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1.
Bioorg Chem ; 115: 105265, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34426160

RESUMEN

In spite of possessing a wide range of pharmacological properties the anti-inflammatory activities of isoquinolin-1(2H)-ones were rarely known or explored earlier. PDE4 inhibitors on the other hand in addition to their usefulness in treating inflammatory diseases have been suggested to attenuate the cytokine storm in COVID-19 especially TNF-α. In our effort, a new class of isoquinolin-1(2H)-ones derivatives containing an aminosulfonyl moiety were designed and explored as potential inhibitors of PDE4. Accordingly, for the first time a CuCl2-catalyzed inexpensive, faster and ligand/additive free approach has been developed for the synthesis of these predesigned isoquinolin-1(2H)-one derivatives via the coupling-cyclization strategy. Thus, the CuCl2-catalyzed reaction of 2-iodobenzamides with appropriate terminal alkynes proceeded with high chemo and regioselectivity affording the desired compounds in 77-84% yield within 1-1.5 h. The methodology also afforded simpler isoquinolin-1(2H)-ones devoid of aminosulfonyl moiety showing a broader generality and scope of this approach. Several of the synthesized compounds especially 3c, 3k and 3s showed impressive inhibition (83-90%) of PDE4B when tested at 10 µM in vitro whereas compounds devoid of aminosulfonyl moiety was found to be less active. In spite of high inhibition showed at 10 µM these compounds did not show proper concertation dependent inhibition below 1 µM that was reflected in their IC50 values e.g. 2.43 ± 0.32, 3.26 ± 0.24 and 3.63 ± 0.80 µM for 3k, 3o and 3s respectively. The anti-inflammatory potential of these compounds was indicated by their TNF-α inhibition (60-50% at 10 µM). The in silico docking studies of these molecules suggested good interactions with PDE4B and selective inhibition of PDE4B by 3k over PDE4D that was supported by in vitro assay results. These observations together with the favorable ADME and safety predicted for 3kin silico not only suggested 3k as an interesting hit molecule for further studies but also reveal the first example of isoquinolin-1(2H)-one based inhibitor of PDE4B.


Asunto(s)
Antiinflamatorios/química , Cobre/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Isoquinolinas/química , Inhibidores de Fosfodiesterasa 4/química , Animales , Antiinflamatorios/síntesis química , Catálisis , Ciclización , Pruebas de Enzimas , Humanos , Isoquinolinas/síntesis química , Ratones , Estructura Molecular , Inhibidores de Fosfodiesterasa 4/síntesis química , Células RAW 264.7 , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores
2.
Eur J Med Chem ; 221: 113514, 2021 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-33992926

RESUMEN

While anti-inflammatory properties of isocoumarins are known their PDE4 inhibitory potential was not explored previously. In our effort the non-PDE4 inhibitor isocoumarins were transformed into the promising inhibitors via introducing an aminosulfonyl/aminocarboxamide moiety to the C-3 benzene ring attached to the isocoumarin framework. This new class of isocoumarins were synthesized via a PdCl2-catalyzed construction of the 4-allyl substituted 3-aryl isocoumarin ring starting from the appropriate 2-alkynyl benzamide derivative. Several compounds showed good inhibition of PDE4B in vitro and the SAR indicated superiority of aminosulfonamide moiety over aminocarboxamide in terms of PDE4B inhibition. Two compounds 3q and 3u with PDE4B IC50 = 0.43 ± 0.11 and 0.54 ± 0.19 µM and ≥ 2-fold selectivity over PDE4D emerged as initial hits. The participation of aminosulfonamide moiety in PDE4B inhibition and the reason for selectivity though moderate shown by 3q and 3u was revealed by the in silico docking studies. In view of potential usefulness of moderately selective PDE4B inhibitors the compound 3u (that showed PDE4 selectivity over other PDEs) was further evaluated in adjuvant induced arthritic rats. At an intraperitoneal dose of 30 mg/kg the compound showed a significant reduction in paw swelling (in a dose dependent manner), inflammation and pannus formation (in the knee joints) as well as pro-inflammatory gene expression/mRNA levels and increase in body weight. Moreover, besides its TNF-α inhibition and no significant toxicity in an MTT assay the compound did not show any adverse effects in a thorough toxicity studies e.g. teratogenicity, hepatotoxicity, cardiotoxicity and apoptosis in zebrafish. Thus, the isocoumarin 3u emerged as a new, safe and moderately selective PDE4B inhibitor could be useful for inflammatory diseases possibly including COVID-19.


Asunto(s)
Antiinflamatorios/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Isocumarinas/uso terapéutico , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Sulfonamidas/uso terapéutico , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/metabolismo , Antiinflamatorios/toxicidad , Artritis Experimental/patología , Catálisis , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Embrión no Mamífero/efectos de los fármacos , Femenino , Isocumarinas/síntesis química , Isocumarinas/metabolismo , Isocumarinas/toxicidad , Articulación de la Rodilla/efectos de los fármacos , Articulación de la Rodilla/patología , Masculino , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Paladio/química , Inhibidores de Fosfodiesterasa 4/síntesis química , Inhibidores de Fosfodiesterasa 4/metabolismo , Inhibidores de Fosfodiesterasa 4/toxicidad , Unión Proteica , Células RAW 264.7 , Ratas Wistar , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/metabolismo , Sulfonamidas/toxicidad , Pez Cebra
3.
Explore (NY) ; 17(2): 127-129, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33046408

RESUMEN

This report provides a perspective on the relevance of saline water gargling and nasal irrigation to the COVID-19 crisis. While there is limited evidence concerning their curative or preventive role against SARS-CoV-2 infection, previous work on their utility against influenza and recent post-hoc analysis of the Edinburgh and Lothians Viral Intervention Study (ELVIS) provide compelling support to their applicability in the current crisis. Saline water gargling and nasal irrigation represent simple, economical, practically feasible, and globally implementable strategies with therapeutic and prophylactic value. These methods, rooted in the traditional Indian healthcare system, are suitable and reliable in terms of infection control and are relevant examples of harmless interventions. We attempt to derive novel insights into their usefulness, both from theoretical and practical standpoints.


Asunto(s)
COVID-19/prevención & control , Lavado Nasal (Proceso)/métodos , Faringe , Solución Salina Hipertónica/uso terapéutico , Solución Salina/uso terapéutico , COVID-19/terapia , Humanos , SARS-CoV-2 , Irrigación Terapéutica/métodos
4.
J Contemp Dent Pract ; 21(11): 1197-1200, 2020 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-33850062

RESUMEN

Vegetarian diets are known to have significant positive effects on personal and planetary health and are likely to curb zoonotic infection transmission. We propose that minimizing meat consumption should become an essential dietary shift in the post-COVID-19 era. To date, however, there is limited knowledge concerning suitable methods that could catalyze this change on a global scale. Meditation and Yoga are practical and easy to implement psychomodulatory strategies that can naturally trigger vegetarianism and related eating behaviors, lowering our reliance on animal meat. Decreasing dependence on animal meat reduces the need for animal markets and may substantially minimize the likelihood of spillover (passage of viruses from animal reservoirs into human populations). Global implementation of these strategies, in our opinion, can add to spiritual wisdom, compassion, and cooperative human behavior, thus reducing the encroachment of wild-life reserves and animal exploitation. The application of these ancient Indian approaches represents a novel and focused strategy toward curbing zoonotic pandemics. Keywords: Coronavirus disease-19, Meat-eating, Meditation, Pandemics, Viral spillover, Yoga.


Asunto(s)
COVID-19 , Meditación , Yoga , Animales , Dieta , Humanos , Pandemias , SARS-CoV-2
5.
MethodsX ; 6: 1-5, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30591915

RESUMEN

The development of gene editing technologies, especially the CRISPR-Cas9 system, has been pivotal for understanding the functional role of proteins. Rapid and efficient genotyping methods are necessary to screen for generated mutations and streamline the isolation of homozygotes. CRISPR-Cas9 system targeting a single site in the gene typically results in small indels. Many genotyping methods utilize the heteroduplex that is formed when wild-type and mutant amplicons with small indels anneal during PCR creating a bubble due to mismatched strands. These methods include T7 endonuclease/Cel-I assay, high resolution melting (HRM) analysis, and heteroduplex mobility assay (HMA). Our protocol explains a simple, two step method of a mixing HMA (mHMA) to identify homozygous mutants, a modification of the previously published HMA. We have utilized the mHMA for screening and genotyping numerous CRISPR generated models. The mHMA method to differentiate homozygous wild type from homozygous mutant animals eliminates - •DNA sequencing, even with small indels that can be difficult to discern on a gel.•additional enzymatic reaction steps, such as with the T7EI/Cel-I assay.•specialized equipment and analysis tools, such as with HRM analysis.

6.
Front Microbiol ; 7: 1347, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27621726

RESUMEN

Zinc metalloprotease-1 (Zmp1) from Mycobacterium tuberculosis (M.tb), the tuberculosis (TB) causing bacillus, is a virulence factor involved in inflammasome inactivation and phagosome maturation arrest. We earlier reported that Zmp1 was secreted under granuloma-like stress conditions, induced Th2 cytokine microenvironment and was highly immunogenic in TB patients as evident from high anti-Zmp1 antibody titers in their sera. In this study, we deciphered a new physiological role of Zmp1 in mycobacterial dissemination. Exogenous treatment of THP-1 cells with 500 nM and 1 µM of recombinant Zmp1 (rZmp1) resulted in necrotic cell death. Apart from inducing secretion of necrotic cytokines, TNFα, IL-6, and IL-1ß, it also induced the release of chemotactic chemokines, MCP-1, MIP-1ß, and IL-8, suggesting its likely function in cell migration and mycobacterial dissemination. This was confirmed by Gap closure and Boyden chamber assays, where Zmp1 treated CHO or THP-1 cells showed ∼2 fold increased cell migration compared to the untreated cells. Additionally, Zebrafish-M. marinum based host-pathogen model was used to study mycobacterial dissemination in vivo. Td-Tomato labeled M. marinum (TdM. marinum) when injected with rZmp1 showed increased dissemination to tail region from the site of injection as compared to the untreated control fish in a dose-dependent manner. Summing up these observations along with the earlier reports, we propose that Zmp1, a multi-faceted protein, when released by mycobacteria in granuloma, may lead to necrotic cell damage and release of chemotactic chemokines by surrounding infected macrophages, attracting new immune cells, which in turn may lead to fresh cellular infections, thus assisting mycobacterial dissemination.

7.
Bioorg Chem ; 67: 139-47, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27388635

RESUMEN

A series of novel 5-benzylidene-2,4-thiazolidinediones were designed as inhibitors of angiogenesis targeting VEGFR-2. In docking study, molecules showed similar way of binding with VEGFR-2 as that of the co-crystallized ligand. Compounds were then synthesized, purified and characterized by spectroscopic techniques. Compounds 3f and 3i were found to be most active in the series showing good inhibition of angiogenesis in both CAM and in zebrafish embryo assays. Compound 3i also exhibited IC50 of 0.5µM against VEGFR-2.


Asunto(s)
Compuestos de Bencilideno/farmacología , Diseño de Fármacos , Neovascularización Patológica/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Tiazoles/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Compuestos de Bencilideno/síntesis química , Compuestos de Bencilideno/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Neovascularización Patológica/patología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/química , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Pez Cebra
8.
Pharmacol Rep ; 66(1): 179-83, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24905326

RESUMEN

BACKGROUND AND METHODS: We describe a method for obtaining pharmacokinetics (PK) and pharmacology data from adult zebrafish in terms of mg/kg using a novel method of oral administration. Using carbamazepine (CBZ) as a test drug, we employed dried blood spot (DBS) cards to enable drug quantification for PK; and we evaluated the pharmacological anxiolytic effect using novel tank test. RESULTS: The PK study confirmed the presence of CBZ in both blood and brain and the behavioural study showed dose dependent anxiolytic effect. The reproducibility of oral dosing was confirmed by the fact that the results obtained in both the experiments had negligible errors. CONCLUSIONS: This report enables a novel approach for optimizing the utility of zebrafish in drug discovery and drug delivery research.


Asunto(s)
Carbamazepina/farmacocinética , Administración Oral , Animales , Ansiolíticos/farmacología , Barrera Hematoencefálica , Carbamazepina/farmacología , Masculino , Pez Cebra
9.
Org Biomol Chem ; 11(39): 6680-5, 2013 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-23986357

RESUMEN

A series of 1,3-disubstituted pyrrolo[2,3-b]quinoxalines has been designed for the potential inhibition of PDE4 without inhibiting luciferase. A ligand/PTC (phase transfer catalyst) free intramolecular Heck cyclization strategy was used to prepare these compounds, some of which showed significant inhibition of PDE4B (IC50≈ 5-14 µM) and growth inhibition of oral cancer cells (CAL 27) but not inhibition of luciferase in vitro. They also showed acceptable safety profiles but no apoptosis in zebrafish embryos.


Asunto(s)
Neoplasias de la Boca/tratamiento farmacológico , Inhibidores de Fosfodiesterasa 4/síntesis química , Inhibidores de Fosfodiesterasa 4/farmacología , Quinoxalinas/síntesis química , Quinoxalinas/farmacología , Animales , Sitios de Unión , Catálisis , Proliferación Celular/efectos de los fármacos , Embrión no Mamífero/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Ligandos , Luciferasas/metabolismo , Estructura Molecular , Inhibidores de Fosfodiesterasa 4/química , Quinoxalinas/química , Pez Cebra/embriología
10.
Org Biomol Chem ; 11(19): 3103-7, 2013 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-23575971
11.
Epilepsy Behav ; 27(1): 212-9, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23466252

RESUMEN

We report the effect of orally administered gabapentin (GBP) on pentylenetetrazole (PTZ)-induced seizure-like activity in adult zebrafish. Zebrafish were pretreated with vehicle or GBP using a novel method of precise oral administration, followed by an intraperitoneal administration of PTZ. Behavioral assessment was carried out using locomotion-based video-tracking analysis and seizure score assignment using visual observation. Cephalic field potential recordings of the zebrafish brain were conducted using an electrical data acquisition system. Orally administered GBP significantly suppressed the seizure-like locomotor activity and strong slow-wave (~3Hz) activity in the cephalic field potential caused by PTZ. This work is the first report of the activity of an orally delivered anticonvulsant in adult zebrafish. Our study provides behavioral and physiological evidence in support of an adult zebrafish model for studying seizures including excitotoxic brain injury and a novel in vivo framework for the evaluation of pharmacological modulators of epilepsy.


Asunto(s)
Potenciales de Acción/efectos de los fármacos , Aminas/administración & dosificación , Anticonvulsivantes/administración & dosificación , Encéfalo/fisiopatología , Ácidos Ciclohexanocarboxílicos/administración & dosificación , Convulsiones/tratamiento farmacológico , Ácido gamma-Aminobutírico/administración & dosificación , Administración Oral , Análisis de Varianza , Animales , Encéfalo/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Gabapentina , Locomoción/efectos de los fármacos , Masculino , Pentilenotetrazol/toxicidad , Convulsiones/inducido químicamente , Factores de Tiempo , Pez Cebra
12.
J Pharmacol Toxicol Methods ; 67(2): 115-20, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23353637

RESUMEN

INTRODUCTION: Recent studies have shown the utility of adult zebrafish ECG (electrocardiogram) in assessing drug-induced QTc prolongation. While the method has significant advantages over current ECG animal models including ethical issues, low compound requirement and expense, adoption of the method into drug discovery programs has been hampered by specific limitations. The limitations include the inability to determine the exact dose of test compound administered, and potential effects due to variables such as flow rate of oral perfusion and immobilization method. We describe a refined method for the reproducible recording of the adult zebrafish ECG and illustrate its application in investigating drug-induced QTc prolongation using the histamine receptor antagonist Terfenadine as a test drug. METHOD: We chose to perform parenteral administration of test drug instead of perfusion on the basis of mg per kg body weight of adult zebrafish. Acclimatization and immobilization methods were optimized to avoid ECG artifacts due to sudden environmental changes. We further modified the formula for QT correction and ensured reproducible recording of stable ECGs. Various concentrations of Terfenadine were used and the resultant proarrhythmic effects were analyzed as compared to the baseline and untreated controls. RESULTS: Normal, stable and reproducible ECGs were recorded in all zebrafish. Terfenadine at the rate of 0.1mg/kg body weight was found to be the NOAEL. We found an excellent correlation between known QTc effects in humans and those observed in adult zebrafish at all concentrations. All Terfenadine-induced proarrhythmic effects observed in zebrafish were dose and time dependent. DISCUSSION: We report a refined method for reproducible recording of stable zebrafish ECGs to facilitate its routine application in preclinical investigation of QTc-prolonging drugs with reliable estimation of NOAEL. Our study is of relevance to the development and use of alternate animal models in drug discovery.


Asunto(s)
Modelos Animales de Enfermedad , Electrocardiografía/métodos , Antagonistas de los Receptores Histamínicos H1 no Sedantes/toxicidad , Síndrome de QT Prolongado/inducido químicamente , Terfenadina/toxicidad , Pez Cebra/fisiología , Animales , Evaluación Preclínica de Medicamentos , Inyecciones Intraperitoneales , Síndrome de QT Prolongado/fisiopatología , Masculino , Reproducibilidad de los Resultados
13.
Zebrafish ; 10(3): 264-74, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23234507

RESUMEN

Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome.


Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Pez Cebra/metabolismo , Animales , Desarrollo Embrionario , Humanos , Pez Cebra/embriología
14.
Chembiochem ; 13(13): 1889-94, 2012 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-22887835

RESUMEN

Glowing fish: Fluorescent organic nanoparticles (FONs) were prepared from pyrene-containing guanine analogues. FONs are able to penetrate into human cells and zebrafish embryos; they are nontoxic, biocompatible and were found to be specific for muscular tissues. This study demonstrates for the first time the possibility of using FONs for in vivo, whole-body, fluorescence imaging of zebrafish.


Asunto(s)
Embrión no Mamífero/ultraestructura , Colorantes Fluorescentes/química , Nanopartículas/química , Imagen de Cuerpo Entero/métodos , Pez Cebra/embriología , Animales , Fluorescencia , Guanina/análogos & derivados , Humanos , Microscopía Fluorescente/métodos , Imagen Óptica , Pirenos/química
15.
Bioorg Med Chem ; 20(5): 1711-22, 2012 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-22316553

RESUMEN

A direct and single-step method has been developed for the synthesis of mono and 2,3-disubstituted quinoxalines by using a AlCl(3) induced (hetero)arylation of 2,3-dichloroquinoxaline. Both symmetrical and unsymmetrical 2,3-disubstituted quinoxalines can be prepared conveniently by using this method under appropriate reaction conditions. The reaction proceeds via C-C bond formation and can be utilized for the preparation of a variety of quinoxaline derivatives from readily available starting materials and reagents. The molecular structure of a representative compound was confirmed by single crystal X-ray diffraction study. Some of the compounds synthesized were tested for chorismate mutase inhibitory properties in vitro and one compound showed promising activity representing one of the few examples of chorismate mutase inhibition by a heteroarene based small molecule.


Asunto(s)
Compuestos de Aluminio/química , Antituberculosos/síntesis química , Cloruros/química , Quinoxalinas/síntesis química , Cloruro de Aluminio , Cristalografía por Rayos X , Modelos Moleculares , Estructura Molecular , Quinoxalinas/farmacología
16.
Zebrafish ; 7(1): 69-81, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20415645

RESUMEN

Zebrafish embryos are well suited as a model system to perform chemical biology experiments effectively in educational settings. We studied the effect of caffeine on heart rate (HR) and other phenotypes of zebrafish embryos using visual microscopy and simple imaging. Acute treatment with millimolar concentrations of caffeine in embryo medium caused a dose-dependent decrease in HR in 2-3-day-old zebrafish embryos, ultimately resulting in complete HR cessation. A characteristic pattern of decrease in HR was observed, with an initial acute drop in HR and a period of stabilization followed by complete cessation. The effects of caffeine were not reversed by cotreatment with ruthenium red and adenosine, agents known to be antagonistic to caffeine, or by changes in calcium concentration in embryo medium. Apparent cardiac arrhythmia and a typical kinking effect in the trunk/tail region were also observed because of caffeine treatment. Our results, taken together with previous reports, raise the possibility that caffeine exerts its effects on embryonic HR of zebrafish by inhibition of ether-a-go-go potassium channels. However, further experimentation is required to dissect the molecular basis of caffeine action. We demonstrate that such experiments can be used to explore the effect of small molecules, such as caffeine, on cardiovascular phenotypes and to encourage experimental design in chemical biology.


Asunto(s)
Cafeína/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Corazón/efectos de los fármacos , Pez Cebra/fisiología , Animales , Cafeína/química , Calcio/metabolismo , Corazón/embriología , Estructura Molecular , Pez Cebra/embriología
17.
Proc Natl Acad Sci U S A ; 105(34): 12463-8, 2008 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-18719096

RESUMEN

Amplification of the receptor tyrosine kinase ErbB2 is frequently observed in breast cancer. Amplification of erbB2 is also associated with multiple genomic gains and losses; however, the importance of these associated changes is largely unknown. We demonstrate that Brk, a cytoplasmic tyrosine kinase, is coamplified and coexpressed with ErbB2 in human breast cancers. ErbB2 interacts with Brk and increases its intrinsic kinase activity. Expression of Brk enhances the ErbB2-induced activation of Ras/MAPK signaling and cyclin E/cdk2 activity to induce cell proliferation of mammary 3-dimensional acini in culture. In a murine model of breast cancer, expression of Brk was found to shorten the latency of ErbB2-induced tumors by promoting cell proliferation, with no effect on protection from apoptosis. Furthermore, overexpression of Brk conferred resistance to the ability of Lapatinib, an ErbB2 kinase inhibitor, to inhibit ErbB2-induced proliferation. Thus, we identified Brk as a drug target for ErbB2-positive cancers.


Asunto(s)
Neoplasias de la Mama/patología , Proliferación Celular , Amplificación de Genes/fisiología , Neoplasias Mamarias Experimentales/patología , Proteínas de Neoplasias/genética , Proteínas Tirosina Quinasas/fisiología , Receptor ErbB-2/fisiología , Familia-src Quinasas/fisiología , Animales , Biopsia , Neoplasias de la Mama/etiología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/análisis , Quinasa 2 Dependiente de la Ciclina/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Neoplasias Mamarias Experimentales/etiología , Neoplasias Mamarias Experimentales/genética , Ratones , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/fisiología , Proteínas Tirosina Quinasas/análisis , Proteínas Tirosina Quinasas/genética , Receptor ErbB-2/análisis , Receptor ErbB-2/genética , Familia-src Quinasas/genética
18.
Biochemistry ; 46(51): 14810-8, 2007 Dec 25.
Artículo en Inglés | MEDLINE | ID: mdl-18052197

RESUMEN

The redox regulation of Janus kinase 2 (JAK2) is poorly understood, and there are contradictory reports as to whether the enzyme's activity is inhibited or stimulated by oxidizing conditions in the cell. Here we demonstrate that multiple cysteine residues within the JAK2 catalytic domain may be crucial for enzymatic activity. The enzyme is catalytically inactive when oxidized; activity can be restored via reduction to the thiol state. A series of recombinant variants of JAK2 were overproduced using the baculoviral expression vector system. A truncated variant of JAK2, GST/(NDelta661)rJAK2, provided evidence that the amino-terminal autoinhibitory domain was not essential for direct redox regulation and that only nine cysteine residues were potentially involved. The effect of individually and combinatorially altering these nine cysteines was examined via cysteine-to-serine mutagenesis. This identified four cysteine residues in the catalytic domain (Cys866, Cys917, Cys1094, and Cys1105) that cooperatively maintain JAK2's catalytic competency. Our data are consistent with a direct mechanism for redox regulation of JAK2 via oxidation and reduction of critical cysteine residues.


Asunto(s)
Cisteína/metabolismo , Janus Quinasa 2/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Cisteína/genética , Activación Enzimática/efectos de los fármacos , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/química , Janus Quinasa 2/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Oxidación-Reducción , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido
19.
Biochemistry ; 43(14): 4272-83, 2004 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-15065871

RESUMEN

The phosphorylation of an "activation loop" within protein kinases is commonly associated with establishing catalytic competence, and phosphorylation of the Tyr(1007) residue in the activation loop of Janus kinase 2 (JAK2) has been shown to be essential for intracellular propagation of cytokine-initiated signaling. We provide evidence for the presence of a basal activity state of JAK2, which was observed in the absence of activation loop phosphorylation. Phosphorylation of the JAK2 activation loop was essential for conversion to the high-activity state, characterized by high-efficiency ATP utilization during autophosphorylation. Mutagenesis of activation loop tyrosine residues Tyr(1007/1008) to phenylalanine residues impaired, but did not abolish, the enzyme's ability to autophosphorylate. The activation loop mutant JAK2 could also transphosphorylate an inactive JAK2 fragment coexpressed in Sf21 cells, providing evidence of exogenous substrate phosphorylation. The mutant enzyme remained in a basal activity state characterized by low-efficiency ATP utilization during autophosphorylation. Mutagenesis of a critical Lys(882) residue to a glutamate residue abolished all evidence of kinase activity, confirming that the observed activity of Tyr-to-Phe mutants was not due to another kinase. Our data are consistent with the proposal that JAK2 is an inefficient but active enzyme in the absence of activation loop phosphorylation and is capable of conversion to a high-activity state by autophosphorylation under physiological ATP concentrations. This theoretically precludes the need for an upstream activating kinase. The activation process of JAK2 may be envisioned as a multistate process involving at least two kinetically distinct states of activity.


Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Tirosina/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/fisiología , Animales , Baculoviridae/genética , Catálisis , Activación Enzimática/genética , Vectores Genéticos , Janus Quinasa 2 , Modelos Químicos , Mutagénesis Sitio-Dirigida , Fosforilación , Fosfotirosina/inmunología , Fosfotirosina/metabolismo , Pruebas de Precipitina , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Quinasas/fisiología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/aislamiento & purificación , Proteínas Tirosina Quinasas/fisiología , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Transducción de Señal/genética , Spodoptera/genética , Tirosina/genética
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