Caroline Nunn

Drug design at peptide receptors

Somatostatin (SRIF, somatotropin release inhibiting factor), discovered for its inhibitory action on growth hormone (GH) secretion from pituitary, is an abundant neuropeptide. Two forms, SRIF14 and SRIF28 exist. Recently, a second family of peptides with very similar sequences and features was described; the cortistatins (CST), CST17 and CST29 which are brain selective. The five cloned SRIF receptors (sst1–5) belong to the G-protein coupled/heptathelical receptor family. Structural and operational features distinguish two classes of receptors; SRIF1-sst2/sst3/sst5 (high affinity for octreotide or seglitide) and SRIF2=sst1/sst4 (very low affinity for the aforementioned ligands). The affinity of SRIF receptors for somatostatins and cortistatins is equally high, and it is not clear whether selective receptors do exist for one or the other of the peptides. Several radiologlands label all SRIF receptors, e.g., [125I]LTT-SRIF28, [125I]CGP23996, [125I]Tyr10cortistatin or [125I]Tyr11SRIF14. In contrast, [125I]Tyr3octreotide, [125I]BIM23027, [125I]MK678 or [125I]D-Trp8SRIF14 label predominantly SRIF1 sites, especially sst2 and possibly sst5 receptors. In brain, [125I]Tyr3octreotide binding equates with sst2 receptor mRNA distribution. Native SRIF2 receptors can be labeled with [125I]SRIF14 in the presence of high NaCl in brain (sst1) or lung (sst4) tissue. Short cyclic or linear peptide analogs show selectivity for sst2/sst5 (octreotide, lanreotide, BIM 23027), sst1 (CH-275), sst3 (sst3-ODN-8), or sst5 receptors (BIM 23268); although claims for selectivity have not always been confirmed. Beta peptides with affinity for SRIF receptors are also reported. The general lack of SRIF receptor antagonists is unique for peptide receptors, although CYN 154806 is a selective and potent sst2 antagonist. Nonpeptide ligands are still rare, although a number of molecules have been reported with selectivity and potency for sst1 (L 757,519), sst2 (L 779,976), sst3 (L 796,778), sst4 (NNC 26-9100, L 803,087) or sst1/sst5 receptors (L 817,018). Such molecules are essential to establish the role of SRIF receptors, e.g., sst1 in hypothalamic glutamate currents: sst2 in inhibiting release of GH, glucagon, TSH, gastric acid secretion, pain, seizures and tumor growth, and sst5 in vascular remodeling and inhibition of insulin and GH release.

Somatostatin receptor subtypes 2 and 4 affect seizure susceptibility and hippocampal excitatory neurotransmission in mice

We have investigated the role of somatostatin receptor subtypes sst2 and sst4 in limbic seizures and glutamate‐mediated neurotransmission in mouse hippocampus. As compared to wild‐type littermates, homozygous mice lacking sst2 receptors showed a 52% reduction in EEG ictal activity induced by intrahippocampal injection of 30 ng kainic acid (P < 0.05). The number of behavioural tonic–clonic seizures was reduced by 50% (P < 0.01) and the time to onset of seizures was doubled on average (P < 0.05). Seizure‐associated neurodegeneration was found in the injected hippocampus (CA1, CA3 and hilar interneurons) and sporadically in the ipsilateral latero‐dorsal thalamus. This occurred to a similar extent in wild‐type and sst2 knock‐out mice. Intrahippocampal injection of three selective sst2 receptor agonists in wild‐type mice (Octreotide, BIM 23120 and L‐779976, 1.5–6.0 nmol) did not affect kainate seizures while the same compounds significantly reduced seizures in rats. L‐803087 (5 nmol), a selective sst4 receptor agonist, doubled seizure activity in wild‐type mice on average. Interestingly, this effect was blocked by 3 nmol octreotide. It was determined, in both radioligand binding and cAMP accumulation, that octreotide had no direct agonist or antagonist action at mouse sst4 receptors expressed in CCl39 cells, up to micromolar concentrations. In hippocampal slices from wild‐type mice, octreotide (2 µm) did not modify AMPA‐mediated synaptic responses while facilitation occurred with L‐803087 (2 µm). Similarly to what was observed in seizures, the effect of L‐803087 was reduced by octreotide. In hippocampal slices from sst2 knock‐out mice, both octreotide and L‐803087 were ineffective on synaptic responses. Our findings show that, unlike in rats, sst2 receptors in mice do not mediate anticonvulsant effects. Moreover, stimulation of sst4 receptors in the hippocampus of wild‐type mice induced excitatory effects which appeared to depend on the presence of sst2 subtypes, suggesting these receptors are functionally coupled.

Pharmacological profile of somatostatin and cortistatin receptors

Somatostatin (SRIF) and cortistatin (CST) are two endogenous peptides with high sequence similarities that act as hormones/neurotransmitters both in the CNS and the periphery; their genes although distinct result from gene duplication. Their receptors appear to be common, since the five known SRIF receptors (sst1-sst5) have similar subnanomolar affinity for SRIF and CST, whether the short (SRIF-14, CST-14, CST-17) or the long versions (SRIF-28, CST-29) of the peptides. Whether CST targets specific receptors not shared by SRIF, is still debated: MrgX2 has been described as a selective CST receptor, with submicromolar affinity for CST but devoid of affinity for SRIF; however the distribution of CST and MrgX2 is largely different, and there is no MrgX2 in rodents. A similar situation arises with the GHS receptor GHS-R1a, which displays some preferential affinity for CST over SRIF, but for which there is no evidence that it is activated by CST in vivo. In both cases, one may argue that submicromolar affinity is not the norm of a GPCR for its endogenous neuropeptide. On the other hand, all receptors known to bind SRIF have similar high affinity for CST and both peptides act as potent agonists at the sst1-sst5 receptors, whichever transduction pathway is considered. In addition, [(125)I][Tyr(10)]CST(14) labels sst1-sst5 receptors with subnanomolar affinity, and [(125)I][Tyr(10)]CST(14) binding in the brain is overlapping with that of [(125)I][Tyr(0)]SRIF(14). The functional differences reported that distinguish CST from SRIF, have not been explained convincingly and may relate to ligand-driven transductional selectivity, and other complicating factors such as receptor dimerisation, (homo or heterodimerisation), and/or the influence of accessory proteins (GIPs, RAMPS), which remain to be studied in more detail.

Pharmacological characterisation of native somatostatin receptors in AtT-20 mouse tumour corticotrophs

The mouse corticotroph tumour cell line AtT‐20 is a useful model to investigate the physiological role of native somatostatin (SRIF, Somatotropin release inhibitory factor) receptor subtypes (sst1 – sst5). The objective of this study was to characterise the pharmacological features and the functional effects of SRIF receptors expressed by AtT‐20 cells using radioligand binding and cAMP accumulation. [125I]LTT‐SRIF‐28, [125I]CGP 23996, [125I]Tyr10‐cortistatin‐14 and [125I]Tyr3‐octreotide labelled SRIF receptor binding sites with high affinity and in a saturable manner (Bmax=315, 274, 239 and 206 fmol mg−1, respectively). [125I]LTT‐SRIF‐28 labels significantly more sites than [125I]Tyr10 ‐cortistatin‐14 and [125I]Tyr3 ‐octreotide as seen previously in cells expressing pure populations of sst2 or sst5 receptors. SRIF analogues displaced the binding of the four radioligands. sst2/5 receptor‐selective ligands showed much higher affinity than sst1/3/4 receptor‐selective ligands. The binding profile of [125I]Tyr3‐octreotide was different from that of [125I]LTT‐SRIF‐28, [125I]CGP 23996 and [125I]Tyr10‐cortistatin‐14. The sst5/1 receptor‐selective ligand L‐817,818 identified two binding sites, one with subnanomolar affinity (sst5 receptors) and one with micromolar affinity (sst2 receptors); however, the proportions were different: 70 – 80% high affinity with [125I]LTT‐SRIF‐28, [125I]CGP 23996, [125I]Tyr10‐cortistatin‐14, but only 20% with [125I]Tyr3‐octreotide. SRIF analogues inhibited the forskolin‐stimulated cAMP levels depending on concentration. sst2/5 receptor‐selective ligands were highly potent, whereas sst1/3/4 receptor‐selective ligands had no significant effects. The sst2 receptor antagonist D‐Tyr8‐CYN 154806 competitively antagonised the effects of SRIF‐14 and sst2 receptor‐preferring agonists, but not those of L‐817,818. The complex binding properties of SRIF receptor analogues indicate that sst2 and sst5 receptors are the predominant SRIF receptors expressed on AtT‐20 cell membranes with no or only negligible presence of sst1, sst3 and sst4 receptors. In the functional studies using cAMP accumulation, only sst2 and sst5 receptors appear to play a role. However, the ‘predominant’ receptor appears to be the sst2 receptor, although sst5 receptors can also mediate the effect, when the ligand is not able to activate sst2 receptors. This clearly adds flexibility to SRIF‐mediated functional effects and suggests that the physiological role of SRIF and its analogues may be mediated preferentially via one subtype over another.

β2/β3-di- and α/β3-tetrapeptide derivatives as potent agonists at somatostatin sst4 receptors

Four linear β2/β3-di- and α/β3-tetrapeptides (1–4) were investigated as somatostatin sst4 receptor agonists on recombinant human and mouse somatostatin receptors. Human somatostatin receptor subtypes 1–5 (sst1–5), and mouse somatostatin receptor subtypes 1,3,4 and 5, were characterised using the agonist radioligands [125I]LTT-SRIF-28, [125I][Tyr10]CST14 and [125I]CGP 23996 in stably transfected Chinese hamster lung fibroblast (CCL39) cells. The peptides bound selectively to sst4 receptors with nanomolar affinity (pKd=5.4–7.8). The peptides were investigated on second messenger systems both as agonists, and as antagonists to SRIF-14-mediated effects in CCL39 cells expressing mouse sst4 receptors, via measurement of inhibition of forskolin-stimulated adenylate cyclase activity, and stimulation of luciferase expression. The peptides showed full agonism or pronounced partial agonism (40 to 100% relative intrinsic activity) in both inhibition of forskolin-stimulated adenylate cyclase activity (pEC50=5.5–6.8), and luciferase expression (pEC50=5.5–6.5). The agonist potential was confirmed since antagonism was very difficult to establish. The data show that β2/β3-di- and α/β3-tetrapeptide derivatives have agonist potential at recombinant somatostatin sst4 receptors. Therefore, they may be used to elucidate physiological and biochemical effects mediated by sst4, and may also have potential as therapeutic agents.

Cloning, expression and pharmacological characterisation of the mouse somatostatin sst5 receptor

Abstract The mouse somatostatin (somatotropin release inhibiting factor, SRIF) sst 5 receptor coding sequence was cloned from a mouse BALB/c genomic library. It shows 97% and 81% homology with the corresponding rat and human receptors, respectively. The msst 5 receptor messenger RNA (mRNA) is present at low levels in the adult mouse brain, with significant expression in a few nuclei only, e.g. in the septum (lateral septal nuclei) or the amygdala (medial amygdaloid nucleus); very few signals were observed in the mesencephalon, metencephalon, and myelencephalon (except the dorsal motor nucleus of the vagus nerve). The msst 5 receptor was stably expressed in the hamster fibroblast cell line CCL39-SRE-Luci, which harbours the luciferase reporter gene driven by the serum responsive element. [ 125 I]LTT-SRIF-28 ([Leu 8 , D-Trp 22 , 125 I-Tyr 25 ]-SRIF-28), [ 125 I]Tyr 10 -CST, [ 125 I]CGP 23996, and [ 125 I]Tyr 3 -octreotide labelled msst 5 receptors with high affinity (pK d values: 11.0, 10.15, 9.75 and 9.43) and in a saturable manner, but defined different Bmax values: 697, 495, 540 and 144 fmoles/mg, respectively. [ 125 I]LTT-SRIF-28-labelled sites displayed the following rank order: SRIF-28> rCST-14> somatuline > CGP-23996= SRIF-14= octreotide, whereas [ 125 I]Tyr 3 -octreotide-labelled sites displayed a different profile: octreotide > SRIF-28> rCST-14= somatuline > SRIF-14> CGP-23996. The pharmacological profiles determined with [ 125 I]LTT-SRIF-28, [ 125 I]CGP 23996 and [ 125 I]Tyr 10 -CST correlated highly significantly (r 2 =0.88–0.99), whereas [ 125 I]Tyr 3 -octreotide binding was rather divergent (r 2 =0.77). Also, human and mouse sst 5 receptor profiles are very different, e.g. r 2 =0.385 for [ 125 I]Tyr 10 -CST and r 2 =0.323 for [ 125 I]LTT-SRIF-28-labelled sites. Somatostatin induces expression of luciferase reporter gene in CCL39-SRE-Luci cells. The profile was consistent with a msst 5 receptor-mediated effect although apparent potency in the luciferase assay was much reduced compared to radioligand binding data: Octreotide = SRIF-28> rCST-14= SRIF-14= CGP-23996. Octreotide, SRIF-28, BIM23052 and D Tyr Cyanamid 154806 behaved as full or nearly full agonists in comparison to SRIF-14, whereas the other compounds had relative efficacies of 40 to 70%. The present study shows that agonists radioligands define apparently different receptor populations in terms of number of sites and pharmacological profile in cells expressing a single recombinant receptor. These variations suggest that the conformation of the ligand receptor complex may vary depending on the agonist. Further, the msst 5 receptor, although primarily coupled to Gi/Go proteins, is able to stimulate luciferase gene expression driven by the serum responsive element. Finally, it is suggested that putative sst 2 selective agonists e.g. octreotide, RC160 or BIM23027 show similar or higher potency at msst 5 receptors than SRIF-14.

SRA880, in vitro characterization of the first non-peptide somatostatin sst1 receptor antagonist

Abstract This report describes the in vitro features of the first somatostatin sst 1 receptor selective non-peptide antagonist, SRA880 ([3R,4aR,10aR]-1,2,3,4,4a,5,10,10a-Octahydro-6-methoxy-1-methyl-benz[g] quinoline-3-carboxylic-acid-4-(4-nitro-phenyl)-piperazine-amide, hydrogen malonate). SRA was evaluated in a number of in vitro systems of various species, both at native and recombinant receptors, using radioligand binding and second messenger/transduction studies. SRA880 has high affinity for native rat, mouse, monkey and human cerebral cortex somatostatin sst 1 receptors (p K d =7.8−8.6) and for human recombinant sst 1 receptors (p K d =8.0−8.1). SRA880 displayed significantly lower affinity for the other human recombinant somatostatin receptors (p K d ≤6.0) or a wide range of neurotransmitter receptors, except for the human dopamine D4 receptors. SRA880 was characterized in various transduction assays: somatotropin release inhibiting factor (SRIF) induced inhibition of forskolin-stimulated cAMP accumulation, SRIF stimulated-GTPγS binding, and SRIF stimulated luciferase gene expression; in all tests, SRA880 was devoid of intrinsic activity and acted as an apparently surmountable antagonist with p K B values of 7.5–7.7. Combined with the data from binding studies, these results suggest that SRA880 acts as a competitive antagonist. Thus, SRA880 is the first non-peptide somatostatin sst 1 receptor antagonist to be reported; SRA880 will be a useful tool for the characterization of somatostatin sst 1 receptor-mediated effects both in vitro and in vivo.

Comparison of functional profiles at human recombinant somatostatin sst2 receptor: simultaneous determination of intracellular Ca2+ and luciferase expression in CHO-K1 cells.

Somatostatin (somatotropin release inhibiting factor; SRIF) acts via five G protein‐coupled receptors (sst1–sst5) that modulate multiple cellular effectors. The aim of this study was to compare two functional effects of the human sst2 receptor stably expressed in CHO‐K1 cells in a single experiment using a duplex assay for intracellular calcium and serum response element (SRE)‐driven luciferase expression. Intracellular calcium was measured using a fluorometric imaging plate reader II (FLIPR II). SRIF‐14 rapidly and transiently increased intracellular calcium with a pEC50 of 8.74±0.03 (n=52). At 5 h after FLIPR II measurements, luciferase expression was determined. SRIF‐14 concentration‐dependently increased luciferase expression (pEC50=9.06±0.03, n=52). Natural and synthetic agonist/antagonist ligands for SRIF receptors were tested in the duplex assay. Correlation of agonist potencies and efficacies between the two responses were significant (r2=0.83 and 0.90, pEC50 and Emax, respectively). Pertussis toxin pretreatment reduced SRIF‐14/octreotide‐mediated intracellular calcium increases by 45–47% and luciferase expression by 95–98%. Thapsigargin pretreatment abolished the SRIF‐14/octreotide‐mediated intracellular calcium increase but had no effect on luciferase expression. In conclusion, SRIF stimulates an increase in intracellular calcium and SRE‐luciferase expression via human sst2 receptors in CHO‐K1 cells. The increase in luciferase is mediated via Gi/Go while intracellular calcium increase is mediated by both Gi/Go proteins and pertussis toxin‐insensitive G proteins, and is mainly via release of calcium from intracellular stores. SRIF ligands display a similar recognition profile suggesting that the ligand/receptor/G protein/effector interaction is similar for the two parameters.

Agonist properties of putative small-molecule somatostatin sst2 receptor-selective antagonists

The availability of antagonist ligands for somatostatin receptors is very limited, with those that are available often displaying agonist properties or limited receptor subtype selectivity. Hay et al. [Bioorg. Med. Chem. Lett. 11 (2001) 2731] recently described the development of small-molecule somatostatin receptor subtype 2 (sst(2)) selective compounds. This study investigates the binding affinity and functional characteristics of two of those antagonists (2 and 3) and the agonist compound, from which they were derived (1). In radioligand binding studies using the agonist radioligands [125I][Tyr(11)]SRIF-14 (Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-(125I-Tyr)-Thr-Ser-Cys]-OH), [125I]LTT-SRIF-28 ([Leu(8),DTrp(22),125I-Tyr(25)]SRIF-28; Ser-Ala-Asn-Ser-Asn-Pro-Ala-Leu-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-DTrp-Lys-Thr-(125I-Tyr)-Thr-Ser-Cys]-OH), [125I]CGP 23996 (c[Lys-Asu-Phe-Phe-Trp-Lys-Thr-(125I-Tyr)-Thr-Ser]), [125I][Tyr(3)]octreotide (DPhe-c[Cys-(125I-Tyr)-DTrp-Lys-Thr-Cys]-Thr-OH) and [125I][Tyr(10)]cortistatin-14 (Pro-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-(125I-Tyr)-Ser-Ser-Cys]-Lys) at human recombinant somatostatin receptors expressed in Chinese hamster lung fibroblast (CCL39) cells and native rat cortex, the compounds bound with high affinity (pK(d) 6.8-9.7) and selectivity to human sst(2) receptors. Some affinity was also observed for sst(5) labelled by [125I][Tyr(3)]octreotide and [125I]CGP 23996. In functional studies at human sst(2) receptors expressed in Chinese hamster ovary (CHO) cells, both the agonist 1 and the two putative antagonists 2 and 3 concentration dependently inhibited forskolin-stimulated adenylate cyclase and stimulated luciferase reporter gene expression, with similar efficacy to the natural ligand somatotropin release inhibiting factor (SRIF)-14. Compound 1 had similar potency to SRIF-14, which was in the nanomolar range, whereas 2 and 3 were 10-100-fold less potent. The intrinsic activity of 2 and 3 was too high to allow antagonist studies to be carried out. In conclusion, in contrast to previous findings, all three compounds are potent agonists at recombinant human sst(2) receptors.