Novel sst(4)-selective somatostatin (SRIF) agonists. 1. Lead identification using a betide scan.
Hypothesizing that structural constraints in somatostatin (SRIF) analogues may result in receptor selectivity, and aiming to characterize the bioactive conformation of somatostatin at each of its five receptors, we carried out an N(beta)-methylated aminoglycine (Agl) scan of the octapeptide H-c[Cys(3)-Phe(6)-Phe(7)-dTrp(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)]-OH (SRIF numbering) (ODT-8) that is potent at all SRIF receptor subtypes (sst's) but sst(1). We found that H-c[Cys-LAgl(N(beta)Me,benzoyl)-Phe-DTrp-Lys-Thr-Phe-Cys]-OH (4), H-c[Cys-Phe-LAgl(N(beta)Me,benzoyl)-Trp-Lys-Thr-Phe-Cys]-OH (6), H-c[Cys-Phe-LAgl(N(beta)Me,benzoyl)-dTrp-Lys-Thr-Phe-Cys]-OH (8), and H-c[DCys-Phe-LAgl(N(beta)Me,benzoyl)-DTrp-Lys-Thr-Phe-Cys]-OH (10) had high affinity (IC(50) = 14.3, 5.4, 5.2, and 3.4 nM, respectively) and selectivity for sst(4) (>50-fold over the other receptors). The l-configuration at positions 7 and 8 (l(7), l(8)) yields greater sst(4) selectivity than the l(7), d(8) configuration (6 versus 8). Peptides with the d(7), l(8) (7) and d(7), d(8) (9) configurations are significantly less potent at all receptors. H-c[Cys-Phe-Phe-DTrp-LAgl(betaAla)-Thr-Phe-Cys]-OH (16), H-c[Cys-Phe-Phe-DTrp-DAgl(betaAla)-Thr-Phe-Cys]-OH (17), and their N(beta)Me derivatives at position 9 (18, 19) were essentially inactive. Potent but less sst(4)-selective were members of the Agl-scan at positions 10, H-c[Cys-Phe-Phe-dTrp-Lys-lAgl(N(beta)Me,HO-Ac)-Phe-Cys]-OH (20, IC(50) = 6.5 nM), and 11, H-c[Cys-Phe-Phe-DTrp-Lys-Thr-LAgl(N(beta)Me,benzoyl)-Cys]-OH (22, IC(50) = 6.9 nM), while the d-configuration at positions 10 (21) and 11 (23) led to reduced affinity. One of our best analogues, 8, is an agonist when tested for its ability to inhibit forskolin-stimulated cAMP accumulation in sst(4)-transfected CCL39 cells (EC(50) = 1.01 nM). All Agl-containing analogues were first synthesized using unresolved Fmoc-Agl(N(beta)Me,Boc)-OH, and the diastereomers were separated using HPLC. Chiral assignment at the Agl-containing residue was subsequently done using enzymatic degradation and by de novo synthesis in the cases of H-c[Cys-Phe-DAgl(N(beta)Me,benzoyl)-DTrp-Lys-Thr-Phe-Cys]-OH (9) and H-c[DCys-Phe-DAgl(N(beta)Me,benzoyl)-DTrp-Lys-Thr-Phe-Cys]-OH (11), starting with the papain-resolved Fmoc-DAgl(Boc). These results suggested that the orientation of side chains at position 6, 7, or 11 with respect to the side chains of residues 8 and 9 may be independently responsible for sst(4) selectivity.
Rivier
Jean
J
The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. jrivier@salk.edu
Erchegyi
Judit
J
Hoeger
Carl
C
Miller
Charleen
C
Low
William
W
Wenger
Sandra
S
Waser
Beatrice
B
Schaer
Jean-Claude
JC
Reubi
Jean Claude
JC
D000818:Animals; D001345:Autoradiography; D001667:Binding, Competitive; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011869:Radioligand Assay; D017481:Receptors, Somatostatin / Q000819:agonists* / Q000737:chemistry*; D013004:Somatostatin / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
D008024:Ligands; D008565:Membrane Proteins; D009842:Oligopeptides; D010456:Peptides, Cyclic; D017481:Receptors, Somatostatin; C480605:cyclo(cysteinyl(3)-phenylalanyl(6)-phenylalanyl(7)-tryptophyl(8)-lysyl(9)-threonyl(10)-phenylalanyl(11)-cysteinyl(14)); C098681:somatostatin receptor subtype-4; D013004:Somatostatin; D000242:Cyclic AMP