N-Methyl Scan of Somatostatin Octapeptide Agonists Produces Interesting Effects on Receptor Subtype Specificity

Abstract

The search for synthetic analogues of somatostatin which exhibit selective affinities for the five receptor subtypes is of considerable basic and therapeutic interest and has generated a large number of potent agonist analogues with a wide spectrum of binding profiles. In the past, conformational restriction of side chain groups and the peptide backbone has yielded the most interesting results. Under the latter category and as part of the present study, we were interested in the potential effects of N-methylation of peptide bond NH groups on binding affinity since this approach had not been systematically examined with these peptides. This was aided by new chemistries for introducing an N-Me group during regular solid-phase peptide synthesis using Boc protection. A number of interesting effects were noted on relative binding affinities of the two series of agonist sequences chosen, DPhe(5)(or Tyr(5))-c[Cys(6)-Phe(7)-DTrp(8)-Lys(9)-Thr(10)-Cys(11)]Thr(12)-NH(2) (SRIF numbering), at the five known human somatostatin receptors transfected into and stably expressed by CHO cells. N-Methylation of residues 7 (Phe), 10 (Thr), 11 (Cys), and 12 (Thr) largely destroyed affinities for all five receptors. N-Methylation of DTrp in the DPhe series gave an analogue with extraordinarily high affinity for the type 5 receptor for which it was also quite selective. N-Methylation of Lys in both series resulted in retention of type 2 affinity despite this residue constituting the "active center" of somatostatin peptides. N-Methylation of either the N-terminal Tyr residue or of Cys(6) in the Tyr series resulted in analogues with extraordinarily high affinity for the type 3 receptor, also with a degree of specificity. N-Methylation of the peptide bond constrains the conformational space of the amino acid and eliminates the possibility of donor hydrogen bond formation from the amide linkage. The beta-bend conformation of the agonists around DTrp-Lys is stabilized by a transannular intramolecular hydrogen bond(s) between Phe(7) and Thr(10) so methylation of these residues eliminates this source of stabilization. It is expected that several of these analogues will provide additional tools for determining some of the physiological roles played by type 3 and 5 somatostatin receptors which are still far from being fully elucidated.

Synthesis of Chemically Modified Bioactive Peptides: Recent Advances, Challenges and Developments for Medicinal Chemistry

“…For this purpose, libraries of peptides with N-methylated amino acids at different positions of the sequence have been synthesized and tested for their affinity and stability. Rajeswaran et al performed an N-methyl scan of somatostatin agonist and antagonist sequences leading in some cases to high affinity and specificity [117,118]. For other early examples of N-methylation on bioactive peptides, refer to Sagan et al [115].…”

Multiple N-Methylation of MT-II Backbone Amide Bonds Leads to Melanocortin Receptor Subtype hMC1R Selectivity: Pharmacological and Conformational Studies

“…When creating peptidic ligands one possible way to obtain these properties is N -methylation of the amide bonds,4–6,42–44 since substitution of amide protons with methyl groups can result in receptor subtype selectivity,45–48 but also other pharmacological properties can be improved, such as metabolic stability6,49,51 lipophilicity,49–51 enhanced potency,52–55 enhanced bioavailability6,56,57 and conformational rigidity 48,51,58. It may also turn an agonist into an antagonist 57…”

Improvement of drug-like properties of peptides: the somatostatin paradigm

“…Thus, over the past years, N-methylation was used in the development of analogs for various bioactive peptides, including somatostatin [89]. Rajeswaran et al have performed N-methylation at every amino-acid residue of truncated SRIF analogs (9), D-Phe-c [Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-NH 2 and 18, Tyr-c[CysPhe-D-Trp-Lys-Thr-Cys]-Thr-NH 2 ) [90]. All analogs were screened for their potency and selectivity at the sst receptors.…”