenilalanina/genética , Pirazóis/farmacologia , Pirimidinas , Transdução de Sinais , Imagem Individual de Molécula , Valina/genéticaRESUMO
Exposure to di (2-ethylhexyl) phthalate (DEHP) induces lipid metabolism disorder and high-fat diet (HD) may have joint effects with DEHP. We aim to clarify the role of JAK2/STAT5 pathway in the process and reveal the effects of HD on the toxicity of DEHP. Wistar rats (160 animals) were fed with HD or normal diet (ND) respectively and exposed to DEHP 0, 5, 50, and 500 mg/kg/day for 8 weeks. Lipid levels, as well as the morphology of liver and adipose, mRNA levels, and protein levels of JAK2, STAT5A, STAT5B, FAS, ap2, and PDK4 were detected. The results showed that DEHP exposure leads to increased weight gain. The JAK2/STAT5 pathway was activated in adipose after DEHP exposure and promoted the expression of FAS, ap2, and PDK4 in ND rats. While in the liver, JAK2 was inhibited, and lipid synthesis and accumulation were increased. However, rats exposed to DEHP in combination with HD showed a complete disorder of lipid metabolism. Therefore, we conclude that DEHP affects lipid metabolism through regulating the JAK2/STAT5 pathway and promotes adipogenesis and lipid accumulation. High-fat diet may have a joint effect with DEHP on lipid metabolism disorder.
Assuntos
Dietilexilftalato , Janus Quinase 2/metabolismo , Metabolismo dos Lipídeos , Ácidos Ftálicos/química , Fator de Transcrição STAT5/química , Fator de Transcrição STAT5/metabolismo , Animais , Dieta Hiperlipídica , Janus Quinase 2/química , Ratos , Ratos WistarRESUMO
Janus kinase 2 (JAK2) inhibitors represent a promising therapeutic class of anticancer agents against many myeloproliferative disorders. Bioactivity data on pIC 50 of 2229 JAK2 inhibitors were employed in the construction of quantitative structure-activity relationship (QSAR) models. The models were built from 100 data splits using decision tree (DT), support vector machine (SVM), deep neural network (DNN) and random forest (RF). The predictive power of RF models were assessed via 10-fold cross validation, which afforded excellent predictive performance with R 2 and RMSE of 0.74 ± 0.05 and 0.63 ± 0.05, respectively. Moreover, test set has excellent performance of R 2 (0.75 ± 0.03) and RMSE (0.62 ± 0.04). In addition, Y-scrambling was utilized to evaluate the possibility of chance correlation of the predictive model. A thorough analysis of the substructure fingerprint count was conducted to provide insights on the inhibitory properties of JAK2 inhibitors. Molecular cluster analysis revealed that pyrazine scaffolds have nanomolar potency against JAK2.