Nonpeptide agonists of each of the five somatostatin receptors were identified in combinatorial libraries constructed on the basis of molecular modeling of known peptide agonists. In vitro experiments using these selective compounds demonstrated the role of the somatostatin subtype-2 receptor in inhibition of glucagon release from mouse pancreatic alpha cells and the somatostatin subtype-5 receptor as a mediator of insulin secretion from pancreatic beta cells. Both receptors regulated growth hormone release from the rat anterior pituitary gland. The availability of high-affinity, subtype-selective agonists for each of the somatostatin receptors provides a direct approach to defining their physiological functions.

Somatostatin (SST) potently inhibits insulin and glucagon release from pancreatic islets. Five distinct membrane receptors (SSTR1-5) for SST are known, and at least two (SSTR2 and SSTR5) have been proposed to regulate pancreatic endocrine function. Our current understanding of SST physiology is limited by the receptor subtype selectivity of peptidyl SST analogs, making it difficult to assign a physiological function to an identified SST receptor subtype. To better understand the physiology of SSTRs we studied the in vitro effects of potent subtype-selective nonpeptidyl SST analogs on the regulation of pancreatic glucagon and insulin secretion in wild-type (WT) and in somatostatin receptor 2 knockout (SSTR2KO) mice. There was no difference in basal glucagon and insulin secretion between islets isolated from SSTR2KO and WT mice; however, potassium/arginine-stimulated glucagon secretion was approximately 2-fold higher in islets isolated from SSTR2KO mice. Neither SST nor any SSTR-selective agonist inhibited basal glucagon or insulin release. SST-14 potently inhibited stimulated glucagon secretion in islets from WT mice and much less effectively in islets from SSTR2KO mice. The SSTR2 selective analog L-779,976 inhibited glucagon secretion in islets from WT, but was inactive in islets from SSTR2KO mice. L-817,818, an SSTR5 selective analog, slightly reduced glucagon release in both animal groups, whereas SSTR1, -3, and -4 selective analogs were inactive. SST and L-817,818 inhibited glucose stimulated insulin release in islets from WT and SSTR2KO mice. L-779,976 much less potently reduced insulin secretion from WT islets. In conclusion, our data demonstrate that SST inhibition of glucagon release in mouse islets is primarily mediated via SSTR2, whereas insulin secretion is regulated primarily via SSTR5.

A series of nonpeptide somatostatin agonists which bind selectively and with high affinity to somatostatin receptor subtype 2 (sst2) have been synthesized. One of these compounds, L-054,522, binds to human sst2 with an apparent dissociation constant of 0.01 nM and at least 3,000-fold selectivity when evaluated against the other somatostatin receptors. L-054,522 is a full agonist based on its inhibition of forskolin-stimulated adenylate cyclase activity in Chinese hamster ovary-K1 cells stably expressing sst2. L-054,522 has a potent inhibitory effect on growth hormone release from rat primary pituitary cells and glucagon release from isolated mouse pancreatic islets. Intravenous infusion of L-054,522 to rats at 50 g/kg per hr causes a rapid and sustained reduction in growth hormone to basal levels. The high potency and selectivity of L-054,522 for sst2 will make it a useful tool to further characterize the physiological functions of this receptor subtype.Somatostatin is widely distributed throughout the central nervous system and various endocrine tissues (1-3). Two biologically active forms of somatostatin are known, a 14-amino acid peptide and an N-terminal extended peptide with 28 amino acids (4 -6). Somatostatin has multiple functions, including modulation of growth hormone, insulin, glucagon, and gastric acid secretion (3, 7-10). Five somatostatin receptors (sst1-5) have been cloned and characterized (11)(12)(13)(14). All five receptors are members of the G protein-linked receptor family (15). Structure-function studies with a large number of peptidal analogs have shown that the Trp 8 -Lys 9 dipeptide of somatostatin is necessary for high-affinity binding (16) and have facilitated the development of potent analogs, including SMS 201-955 (Sandostatin or octreotide) which is clinically used for the treatment of acromegaly and certain endocrine tumors (17-19). We describe here strategies that were successful in designing small molecule subtype 2-selective agonists whose potencies on this receptor exceed somatostatin. Studies utilizing one of these subtype-selective somatostatin peptidomimetics, L-054,522, in both in vivo and in vitro experiments demonstate that somatostatin receptor subtype 2 (sst2) mediates the inhibition of growth hormone release from the rat anterior pituitary as well as glucagon from the rat pancreas. Insulin release is only inhibited at significantly higher concentrations of the compound. These results suggest possible therapeutic applications of selective sst2 agonists. Compound 4 was initially synthesized in a combinatorial library consisting of 20 diamines, 20 amino acids, and 79 amines. The diamines were linked to 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid functionalized Tentagel resin via urethane chemistry in a regiorandom way. 9-Fluorenylmethoxycarbonyl amino acids were coupled to the amine resins using standard 1,3-diisopropylcarbodiimide coupling. The 9-fluorenylmethoxycarbonyl groups were removed with piperidine and activated with p-nitrophenyl chloroformate before ...

Somatostatin (SRIH) regulates pituitary adrenocorticotropin (ACTH) secretion by interacting with a family of homologous G protein-coupled membrane receptors. The SRIH receptor subtypes (sst₁–sst₅) that control ACTH release remain unknown. Using novel, subtype-selective SRIH analogs, we have identified the SRIH receptor subtypes involved in regulating ACTH release from AtT-20 cells, a model for cell line pituitary corticotropes. Radioligand-binding studies with ¹²⁵I-SRIH-14 and ¹²⁵I-SRIH-28 showed that SRIH-14 and SRIH-28 recognized specific, high-affinity and saturable membrane-binding sites. Nonpeptidyl agonists with selectivity for the sst₂ (L-779,976; compound 2) or sst₁/sst₅) (L-817,818; compound 5) receptor subtypes potently displaced ¹²⁵I-SRIH-28 from AtT-20 cell membranes, while agonists selective for the sst₁ (L-779,591; compound 1), sst₃ (L-796,778; compound 3) or sst₄ (L-803,087; compound 4) subtypes were inactive. Tyr¹¹-SRIH-14, compound 2 (sst₂) or compound 5 (sst₅) inhibited forskolin and corticotropin-releasing hormone (CRH)-induced increases in intracellular cAMP. Furthermore, the sst₂ and sst₅ agonists potently inhibited CRH-induced ACTH release from AtT-20 cells. These results provide the first evidence that sst₂ and sst₅ receptor subtypes, but not sst₁, sst₃ or sst₄, inhibit cAMP accumulation and regulate ACTH secretion in the AtT-20 cell model of the rodent corticotrope.

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