RESUMEN
Single-stranded silencing RNAs (ss siRNA), while not as potent as duplex RNAs, have the potential to become a novel platform technology in RNA interference based gene silencing by virtue of their simplicity and plausibly favorable characteristics in pharmacokinetics and biodistribution. Like other therapeutic pharmaceutical agents, ss siRNA can be optimized to achieve higher potency through a structure-activity based approach. Systematic chemical modification at each position of a 21-mer oligonucleotide identified 2',5'-linked 3'-deoxythymidine (3dT) at position 1 and locked nucleic acids (LNAs) at the seed region as key components to afford significant enhancement in knockdown activity both in vitro and in vivo. Further optimization by additional chemical modifications should enable ss siRNA as an alternative gene silencing modality.
Asunto(s)
Silenciador del Gen , ARN Mensajero/genética , ARN Interferente Pequeño/genética , beta Catenina/genética , Células HEK293 , HumanosRESUMEN
The inhibition of KSP causes mitotic arrest by activating the spindle assembly checkpoint. While transient inhibition of KSP leads to reversible mitotic arrest, prolonged exposure to a KSP inhibitor induces apoptosis. Induction of apoptosis by the KSP inhibitor couples with mitotic slippage. Slippage-refractory cells show resistance to KSP inhibitor-mediated lethality, whereas promotion of slippage after mitotic arrest enhances apoptosis. However, attenuation of the spindle checkpoint confers resistance to KSP inhibitor-induced apoptosis. Furthermore, sustained KSP inhibition activates the proapoptotic protein, Bax, and both activation of the spindle checkpoint and subsequent mitotic slippage are required for Bax activation. These studies indicate that in response to KSP inhibition, activation of the spindle checkpoint followed by mitotic slippage initiates apoptosis by activating Bax.
Asunto(s)
Apoptosis , Genes cdc/fisiología , Cinesinas/antagonistas & inhibidores , Mitosis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Huso Acromático/fisiología , Caspasa 3 , Caspasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Resistencia a Medicamentos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Estructura Molecular , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Pirroles/farmacología , Proteína X Asociada a bcl-2RESUMEN
Mouse models with liver-specific expression of firefly luciferase were developed that enable a noninvasive and longitudinal assessment of small-interfering RNA (siRNA)-mediated gene silencing in hepatocytes of live animals via bioluminescence imaging. Using these models, a set of lipid nanoparticles (LNPs) with different compositions of cationic lipids, polyethylene glycol (PEG), and cholesterol, were tested for their abilities in delivering a luciferase siRNA to the liver via systemic administration. A dose-dependent luciferase knockdown by LNP/siRNA assemblies was measured by in vivo bioluminescence imaging, which correlated well with the results from parallel ex vivo analyses of luciferase mRNA and protein levels in the liver. RNA interference (RNAi)-mediated target silencing was further confirmed by the detection of RNAi-specific target mRNA cleavage. A single dose of LNP02L at 3 mg/kg (siRNA) caused 90% reduction of luciferase expression and the target repression lasted for at least 10 days. With identical components, LNPs containing 2% PEG are more potent than those with 5.4% PEG. Our results demonstrate that these liver-luciferase mouse models provide a powerful tool for a high-throughput evaluation of hepatic delivery platforms by noninvasive imaging and that the molar ratio of PEG lipid can affect the efficacy of LNPs in silencing liver targets via systemic administration.
Asunto(s)
Lípidos/química , Hígado/metabolismo , Nanopartículas/administración & dosificación , Nanopartículas/química , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Animales , Técnica del Anticuerpo Fluorescente , Silenciador del Gen/fisiología , Hígado/enzimología , Luciferasas/genética , RatonesRESUMEN
The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/biosíntesis , Apoptosis/fisiología , Caspasa 3/metabolismo , Cinesinas/fisiología , Proteínas Proto-Oncogénicas/biosíntesis , Proteína p53 Supresora de Tumor/fisiología , Proteína X Asociada a bcl-2/biosíntesis , Antibióticos Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Doxorrubicina/farmacología , Humanos , Cinesinas/antagonistas & inhibidores , Paclitaxel/farmacología , Procesamiento Proteico-Postraduccional , Transducción de Señal , Huso AcromáticoRESUMEN
Growth differentiation factor 15 (GDF15), a distant member of the transforming growth factor (TGF)-ß family, is a secreted protein that circulates as a 25-kDa dimer. In humans, elevated GDF15 correlates with weight loss, and the administration of GDF15 to mice with obesity reduces body weight, at least in part, by decreasing food intake. The mechanisms through which GDF15 reduces body weight remain poorly understood, because the cognate receptor for GDF15 is unknown. Here we show that recombinant GDF15 induces weight loss in mice fed a high-fat diet and in nonhuman primates with spontaneous obesity. Furthermore, we find that GDF15 binds with high affinity to GDNF family receptor α-like (GFRAL), a distant relative of receptors for a distinct class of the TGF-ß superfamily ligands. Gfral is expressed in neurons of the area postrema and nucleus of the solitary tract in mice and humans, and genetic deletion of the receptor abrogates the ability of GDF15 to decrease food intake and body weight in mice. In addition, diet-induced obesity and insulin resistance are exacerbated in GFRAL-deficient mice, suggesting a homeostatic role for this receptor in metabolism. Finally, we demonstrate that GDF15-induced cell signaling requires the interaction of GFRAL with the coreceptor RET. Our data identify GFRAL as a new regulator of body weight and as the bona fide receptor mediating the metabolic effects of GDF15, enabling a more comprehensive assessment of GDF15 as a potential pharmacotherapy for the treatment of obesity.
Asunto(s)
Ingestión de Alimentos/efectos de los fármacos , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor 15 de Diferenciación de Crecimiento/genética , Obesidad/metabolismo , Pérdida de Peso/efectos de los fármacos , Animales , Dieta Alta en Grasa , Ingestión de Alimentos/genética , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Factor 15 de Diferenciación de Crecimiento/metabolismo , Factor 15 de Diferenciación de Crecimiento/farmacología , Humanos , Macaca fascicularis , Ratones , Ratones Noqueados , Pérdida de Peso/genéticaRESUMEN
The evolution of 2,4-diaryl-2,5-dihydropyrroles as inhibitors of KSP is described. Introduction of basic amide and urea moieties to the dihydropyrrole nucleus enhanced potency and aqueous solubility, simultaneously, and provided compounds that caused mitotic arrest of A2780 human ovarian carcinoma cells with EC(50)s<10nM. Ancillary hERG activity was evaluated for this series of inhibitors.