CDB15:0001411 SST — SSTR2

ABSTRACT: Recent reports (Raynor et al) have claimed the identification of potent somatostatin (SST) agonists exhibiting binding affinities of 1-2 pM and up to 30,000 fold binding selectivity for several of the 5 cloned sstr subtypes. These conclusions, however, are based on binding comparisons of sstr subtypes from different species expressed in different cell lines and studied with different radioligands. To eliminate the effect of species and/or methodological variations, we have investigated agonist selectivity of 32 synthetic SST analogs for all 5 hsstrs stably expressed in CHO-K1 cells under identical binding conditions. We show that hsstr2, 3, 5 react potently with hexapeptide as well as cyclic and linear octapeptide analogs and belong to a similar sstr subclass. hsstr1 and 4 react poorly with these analogs and belong to a separate subclass. The present generation of SST analogs exhibit a modest-50 fold increase in binding potency compared to SST-14 for 2 subtypes (hsstr2, 3), and relative selectivity for only 1 subtype (hsstr2) which is at best only 35 fold. The potency and degree of selectivity of these analogs is several orders of magnitude less than that reported earlier and suggests the need for caution in using these compounds as putative superagonists or subtype selective compounds for any of the individual sstrs.

ABSTRACT: The somatostatin (SRIF) receptors (SSTRs) 1 and 2 bind SRIF and SRIF 28 with high affinity, although a number of synthetic hexapeptide and octapeptide analogs of SRIF bind selectively to SSTR2. Extracellular loop three and its adjoining trans-membrane-spanning regions contain elements essential for the binding of such analogs to murine SSTR2. In particular, a stretch of amino acids from residues 294-297 (FDFV) in murine SSTR2 in trans-membrane domain seven can determine affinity for the SSTR2-selective analogs. Within this region, Phe294 has previously been predicted to be essential for the binding of octapeptides (Kaupmann, K., Bruns, C., Raulf, F., Weber, H., Mattes, H., and Lubbert, H. (1995) EMBO J. 14, 727-735) based on the observation that SSTR1 can bind the octapeptide SMS-201-995 with reasonable affinity after a Ser-to-Phe conversion in the analogous region of this receptor (SSTR1S305F). We find that SSTR1S305F has low affinity for a number of SSTR2-selective hexapeptides, suggesting that these analogs have different binding requirements than SMS-201-995. A correlation is seen between the ability of SSTR1S305F to bind hexapeptide analogs and the presence of a phenylalanine, but not tyrosine, at position two in these small cyclic molecules. Thus, a single hydroxyl group in hexapeptides can play a critical role in determining receptor binding to these receptor mutants. We also find that the second extracellular loop of SSTR1 is important for the selectivity of certain SRIF agonists for binding to SSTR1. Taken together, our data indicate that there are multiple elements in the somatostatin receptors that can determine the binding affinity and selectivity of peptide analogs.

ABSTRACT: Human somatostatin (somatotropin release inhibiting factor = SRIF) receptor subtypes sst2 and sst5 were stably expressed in Chinese hamster lung fibroblast (CCL39) cells. [125I][Tyr3]octreotide labelled with high affinity and in a saturable manner both sst2 (pKd = 9.89+/-0.02, Bmax = 210+/-10 fmol/mg, n = 3) and sst5 sites (pKd = 9.64+/-0.04, Bmax = 920+/-170 fmol/mg, n = 3). The pharmacological profile of sst2 sites established in CCL39 cells using SRIF and various peptide analogues was very similar to that described previously in CHO cells and in human cortex: SRIF14 = SRIF28 > or = seglitide > BIM 23014 = RC 160 > octreotide > CGP 23996 > or = L362,855 > BIM 23052 > L361,301 = cortistatin14 > BIM 23030 > BIM 23056 > cycloantagonist SA. However, peptides classically perceived as sst2 receptor selective (e.g., seglitide, octreotide, vapreotide) showed also high affinity for human sst5 receptors labelled with [125I][Tyr3]octreotide: SRIF28 > seglitide > SRIF14 > L361,301 = octreotide > cortistatin14 = BIM 23014 = BIM 23052 > L362,855 = RC160 > CGP 23996 > BIM 23056 > cycloantagonist SA > BIM 23030. Further radioligand binding studies were performed with [Leu8,D-Trp22,125I-Tyr25]SRIF28 ([125I]LTT-SRIF28) and [125I]CGP 23996. At sst2 receptors, Bmax values determined with [125I][Tyr3]octreotide, [125I]LTT-SRIF28 and [125I]CGP 23996 were in the same range (180-370 fmol/mg). 5'-Guanylyl-imidodiphosphate (GppNHp) displaced all three radioligands to the same extent (85%) and the pharmacological profiles were superimposable. By contrast, at sst5 receptors Bmax values were very different: [125I][Tyr3]octreotide (920 fmol/mg), [125I]CGP 23996 (3530 fmol/mg) and [125I]LTT-SRIF28 (6950 fmol/mg). GppNHp affected [125I][Tyr3]octreotide more than [125I]CGP 23996 binding, whereas [125I]LTT-SRIF28 was much less affected. In addition, the affinity values determined in competition experiments at sst5 receptors, varied markedly; whereas SRIF14, cortistatin14 and SRIF28 showed 2-, 4- and 8-fold differences in affinity at sst5 receptors labelled with [125I][Tyr3]octreotide and [125I]LTT-SRIF28 compounds such as RC160, L363,301, L362,855, octreotide or CGP 23996 showed between 42- and 123-fold lower affinity when sst5 sites were labelled with [125I]LTT-SRIF28. The present data suggest caution to be used when comparing affinity profiles determined in binding studies using different radioligands. In addition, the present results suggest that effects produced by octreotide and related short chain SRIF analogues on hormone release, modulation of tumour growth and central effects may be mediated by either sst2 and/or sst5 receptors.