RESUMEN
P2Y14 is a member of the pyrimidinergic GPCR family. UDP-Glc has been previously shown to activate human P2Y14, whereas UDP was unable to activate the receptor. In this study, the authors used conventional and nonconventional methods to further characterize P2Y14 and its ligands. Conventional calcium mobilization and nonconventional cellular impedance functional assays revealed that UMP and UDP selectively activated HEK cells coexpressing P2Y14 and Gα(qi5). In the impedance assays, the presence of exogenous Gα(qi5) resulted in agonist-induced Gq signaling, whereas in the absence of exogenous Gα(qi5), the signal was indicative of Gi. The authors established the first P2Y14 membrane filtration binding assay using a novel optimized expression vector and [(3)H]UDP as radioligand. UDP-Glc, UMP, and UDP dose dependently inhibited [(3)H]UDP binding in the binding assay, and saturation analysis revealed that UDP bound P2Y14 with a K(D) = 10 nM and a B(max) = 110 pmol/mg. The authors screened a phosphonate library and identified compound A, which inhibited UDP-Glc-mediated calcium signaling in the fluorometric imaging plate reader assay (IC(50) = 2.3 µM) and competed for [(3)H]UDP binding in the novel binding assay with a K(i) = 1280 nM.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Agonistas del Receptor Purinérgico P2/farmacología , Antagonistas del Receptor Purinérgico P2/farmacología , Receptores Purinérgicos P2/metabolismo , Animales , Línea Celular Transformada , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Ligandos , Ratones , Pan troglodytes , Unión Proteica , Receptores Purinérgicos P2/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Two new series of EP(4) antagonists based on naphthalene/quinoline scaffolds have been identified as part of our on-going efforts to develop treatments for inflammatory pain. One series contains an acidic sulfonylurea pharmacophore, whereas the other is a neutral amide. Both series show subnanomolar intrinsic binding potency towards the EP(4) receptor, and excellent selectivity towards other prostanoid receptors. While the amide series generally displays poor pharmacokinetic parameters, the sulfonylureas exhibit greatly improved profile. MF-592, the optimal compound from the sulfonylurea series, has a desirable overall preclinical profile that suggests it is suitable for further development.
Asunto(s)
Amidas/química , Indoles/química , Naftalenos/química , Quinolinas/química , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Compuestos de Sulfonilurea/química , Amidas/síntesis química , Amidas/farmacocinética , Animales , Perros , Evaluación Preclínica de Medicamentos , Humanos , Indoles/síntesis química , Indoles/farmacocinética , Ratas , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Relación Estructura-Actividad , Compuestos de Sulfonilurea/síntesis química , Compuestos de Sulfonilurea/farmacocinéticaRESUMEN
A genetic contribution to asthma susceptibility is well recognized, and linkage studies have identified a large number of genes associated with asthma pathogenesis. Recently, a locus encoding a seven-transmembrane protein was shown to be associated with asthma in founder populations. The expression of the protein GPRA (G protein-coupled receptor for asthma susceptibility) in human airway epithelia and smooth muscle, and its increased expression in a mouse model of asthma, suggested that a gain-of-function mutation in this gene increased the disease risk. However, we report here that the development of allergic lung disease in GPRA-deficient mice is unaltered. A possible explanation for this finding became apparent upon reexamination of the expression of this gene. In contrast to initial studies, our analyses failed to detect expression of GPRA in human lung tissue or in mice with allergic lung disease. We identify a single parameter that distinguishes GPRA-deficient and wild-type mice. Whereas the change in airway resistance in response to methacholine was identical in control and GPRA-deficient mice, the mutant animals showed an attenuated response to thromboxane, a cholinergic receptor-dependent bronchoconstricting agent. Together, our studies fail to support a direct contribution of GPRA to asthma pathogenesis. However, our data suggest that GPRA may contribute to the asthmatic phenotype by altering the activity of other pathways, such as neurally mediated mechanisms, that contribute to disease. This interpretation is supported by high levels of GPRA expression in the brain and its recent identification as the neuropeptide S receptor.