RESUMEN
We present two high-throughput compatible methods to detect the interaction of ectopically expressed (RT-Bind) or endogenously tagged (EndoBind) proteins of interest. Both approaches provide temporal evaluation of dimer formation over an extended duration. Using examples of the Nrf2-KEAP1 and the CRAF-KRAS-G12V interaction, we demonstrate that our method allows for the detection of signal for more than 2 days after substrate addition, allowing for continuous monitoring of endogenous protein-protein interactions in real time.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Proteína 1 Asociada A ECH Tipo Kelch/química , Factor 2 Relacionado con NF-E2/química , Proteínas Proto-Oncogénicas p21(ras)/química , Células HEK293 , Humanos , Unión ProteicaRESUMEN
Autoimmune deficiency and destruction in either ß-cell mass or function can cause insufficient insulin levels and, as a result, hyperglycemia and diabetes. Thus, promoting ß-cell proliferation could be one approach toward diabetes intervention. In this report we describe the discovery of a potent and selective DYRK1A inhibitor GNF2133, which was identified through optimization of a 6-azaindole screening hit. In vitro, GNF2133 is able to proliferate both rodent and human ß-cells. In vivo, GNF2133 demonstrated significant dose-dependent glucose disposal capacity and insulin secretion in response to glucose-potentiated arginine-induced insulin secretion (GPAIS) challenge in rat insulin promoter and diphtheria toxin A (RIP-DTA) mice. The work described here provides new avenues to disease altering therapeutic interventions in the treatment of type 1 diabetes (T1D).
Asunto(s)
Compuestos Aza/química , Compuestos Aza/farmacología , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Indoles/química , Indoles/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Compuestos Aza/farmacocinética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Humanos , Hipoglucemiantes/farmacocinética , Indoles/farmacocinética , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Simulación del Acoplamiento Molecular , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Quinasas DyrKRESUMEN
NOD2 (nucleotide-binding oligomerization domain-containing protein 2) is an internal pattern recognition receptor that recognizes bacterial peptidoglycan and stimulates host immune responses. Dysfunction of NOD2 pathway has been associated with a number of autoinflammatory disorders. To date, direct inhibitors of NOD2 have not been described due to technical challenges of targeting the oligomeric protein complex. Receptor interacting protein kinase 2 (RIPK2) is an intracellular serine/threonine/tyrosine kinase, a key signaling partner, and an obligate kinase for NOD2. As such, RIPK2 represents an attractive target to probe the pathological roles of NOD2 pathway. To search for selective RIPK2 inhibitors, we employed virtual library screening (VLS) and structure based design that eventually led to a potent and selective RIPK2 inhibitor 8 with excellent oral bioavailability, which was used to evaluate the effects of inhibition of RIPK2 in various in vitro assays and ex vivo and in vivo pharmacodynamic models.
RESUMEN
The current treatment regimens for HIV include over 20 anti-retrovirals. However, adverse drug effects and the emergence of drug resistance necessitates the continued improvement of the existing drug classes as well as the development of novel drugs that target as yet therapeutically unexploited viral and cellular pathways. Here we demonstrate a strategy for the discovery of protein-protein interaction inhibitors of the viral pathogenicity factor HIV-1 Nef and its interaction with the host factor SH3. A combination of a time-resolved fluorescence resonance energy resonance energy transfer-based assay and a label-free resonant waveguide grating-based assay was optimized for high-throughput screening formats.
RESUMEN
Two series of fused tricyclic indoles were identified as potent and selective S1P(1) agonists. In vivo these agonists produced a significant reduction in circulating lymphocytes which translated into robust efficacy in several rodent models of autoimmune disease. Importantly, these agonists were devoid of any activity at the S1P(3) receptor in vitro, and correspondingly did not produce S1P(3) mediated bradycardia in telemeterized rat.
Asunto(s)
Factores Inmunológicos/química , Indoles/química , Receptores de Lisoesfingolípidos/agonistas , Animales , Enfermedades Autoinmunes/tratamiento farmacológico , Modelos Animales de Enfermedad , Femenino , Humanos , Factores Inmunológicos/farmacocinética , Factores Inmunológicos/uso terapéutico , Indoles/farmacocinética , Indoles/uso terapéutico , Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Lisoesfingolípidos/metabolismo , Relación Estructura-ActividadRESUMEN
S1P(1) receptor driven lymphopenia has proven utility in the treatment of an array of autoimmune disease states. As a part of our efforts to develop potent and selective S1P(1) receptor agonists, we have identified a novel chemical series of 4-oxo-4-(5-(5-phenyl-1,2,4-oxadiazol-3-yl)indolin-1-yl)butanoic acid S1P(1) receptor agonists.