Adriforant is a functional antagonist of histamine receptor 4 and attenuates itch and skin inflammation in mice.

BACKGROUND: Histamine has been postulated to play a role in atopic dermatitis via histamine receptor 4, mediating pruritic and inflammatory effects. The H4R antagonist adriforant (PF-3893787 or ZPL389) indicated clinical efficacy in a Ph2a study in atopic dermatitis. Preclinical investigations of adriforant had been scarce as experiments in transfectants with H4R from several species suggested partial agonism, not seen in human cells. OBJECTIVE: During the Ph2b trial in AD, we performed experiments to understand the pharmacology of adriforant in primary murine cells and in vivo models. We assessed its effects on ERK phosphorylation and transcriptional changes in bone marrow-derived mast cells, histamine-dependent Ca2+ flux in neurons and histamine-induced itch response. In addition, its impact on MC903-induced skin inflammation was evaluated. RESULTS: We show that, contrary to transfectants, adriforant is a competitive antagonist of the murine histamine receptor 4, antagonizes histamine-induced ERK phosphorylation, normalizes histamine-induced transcriptional changes in mast cells and reduces histamine-dependent Ca2+ flux in neurons. Administration to mice reduces acute histamine-induced itch response. In addition, adriforant ameliorates inflammation in the mouse MC903 model. CONCLUSIONS: Our results suggest that functional inhibition of histamine receptor 4 by adriforant reduces itch and inflammation in vivo. The effects observed in mice, however, did not translate to clinical efficacy in patients as the Ph2b clinical trial with adriforant did not meet pre-specified efficacy endpoints. Given the complex pathogenesis of AD, antagonism of histamine receptor 4 alone appears insufficient to reduce disease severity in AD patients, despite the effects seen in mouse models.

Discovery and Optimization of Novel SUCNR1 Inhibitors: Design of Zwitterionic Derivatives with a Salt Bridge for the Improvement of Oral Exposure.

G-protein-coupled receptor SUCNR1 (succinate receptor 1 or GPR91) senses the citric cycle intermediate succinate and is implicated in various pathological conditions such as rheumatoid arthritis, liver fibrosis, or obesity. Here, we describe a novel SUCNR1 antagonist scaffold discovered by high-throughput screening. The poor permeation and absorption properties of the most potent compounds, which were zwitterionic in nature, could be improved by the formation of an internal salt bridge, which helped in shielding the two opposite charges and thus also the high polarity of zwitterions with separated charges. The designed compounds containing such a salt bridge reached high oral bioavailability and oral exposure. We believe that this principle could find a broad interest in the medicinal chemistry field as it can be useful not only for the modulation of properties in zwitterionic compounds but also in acidic or basic compounds with poor permeation.

Structural basis of species-selective antagonist binding to the succinate receptor.

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3-5. Because SUCNR1 senses succinate as an immunological danger signal6-which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.

Blunted 5-HT1A receptor-mediated responses and antidepressant-like behavior in mice lacking the GABAB1a but not GABAB1b subunit isoforms.

RATIONALE: There is accumulating evidence for a role of GABAB receptors in depression. GABAB receptors are heterodimers of GABAB1 and GABAB2 receptor subunits. The predominant GABAB1 subunit isoforms are GABAB1a and GABAB1b. GABAB1 isoforms in mice differentially influence cognition, conditioned fear, and susceptibility to stress, yet their influence in tests of antidepressant-like activity has not been fully investigated. OBJECTIVES: Given the interactions between GABAB receptors and the serotonergic system and the involvement of 5-HT1A receptors (5-HT1AR) in antidepressant action, we sought to evaluate 5-HT1AR function in GABAB1a-/- and GABAB1b-/- mice. METHODS: GABAB1a-/- and GABAB1b-/- mice were assessed in the forced swim test (FST), and body temperature and hypothalamic-pituitary-adrenal (HPA) responses to the 5-HT1AR agonist 8-OH-DPAT were determined. Brain 5-HT1AR expression was assessed by [3H]-MPPF and [3H]-8-OH-DPAT autoradiography and 5-HT1AR G-protein coupling by [35S]GTP-γ-S autoradiography. RESULTS: As previously described, GABAB1a-/- mice showed an antidepressant-like profile in the FST. GABAB1a-/- mice also demonstrated profoundly blunted hypothermic and motoric responses to 8-OH-DPAT. Furthermore, 8-OH-DPAT-induced corticosterone and adrenocorticotropic hormone (ACTH) release were both attenuated in GABAB1a-/- mice. Interestingly, [3H]-MPPF and [3H]-8-OH-DPAT binding was largely unaffected by genotype. [35S]GTP-γ-S autoradiography suggested that altered 5-HT1AR G-protein coupling only partially contributes to the functional presynaptic 5-HT1AR desensitization, and not at all to the blunted postsynaptic 5-HT1AR-mediated responses, seen in GABAB1a-/- mice. CONCLUSION: These data demonstrate distinct functional links between 5-HT1ARs and the GABAB1a subunit isoform and suggest that the GABAB1a isoform may be implicated in the antidepressant-like effects of GABAB receptor antagonists and in neurobiological mechanisms underlying depression.

Antidepressants influence somatostatin levels and receptor pharmacology in brain.

This study investigated how the administration (acute and chronic) of the antidepressants citalopram and desmethylimipramine (DMI) influences somatostatin (somatotropin release inhibitory factor, SRIF) levels and SRIF receptor density (sst(1-5)) in rat brain. Animals received either of the following treatments: (1) saline for 21 days (control group), (2) saline for 20 days and citalopram or DMI for 1 day (citalopram or DMI acute groups), (3) citalopram or DMI for 21 days (citalopram or DMI chronic groups). Somatostatin levels were determined by radioimmunoassay. [(125)I]LTT SRIF-28 binding in the absence (labeling of sst(1-5)) or presence of 3 nM MK678 (labeling of sst(1/4)) and [(125)I]Tyr(3) octreotide (labeling of sst(2/5)) binding with subsequent autoradiography was performed in brains of rats treated with both antidepressants. Somatostatin levels were increased after citalopram, but not DMI administration, in the caudate-putamen, hippocampus, nucleus accumbens, and prefrontal cortex. Autoradiography studies illustrated a significant decrease in receptor density in the superficial and deep layers of frontal cortex (sst(2)), as well as a significant increase in the CA1 (sst(1/4)) hippocampal field in brains of chronically citalopram-treated animals. DMI administration increased sst(1/4) receptors levels in the CA1 hippocampal region. These results suggest that citalopram and to a lesser extent DMI influence the function of the somatostatin system in brain regions involved in the emotional, motivational, and cognitive aspects of behavior.

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.

Native somatostatin sst2 and sst5 receptors functionally coupled to Gi/o-protein, but not to the serum response element in AtT-20 mouse tumour corticotrophs.

Of the five cloned somatostatin (SRIF: somatotropin release inhibitory factor) receptors (sst1-5), only sst2 and sst5 receptors appear to be endogenously expressed and functionally active in AtT-20 mouse anterior pituitary tumour cells. In this study, the presence and the functional coupling of SRIF receptors to G-protein in AtT-20 cells was evaluated by receptor autoradiography and guanosine-5'-Omicron-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding, respectively. In addition, transcriptional effects via the serum response element (SRE) were assessed in AtT-20-SRE-luci cells, engineered to express constitutively SRE upstream of the luciferase reporter gene. [125I]LTT-SRIF-28, [125I]CGP 23996 and [125I]Tyr3-octreotide binding illustrates the high level of sst2/5 receptor in AtT-20 cell membranes. SRIF-14 and SRIF-28 produced a concentration-dependent increase in [35S]GTPgammaS binding (pEC50=6.72 and 7.45; Emax=79 and 74.9, respectively) which was completely abolished by pertussis toxin. sst2/5 receptor-selective ligands caused a concentration-dependent increase in [35S]GTPgammaS binding (pEC50=7.74-5.84; Emax=76.6-20.2) while sst1/3/4 receptor-selective ligands were devoid of activity. The binding profiles of [125I]LTT-SRIF-28 and the inhibition of cAMP accumulation correlated highly significantly with their corresponding [35S]GTPgammaS binding profiles (r=0.862 and 0.874, respectively). The effects of the sst2 receptor-preferring agonists Tyr3-octreotide and BIM 23027 on [35S]GTPgammaS binding, but not those of SRIF-14 and the sst5/1 receptor selective-agonist L-817,818, were competitively antagonised by the sst2 receptor antagonist d-Tyr8-CYN 154806 (pKB=7.36 and 7.72, respectively; slope factors not significantly different from unity). In AtT-20-SRE-luci cells, which carry a SRE-luciferase construct functioning in a very efficient manner, SRIF and its analogues did not affect luciferase activity. Taken together, these results demonstrate that in AtT-20 cells the expression of sst2 and sst5 receptors fit with their functional coupling to G(i/o)-proteins. The pharmacological implications of the existence of different ligand/receptor complexes are discussed. However, the intracellular pathways coupled to the activation of sst2 and sst5 receptors appear not to modulate the SRE-mediated transcriptional activity, suggesting that SRIF effects on gene expression coupled to mechanisms that have promoters other than SRE.

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.