RESUMO
Using Cu(I)-catalyzed azide-alkyne cycloaddition in a mixed classical organic phase and solid phase peptide synthesis approach, we synthesized four analogs of Leu-enkephalin to systematically replace amides by 1,4-disubstituted[1,2,3]triazoles. The peptidomimetics obtained were characterized by competitive binding, contractility assays and ERK1/2 phosphorylation. The present study reveals that the analog bearing a triazole between Phe and Leu retains some potency, more than all the others, suggesting that the hydrogen bond acceptor capacity of the last amide of Leu-enkephalin is essential for the biological activity of the peptide.
Assuntos
Amidas/química , Encefalina Leucina/química , Receptores Opioides delta/química , Triazóis/química , Ligação Competitiva , Encefalina Leucina/farmacologia , Ligação de Hidrogênio , Concentração Inibidora 50 , Estrutura Molecular , Peptidomiméticos , Receptores Opioides delta/efeitos dos fármacosRESUMO
Leu-enkephalin analogues, in which the amide bonds were sequentially and systematically replaced either by ester or N-methyl amide bonds, were prepared using classical organic chemistry as well as solid phase peptide synthesis (SPPS). The peptidomimetics were characterized using competition binding, ERK1/2 phosphorylation, receptor internalization, and contractility assays to evaluate their pharmacological profile over the delta opioid receptor (DOPr). The lipophilicity (LogD7.4) and plasma stability of the active analogues were also measured. Our results revealed that the last amide bond can be successfully replaced by either an ester or an N-methyl amide bond without significantly decreasing the biological activity of the corresponding analogues when compared to Leu-enkephalin. The peptidomimetics with an N-methyl amide function between residues Phe and Leu were found to be more lipophilic and more stable than Leu-enkephalin. Findings from the present study further revealed that the hydrogen-bond donor properties of the fourth amide of Leu-enkephalin are not important for its biological activity on DOPr. Our results show that the systematic replacement of amide bonds by isosteric functions represents an efficient way to design and synthesize novel peptide analogues with enhanced stability. Our findings further suggest that such a strategy can also be useful to study the biological roles of amide bonds.
Assuntos
Encefalina Leucina/análogos & derivados , Peptidomiméticos/farmacologia , Receptores Opioides delta/metabolismo , Amidas/farmacologia , Animais , Depsipeptídeos/síntese química , Depsipeptídeos/farmacologia , Encefalina Leucina/síntese química , Ésteres/síntese química , Ésteres/farmacologia , Ligação de Hidrogênio , Masculino , Camundongos , Peptidomiméticos/síntese química , Receptores Opioides delta/efeitos dos fármacos , Técnicas de Síntese em Fase Sólida/métodos , Ducto Deferente/efeitos dos fármacosRESUMO
The role of each of the four amide bonds in Leu(5)-enkephalin was investigated by systematically and sequentially replacing each with its corresponding trans-alkene. Six Leu(5)-enkephalin analogs based on six dipeptide surrogates and two Met(5)-enkephalin analogs were synthesized and thoroughly tested using a δ-opioid receptor internalization assay, an ERK1/2 activation assay, and a competition binding assay to evaluate their biological properties. We observed that an E-alkene can efficiently replace the first amide bond of Leu(5)- and Met(5)-enkephalin without significantly affecting biological activity. By contrast, the second amide bond was found to be highly sensitive to the same modification, suggesting that it is involved in biologically essential intra- or intermolecular interactions. Finally, we observed that the affinity and activity of analogs containing an E-alkene at either the third or fourth position were partially reduced, indicating that these amide bonds are less important for these intra- or intermolecular interactions. Overall, our study demonstrates that the systematic and sequential replacement of amide bonds by E-alkene represents an efficient way to explore peptide backbones.