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1.
Cell ; 165(2): 382-95, 2016 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-27040500

RESUMO

Gene duplication is a major evolutionary force driving adaptation and speciation, as it allows for the acquisition of new functions and can augment or diversify existing functions. Here, we report a gene duplication event that yielded another outcome--the generation of antagonistic functions. One product of this duplication event--UPF3B--is critical for the nonsense-mediated RNA decay (NMD) pathway, while its autosomal counterpart--UPF3A--encodes an enigmatic protein previously shown to have trace NMD activity. Using loss-of-function approaches in vitro and in vivo, we discovered that UPF3A acts primarily as a potent NMD inhibitor that stabilizes hundreds of transcripts. Evidence suggests that UPF3A acquired repressor activity through simple impairment of a critical domain, a rapid mechanism that may have been widely used in evolution. Mice conditionally lacking UPF3A exhibit "hyper" NMD and display defects in embryogenesis and gametogenesis. Our results support a model in which UPF3A serves as a molecular rheostat that directs developmental events.


Assuntos
Desenvolvimento Embrionário , Genes Duplicados , Degradação do RNAm Mediada por Códon sem Sentido , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Evolução Molecular , Gametogênese , Células HeLa , Humanos , Camundongos
2.
Mol Cell ; 43(6): 950-61, 2011 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-21925383

RESUMO

Nonsense-mediated mRNA decay (NMD) is a conserved RNA decay pathway that degrades aberrant mRNAs and directly regulates many normal mRNAs. This dual role for NMD raises the possibility that its magnitude is buffered to prevent the potentially catastrophic alterations in gene expression that would otherwise occur if NMD were perturbed by environmental or genetic insults. In support of this, here we report the existence of a negative feedback regulatory network that directly acts on seven NMD factors. Feedback regulation is conferred by different branches of the NMD pathway in a cell type-specific and developmentally regulated manner. We identify feedback-regulated NMD factors that are rate limiting for NMD and demonstrate that reversal of feedback regulation in response to NMD perturbation is crucial for maintaining NMD. Together, our results suggest the existence of an intricate feedback network that maintains both RNA surveillance and the homeostasis of normal gene expression in mammalian cells.


Assuntos
Estabilidade de RNA , RNA Mensageiro/metabolismo , Fator 3 Ativador da Transcrição/metabolismo , Western Blotting , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Células HeLa , Homeostase , Humanos , RNA Helicases , Interferência de RNA , Transativadores/antagonistas & inibidores
3.
Bioorg Med Chem Lett ; 24(21): 4969-75, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25262541

RESUMO

The discovery of a novel series of pyrrolopyrazines as JAK inhibitors with comparable enzyme and cellular activity to tofacitinib is described. The series was identified using a scaffold hopping approach aided by structure based drug design using principles of intramolecular hydrogen bonding for conformational restriction and targeting specific pockets for modulating kinase activity.


Assuntos
Janus Quinase 3/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/química , Pirróis/química , Desenho de Fármacos , Humanos , Janus Quinase 3/metabolismo , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Fosforilação , Piperidinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Relação Estrutura-Atividade
4.
Bioorg Med Chem Lett ; 23(9): 2522-6, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23541670

RESUMO

We report the discovery of a novel series of ATP-competitive Janus kinase 3 (JAK3) inhibitors based on the 5H-pyrrolo[2,3-b]pyrazine scaffold. The initial leads in this series, compounds 1a and 1h, showed promising potencies, but a lack of selectivity against other isoforms in the JAK family. Computational and crystallographic analysis suggested that the phenyl ether moiety possessed a favorable vector to achieve selectivity. Exploration of this vector resulted in the identification of 12b and 12d, as potent JAK3 inhibitors, demonstrating improved JAK family and kinase selectivity.


Assuntos
Janus Quinase 3/antagonistas & inibidores , Éteres Fenílicos/química , Inibidores de Proteínas Quinases/química , Piridazinas/química , Pirróis/química , Sítios de Ligação , Domínio Catalítico , Avaliação Pré-Clínica de Medicamentos , Janus Quinase 3/metabolismo , Simulação de Acoplamento Molecular , Éteres Fenílicos/síntese química , Éteres Fenílicos/metabolismo , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Relação Estrutura-Atividade
5.
Bioorg Med Chem Lett ; 23(12): 3565-9, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23664880

RESUMO

A novel series of indole/indazole-aminopyrimidines was designed and synthesized with an aim to achieve optimal potency and selectivity for the c-Jun kinase family or JNKs. Structure guided design was used to optimize the series resulting in a significant potency improvement. The best compound (17) has IC50 of 3 nM for JNK1 and 20 nM for JNK2, with greater than 40-fold selectivity against other kinases with good physicochemical and pharmacokinetic properties.


Assuntos
Indóis/química , Indóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Cristalografia por Raios X , Indazóis/química , Indazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/química , Fosforilação , Relação Estrutura-Atividade
6.
Bioorg Med Chem Lett ; 23(5): 1486-92, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23352510

RESUMO

A series of amino-pyrimidines was developed based upon an initial kinase cross-screening hit from a CDK2 program. Kinase profiling and structure-based drug design guided the optimization from the initial 1,2,3-benzotriazole hit to a potent and selective JNK inhibitor, compound 24f (JNK1 and 2 IC(50)=16 and 66 nM, respectively), with bioavailability in rats and suitable for further in vivo pharmacological evaluation.


Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Triazóis/química , Triazóis/farmacologia , Animais , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Inibidores de Proteínas Quinases/síntese química , Pirimidinas/síntese química , Ratos , Relação Estrutura-Atividade , Triazóis/síntese química
7.
Acta Crystallogr D Biol Crystallogr ; 68(Pt 8): 893-900, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22868754

RESUMO

Focused acoustic energy allows accurate and precise liquid transfer on scales from picolitre to microlitre volumes. This technology was applied in protein crystallization, successfully transferring a diverse set of proteins as well as hundreds of precipitant solutions from custom and commercial crystallization screens and achieving crystallization in drop volumes as small as 20 nl. Only higher concentrations (>50%) of 2-methyl-2,4-pentanediol (MPD) appeared to be systematically problematic in delivery. The acoustic technology was implemented in a workflow, successfully reproducing active crystallization systems and leading to the discovery of crystallization conditions for previously uncharacterized proteins. The technology offers compelling advantages in low-nanolitre crystallization trials by providing significant reagent savings and presenting seamless scalability for those crystals that require larger volume optimization experiments using the same vapor-diffusion format.


Assuntos
Cristalização , Cristalografia por Raios X/métodos , Acústica , Animais , Galinhas , Clara de Ovo/química , Glicóis/química , Transcriptase Reversa do HIV/química , Hepacivirus/metabolismo , Humanos , Muramidase/química , Nanopartículas , Nanotecnologia/métodos , Proteínas Tirosina Quinases/química , Proteínas/química , Albumina Sérica/química , Proteínas Virais/química , Viscosidade
8.
9.
Acta Crystallogr D Biol Crystallogr ; 66(Pt 5): 568-76, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20445232

RESUMO

A crystal seeding technique is introduced that uses acoustic waves to deliver nanolitre volumes of seed suspension into protein drops. The reduction in delivery volume enables enhanced crystal growth in matrix-seeding experiments without concern for bias from chemical components in the seed-carrying buffer suspension. Using this technique, it was found that while buffer components alone without seed can marginally promote crystal growth in some cases, crystal seeding is far more effective in boosting the number of sparse-matrix conditions that yield protein crystals.


Assuntos
Cristalização/métodos , Proteínas/química , Humanos
10.
EMBO Mol Med ; 6(12): 1518-20, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25378479

RESUMO

In this issue of EMBO Molecular Medicine, Bhuvanagiri et al report on a chemical means to convert molecular junk into gold. They identify a chemical inhibitor of a quality control pathway that is best known for its ability to clear cells of rubbish, but that in certain cases can be detrimental because it eliminates "useful" garbage. The chemical inhibitor identified by Bhuvanagiri et al perturbs Nonsense­Mediated RNA Decay (NMD), a RNA surveillance pathway that targets mRNAs harboring premature termination codons (PTCs) for degradation (Kervestin & Jacobson, 2012).


Assuntos
Azacitidina/farmacologia , Degradação do RNAm Mediada por Códon sem Sentido/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/metabolismo , Humanos
11.
Cell Rep ; 6(4): 748-64, 2014 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-24529710

RESUMO

The mechanisms dictating whether a cell proliferates or differentiates have undergone intense scrutiny, but they remain poorly understood. Here, we report that UPF1, a central component in the nonsense-mediated RNA decay (NMD) pathway, plays a key role in this decision by promoting the proliferative, undifferentiated cell state. UPF1 acts, in part, by destabilizing the NMD substrate encoding the TGF-ß inhibitor SMAD7 and stimulating TGF-ß signaling. UPF1 also promotes the decay of mRNAs encoding many other proteins that oppose the proliferative, undifferentiated cell state. Neural differentiation is triggered when NMD is downregulated by neurally expressed microRNAs (miRNAs). This UPF1-miRNA circuitry is highly conserved and harbors negative feedback loops that act as a molecular switch. Our results suggest that the NMD pathway collaborates with the TGF-ß signaling pathway to lock in the stem-like state, a cellular state that is stably reversed when neural differentiation signals that induce NMD-repressive miRNAs are received.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Neurais/metabolismo , Neurogênese , Degradação do RNAm Mediada por Códon sem Sentido , Animais , Sequência de Bases , Linhagem Celular Tumoral , Células Cultivadas , Retroalimentação Fisiológica , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Células-Tronco Neurais/citologia , Proteína Smad7/genética , Proteína Smad7/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Xenopus
12.
J Med Chem ; 56(1): 345-56, 2013 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-23214979

RESUMO

The Janus kinases (JAKs) are involved in multiple signaling networks relevant to inflammatory diseases, and inhibition of one or more members of this class may modulate disease activity or progression. We optimized a new inhibitor scaffold, 3-amido-5-cyclopropylpyrrolopyrazines, to a potent example with reasonable kinome selectivity, including selectivity for JAK3 versus JAK1, and good biopharmaceutical properties. Evaluation of this analogue in cellular and in vivo models confirmed functional selectivity for modulation of a JAK3/JAK1-dependent IL-2 stimulated pathway over a JAK1/JAK2/Tyk2-dependent IL-6 stimulated pathway.


Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Ciclopropanos/síntese química , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 3/antagonistas & inibidores , Pirazinas/síntese química , Pirróis/síntese química , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/farmacocinética , Anti-Inflamatórios não Esteroides/farmacologia , Células CACO-2 , Cristalografia por Raios X , Ciclopropanos/farmacocinética , Ciclopropanos/farmacologia , Técnicas de Silenciamento de Genes , Ensaios de Triagem em Larga Escala , Humanos , Interleucina-2/fisiologia , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Camundongos , Modelos Moleculares , Pirazinas/farmacocinética , Pirazinas/farmacologia , Pirróis/farmacocinética , Pirróis/farmacologia , RNA Interferente Pequeno/genética , Ratos , Receptores de Interleucina-6/fisiologia , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
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