RESUMEN
Traceable truncated Neuropeptide Y (NPY) analogues with Y1 receptor (Y1R) affinity and selectivity are highly desirable tools in studying receptor location, regulation, and biological functions. A range of fluorescently labeled analogues of a reported Y1R/Y4R preferring ligand BVD-15 have been prepared and evaluated using high content imaging techniques. One peptide, [Lys(2)(sCy5), Arg(4)]BVD-15, was characterized as an Y1R antagonist with a pKD of 7.2 measured by saturation analysis using fluorescent imaging. The peptide showed 8-fold lower affinity for Y4R (pKD = 6.2) and was a partial agonist at this receptor. The suitability of [Lys(2)(sCy5), Arg(4)]BVD-15 for Y1R and Y4R competition binding experiments was also demonstrated in intact cells. The nature of the label was shown to be critical with replacement of sCy5 by the more hydrophobic Cy5.5 resulting in a switch from Y1R antagonist to Y1R partial agonist.
Asunto(s)
Carbocianinas/química , Colorantes/química , Neuropéptido Y/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Receptores de Neuropéptido Y/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Células HEK293 , Humanos , Ligandos , Coloración y EtiquetadoRESUMEN
The cyclic dimeric peptide 1229U91 (GR231118) has an unusual structure and displays potent, insurmountable antagonism of the Y1 receptor. To probe the structural basis for this activity, we have prepared ring size variants and heterodimeric compounds, identifying the specific residues underpinning the mechanism of 1229U91 binding. The homodimeric structure was shown to be dispensible, with analogues lacking key pharmacophoric residues in one dimer arm retaining high antagonist affinity. Compounds 11d-h also showed enhanced Y1R selectivity over Y4R compared to 1229U91.
Asunto(s)
Neuropéptidos/química , Neuropéptidos/metabolismo , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Receptores de Neuropéptido Y/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Neuropéptidos/farmacología , Péptidos Cíclicos/farmacología , Multimerización de Proteína/efectos de los fármacos , Multimerización de Proteína/fisiología , Receptores de Neuropéptido Y/antagonistas & inhibidoresRESUMEN
The dimeric peptide 1 (BVD-74D, as a diastereomeric mixture) is a potent and selective neuropeptide Y Y4 receptor agonist. It represents a valuable candidate in developing traceable ligands for pharmacological studies of Y4 receptors and as a lead compound for antiobesity drugs. Its optically pure stereoisomers along with analogues and fluorescently labeled variants were prepared by exploiting alkene metathesis reactions. The (2R,7R)-diaminosuberoyl containing peptide, (R,R)-1, had markedly higher affinity and agonist efficacy than its (S,S)-counterpart. Furthermore, the sulfo-Cy5 labeled (R,R)-14 retained high agonist potency as a novel fluorescent ligand for imaging Y4 receptors.