Structural requirements for B2-agonists with improved degradation stability.

Studies on bradykinin (BK) have been impeded by the fact that this peptide is rapidly degraded by various kininases. Modifications enacted to stabilize the BK sequence have usually resulted in a loss of agonistic activity. In this study, new structural modifications were investigated with the aim to identify degradation-resistant agonists on the bradykinin B2-receptor. The efficacy and degradation stability of several potentially agonistic derivatives were examined using a B2-receptor model (FURA-stained rat fibroblasts) and rat serum kininases. Modifications of the investigated BK analogues included amino-terminal (D-Arg) or carboxy-terminal (Ile-Tyr) prolongation, various substitutions at positions 2, 5, 7, 8 (tetrahydroisoquinoline-3-carboxylic acid, octahydroindole-2-carboxylic acid, hydroxy-proline, beta-2-thienylalanine, 2,3-dehydro-phenylalanine, erythro-beta-phenylserine, erythro-alpha-amino-beta-phenyl-butyric acid, N-methyl-phenylalanine), or intramolecular cyclization via lactam bridges. Kinin inactivation was investigated in rat serum, where the activities of angiotensin I-converting enzyme (ACE), carboxypeptidase N (CPN), aminopeptidase P (APP) and aminopeptidase M (APM) could be differentiated by selective inhibitors. Analogues derived from phyllokinin (BK-Ile-Tyr-SO4) and cyclic peptides had no receptor affinity. Useful modifications compatible with agonistic activity included D-Arg0 (protects against APP), D-N-methyl-Phe7 and dehydro-Phe5 (protect against ACE), and erythro-phenylserine or erythro-amino-phenyl-butyric acid at position 8 (protect against ACE and CPN). Finally, the kinin derivatives D-Arg0-[Hyp3, Thi5, epsilonSer(betaPh)8]-BK and D-Arg0-[Hyp3, Thi5, epsilonAbu(betaPh)8]-BK proved to be potent B2-agonists with extensive stability against rat serum kininases.