Novel sst₄-Selective Somatostatin (SRIF) Agonists. 1. Lead Identification Using a Betide Scan

Abstract

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.

Novel sst2-Selective Somatostatin Agonists. Three-Dimensional Consensus Structure by NMR

“…The sst 1 and sst 4 receptors have maximum sequence similarity and hence were assumed to be part of one close family, different from the family of sst 2/3/5 receptors. Yet, the proposed sst 1 and sst 4 pharmacophores are completely distinct from each other.…”

“…Yet, the proposed sst 1 and sst 4 pharmacophores are completely distinct from each other. For example, the position and the number of aromatic rings involved in binding are different for the two ligands, suggesting that these hydrophobic residues interact with different regions of the sst 1 and sst 4 receptors, respectively ( Figure 3D and 3E). On the other hand, most of the previously published analogues binding with high affinity to sst 2 receptor were also binding to sst 5 receptors with nM affinity and sometimes to sst 3 receptors as well.…”

“…On the other hand, the sst 1 pharmacophore has two aromatic side chains, at position 6 or 7 and 11, involved in binding in addition to the DTrp 8 and IAmp 9 pair. The positioning of the two aromatic side chains at the back of the peptide plane has to be noted, which is opposite to that of the sst 4 and sst 2/3/5 pharmacophores. The sst 1 receptor selectivity is mainly achieved by the amino acid IAmp, which replaces Lys at position 9.…”

“…Recently, we have proposed models of the sst 4 and the sst 1 pharmacophores derived from the NMR consensus structures of several receptor selective analogues. 20,21 These models of the pharmacophores are different from that of the reported sst 2/3/5 selective pharmacophore in the following manner.…”

“…20,21 These models of the pharmacophores are different from that of the reported sst 2/3/5 selective pharmacophore in the following manner. The sst 4 pharmacophore has one aromatic side chain, either Phe 6 or Phe 11 involved in binding in addition to the DTrp 8 and Lys 9 pair. The distance between DTrp 8 and Lys 9 is 4.5−6.5 Å, between DTrp 8 and Phe 6/11 is 5.5−9.5 Å and between Lys 9 and Phe 6/11 is 4.5−6.5 Å.…”