RESUMO
Glucagon-like peptide 2 (GLP-2) is an intestinotropic peptide that binds to GLP-2 receptor (GLP-2R), a class-B G protein-coupled receptor. The GLP-2R antagonist GLP-2(3-33) has relatively high partial agonistic activity, and there are as yet no ideal known potent GLP-2R antagonists. We therefore prepared several truncated forms of human GLP-2 and characterized them by binding and reporter assays to find antagonists more potent than GLP-2(3-33). We found that GLP-2(11-33) was the most potent orthosteric GLP-2R antagonist, with binding activity almost equal to those of GLP-2 and GLP-2(3-33) and weaker intrinsic agonistic activity than GLP-2(3-33). GLP-2(11-33) retained weak agonistic activity toward human, cynomolgus monkey, dog, and Syrian hamster GLP-2Rs. However, it had no agonistic activity toward rat GLP-2R. GLP-2(11-33) potentiated the agonistic activity of an ago-allosteric modulator of GLP-2R, compound 1 (N-[1-(2,5-dichlorothiophen-3-yl)-2-(phenylsulfanyl)ethylidene]hydroxylamine), synergistically toward human GLP-2R. In the case of rat GLP-2R, GLP-2(11-33) decreased the agonistic activity of compound 1, although GLP-2 and GLP-2(3-33) increased this activity additively. These findings suggest that the binding sites of the ago-allosteric modulator and GLP-2 overlap, at least in rat GLP-2R. GLP-2(11-33) is a novel, useful tool for analyzing the mode of action of agonists and ago-allosteric modulators of GLP-2R.
Assuntos
Peptídeo 2 Semelhante ao Glucagon/química , Fragmentos de Peptídeos/química , Receptores de Glucagon/agonistas , Receptores de Glucagon/antagonistas & inibidores , Fosfatase Alcalina/metabolismo , Regulação Alostérica , Animais , Cricetinae , AMP Cíclico/metabolismo , Cães , Peptídeo 2 Semelhante ao Glucagon/genética , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 2 , Células HEK293 , Humanos , Hidroxilamina/síntese química , Hidroxilamina/farmacologia , Cinética , Macaca fascicularis , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Ratos , Receptores de Glucagon/metabolismo , Especificidade da Espécie , Tiofenos/síntese química , Tiofenos/farmacologiaRESUMO
Glucagon-like peptide 2 (GLP-2) is an intestinotropic peptide that binds to GLP-2 receptor (GLP- 2R), a class-B G protein-coupled receptor (GPCR) coupled with Gα(s). Few small-molecule agonists had been reported for class-B GPCRs, but we recently reported the first scaffold compounds of ago-allosteric modulators for human GLP-2R. Methyl 2-{[(2Z)-2-(2,5-dichlorothiophen- 3-yl)-2-(hydroxyimino)ethyl]sulfanyl}benzoate (compound 1) and its de-esterified derivative (compound 2) induced placental alkaline phosphatase (PLAP) activity in HEK293 cells overexpressing human GLP-2R and PLAP driven by cAMP response element. In this study, we observed that rat, Syrian hamster, and dog GLP-2Rs also responded to compounds 1 and 2 in the same reporter system. However, no agonistic activity of the compounds toward mouse GLP-2R was detected. Mutagenesis studies showed that mutant human GLP-2Rs with Pro392Leu substitution of mouse GLP-2R for human GLP-2R amino acid residues nullified the PLAP activity of compound 2, although these mutant receptors responded to GLP-2. This finding suggests that the Pro392 residue of human GLP-2R is essential for the agonistic activity of compound 2.
Assuntos
Peptídeo 2 Semelhante ao Glucagon/farmacologia , Receptores de Glucagon/agonistas , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Cães , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Receptor do Peptídeo Semelhante ao Glucagon 2 , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação , Ratos , Receptores de Glucagon/química , Receptores de Glucagon/genética , Alinhamento de Sequência , Especificidade da EspécieRESUMO
Glucagon-like peptide 2 (GLP-2) is an intestinotropic peptide that binds to GLP-2 receptor (GLP-2R), a class-B G protein-coupled receptor (GPCR). Few synthetic agonists have been reported so far for class-B GPCRs. Here, we report the first scaffold compounds of ago-allosteric modulators for human GLP-2R, derived from methyl 2-{[(2Z)-2-(2,5-dichlorothiophen-3-yl)-2-(hydroxyimino)ethyl]sulfanyl}benzoate (compound 1).
