Kinetic properties of "dual" orexin receptor antagonists at OX1R and OX2R orexin receptors.

Orexin receptor antagonists represent attractive targets for the development of drugs for the treatment of insomnia. Both efficacy and safety are crucial in clinical settings and thorough investigations of pharmacokinetics and pharmacodynamics can predict contributing factors such as duration of action and undesirable effects. To this end, we studied the interactions between various "dual" orexin receptor antagonists and the orexin receptors, OX1R and OX2R, over time using saturation and competition radioligand binding with [(3)H]-BBAC ((S)-N-([1,1'-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide). In addition, the kinetics of these compounds were investigated in cells expressing human, mouse and rat OX1R and OX2R using FLIPR® assays for calcium accumulation. We demonstrate that almorexant reaches equilibrium very slowly at OX2R, whereas SB-649868, suvorexant, and filorexant may take hours to reach steady state at both orexin receptors. By contrast, compounds such as BBAC or the selective OX2R antagonist IPSU ((2-((1H-Indol-3-yl)methyl)-9-(4-methoxypyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-1-one) bind rapidly and reach equilibrium very quickly in binding and/or functional assays. Overall, the "dual" antagonists tested here tend to be rather unselective under non-equilibrium conditions and reach equilibrium very slowly. Once equilibrium is reached, each ligand demonstrates a selectivity profile that is however, distinct from the non-equilibrium condition. The slow kinetics of the "dual" antagonists tested suggest that in vitro receptor occupancy may be longer lasting than would be predicted. This raises questions as to whether pharmacokinetic studies measuring plasma or brain levels of these antagonists are accurate reflections of receptor occupancy in vivo.

Reaction of Fe3(CO)12 with octreotide--chemical, electrochemical and biological investigations.

In search for peptidic [FeFe] hydrogenase mimics, the cyclic disulfide Sandostatin (octreotide) was allowed to react with Fe(3)(CO)(12). An octreotide-Fe(2)(CO)(6) complex was isolated and characterized spectroscopically as well as by elemental and thermochemical analysis. The complex catalyzes the electrochemical reduction of H(+) to H(2). It is suggested by radioligand binding assays that the complex retains much of the binding affinity for the somatostatin hsst(1-5) receptors of octreotide.

Decahydroisoquinoline derivatives as novel non-peptidic, potent and subtype-selective somatostatin sst(3) receptor antagonists.

Starting from non-peptidic sst(1)-selective somatostatin receptor antagonists, first compounds with mixed sst(1)/sst(3) affinity were identified by directed structural modifications. Systematic optimization of these initial leads afforded novel, enantiomerically pure, highly potent and sst(3)-subtype selective somatostatin antagonists based on a (4S,4aS,8aR)-decahydroisoquinoline-4-carboxylic acid core moiety. These compounds can efficiently be synthesized and show promising PK properties in rodents.

Discovery of novel non-peptidic beta-alanine piperazine amide derivatives and their optimization to achiral, easily accessible, potent and selective somatostatin sst1 receptor antagonists.

Structural simplification of the core moieties of obeline and ergoline somatostatin sst(1) receptor antagonists, followed by systematic optimization, led to the identification of novel, highly potent and selective sst(1) receptor antagonists. These achiral, non-peptidic compounds are easily prepared and show promising PK properties in rodents.

The enantiomer of octreotate binds to all five somatostatin receptors with almost equal micromolar affinity--a comparison with SANDOSTATIN.

Octreotate (1b) is the octreotide (SANDOSTATIN; 1a) analogue, carrying a C-terminal CO(2)H (Thr) instead of the CH(2)OH (threoninol) group. In pursuit of our interest in unnatural peptides, we have now synthesized (by the solid-phase Fmoc method) the enantiomeric form 2 of octreotate and determined its affinity for the five human somatostatin (SRIF) receptors (hsst(1-5)). The binding was found to be 9.1, 4.1, 1.0, 1.4, and 4.2 microM, respectively. This almost equal one-digit micromolar affinity of ent-octreotate (2) to all five receptors contrasts with the behavior of most other somatostatin mimics including SANDOSTATIN (octreotide; 1a) and [Tyr(3)]-octreotate (1c), which have affinities for the various receptors differing up to and above 10(4)-fold. Thus, the structure of the new compound does not prevent binding, albeit more weakly than its pseudo-enantiomer octreotide, and there is hardly any selectivity of the peptide-protein interaction (PPI) for any one of the five SRIF G-protein coupled receptors (GPCRs). Since the detailed structure(s) of these membrane-embedded receptors is unknown (no X-ray structure!), the result described here may be useful for modeling structures by comparing the affinities of the numerous known somatostatin mimics.

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.

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.

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.

Compensatory changes in the hippocampus of somatostatin knockout mice: upregulation of somatostatin receptor 2 and its function in the control of bursting activity and synaptic transmission.

Somatostatin-14 (SRIF) co-localizes with gamma-aminobutyric acid (GABA) in the hippocampus and regulates neuronal excitability. A role of SRIF in the control of seizures has been proposed, although its exact contribution requires some clarification. In particular, SRIF knockout (KO) mice do not exhibit spontaneous seizures, indicating that compensatory changes may occur in KO. In the KO hippocampus, we examined whether specific SRIF receptors and/or the cognate peptide cortistatin-14 (CST) compensate for the absence of SRIF. We found increased levels of both sst2 receptors (sst2) and CST, and we explored the functional consequences of sst2 compensation on bursting activity and synaptic responses in hippocampal slices. Bursting was decreased by SRIF in wild-type (WT) mice, but it was not affected by either CST or sst2 agonist and antagonist. sst4 agonist increased bursting frequency in either WT or KO. In WT, but not in KO, its effects were blocked by agonizing or antagonizing sst2, suggesting that sst2 and sst4 are functionally coupled in the WT hippocampus. Bursting was reduced in KO as compared with WT and was increased upon application of sst2 antagonist, while SRIF, CST and sst2 agonist had no effect. At the synaptic level, we observed that in WT, SRIF decreased excitatory postsynaptic potentials which were, in contrast, increased by sst2 antagonist in KO. We conclude that sst2 compensates for SRIF absence and that its upregulation is responsible for reduced bursting and decreased excitatory transmission in KO mice. We suggest that a critical density of sst2 is needed to control hippocampal activity.

Binding and functional properties of the novel somatostatin analogue KE 108 at native mouse somatostatin receptors.

Clinically used somatostatin (SRIF) analogs, octreotide and lanreotide, act primarily by binding to SRIF receptor subtype 2 (sst2). In contrast, the recently described multiligand SOM230 binds with high affinity to sst(1-3) and sst5 and KE 108 is characterised as a high affinity ligand for all five SRIF receptors. In tumoural mouse corticotrophs (AtT-20 cells) and in mouse hippocampus, binding and functional features of KE 108 were examined and compared to SRIF-14, octreotide and SOM230. In AtT-20 cells, KE 108 bound with high affinity at [125I]LTT-SRIF-28-labelled sites similarly to SRIF-14, octreotide and SOM230. At the functional level, all four ligands increased guanosine-5'-O-(3-[35S]thio)-triphosphate binding and decreased cAMP accumulation or intracellular Ca2+ concentration through G(i/o) proteins. In hippocampal slices, KE 108, octreotide and SOM230 also bound with high affinity at [125I]LTT-SRIF-28-labelled sites similarly to SRIF-14, but KE 108, octreotide or SOM230 did not influence spontaneous epileptiform activity which was, in contrast, inhibited by SRIF-14. In conclusion, this study demonstrates that KE 108 has high affinity for native mouse SRIF receptors. Functionally, KE 108 mediates SRIF action at sst(2/5) in corticotrophs whereas it does not mimic the SRIF-induced inhibition of hippocampal excitation suggesting that the high potency and efficacy of a synthetic ligand to all known SRIF receptors may not reproduce entirely the effects of the natural SRIF.

Somatostatin receptors differentially affect spontaneous epileptiform activity in mouse hippocampal slices.

Somatostatin-14 [somatotropin release-inhibiting factor (SRIF)] reduces hippocampal epileptiform activity but the contribution of its specific receptors (sst1-5) is poorly understood. We have focused on the role of sst1 and sst2 in mediating SRIF modulation of epilepsy using hippocampal slices of wild-type (WT) and sst1 or sst2 knockout (KO) mice. Recordings of epileptiform discharge induced by Mg2+ -free medium with 4-aminopyridine were performed from the CA3 region before and after the application of SRIF compounds. In WT mice, SRIF and the sst1 agonist CH-275 reduce epilepsy whereas sst1 blockade with its antagonist SRA-880 increases the bursting discharge. Activation of sst2 does not affect the bursting frequency unless its agonist octreotide is applied with SRA-880, indicating that sst1 masks sst2-mediated modulation of epilepsy. In sst1 KO mice: (i) the bursting frequency is lower than in WT; (ii) SRIF, CH-275 and SRA-880 are ineffective on epilepsy and (iii) octreotide is also devoid of effects, whereas blockade of sst2 with the antagonist D-Tyr8 Cyn 154806 increases the bursting frequency. In sst2 KO mice, the SRIF ligand effects are similar to those in WT. In the whole hippocampus of sst1 KO mice, sst2 mRNA, protein and binding are higher than in WT and reverse transcription-polymerase chain reaction of the CA3 subarea confirms an increase of the sst2 messenger. We conclude that sst1 mediates inhibitory actions of SRIF and that interactions between sst1 and sst2 may prevent sst2 modulation of epilepsy. We suggest that, in sst1 KO mice, activation of over-expressed sst2 reduces the bursting frequency, indicating that sst2 density represents the rate-limiting factor for ss(2-mediated modulation of epilepsy.

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

1. Somatostatin (somatotropin release inhibiting factor; SRIF) acts via five G protein-coupled receptors (sst(1)-sst(5)) that modulate multiple cellular effectors. The aim of this study was to compare two functional effects of the human sst(2) 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. 2. Intracellular calcium was measured using a fluorometric imaging plate reader II (FLIPR II). SRIF-14 rapidly and transiently increased intracellular calcium with a pEC(50) 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 (pEC(50)=9.06+/-0.03, n=52). 3. 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 (r(2)=0.83 and 0.90, pEC(50) and E(max), respectively). 4. Pertussis toxin pretreatment reduced SRIF-14/octreotide-mediated intracellular calcium increases by 45-47% and luciferase expression by 95-98%. 5. Thapsigargin pretreatment abolished the SRIF-14/octreotide-mediated intracellular calcium increase but had no effect on luciferase expression. 6. In conclusion, SRIF stimulates an increase in intracellular calcium and SRE-luciferase expression via human sst(2) receptors in CHO-K1 cells. The increase in luciferase is mediated via G(i)/G(o) while intracellular calcium increase is mediated by both G(i)/G(o) 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.

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

1. 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 (sst(1) - sst(5)). 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. 2. [(125)I]LTT-SRIF-28, [(125)I]CGP 23996, [(125)I]Tyr(10)-cortistatin-14 and [(125)I]Tyr(3)-octreotide labelled SRIF receptor binding sites with high affinity and in a saturable manner (B(max)=315, 274, 239 and 206 fmol mg(-1), respectively). [(125)I]LTT-SRIF-28 labels significantly more sites than [(125)I]Tyr(10) -cortistatin-14 and [(125)I]Tyr(3) -octreotide as seen previously in cells expressing pure populations of sst(2) or sst(5) receptors. 3. SRIF analogues displaced the binding of the four radioligands. sst(2/5) receptor-selective ligands showed much higher affinity than sst(1/3/4) receptor-selective ligands. The binding profile of [(125)I]Tyr(3)-octreotide was different from that of [(125)I]LTT-SRIF-28, [(125)I]CGP 23996 and [(125)I]Tyr(10)-cortistatin-14. The sst(5/1) receptor-selective ligand L-817,818 identified two binding sites, one with subnanomolar affinity (sst(5) receptors) and one with micromolar affinity (sst(2) receptors); however, the proportions were different: 70 - 80% high affinity with [(125)I]LTT-SRIF-28, [(125)I]CGP 23996, [(125)I]Tyr(10)-cortistatin-14, but only 20% with [(125)I]Tyr(3)-octreotide. 4. SRIF analogues inhibited the forskolin-stimulated cAMP levels depending on concentration. sst(2/5) receptor-selective ligands were highly potent, whereas sst(1/3/4) receptor-selective ligands had no significant effects. The sst(2) receptor antagonist D-Tyr(8)-CYN 154806 competitively antagonised the effects of SRIF-14 and sst(2) receptor-preferring agonists, but not those of L-817,818. 5. The complex binding properties of SRIF receptor analogues indicate that sst(2) and sst(5) receptors are the predominant SRIF receptors expressed on AtT-20 cell membranes with no or only negligible presence of sst(1), sst(3) and sst(4) receptors. In the functional studies using cAMP accumulation, only sst(2) and sst(5) receptors appear to play a role. However, the "predominant" receptor appears to be the sst(2) receptor, although sst(5) receptors can also mediate the effect, when the ligand is not able to activate sst(2) 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.

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.

Functional characterisation of the putative somatostatin sst2 receptor antagonist CYN 154806.

The two forms (DTyr8 and LTyr8) of the putative somatostatin sst2 receptor antagonist CYN 154806 (Ac-4NO2-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-D/LTyr-NH2) were investigated on recombinant human somatostatin receptors and endogenous guinea-pig ileum receptors. In radioligand binding studies using the agonist radioligands [125I]LTT-SRIF-28, [125I][Tyr10]cortistatin-14, [125I]CGP 23996 and [125I][Tyr3]octreotide in Chinese hamster lung fibroblast (CCL39) and Chinese hamster ovary (CHO) cells expressing human somatostatin receptors (hsst1-5), CYN 154806 binds to sst2 receptors with nanomolar affinity (pKD=8.14-8.89), 40- to 4500-fold higher than for sst1, sst3 or sst4. High affinity was also demonstrated for sst5 receptors, particularly for LTyr8CYN 154806 where the sst5 affinity was higher than for sst2 receptors when using [125I]CGP 23996 and [125I][Tyr3]octreotide. Functional properties of the compounds were examined in Chinese hamster ovary (CHO) cells expressing human sst2 receptors, in (1) inhibition of forskolin-stimulated adenylate cyclase, (2) stimulation of serum response element-driven luciferase expression and (3) [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPS) binding. L- and DTyr8CYN 154806 showed full agonism at inhibition of forskolin-stimulated cAMP accumulation (pEC50=7.73 for both, Emax 104% and 78%, respectively), partial agonism at luciferase expression (pEC50=7.85 and 8.16, Emax=50% and 29%, respectively) and behaved as apparently silent antagonists at [35S]GTPS binding (no agonism observed, pKB=6.88 and 7.50, respectively). The agonist potential was confirmed in isolated guinea-pig ileum preparations via measurement of SRIF-induced inhibition of neurotransmission, where the L-isoform had marked agonism (pEC50=8.23, Emax=32%) whereas the D-isoform was apparently devoid of agonism. The present data suggest that CYN 154806 should be used with caution as an sst2 receptor antagonist tool, since it possesses intrinsic activity at sst2, and high affinity for both sst2 and sst5 receptors. The DTyr form, having lower intrinsic activity, especially in natural tissues, and greater selectivity for sst2 receptors, may be more reliable than LTyr CYN 154806.

Beta(2)/beta(3)-di- and alpha/beta(3)-tetrapeptide derivatives as potent agonists at somatostatin sst(4) receptors.

Four linear beta(2)/beta(3)-di- and alpha/beta(3)-tetrapeptides (1-4) were investigated as somatostatin sst(4) receptor agonists on recombinant human and mouse somatostatin receptors. Human somatostatin receptor subtypes 1-5 (sst(1-5)), and mouse somatostatin receptor subtypes 1,3,4 and 5, were characterised using the agonist radioligands [(125)I]LTT-SRIF-28, [(125)I][Tyr(10)]CST(14) and [(125)I]CGP 23996 in stably transfected Chinese hamster lung fibroblast (CCL39) cells. The peptides bound selectively to sst(4) receptors with nanomolar affinity (pK(d)=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 sst(4 )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 (pEC(50)=5.5-6.8), and luciferase expression (pEC(50)=5.5-6.5). The agonist potential was confirmed since antagonism was very difficult to establish. The data show that beta(2)/beta(3)-di- and alpha/beta(3)-tetrapeptide derivatives have agonist potential at recombinant somatostatin sst(4) receptors. Therefore, they may be used to elucidate physiological and biochemical effects mediated by sst(4), and may also have potential as therapeutic agents.