Highly Selective Bradykinin Agonists and Antagonists with Replacement of Proline Residues byN-Methyl-d- andl-phenylalanine

Abstract

For further studies on the structural and conformational requirements of positions 2,3, and 7 in the bradykinin sequence, we replaced the proline residues by the more hydrophobic and conformationally restricted N-methyl-L- and D-phenylalanine (NMF). The biological activities of the new analogs were evaluated on rat uterus, guinea pig ileum, and guinea pig lung strip. Receptor binding of the analogs was studied in membranes from rat uterus and guinea pig ileum. Influence of bradykinin analogs on the release of cytokines from mouse spleen cell cultures was also measured. Bradykinin analogs were synthesized by the solid phase method, using Boc strategy on PAM or Merrifield resins. The best results in the formation of the N-methylamide bond were obtained with the coupling reagent PyBrop. In position 7 the substitution of D-Phe by D-NMF, retaining the configuration of the amino acid, converts bradykinin antagonists into agonists. The bradykinin analogs with D-NMF at position 7 gave the highest known tissue selectivity for rat uterus among agonists. [L-NMF(2)]bradykinin has moderate agonist activity on rat uterus but antagonist activity on guinea pig lung strip. It represents a new antagonist for B(2) receptors without any replacement at position 7. The same analog completely inhibits bradykinin-evoked cytokine expression by mononuclear cells.

Potentiation of Kinin Analogues by Ramiprilat Is Exclusively Related to Their Degradation

“…[ 5 ]-BK were synthesized by a solid-phase method by means of the Boc-strategy. 15 The specific activation of B 2 -receptors by these kinin analogues has been reported. 11,16,17 Ramiprilat was kindly donated by Hoechst Marion Roussel.…”

Amino Acid-Protecting Groups

“…414,415 Nevertheless, there are also some reports of the succesful use of unprotected Ser in solution phase synthesis, but care must be taken when choosing the activating agents. 416,417 In peptide synthesis, hydroxyl funcionalities are protected as ethers, which are more stable than the corresponding carbamates and esters.…”

Identification of Bombesin Receptor Subtype-Specific Ligands: Effect ofN-Methyl Scanning, Truncation, Substitution, and Evaluation of Putative Reported Selective Ligands

“…Because with a number of other receptors the insertion of N-methyl groups in ligands results in subtype-selective analogs (Lin et al, 1990;Reissmann et al, 1996;Pradhan et al, 1998) (Mantey et al, 1997(Mantey et al, , 2001Pradhan et al, 1998;Ryan et al, 1998a) (Benya et al, 1995;Ryan et al, 1998a,b -stimulated values were 1510 Ϯ 98 and 8200 Ϯ 210 dpm, respectively, for hGRPR; 1950 Ϯ 82 and 22820 Ϯ 540 dpm, respectively, for hNMBR; and 2510 Ϯ 120 and 12600 Ϯ 2305 dpm, respectively, for hBRS-3. Results are the mean Ϯ S.E.M.…”

“…N-Methyl substitutions should introduce conformational restriction, disrupt hydrogen bonding, and thus have pronounced effects on the conformation of the COOH terminus (Lin et al, 1995(Lin et al, , 1996. Substitution of N-methyl groups to produce conformationally restricted analogs has been widely used with analogs of somatostatin (Rajeswaran et al, 2001), bradykinin (Reissmann et al, 1996), cholecystokinin (Pradhan et al, 1998), endothelin (Cody et al, 1997), tachykinins (Wormser et al, 1986), glucose-dependent insulinotropic polypeptide (Hinke et al, 2003), enkephalins (Penkler et al, 1993), angiotensin (Khosla et al, 1976), insulin (Ogawa et al, 1987), and galanin (Rivera et al, 1994). N-Methyl substitution can result in analogs with enhanced selectivity, potency, or enhanced stability (Wormser et al, 1986;Lin et al, 1990;Schmidt et al, 1995;Cody et al, 1997;Pradhan et al, 1998).…”

“…]Bn(6 -14), because this analog has been shown to be metabolically stable and to have high affinity and potency for activating all Bn receptor subtypes (Mantey et al, 1997;Pradhan et al, 1998;Ryan et al, 1998a,b;Reubi et al, 2002;Moody et al, 2004). This approach was used because it has successfully yielded highly selective peptide ligands with a number of other receptors (Reissmann et al, 1996;Pradhan et al, 1998;Rajeswaran et al, 2001 (6 -14) that were N-terminally truncated or had selective substitutions, because this was reported to result in increasing selectivity for one type of Bn receptor in a recent study (Darker et al, 2001). The final approach used was to synthesize various shortened , ␤Ala ]Bn (6 -14) analogs recently reported to be selective for a Bn receptor subtype and fully characterize them pharmacologically by binding studies and assays assessing receptor activation at each of the three human Bn receptor subtypes.…”