Lanthionine−Somatostatin Analogs:  Synthesis, Characterization, Biological Activity, and Enzymatic Stability Studies

Abstract

A series of cyclic somatostatin analogs containing a lanthionine bridge have been subjected to studies of structure-activity relationships. A direct synthesis of the thioether bridged analog (1) of sandostatin (SMS 201,995) and several lanthionine hexa-, hepta-, and octapeptides was carried out by using the method of cyclization on an oxime resin (PCOR) followed by condensation reactions in solution. The structures of the target peptides were analyzed by liquid secondary ion mass spectrometry (LSIMS) and subjected to high-energy collision-induced dissociation (CID) studies after opening of the peptide ring by proteolytic cleavage. The biological activities of these compounds have been evaluated by assaying their inhibitory potencies for the release of growth hormone (GH) from primary cultures of rat anterior pituitary cells, as well as by their binding affinities to cloned somatostatin receptors (SSTR1-5). The structural modification of sandostatin by introducing a lanthionine bridge resulted in a significantly increased receptor binding selectivity. The lanthionine octapeptide with C-terminal Thr-ol (1) showed similar high affinity for rat SSTR5 compared to somatostatin[1-14] and sandostatin. However, it exhibits about 50 times weaker binding affinity for mSSTR2b than sandostatin. Similarly, the lanthionine octapeptide with the C-terminal Thr-NH2 residue (2) has higher affinity for rSSTR5 than for mSSTR2B. Both peptides (compounds 1 and 2) have much lower potencies for inhibition of growth hormone secretion than sandostatin. This is consistent with their affinities to SSTR2, the receptor which is believed to be linked to the inhibition of growth hormone release by somatostatin and its analogs. The metabolic stability of lanthionine-sandostatin and sandostatin have been studied in rat brain homogenates. Although both compounds have a high stability toward enzymatic degradation, the lanthionine analog has a 2.4 times longer half-life than sandostatin. The main metabolites of both compounds have been isolated and identified by using an in vivo technique (cerebral microdialysis) and mass spectrometry.

Ring Size in Octreotide Amide Modulates Differently Agonist versus Antagonist Binding Affinity and Selectivity

“…Increasing the number of atoms in the cycle had different effects for the agonist and the antagonist. While Hcy at position 3 enhanced selectivity for sst 2 , DHcy replacement at position 3 resulted in dramatic loss in affinity compared to the parent compound. The 3D NMR structures identified the presence and absence of the sst 2 -selective pharmacophore in the analogues which explains the binding data.…”

“…The shorter side chain of Ncy constrains the peptide backbone so tightly that the analogue lost binding affinity to all of the receptors. Introduction of Hcy, which has a longer side chain than Cys, resulted in 3 with partial selectivity for sst 2 . In contrast, DHcy with a different chirality at position 3, resulted in 5 with a complete loss of binding to sst 2 .…”

“…1 This conformational transition complicates the interpretation of a particular structure as the bioactive conformation for a particular receptor (i.e., complicates the definition of the pharmacophore). In fact, it suggests that two conformations are necessary for the analogue to fit into the two different subtype-selective pharmacophores for sst 2 and sst 5 , respectively. Recently, we published that the replacement of Phe at position 7 by an Ala in the octreotide scaffold resulted in an agonist (H-DPhe 2 -c[Cys 3 -Ala 7 -DTrp 8 -Lys 9 -Thr 10 -Cys 14 ]-Thr 15 -NH 2 ), which showed unique sst 2 selectivity.…”

“…Recently, we published that the replacement of Phe at position 7 by an Ala in the octreotide scaffold resulted in an agonist (H-DPhe 2 -c[Cys 3 -Ala 7 -DTrp 8 -Lys 9 -Thr 10 -Cys 14 ]-Thr 15 -NH 2 ), which showed unique sst 2 selectivity. 4 SAR studies of such analogues suggested that the aromatic side chain at position 7 was not necessary for sst 2 binding, but was crucial for sst 3 and sst 5 binding. The 3D structure of this peptide also had a β-turn of type-II' for the backbone conformation, which oriented the side chain of DTrp 8 , the amino alkyl group of Lys 9 and the aromatic ring of DPhe 2 outside the cycle in their respective positions, which were crucial for effective receptor-ligand binding.…”

The cyclization of peptides and depsipeptides

“…The only reported total synthesis in the solution phase is by Shiba et al [212], with the cyclized sulfur bridges being formed by extrusion of sulfur from cystine, followed by fragment condensation. In progressing to solid phase techniques, lanthionine bridges have been made compatible with peptide cyclization on an oxime resin [213,214], and using a biomimetic approach of assembling linear peptides containing cysteine and dehydroalanine residues, and forming the sulfur bridges by intramolecular 1,4-addition of the cysteine SH to the dehydroalanine [215,216]. In the more recent solid phase approach [217], an analogue of the ring C of nisin has been introduced via a pre-formed sulfur bridge as part of a triply orthogonal protecting group strategy summarized in Scheme 23.…”