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1.
J Med Chem ; 67(12): 10012-10024, 2024 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-38843875

RESUMEN

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.


Asunto(s)
Janus Quinasa 2 , Inhibidores de Proteínas Quinasas , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Janus Quinasa 2/química , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Modelos Moleculares , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinas/síntesis química , Termodinámica , Sitios de Unión , Pirazoles/química , Pirazoles/farmacología , Pirazoles/síntesis química
2.
J Mol Model ; 28(6): 163, 2022 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-35599265

RESUMEN

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 kcal•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.


Asunto(s)
Antineoplásicos , Inhibidores de las Cinasas Janus , Janus Quinasa 2/química , Janus Quinasa 2/metabolismo , Quinasas Janus , Ligandos , Inhibidores de Proteínas Quinasas/química
3.
Int J Mol Sci ; 23(3)2022 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-35162937

RESUMEN

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.


Asunto(s)
Sustitución de Aminoácidos , Janus Quinasa 2/metabolismo , Trastornos Mieloproliferativos/patología , Epigénesis Genética , Humanos , Janus Quinasa 2/química , Janus Quinasa 2/genética , Sistema de Señalización de MAP Quinasas , Modelos Moleculares , Trastornos Mieloproliferativos/genética , Dominios Proteicos
4.
PLoS One ; 17(1): e0261098, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35089929

RESUMEN

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.


Asunto(s)
Proliferación Celular , Centrosoma/metabolismo , Janus Quinasa 2/metabolismo , Dominios Homologos src/genética , Animales , Células COS , Puntos de Control del Ciclo Celular , Línea Celular , Chlorocebus aethiops , Humanos , Interferón gamma/farmacología , Janus Quinasa 2/química , Janus Quinasa 2/genética , Mutagénesis Sitio-Dirigida , Transporte de Proteínas/efectos de los fármacos , Factor de Transcripción STAT5/metabolismo
5.
Nat Commun ; 12(1): 6110, 2021 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-34671038

RESUMEN

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.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Humanos , Janus Quinasa 2/química , Janus Quinasa 2/metabolismo , Janus Quinasa 3/química , Janus Quinasa 3/metabolismo , Ratones , Mutación , Trastornos Mieloproliferativos/genética , Fosfotirosina , Unión Proteica , Proteínas Proto-Oncogénicas c-kit/química , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores de Eritropoyetina/química , Receptores de Eritropoyetina/metabolismo , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/química , Tirosina Quinasa 3 Similar a fms/metabolismo , Dominios Homologos src
6.
Nat Commun ; 12(1): 3651, 2021 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-34131122

RESUMEN

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.


Asunto(s)
Autofagia/fisiología , Beclina-1/metabolismo , Quimioterapia , Interleucina-6/metabolismo , Autofagia/efectos de los fármacos , Proteínas Relacionadas con la Autofagia/metabolismo , Beclina-1/química , Beclina-1/genética , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-6/farmacología , Janus Quinasa 2/química , Janus Quinasa 2/metabolismo , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos
7.
Int Immunopharmacol ; 96: 107639, 2021 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-34162128

RESUMEN

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.


Asunto(s)
Cardiomiopatías/prevención & control , Cardiotónicos/farmacología , Janus Quinasa 2/antagonistas & inhibidores , Sepsis/complicaciones , Estilbenos/farmacología , Animales , Apoptosis/efectos de los fármacos , Cardiomiopatías/etiología , Cardiomiopatías/patología , Cardiotónicos/química , Cardiotónicos/uso terapéutico , Línea Celular , Modelos Animales de Enfermedad , Inflamación/tratamiento farmacológico , Inflamación/etiología , Inflamación/metabolismo , Janus Quinasa 2/química , Janus Quinasa 2/metabolismo , Masculino , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Miocitos Cardíacos/efectos de los fármacos , Ratas , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Sepsis/tratamiento farmacológico , Sepsis/metabolismo , Transducción de Señal/efectos de los fármacos , Estilbenos/química , Estilbenos/uso terapéutico , Tirfostinos/farmacología
8.
Clin Exp Dermatol ; 46(5): 880-887, 2021 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-33548083

RESUMEN

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.


Asunto(s)
Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/metabolismo , Interleucina-33/metabolismo , Factor de Transcripción STAT2/metabolismo , Ubiquitinas/farmacología , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Inmunoprecipitación de Cromatina/métodos , Dermatitis Atópica/inmunología , Regulación hacia Abajo , Femenino , Técnica del Anticuerpo Fluorescente/métodos , Células HaCaT/metabolismo , Células HaCaT/patología , Humanos , Inmunohistoquímica/métodos , Inflamación/metabolismo , Janus Quinasa 2/química , Janus Quinasa 2/metabolismo , Masculino , Persona de Mediana Edad , Nitrilos/uso terapéutico , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Células Th2/inmunología , Células Th2/metabolismo , Ubiquitinas/metabolismo , Adulto Joven
9.
Chembiochem ; 22(5): 861-864, 2021 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-33103835

RESUMEN

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.


Asunto(s)
Hemo/química , Hemo/metabolismo , Janus Quinasa 2/química , Janus Quinasa 2/metabolismo , Leucemia Mieloide/patología , Tirosina/química , Humanos , Leucemia Mieloide/metabolismo , Fosforilación , Conformación Proteica , Transducción de Señal , Células Tumorales Cultivadas , Tirosina/metabolismo
10.
Molecules ; 25(21)2020 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-33182318

RESUMEN

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.


Asunto(s)
Antineoplásicos/síntesis química , Neoplasias/tratamiento farmacológico , Pteridinas/síntesis química , Piridinas/síntesis química , Pirimidinas/síntesis química , Xantina/síntesis química , Células A549 , Antineoplásicos/farmacología , Sitios de Unión , Técnicas de Química Sintética , Quinasa 2 Dependiente de la Ciclina/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Ácido Fólico/química , Humanos , Concentración 50 Inhibidora , Janus Quinasa 2/química , Células MCF-7 , Simulación del Acoplamiento Molecular , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-mdm2/química , Pteridinas/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Tetrahidrofolato Deshidrogenasa/química , Proteína p53 Supresora de Tumor/química , Xantina/farmacología
11.
J Chem Theory Comput ; 16(11): 7184-7194, 2020 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-33048555

RESUMEN

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.


Asunto(s)
Teoría Funcional de la Densidad , Modelos Moleculares , Furanos/química , Janus Quinasa 2/química , Dominios Proteicos , Termodinámica , Agua/química
12.
Cell Rep ; 32(11): 108158, 2020 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-32937124

RESUMEN

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.


Asunto(s)
Artritis Reumatoide/patología , Cumarinas/farmacología , Inflamación/patología , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 3/antagonistas & inhibidores , Animales , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/enzimología , Artritis Experimental/patología , Artritis Experimental/prevención & control , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/enzimología , Productos Biológicos/administración & dosificación , Productos Biológicos/química , Productos Biológicos/farmacología , Productos Biológicos/uso terapéutico , Quimiocinas/metabolismo , Cumarinas/administración & dosificación , Cumarinas/química , Cumarinas/uso terapéutico , Etanercept/farmacología , Inflamación/tratamiento farmacológico , Inflamación/enzimología , Mediadores de Inflamación/metabolismo , Interferón gamma/farmacología , Janus Quinasa 2/química , Janus Quinasa 3/metabolismo , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Dominios Proteicos , Factores de Transcripción STAT/metabolismo , Factor de Necrosis Tumoral alfa/farmacología
13.
Int J Biochem Cell Biol ; 125: 105777, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32504672

RESUMEN

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.


Asunto(s)
Adenocarcinoma/metabolismo , Antineoplásicos Fitogénicos/farmacología , Neoplasias del Colon/metabolismo , Cumarinas/farmacología , Janus Quinasa 2/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/enzimología , Adenocarcinoma/genética , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Cumarinas/química , Cumarinas/uso terapéutico , Fraxinus/química , Humanos , Janus Quinasa 2/química , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Simulación del Acoplamiento Molecular , Fosforilación , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacos , Proteínas Quinasas Asociadas a Fase-S/genética , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Transducción de Señal/genética , Ensayos Antitumor por Modelo de Xenoinjerto
14.
Mol Cell Biochem ; 469(1-2): 143-157, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32356241

RESUMEN

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.


Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Interleucina-6/metabolismo , Janus Quinasa 2/metabolismo , Factor de Transcripción STAT3/metabolismo , Tiazoles/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Antiprotozoarios/farmacología , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Citocromos c/metabolismo , Fluorouracilo/química , Fluorouracilo/farmacología , Células HCT116 , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Janus Quinasa 2/química , Simulación del Acoplamiento Molecular , Nitrocompuestos , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos , Tiazoles/química , Factor A de Crecimiento Endotelial Vascular/metabolismo
15.
J Med Chem ; 63(10): 5324-5340, 2020 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-32329617

RESUMEN

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.


Asunto(s)
Amitrol (Herbicida)/química , Amitrol (Herbicida)/metabolismo , Activadores de Enzimas/química , Activadores de Enzimas/metabolismo , Janus Quinasa 2/química , Janus Quinasa 2/metabolismo , Animales , Células HEK293 , Humanos , Ligandos , Unión Proteica/fisiología , Células Sf9 , Difracción de Rayos X/métodos
16.
Science ; 367(6478): 643-652, 2020 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-32029621

RESUMEN

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.


Asunto(s)
Carcinogénesis/genética , Membrana Celular/química , Janus Quinasa 2/química , Janus Quinasa 2/genética , Multimerización de Proteína , Receptores de Eritropoyetina/química , Receptores de Somatotropina/química , Receptores de Trombopoyetina/química , Sustitución de Aminoácidos/genética , Células HeLa , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Ligandos , Microscopía Fluorescente , Modelos Moleculares , Mutación , Nitrilos , Fenilalanina/genética , Pirazoles/farmacología , Pirimidinas , Transducción de Señal , Imagen Individual de Molécula , Valina/genética
17.
Environ Sci Pollut Res Int ; 27(4): 3837-3848, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31732953

RESUMEN

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.


Asunto(s)
Dietilhexil Ftalato , Janus Quinasa 2/metabolismo , Metabolismo de los Lípidos , Ácidos Ftálicos/química , Factor de Transcripción STAT5/química , Factor de Transcripción STAT5/metabolismo , Animales , Dieta Alta en Grasa , Janus Quinasa 2/química , Ratas , Ratas Wistar
18.
Molecules ; 24(23)2019 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-31805692

RESUMEN

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.


Asunto(s)
Inhibidores Enzimáticos/química , Janus Quinasa 2/química , Minería de Datos , Relación Estructura-Actividad Cuantitativa
19.
J Med Chem ; 62(22): 10305-10320, 2019 11 27.
Artículo en Inglés | MEDLINE | ID: mdl-31670517

RESUMEN

Herein, we describe the design, synthesis, and structure-activity relationships of a series of unique 4-(1H-pyrazol-4-yl)-pyrimidin-2-amine derivatives that selectively inhibit Janus kinase 2 (JAK2) and FLT3 kinases. These screening cascades revealed that 18e was a preferred compound, with IC50 values of 0.7 and 4 nM for JAK2 and FLT3, respectively. Moreover, 18e was a potent JAK2 inhibitor with 37-fold and 56-fold selectivity over JAK1 and JAK3, respectively, and possessed an excellent selectivity profile over the other 100 representative kinases. In a series of cytokine-stimulated cell-based assays, 18e exhibited a higher JAK2 selectivity over other JAK isoforms. The oral administration of 60 mg/kg of 18e could significantly inhibit tumor growth, with a tumor growth inhibition rate of 93 and 85% in MV4-11 and SET-2 xenograft models, respectively. Additionally, 18e showed an excellent bioavailability (F = 58%), a suitable half-life time (T1/2 = 4.1 h), a satisfactory metabolic stability, and a weak CYP3A4 inhibitory activity, suggesting that 18e might be a potential drug candidate for JAK2-driven myeloproliferative neoplasms and FLT3-internal tandem duplication-driven acute myelogenous leukemia.


Asunto(s)
Antineoplásicos/farmacología , Janus Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Perros , Relación Dosis-Respuesta a Droga , Femenino , Semivida , Humanos , Janus Quinasa 2/química , Janus Quinasa 2/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucocitos Mononucleares/efectos de los fármacos , Masculino , Ratones SCID , Simulación del Acoplamiento Molecular , Trastornos Mieloproliferativos/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas Sprague-Dawley , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/química , Tirosina Quinasa 3 Similar a fms/metabolismo
20.
Int J Biol Macromol ; 137: 1030-1040, 2019 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-31299252

RESUMEN

Activating mutations in JAK2 have been described in patients with various hematologic malignancies including acute myeloid leukemia (AML) and myeloproliferative neoplasms. However, mechanism of these mutations in JAK2's activity, structural stability and pathology of AML remains poorly understood. The JAK2 T875N somatic mutation has been detected in about 5.2% of AML patients. But the structural basis and mechanism of JAK2 T875N mutation in the pathology of AML is still unclear. Our results suggested that JAK2 T875N mutation disrupted the T875 and D873 interaction which destroyed the compact structure of JH1 domain, forced it into the active conformation, facilitated the entrance of substrate and thus led to JAK2 hyperactivation. Mutations (T875N, T875A, D873A and D873G) disrupted the T875 and D873 interaction enhanced JAK2's activity, decreased its structural stability and JH2 domain's activity which further enhanced JAK2's activity, while mutations (T875R, D873E, T875R/D873E) repaired this interaction displayed opposite results. Moreover, JAK2 T875N mutation enhanced the activity of JAK2-STAT5 pathway, promoted the proliferation and transformation of OCI-AML3 cells. This study provides clues in understanding structural basis of T875N mutation caused JAK2 hyperactivation and its roles in the pathology of AML.


Asunto(s)
Transformación Celular Neoplásica/genética , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Leucemia Mieloide Aguda/patología , Mutación , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Activación Enzimática/genética , Estabilidad de Enzimas/genética , Humanos , Janus Quinasa 2/química , Modelos Moleculares , Conformación Proteica
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