RESUMO
Preptin, a 34-amino acid residue peptide hormone is co-secreted with insulin from the ß-pancreatic cells and is active in fuel metabolism. We have previously established that a shorter fragment of preptin, namely preptin-(116), stimulates bone growth by proliferation and increasing the survival rate of osteoblasts. This was demonstrated in both in vitro and in vivo models. These findings suggest that preptin-(116) could play an important role in the anabolic therapy of osteoporosis. However, due to the large size of the peptide it is not an ideal therapeutic agent. The aim of this study was to identify the shortest preptin analogue that retains or even increases the bone anabolic activity as compared to the parent preptin-(116) peptide. Truncations were made in a methodical manner from both the N-terminus and the C-terminus of the peptide, and the effect of these deletions on the resulting biological activity was assessed. In order to improve the enzymatic stability of the shortest yet active analogue identified, ruthenium-catalysed ring closing metathesis was used to generate a macrocyclic peptide using allylglycine residues as handles for ring formation. We have successfully identified a short 8-amino acid preptin (18) fragment that retains an anabolic effect on the proliferation of primary rat osteoblasts and enhances bone nodule formation. Preptin (18) is a useful lead compound for the development of orally active therapeutics for the treatment of osteoporosis.
Assuntos
Fator de Crescimento Insulin-Like II/farmacologia , Osteoblastos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fator de Crescimento Insulin-Like II/síntese química , Fator de Crescimento Insulin-Like II/química , Estrutura Molecular , Osteoblastos/citologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Ratos , Relação Estrutura-AtividadeRESUMO
Peptide hormones that modulate insulin secretion have been recognized to have therapeutic potential, with peptides such as amylin (pramlintide acetate, Symlin) and exendin-4 (exenatide, Byetta) now commercially available. Preptin is a peptide that has been shown to increase insulin secretion in vitro and in vivo. Here, we describe the first chemical synthesis and analysis of a short series of preptin analogues based on the rat preptin sequence. Phe 21 in the preptin sequence was substituted with the non-protein amino acids D-Phe, D-Hphe, 3-aminobenzoic acid and 1-aminocyclooctane-1-carboxylic acid, which rendered the preptin analogues resistant to chymotryptic protease hydrolysis at this position. Substitution of Phe 21 with these non-protein amino acids did not abrogate the insulin secretory effect of preptin, with analogues showing a similar dose-dependent effect on insulin secretion from ßTC6-F7 mouse ß-cells in both the presence and absence of glucose as unmodified rat preptin. Further studies on the stability of the preptin analogues and their effect on insulin secretion are in progress.
Assuntos
Fator de Crescimento Insulin-Like II/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Humanos , Fator de Crescimento Insulin-Like II/síntese química , Fator de Crescimento Insulin-Like II/farmacologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Estrutura Secundária de Proteína , RatosAssuntos
Proteínas de Transporte/metabolismo , Fator de Crescimento Insulin-Like II/análogos & derivados , Fator de Crescimento Insulin-Like II/síntese química , Receptor IGF Tipo 2/metabolismo , Sequência de Aminoácidos , Líquido Amniótico/metabolismo , Animais , Linhagem Celular , Feminino , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Fator de Crescimento Insulin-Like II/química , Cinética , Masculino , Dados de Sequência Molecular , Placenta/metabolismo , Gravidez , Próstata , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/metabolismoAssuntos
Fator de Crescimento Epidérmico/síntese química , Engenharia Genética , Fator de Crescimento Insulin-Like II/síntese química , Animais , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/isolamento & purificação , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/isolamento & purificação , Plasmídeos , Sinais Direcionadores de Proteínas , Recombinação GenéticaRESUMO
Human insulin-like growth factor II (IGF-II) with 67 amino acids and three disulfide bridges has been synthesized by the solid-phase method. The synthetic hormone is shown to be homogeneous in high performance liquid chromatography (HPLC), high performance partition chromatography (HPPC), and chromatofocusing. It is indistinguishable from natural hormone in HPLC, peptide map of thermolysin digests, amino acid composition and radioreceptor binding assay. Thus, synthetic and natural IGF-II are identical.