RESUMO
The Agouti Related Protein (AGRP) is an endogenous antagonist of melanocortin-3 and -4 receptors, each of which plays a key role in body weight homeostasis. We designed a peptoid trimer based on AGRP 111-113 in which a single chiral atom is used to partially restrain the backbone structure. Peptoid 5 displaced both radiolabeled Nle4-alpha-MSH (IC(50)=3.1 microM) and AGRP (86-132) (IC(50)=1.9 microM) from the human melanocortin-4 receptor and functioned as an antagonist of alpha-MSH stimulated cAMP generation, thus providing an important lead in the development of AGRP mimetics.
Assuntos
Fragmentos de Peptídeos/química , Peptoides/química , Proteínas/química , Proteína Relacionada com Agouti , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Peptídeos e Proteínas de Sinalização Intercelular , Fragmentos de Peptídeos/farmacologia , Peptoides/farmacologia , Proteínas/fisiologia , Ensaio Radioligante , Receptores de Melanocortina/agonistas , Receptores de Melanocortina/antagonistas & inibidores , Receptores de Melanocortina/metabolismo , alfa-MSH/antagonistas & inibidores , alfa-MSH/farmacologiaRESUMO
A synthetic gene for the human motilin receptor containing 33 unique restriction sites was designed and stably coexpressed in HEK293 cells with the bioluminescent Ca(2+) indicator protein aequorin. The dose-dependent response of the receptor to motilin was demonstrated using transient transfections, and a stable cell line was selected. [(125)I]Motilin binding was used to estimate receptor expression level for the stable cell line, and titration of a membrane preparation indicated a K(d) value of 0.8 nM. The same cell line was used to evaluate a panel of erythromycin-derived agonists and provided EC(50) values for receptor activation that agree closely with data obtained in contractility assays. The peptide antagonist ANQ11125 (Phe3Leu13 motilin 1-14) inhibited motilin induced response with a K(i) value of 10 nM. The system is well-suited for the screening of compound libraries and receptor mutagenesis studies.
