Identification and SAR of potent and selective non-peptide obeline somatostatin sst1 receptor antagonists

A novel class of non-peptide somatostatin receptor ligands bearing the octahydrobenzo[g]quinoline (obeline) structural element has been identified. SAR studies have been performed that led to the discovery of derivatives with high affinity (pK(d) r sst(1) > or = 9) and selectivity (> or = 150-fold for h sst(1) over h sst(2)-h sst(5)) for somatostatin receptor subtype sst(1). In a functional assay, the compounds act as antagonists at human recombinant sst(1) receptors.     

Ergoline derivatives as highly potent and selective antagonists at the somatostatin sst 1 receptor

Non-peptidic compounds containing the octahydro-indolo[4,3-fg]quinoline (ergoline) structural element have been optimized into derivatives with high affinity (pK(d) r sst(1)>9) and selectivity (>1000-fold for h sst(1) over h sst(2)-h sst(5)) for the somatostatin sst(1) receptor. In functional assays, these ergolines act as antagonists at human recombinant sst(1) receptors. Pharmacokinetic studies in rodents reveal good oral bioavailability and brain penetration for some of these compounds.     

Urotensin II and urotensin II-related peptide activate somatostatin receptor subtypes 2 and 5

The UII and urotensin II-related peptide (URP) genes belong to the same superfamily as the somatostatin gene. It has been previously shown that somatostatin activates the UII-receptor (UTR). In contrast, the possible interaction between UII and URP and somatostatin receptors has remained scarcely analyzed. Herein, we have investigated the effects of UII and URP on cell proliferation and free cytosolic Ca2+ concentration ([Ca2+]i) in CHO-K1 cells stably expressing the porcine somatostatin receptor subtypes sst2 and sst5. Results show that both UII and URP induce stimulation of cell proliferation mediated by sst2 receptors and UII provokes inhibition of cell proliferation mediated by sst5 receptors. UII and URP also provoked an increase of [Ca2+]i in both sst2- and sst5-transfected cells. Together, our present data demonstrate that UII and URP directly activate sst2 and sst5 and thus mimic the effect of somatostatin on its cognate receptors.     

Localization of five somatostatin receptors in the rat central nervous system using subtype-specific antibodies.

The cloning of five members of the somatostatin receptor family, sst1-sst5, as well as two isoforms of the somatostatin receptor 2, sst2A and sst2B, enabled us to generate specific anti-peptide antisera against unique sequences in the carboxyl-terminal tail of each somatostatin receptor subtype. We used these antibodies in multicolor immunofluorescent studies aimed to examine the regional and subcellular distribution of somatostatin receptors in adult rat brain. Several findings are notable: The cloned sst1 receptor is primarily localized to axons, and therefore most likely functions in a presynaptic manner. The cloned sst2 receptor isoforms exhibit strikingly different distributions, however, both sst2A and sst2B are confined to the plasma membrane of neuronal somata and dendrites, and therefore most likely function in a postsynaptic manner. The cloned sst3 receptor appears to be excluded from 'classical' pre- or postsynaptic sites but is selectively targeted to neuronal cilia. The cloned sst4 receptor is preferentially distributed to distal dendrites, and therefore most likely functions postsynaptically. The cloned sst5 receptor was not detectable in the adult rat brain, however, prominent sst5 expression was found in the pituitary. Furthermore, sst1-containing axons either co-contained somatostatin or were closely apposed by somatostatin-positive terminals in a regional-specific manner. Neuronal somata and dendrites containing either sst2A, sst2B or sst4 were found to exist in close proximity, although not necessarily synaptically linked, to somatostatin-positive terminals. Together, in the central nervous system the effects of somatostatin are mediated by several different receptor proteins which are distributed with considerable regional overlap. However, there appears to be a high degree of specialization among somatostatin receptor subtypes with regard to their subcellular targeting. This subtype-selective targeting may be the underlying principal of organization that allows somatostatinergic modulation of neuronal activity via both pre- and postsynaptic mechanisms.     

Homologous and heterologous regulation of somatostatin receptor 2

We previously demonstrated that phosphorylation of somatostatin receptor 2A (sst2A) is rapidly increased in transfected cells both by agonist and by the protein kinase C (PKC) activator phorbol myristate acetate (PMA). Here, we investigate whether PKC-mediated receptor phosphorylation is involved in the homologous or heterologous regulation of endogenous sst2 receptors in AR42J pancreatic acinar cells upon stimulation by agonist or by cholecystokinin (CCK) or bombesin (BBS). Somatostatin, PMA, CCK, and BBS all increased sst2A receptor phosphorylation 5- to 10-fold within minutes. Somatostatin binding also caused rapid internalization of the ligand-receptor complex, and PMA, CCK, and BBS all stimulated this internalization further. Additionally, sst2 receptor-mediated inhibition of adenylyl cyclase was desensitized by all treatments. Somatostatin, as well as peptidic (SMS201-995) and nonpeptidic (L-779,976) sst2 receptor agonists increased the EC(50) for somatostatin inhibition 20-fold. In contrast, pretreatment with BBS, CCK, or PMA caused a modest 2-fold increase in the EC(50) for cyclase inhibition. Whereas the PKC inhibitor GF109203X abolished sst2A receptor phosphorylation by CCK, BBS, and PMA, it did not alter the effect of somatostatin, demonstrating that these reactions were catalyzed by different kinases. Consistent with a functional role for PKC-mediated receptor phosphorylation, GF109203X prevented PMA stimulation of sst2 receptor internalization. Surprisingly, however, GF109203X did not inhibit BBS and CCK stimulation of sst2A receptor endocytosis. These results demonstrate that homologous and heterologous hormones induce sst2A receptor phosphorylation by PKC-independent and -dependent mechanisms, respectively, and produce distinct effects on receptor signaling and internalization. In addition, the heterologous hormones also modulate sst2 receptor internalization by a novel mechanism that is independent of receptor phosphorylation.     

Distinct functional properties of native somatostatin receptor subtype 5 compared with subtype 2 in the regulation of ACTH release by corticotroph tumor cells

In a series of human corticotroph adenomas, we recently found predominant mRNA expression of somatostatin (SS) receptor subtype 5 (sst5). After 72 h, the multiligand SS analog SOM230, which has a very high sst5 binding affinity, but not Octreotide (OCT), significantly inhibited basal ACTH release. To further explore the role of sst5 in the regulation of ACTH release, we conducted additional studies with mouse AtT-20 cells. SOM230 showed a 7-fold higher ligand binding affinity and a 19-fold higher potency in stimulating guanosine 5'-O-(3-thiotriphosphate) binding in AtT-20 cell membranes compared with OCT. SOM230 potently suppressed CRH-induced ACTH release, which was not affected by 48-h dexamethasone (DEX) pretreatment. However, DEX attenuated the inhibitory effects of OCT on ACTH release, whereas it increased the inhibitory potency of BIM-23268, an sst5-specific analog, on ACTH release. Quantitative PCR analysis showed that DEX lowered sst(2A+2B) mRNA expression significantly after 24 and 48 h, whereas sst5 mRNA levels were not significantly affected by DEX treatment. Moreover, Scatchard analyses showed that DEX suppressed maximum binding capacity (B(max)) by 72% when 125I-Tyr3-labeled OCT was used as radioligand, whereas B(max) declined only by 17% when AtT-20 cells were treated with [125I-Tyr11]SS-14. These data suggest that the sst5 protein, compared with sst2, is more resistant to glucocorticoids. Finally, after SS analog preincubation, compared with OCT both SOM230 and BIM-23268 showed a significantly higher inhibitory effect on CRH-induced ACTH release. In conclusion, our data support the concept that the sst5 receptor might be a target for new therapeutic agents to treat Cushing's disease.     

SAR of the arylpiperazine moiety of obeline somatostatin sst1 receptor antagonists

The SAR of over 50 derivatives of octahydrobenzo[g]quinoline (obeline)-type somatostatin sst(1) receptor antagonist 1 is presented, focusing on the modification of its arylpiperazine moiety. Sst(1) affinities in this series cover a range of five orders of magnitude with the best derivatives displaying subnanomolar sst(1) affinities and >10,000-fold selectivities over the sst(2) receptor subtype as well as promising pharmacokinetic properties.     

Somatostatin receptor gene expression in neuroblastoma

Somatostatin receptor expression is a favorable prognostic factor in human neuroblastoma. Somatostatin receptors have been demonstrated in vitro by pharmacologic analysis of tumor tissue and in vivo by diagnostic radioreceptor scintigraphy. However, which receptor subtypes (sst(1), sst(2), sst(3), sst(4), and sst(5)) are expressed in these tumors has not yet been delineated. We used RT-PCR to analyze expression of the five somatostatin receptor genes in 32 neuroblastoma tumor specimens. All 32 tumor specimens expressed mRNA for c-abl and sst(1); sst(2) mRNA was detected in 27/32 samples and somatostatin mRNA was detected in 30/32 tumor specimens. The remaining receptor subtypes, sst(3), sst(4), and sst(5) were variably expressed. Receptor protein for sst(1) and sst(2) was visualized in tumor neuroblasts as well as in endothelial cells of tumor vessels using immunostaining with specific anti-receptor antibodies. The effect of high expression of somatostatin receptors on cell proliferation was examined in SKNSH neuroblastoma cells transfected with sst(1) and sst(2). SS(14) binding to wild-type SKNSH cells was undetectable; but the native peptide bound with high affinity to the SKNSH/sst(1) and SKNSH/sst(2) neuroblastoma cell lines. Pharmacologic analysis of binding with two long-acting analogues, CH275 and octreotide, confirmed selective expression of sst(1) and sst(2) in stably transfected SKNSH cells. Formation of neuroblastoma xenograft tumors in nude mice was significantly delayed for both SKNSH/sst(1) (P<0.001) and SKNSH/sst(2) (P<0.05) cells compared to wild-type SKNSH. We conclude that: (1) Somatostatin receptors, sst(1) and sst(2), are expressed in the majority of neuroblastomas at diagnosis; and (2) upregulation of functional sst(1) or sst(2) in neuroblastoma cell lines suppresses tumorigenicity in a xenograft model. These observations suggest that somatostatin receptors may be a useful therapeutic target in neuroblastoma.     

Regulation of insulin and glucagon secretion from rat pancreatic islets in vitro by somatostatin analogues

Somatostatin is an inhibitor of hormone secretion through specific receptors (sst1-5). The aim of this study was to investigate the putative regulatory role of somatostatin analogues on the secretion of insulin and glucagon by rat pancreatic islets. After 48 h exposure only the non-selective agonists (somatostatin, octreotide and SOM-230) inhibited insulin accumulation. The inhibition of insulin secretion was accompanied by increased islet insulin contents. None of the analogues showed a consistent effect on the glucagon accumulation in the medium after 48 h. Since we observed a difference in the regulatory effect between the non-selective and selective analogues, combinations of selective analogues were studied. Combination of sst2+sst5 agonists inhibited the medium insulin accumulation, while combination of sst1+sst2 analogues caused a decrease in glucagon accumulation. After removal of somatostatin a rebound effect with increased insulin secretion were observed. This effect was reversed after 6 h. For SOM-230 insulin secretion continued to be suppressed even after the analogue was removed and returned to control values after 3 h. As for glucagon secretion there was an initial decline after culture with octreotide, while the other substances failed to induce any changes. In summary, non-selective somatostatin analogues or combinations of receptor selective analogues may cause inhibition of hormone secretion from rat pancreatic islets. For insulin and glucagon, combinations of sst2+sst5 and sst1+sst2, respectively may exert this effects. Thus, our data suggest that more than one sst must be involved to down-regulate islet glucagon and insulin secretion.     

The Truncated Isoform of Somatostatin Receptor5 (sst5TMD4) Is Associated with Poorly Differentiated Thyroid Cancer

Somatostatin receptors (ssts) are expressed in thyroid cancer cells, but their biological significance is not well understood. The aim of this study was to assess ssts in well differentiated (WDTC) and poorly differentiated thyroid cancer (PDTC) by means of imaging and molecular tools and its relationship with the efficacy of somatostatin analog treatment. Thirty-nine cases of thyroid carcinoma were evaluated (20 PDTC and 19 WDTC). Depreotide scintigraphy and mRNA levels of sst-subtypes, including the truncated variant sst5TMD4, were carried out. Depreotide scans were positive in the recurrent tumor in the neck in 6 of 11 (54%) PDTC, and in those with lung metastases in 5/11 cases (45.4%); sst5TMD4 was present in 18/20 (90%) of PDTC, being the most densely expressed sst-subtype, with a 20-fold increase in relation to sst2. In WDTC, sst2 was the most represented, while sst5TMD4 was not found; sst2 was significantly increased in PDTC in comparison to WDTC. Five depreotide positive PDTC received octreotide for 3–6 months in a pilot study with no changes in the size of the lesions in 3 of them, and a significant increase in the pulmonary and cervical lesions in the other 2. All PDTC patients treated with octreotide showed high expression of sst5TMD4. ROC curve analysis demonstrated that only sst5TMD4 discriminates between PDTC and WDTC. We conclude that sst5TMD4 is overexpressed in PDTC and may be involved in the lack of response to somatostatin analogue treatment.     

Small molecule somatostatin receptor subtype-2 antagonists

The first potent small molecule sst2 antagonists are reported. Altering known sst2 agonist molecules yielded compounds with high sst2 binding affinity and full antagonist activity. Compound 7a, for example, displaced somatostatin binding to the sst2 receptor with an IC(50)=2.9 nM and antagonized somatostatin action with an IC(50)=29nM.     

Receptor-mediated internalization is critical for the inhibition of the expression of growth hormone by somatostatin in the pituitary cell line AtT-20.

The inhibitory effect of the neuropeptide somatostatin on the expression of growth hormone was measured by quantitative polymerase chain reaction in the pituitary cell line AtT-20. We demonstrate that this effect is dependent on the internalization of somatostatin-receptor complexes and that it is totally independent from the peptide-induced inhibition of adenylate cyclase. Indeed, the inhibitory effect of the peptide on growth hormone mRNA levels was totally insensitive to pertussis toxin treatment but was totally abolished under conditions which block somatostatin receptor internalization. Comparative confocal microscopic imaging of fluorescent somatostatin sequestration and fluorescence immunolabeling of sst1, sst2A, and sst5 receptors suggests that sst2A is most probably responsible of the inhibitory effect of somatostatin on growth hormone expression.     

Somatostatin receptor subtype 5 regulates insulin secretion and glucose homeostasis

Somatostatin (SRIF) regulates pancreatic insulin and glucagon secretion. In the present study we describe the generation of SRIF receptor subtype 5 knockout (sst(5) KO) mice to examine the role of SRIF receptor subtypes (sst) in regulating insulin secretion and glucose homeostasis. Mice deficient in sst(5) were viable, fertile, appeared healthy, and displayed no obvious phenotypic abnormalities. Pancreatic islets isolated from sst(5) KO mice displayed increased total insulin content as compared with islets obtained from wild-type (WT) mice. Somatostatin-28 (SRIF-28) and the sst(5)/sst(1)-selective agonist compound 5/1 potently inhibited glucose-stimulated insulin secretion from WT islets. SRIF-28 inhibited insulin secretion from sst(5) KO islets with 16-fold less potency while the maximal effect of compound 5/1 was markedly diminished when compared with its effects in WT islets. sst(5) KO mice exhibited decreased blood glucose and plasma insulin levels and increased leptin and glucagon concentrations compared with WT mice. Furthermore, sst(5) KO mice displayed decreased susceptibility to high fat diet-induced insulin resistance. The results of these studies suggest sst(5) mediates SRIF inhibition of pancreatic insulin secretion and contributes to the regulation of glucose homeostasis and insulin sensitivity. Our findings suggest a potential beneficial role of sst(5) antagonists for alleviating metabolic abnormalities associated with obesity and insulin resistance.     

Differential beta-arrestin trafficking and endosomal sorting of somatostatin receptor subtypes

The physiological responses of somatostatin are mediated by five different G protein-coupled receptors. Although agonist-induced endocytosis of the various somatostatin receptor subtypes (sst(1)-sst(5)) has been studied in detail, little is known about their postendocytic trafficking. Here we show that somatostatin receptors profoundly differ in patterns of beta-arrestin mobilization and endosomal sorting. The beta-arrestin-dependent trafficking of the sst(2A) somatostatin receptor resembled that of a class B receptor in that upon receptor activation, beta-arrestin and the receptor formed stable complexes and internalized together into the same endocytic vesicles. This pattern was dependent on GRK2 (G protein-coupled receptor kinase 2)-mediated phosphorylation of a cluster of phosphate acceptor sites within the cytoplasmic tail of the sst(2A) receptor. Unlike other class B receptors, however, the sst(2A) receptor was rapidly resensitized and recycled to the plasma membrane. The beta-arrestin mobilization of the sst(3) and the sst(5) somatostatin receptors resembled that of a class A receptor in that upon receptor activation, beta-arrestin and the receptor formed relatively unstable complexes that dissociated at or near the plasma membrane. Consequently, beta-arrestin was excluded from sst(3)-containing vesicles. Unlike other class A receptors, a large proportion of sst(3) receptors was subject to ubiquitin-dependent lysosomal degradation and did not rapidly recycle to the plasma membrane. The sst(4) somatostatin receptor is unique in that it did not exhibit agonist-dependent receptor phosphorylation and beta-arrestin recruitment. Together, these findings may provide important clues about the regulation of receptor responsiveness during long-term administration of somatostatin analogs.     

A transplantable phosphorylation probe for direct assessment of G protein-coupled receptor activation

The newly developed multireceptor somatostatin analogs pasireotide (SOM230), octreotide and somatoprim (DG3173) have primarily been characterized according to their binding profiles. However, their ability to activate individual somatostatin receptor subtypes (sst) has not been directly assessed so far. Here, we transplanted the carboxyl-terminal phosphorylation motif of the sst(2) receptor to other somatostatin receptors and assessed receptor activation using a set of three phosphosite-specific antibodies. Our comparative analysis revealed unexpected efficacy profiles for pasireotide, octreotide and somatoprim. Pasireotide was able to activate sst(3) and sst(5) receptors but was only a partial agonist at the sst(2) receptor. Octreotide exhibited potent agonistic properties at the sst(2) receptor but produced very little sst(5) receptor activation. Like octreotide, somatoprim was a full agonist at the sst(2) receptor. Unlike octreotide, somatoprim was also a potent agonist at the sst(5) receptor. Together, we propose the application of a phosphorylation probe for direct assessment of G protein-coupled receptor activation and demonstrate its utility in the pharmacological characterization of novel somatostatin analogs.     

Differential distribution of somatostatin sst2 receptor splice variants in rat gastric mucosa

The tetradecapeptide somatostatin (SRIF) has an inhibitory action on acid secretion in the stomach. It has been suggested that somatostatin may act directly on parietal cells as well as indirectly via histamine-producing cells. A family of high affinity membrane-bound receptors, which are termed sst1-sst5 receptors, mediates the physiological effects of somatostatin. On the basis of functional studies it has been suggested that somatostatin may mediate its actions in the stomach by activation of a somatostatin sst2 receptor type. Two splice variants of the rat sst2 receptor exist, sst2(a) and sst2(b), which differ in length and composition of their intracellular carboxy termini. To date, little information is available on the distribution of the somatostatin sst2(b) receptor in any peripheral tissue. Here we show for the first time the localisation of this receptor isoform in the rat oxyntic mucosa, where the receptor protein was found to be present in parietal cells. This is in contrast to sst2(a) receptor, which was localised to enterochromaffin-like cells and nerve fibres. The differential localisation of the receptor isoforms to two key cell types, parietal cells and enterochromaffin-like cells, may explain how somatostatin inhibits acid secretion by more than one mechanism.     

Effects of somatostatin-14 and the receptor-specific somatostatin analogs on chromogranin A and alpha-subunit (alpha-SU) release from "clinically nonfunctioning" pituitary adenoma cells incubated in vitro.

The aim of the study was to examine the effect of somatostatin (SST) and its analogs on the release of chromogranin A (CgA) and alpha-subunit (alpha-SU) from clinically non-functioning pituitary adenomas incubated in vitro. Seven pituitary macroadenomas surgically removed were investigated. All of the tumors were diagnosed before surgery as non-functioning, but they expressed either gonadotropins or their subunits as detected by immunohistochemistry. Two tumors additionally expressed prolactin and growth hormone. All adenomas also expressed chromogranin A (CgA) and at least 3 of 5 subtypes of somatostatin receptors. The cells isolated from the examined tumors were exposed in vitro to either native SST-14 or the following receptor-specific SST analogs: BIM-23926 (agonist of sst1 receptor), BIM-23120 (agonist of sst2 receptor), BIM-23206 (agonist of sst5 receptor) and BIM23A387 (somatostatin/dopamine chimera). The concentration of CgA was measured by means of ELISA method and of alpha-SU was measured by an immunoradiometric method. It was found that the exposure on SST-14 resulted in the decrease of CgA and alpha-SU release from tumor cells in majority of samples, and the effect on CgA was positively correlated with the expression of sst3 and also with the sst2A/sst2B expressions ratio. The inhibitory effect of SST-14 on CgA and alpha-SU seems also to correlate negatively with the expression of sst2B. CgA inhibition also correlates positively with sst5 expression. Among the other compounds studied, only the sst2 agonist decreased the release in all the investigated samples. The remaining substances (agonists of sst1 and sst5 and SST/DA chimera) produced the divergent changes (increased or decreased release, depending on the sample). The data suggest that the inhibition of CgA (and possibly of alpha-SU) release by SST is mediated via subtypes sst2A, sst3 and sst5, whereas sst2B subtype may induce the opposite effect.