RESUMEN
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.
Asunto(s)
Desarrollo Embrionario , Genes Duplicados , Degradación de ARNm Mediada por Codón sin Sentido , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular Tumoral , Evolución Molecular , Gametogénesis , Células HeLa , Humanos , RatonesRESUMEN
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.
Asunto(s)
Estabilidad del ARN , ARN Mensajero/metabolismo , Factor de Transcripción Activador 3/metabolismo , Western Blotting , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Células HeLa , Homeostasis , Humanos , ARN Helicasas , Interferencia de ARN , Transactivadores/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Janus Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/química , Pirroles/química , Diseño de Fármacos , Humanos , Janus Quinasa 3/metabolismo , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Fosforilación , Piperidinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Janus Quinasa 3/antagonistas & inhibidores , Éteres Fenílicos/química , Inhibidores de Proteínas Quinasas/química , Piridazinas/química , Pirroles/química , Sitios de Unión , Dominio Catalítico , Evaluación Preclínica de Medicamentos , Janus Quinasa 3/metabolismo , Simulación del Acoplamiento Molecular , Éteres Fenílicos/síntesis química , Éteres Fenílicos/metabolismo , Unión Proteica , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Indoles/química , Indoles/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Cristalografía por Rayos X , Indazoles/química , Indazoles/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/química , Fosforilación , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Triazoles/química , Triazoles/farmacología , Animales , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Modelos Moleculares , Inhibidores de Proteínas Quinasas/síntesis química , Pirimidinas/síntesis química , Ratas , Relación Estructura-Actividad , Triazoles/síntesis químicaRESUMEN
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.
Asunto(s)
Cristalización , Cristalografía por Rayos X/métodos , Acústica , Animales , Pollos , Clara de Huevo/química , Glicoles/química , Transcriptasa Inversa del VIH/química , Hepacivirus/metabolismo , Humanos , Muramidasa/química , Nanopartículas , Nanotecnología/métodos , Proteínas Tirosina Quinasas/química , Proteínas/química , Albúmina Sérica/química , Proteínas Virales/química , ViscosidadRESUMEN
A novel series of highly selective JNK inhibitors based on the 4-quinolone scaffold was designed and synthesized. Structure based drug design was utilized to guide the compound design as well as improvements in the physicochemical properties of the series. Compound (13c) has an IC(50) of 62/170 nM for JNK1/2, excellent kinase selectivity and impressive efficacy in a rodent asthma model.
Asunto(s)
4-Quinolonas/farmacología , Descubrimiento de Drogas , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , 4-Quinolonas/síntesis química , 4-Quinolonas/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Cristalización/métodos , Proteínas/química , HumanosRESUMEN
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).
Asunto(s)
Azacitidina/farmacología , Degradación de ARNm Mediada por Codón sin Sentido/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/metabolismo , HumanosRESUMEN
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.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Células-Madre Neurales/metabolismo , Neurogénesis , Degradación de ARNm Mediada por Codón sin Sentido , Animales , Secuencia de Bases , Línea Celular Tumoral , Células Cultivadas , Retroalimentación Fisiológica , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Datos de Secuencia Molecular , Células-Madre Neurales/citología , Proteína smad7/genética , Proteína smad7/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , XenopusRESUMEN
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.