Highly Potent and Subtype Selective Ligands Derived byN-Methyl Scan of a Somatostatin Antagonist

Abstract

The search for synthetic peptide analogues of somatostatin (SRIF) which exhibit selective affinities for the five known receptor subtypes (sst1-5) has generated a large number of potent agonists. Some of these agonists display good subtype selectivities and affinities for the subtypes 1, 2, 3, and 5, including analogues created by N-methyl amino acid substitutions in a standard octapeptide analogue format. We have now extended this peptide backbone N-methylation approach to a potent somatostatin receptor antagonist series using the antagonist Cpa-cyclo(DCys-Pal-DTrp-Lys-Thr-Cys)-Nal-NH2 9 reported from this laboratory as the lead structure. Synthetic analogues were tested for their ability to inhibit somatostatin-stimulated GH release from rat pituitary cells in culture and to displace 125I-labeled somatostatin from CHO cells transfected with the five known human somatostatin receptors. Several interesting observations resulted from the study. N-Methylation at the Lys(9) residue (5) increased the rat GH release inhibitory potency nearly 4-fold to 0.73 nM but resulted in little change in the binding affinity for human type 2 receptor. This analogue also had a high affinity of 5.98 nM for sst5 receptor (compared to 1.4 nM for somatostatin itself) and is the first antagonist analogue to be reported with high affinity for sst5. It also had high potency on in vitro inhibition of sst5 mediated intracellular calcium mobilization. These results were considered surprising, since the Lys(9) residue has long been considered to constitute the active center of somatostatin, important both for receptor binding and activation, and suggests important conformational differences between D-Cys(9) somatostatin antagonists and normal agonist structures. More modifications were carried out on this analogue with the aim of improving antagonist potency and/or specificity. Tyr(7) substitution of 5 resulted in an analogue, which had the highest affinity in the series for hsst2 (K(I) 5.51 nM) and an extraordinarily low IC50 of 0.53 nM in the rat pituitary cell assay. However, this analogue lost considerable affinity for sst5 relative to analogue 5. Analogue 16 with DTrp(12) at C-terminus had the highest affinity for hsst2, however, the IC50 in the rat GH release assay was only 11.6 nM. Replacement of Lys(9) in 9 with Dab(9) gave 11 which displayed high binding affinity for sst3, and it was also quite selective for that receptor. Both the sst3 and sst5 antagonists should be of value in assigning the physiological roles to type 3 and 5 receptor, respectively.

Receptor Signaling and Endocytosis Are Differentially Regulated by Somatostatin Analogs

“…We examined two compounds, both of which have been reported to behave as sst2 antagonists. Confirming previous results (Rajeswaran et al, 2001), Coy-14 did not inhibit cAMP production in our experiments (data not shown). Furthermore, it did not induce sst2A receptor internalization and instead blocked agonist stimulation (Figs.…”

“…In contrast to agonists, the sst2 antagonists Coy-14 (Rajeswaran et al, 2001) and CYN-154806 (Bass et al, 1996) did not induce receptor endocytosis in either CHO-sst2 cells (Fig. 4) or HEK-sst2 cells (data not shown).…”

“…Analogs currently used for the treatment of acromegaly and gastroenterological tumors include octreotide and similar short, stable peptides that bind to sst2 with high affinity and to sst3 and sst5 with lower affinity. Although there are few sst receptor subtype-selective antagonists, such compounds have now been identified (Bass et al, 1996;Rajeswaran et al, 2001). Nevertheless, there are no systematic studies to quantitatively compare the effects of somatostatin analogs on signaling and endocytosis.…”

Parallel synthesis and biological evaluation of different sizes of bicyclo[2,3]‐Leu‐enkephalin analogues

“…In fact, in some cases the N ␣ -methyl group can significantly increase the biological potency. 30,31 There are several strategies to synthesize the N ␣ -methyl amino acid. Considering the Fmoc protection of amino acids, we have selected the reported method 32 so that the N ␣ -Fmoc is orthogonally protected (Scheme 1).…”

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

“…]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.…”