Use of Bioluminescent Aequorin for the Pharmacological Characterization of 5ht Receptors

Abstract

A convenient functional assay for 5HT2a and 5HT2c receptors is reported utilizing the bioluminescent aequorin to detect intracellular calcium changes. Using this assay, the pharmacological properties of many 5HT ligands can be determined in a 96-well format. The data indicate that the aequorin detection method is superior to the inositol phosphate assay with regard to speed and scope. This system is also appropriate for kinetic studies of receptor desensitization. We showed that the human 5HT2c receptor desensitizes in a biphasic manner, with a fast desensitization of approximately 90% of the total response occurring within 15 minutes while the remaining 10% response remains for at least 3 hours.     

G protein-coupled receptor kinase and beta-arrestin-mediated desensitization of the angiotensin II type 1A receptor elucidated by diacylglycerol dynamics

Receptor desensitization progressively limits responsiveness of cells to chronically applied stimuli. Desensitization in the continuous presence of agonist has been difficult to study with available assay methods. Here, we used a fluorescence resonance energy transfer-based live cell assay for the second messenger diacylglycerol to measure desensitization of a model seven-transmembrane receptor, the Gq-coupled angiotensin II type 1(A) receptor, expressed in human embryonic kidney 293 cells. In response to angiotensin II, we observed a transient diacylglycerol response reflecting activation and complete desensitization of the receptor within 2-5 min. By utilizing a variety of approaches including graded tetracycline-inducible receptor expression, mutated receptors, and overexpression or short interfering RNA-mediated silencing of putative components of the cellular desensitization machinery, we conclude that the rate and extent of receptor desensitization are critically determined by the following: receptor concentration in the plasma membrane; the presence of phosphorylation sites on the carboxyl terminus of the receptor; kinase activity of G protein-coupled receptor kinase 2, but not of G protein-coupled receptor kinases 3, 5, or 6; and stoichiometric expression of beta-arrestin. The findings introduce the use of the biosensor diacylglycerol reporter as a powerful means for studying Gq-coupled receptor desensitization and document that, at the levels of receptor overexpression commonly used in such studies, the properties of the desensitization process are markedly perturbed and do not reflect normal cellular physiology.     

Aequorin luminescence-based assay for 5-hydroxytryptamine (serotonin) type 3 receptor characterization

The classical electrophysiological method to measure the function of the 5-hydroxytryptamine (serotonin) type 3 (5-HT(3)) receptor, a cation-permeable ligand-gated ion channel, is time-consuming and not suitable for high-throughput screening. Therefore, we have optimized the conditions for a sensitive assay suitable to measure 5-HT(3) receptor responses in cell suspension based on aequorin bioluminescence caused by Ca(2+) influx. The assay, carried out in 96-well plates, was applied for the pharmacological characterization of 5-HT(3) receptors on human embryonic kidney (HEK) 293 cells transiently coexpressing apoaequorin and either the human homopentameric 5-HT(3A) receptor or the human heteromeric 5-HT(3A/B) receptor in the same subset of cells. Thus, the luminescence signal originates exclusively from transfected cells, leading to a high signal/noise ratio, a major advantage compared with fluorescence techniques using Ca(2+)-sensitive dyes. The potencies of two 5-HT(3A) receptor agonists and two antagonists as well as the potency and efficacy of serotonin at the heteromeric 5-HT(3A/B) receptor were comparable to those reported using other functional methods. In conclusion, the aequorin assay described here provides a convenient and highly sensitive method for functional characterization of 5-HT(3) receptors that is well suited for high-throughput screening.     

Opposite effects of PSD-95 and MPP3 PDZ proteins on serotonin 5-hydroxytryptamine2C receptor desensitization and membrane stability

PSD-95/Disc large/Zonula occludens 1 (PDZ) domain-containing proteins (PDZ proteins) play an important role in the targeting and the trafficking of transmembrane proteins. Our previous studies identified a set of PDZ proteins that interact with the C terminus of the serotonin 5-hydroxytryptamine (5-HT)(2C) receptor. Here, we show that the prototypic scaffolding protein postsynaptic density-95 (PSD-95) and another membrane-associated guanylate kinase, MAGUK p55 subfamily member 3 (MPP3), oppositely regulate desensitization of the receptor response in both heterologous cells and mice cortical neurons in primary culture. PSD-95 increased desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response, whereas MPP3 prevented desensitization of the Ca(2+) response. The effects of the PDZ proteins on the desensitization of the Ca(2+) response were correlated with a differential regulation of cell surface expression of the receptor. Additional experiments were performed to assess how PDZ proteins globally modulate desensitization of the 5-HT(2C) receptor response in neurons, by using a peptidyl mimetic of the 5-HT(2C) receptor C terminus fused to the human immunodeficiency virus type-1 Tat protein transduction domain, which disrupts interaction between the 5-HT(2C) receptor and PDZ proteins. Transduction of this peptide inhibitor into cultured cortical neurons increased the desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response. This indicates that, overall, interaction of 5-HT(2C) receptors with PDZ proteins inhibits receptor desensitization in cortical neurons.     

Deletion of the serotonin 2c receptor from transgenic mice overexpressing leptin does not affect their lipodystrophy but exacerbates their diet-induced obesity

The binding of leptin to hypothalamic neurons elicits inhibition of orexigenic NPY/AgRP neurons and stimulation of anorexigenic POMC/CART neurons. Projections of serotonergic neurons onto POMC neurons suggest that leptin and serotonin converge onto POMC neurons to regulate body weight. We probed the interaction of these pathways by generating transgenic mice overexpressing leptin (LepTg) without 5HT2c receptors. On a chow diet, the lean phenotype of LepTg mice was unaffected by the absence of 5HT2c receptors, whereas on a high fat diet, LepTg/5HT2c receptors knockout mice showed an exacerbation of diet-induced obesity. POMC mRNA levels were low in LepTg, 5HT2c receptors knockout and LepTg/5HT2c receptors knockout mice, demonstrating that perturbations of the 5HT2c receptor and leptin pathways, either alone or in combination, negatively impact on POMC expression. Thus, on a chow diet, leptin action is independent of 5HT2c receptors whereas on a high fat diet 5HT2c receptors are required for the attenuation of obesity.     

Rapid desensitization and resensitization of 5-HT2 receptor mediated phosphatidyl inositol hydrolysis by serotonin agonists in quiescent calf aortic smooth muscle cells

Agonist regulation of 5-hydroxytryptamine2 (5-HT2) receptors was studied in calf aortic smooth muscle cultures incubated in a quiescent, defined synthetic medium that does not stimulate cell proliferation, but that provides cells with supplements that maintain cell viability. In these cells, 5-hydroxytryptamine (5-HT)-induced [3H]inositol phosphates accumulation showed the characteristics of a 5-HT2 receptor coupled transducing system according to the inhibition of the response by 5-HT2 antagonists at nanomolar concentrations. The 5-HT2 receptor coupled response became rapidly desensitized during continued incubation with 5-HT and 1-(2,5-dimethoxy-4-methylphenyl)-2- aminopropane (DOM); nearly full desensitization was obtained in two hours with 10 microM 5-HT and DOM pretreatment. The recovery of the response had a half-live of 5 hours after 2 hours pretreatment and of 9.5 to 12.5 hours after 24 to 96 hours agonist pretreatment. The DOM-induced desensitization of the 5-HT2 receptor coupled response was fully blocked by 0.1 microM cinanserin. Cinanserin alone did not induce desensitization or up-regulation of the 5-HT2 receptor coupled response at 0.1 microM. It may be that the down-regulation of central 5-HT2 receptors by antagonists in vivo is a heterologous process due to mediators which are triggered by 5-HT2 antagonistic action.     

A high-throughput glow-type aequorin assay for measuring receptor-mediated changes in intracellular calcium levels

A glow-type aequorin luminescence assay for measuring receptor-mediated stimulation of intracellular calcium levels is described and characterized. The human 5-hydroxytryptamine(2A) receptor stably coexpressed in human embryonic kidney cells with apoaequorin was used to characterize the system and showed that following the flash reaction, a stable luminescence signal could be measured using a microplate scintillation counter for between 3 and 7 h after the addition of receptor agonist. Furthermore, this luminescence was dependent on the concentration of agonist used and gave potency values that were stable over this time period. Testing a range of 5-hydroxytryptamine(2A) receptor agonists gave the expected rank order of potency for this receptor. The glow luminescence could also be inhibited by 5-hydroxytryptamine(2A) receptor antagonists, generating affinity values that directly correlated with those determined for inhibition of the flash reaction carried out under the same buffer conditions. The assay therefore gave pharmacologically relevant data and allows a significant improvement of throughput over the traditional flash-type measurements made using an injecting luminometer.     

Effects of serotonergic agonists and antagonists on corticotropin-releasing hormone secretion by explanted rat hypothalami

Experimental evidence suggests that serotonin (5HT) is excitatory to the hypothalamic-pituitary-adrenal axis and that this effect involves activation of both hypothalamic corticotropin-releasing hormone (CRH) and pituitary ACTH secretion. The present study was undertaken to examine the mechanism by which 5HT stimulates the central component of the HPA axis. To accomplish this we employed an in vitro rat hypothalamic organ culture system in which CRH secretion from single explanted hypothalami was measured by specific radioimmunoassay (IR-rCRH). All experiments were performed after an overnight (15-18 hr) preincubation. Serotonin stimulated IR-rCRH secretion in a dose-dependent fashion. The response was bell-shaped and the peak effect was observed at the concentration of 10(-9) M. The stimulatory effect of 10(-9) M 5HT was antagonized by the 5HT1 and 5HT2 receptor metergoline and by the selective 5HT2 receptor antagonists ketanserin and ritanserin. The muscarinic antagonist atropine, the nicotinic antagonist hexamethonium and the alpha-adrenergic receptor antagonist phentolamine, on the other hand, did not inhibit 5HT-induced IR-rCRH secretion. The specific 5HT2 receptor agonist 1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI) stimulated IR-rCRH secretion in a dose-dependent fashion. The response was bell-shaped with peak of effect reached at the concentration of 10(-9) M. We also tested the ability of the 5HT agonist meta-chlorophenylpiperazine (m-CPP) and of the selective 5HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) to cause CRH secretion. Although both m-CPP and 8-OH-DPAT stimulated IR-rCRH secretion in a dose-dependent fashion, several differences were observed when their effect was compared to that of 5HT. These included a different shape of the dose-response curve, a lower maximal stimulatory effect and a different maximal stimulatory concentration. These findings suggest that serotonin stimulates CRH secretion by explanted rat hypothalami and that this effect appears to be mediated mainly through a 5HT2 receptor mechanism.     

Involvement of Receptor Cycling and Receptor Reserve in Resensitization of Muscarinic Responses in SH-SY5Y Human Neuroblastoma Cells

Abstract: Preexposure of SH-SY5Y cells to the muscarinic agonist carbachol caused a rapid desensitization of subsequent carbachol-stimulated intracellular Ca²⁺ responses and a slower decrease in the number of receptors at the plasma membrane. Desensitization (to 30% of the control response) was maximal after 1 min of exposure to agonist, whereas the number of cell surface receptors reached a minimum (33% of control) only after 5 min. Following agonist washout, the recovery of response was complete within 12 min, whereas the recovery of surface receptor number reached a plateau at 65% of control after 30 min. Treatment with inhibitors of endocytosis (concanavalin A) or recycling (nigericin) did not affect rapid desensitization but did decrease resensitization, suggesting that receptor cycling is involved in resensitization. Experiments with the irreversible antagonist propylbenzilylcholine mustard demonstrated that the receptor reserve for the Ca²⁺ response to 1 m M carbachol is ∼50%. Removal of this receptor reserve led to a decrease in the rate of resensitization. We propose that the existence of a receptor reserve might explain the poor correlation between functional response and surface receptor number, and that one of its roles might be to permit rapid resensitization after a significant agonist-induced decrease in surface receptor number. The purpose of receptor cycling might be to allow dephosphorylation (and reactivation) of receptors that have become phosphorylated (and inactivated) in response to agonist stimulation, because the protein phosphatase inhibitor calyculin A significantly reduced resensitization.     

Vasoactive intestinal peptide (VIP) stimulates [Ca2+]i and cyclic AMPin CHO cells expressing Galpha16

The stimulatory effect of vasoactive intestinal peptide (VIP) and analogues on [Ca2+]i has been investigated in chinese hamster ovary (CHO) cells stably transfected with the reporter gene aequorin, and expressing either the human VPAC1or VPAC2 receptor in absence or in presence of the Galpha16. In cells that were not transfected with Galpha16 and expressed a similar density of receptors, the VIP induced [Ca2+]i ncrease was higher in VPAC1 than in VPAC2 receptor expressing cells. In aequorin/Galpha16 cotransfected cells, the VIP-induced response was higher, reaching 70 to 80% of the maximal calcium response, obtained after digitonin treatment, in response to both VPAC1 and VPAC2 receptor stimulation.The results suggest that in hematopoietic cells, which express both VIP receptors and Galpha16, the signalling pathway of VIP could be mediated through both cyclic AMP and [Ca2+]i increase.     

Design, synthesis, and evaluation of linear and cyclic peptide ligands for PDZ10 of the multi-PDZ domain protein MUPP1

PDZ10 is the 10th of 13 PDZ domains found within MUPP1, a cytoplasmic scaffolding protein first identified as an endogenous binding partner of serotonin receptor type 2c (5HT2c). This association, as with those of several other interacting proteins that have subsequently been identified, is mediated through the C-terminal tail of the PDZ domain partner. Using isothermal titration calorimetry (ITC), we measured the thermodynamic binding parameters [changes in Gibbs free energy (DeltaG), enthalpy (DeltaH) and entropy (TDeltaS)] of the isolated PDZ10 domain for variable-length N-acetylated peptides from the 5HT2c serotonin receptor C-terminal sequence, as well as for octapeptides of eight other putative partner proteins of PDZ10 (5HT2a, hc-kit, hTapp1, mTapp2, TARP, NG2, claudin-1, and HPV-18 E6). In length dependence studies of the 5HT2c sequence, the maximal affinity of the peptides leveled off rapidly and further elongation did not significantly improve the dissociation constant (Kd) of 11 microM observed with the pentapeptide. Among the native partners of PDZ10, octapeptides derived from the hc-kit and 5HT2c proteins were the strongest binders, with Kd values of 5.2 and 8.5 microM, respectively. The heat capacity change (DeltaCp) for the 5HT2c octapeptide was determined to be -94 cal/mol, and a calculated estimate indicates burial of polar and apolar surface areas in equal measure upon ligand binding. Peptides with phosphoserine at either the P-1 or P-2 position experienced decreased affinity, which is in accord with the hypothesis that reversible phosphorylation is a possible mechanism for regulating PDZ domain-mediated interactions. Additionally, two conformationally constrained side chain-bridged cyclic peptide ligands were also designed, prepared, evaluated by ITC, and shown to bind PDZ10 primarily through a favorable change in entropy.     

Desensitization of the Y1 cell adrenocorticotropin receptor: evidence for a restricted heterologous mechanism implying a role for receptor-effector complexes

Receptor desensitization provides a potential mechanism for the regulation of adrenocortical adrenocorticotropin (ACTH) responsiveness. Using the mouse adrenocortical Y1 cell line we demonstrate that ACTH effectively desensitizes the cAMP response of its own receptor, the melanocortin 2 receptor (MC2R), in these cells with a maximal effect between 30 and 60 min. Neither forskolin nor isoproterenol (in Y1 cells stably transfected with the beta(2)-adrenergic receptor) desensitize this ACTH response. ACTH desensitizes its receptor at concentrations at which only a fraction of receptors are occupied, implying that this mechanism acts on agonist-unoccupied receptors. Y1 cells express G protein-coupled receptor kinase (GRK) 2 and 5, but stable expression of a dominant negative GRK2 (K220W) only marginally reduces the desensitization by ACTH. The protein kinase A (PKA) inhibitor, H89, extinguishes almost the entire desensitization response over the initial 30-min period at all concentrations of ACTH. A mutant MC2R in which the single consensus PKA phosphorylation site has been mutated (S208A) when expressed in MC2R-negative Y6 cells is also unable to desensitize. These data imply a heterologous, PKA-dependent, mode of desensitization, which is restricted to agonist-occupied and -unoccupied MC2R, possibly as a consequence of receptor/effector complexes that functionally compartmentalize this receptor.     

Simple luminometric assay to detect phosphoinositol-linked receptor expression in Xenopus oocytes.

A simple and sensitive method to measure the expression of phosphoinositol-linked receptors in Xenopus laevis oocytes is described. Oocytes are co-injected with the calcium photoprotein aequorin and RNA, encoding the receptor of interest. The binding of ligand to the expressed receptor increases intracellular calcium that induces the aequorin to luminesce. With an autosampler-equipped luminometer, this provides a fully automated assay of receptor expression of oocytes. This method was applied to cloning the bombesin/GRP receptor expressed in Swiss 3T3 fibroblasts. Oocytes expressing the cloned BR showed up to a 10,000-fold increase in light emission in response to bombesin. The sensitivity of this procedure allowed detection of positive luminometer signals in single oocytes injected with RNA transcribed from cDNA pools as large as 25,000 clones. These findings show the potential value of this procedure for rapid screening of expression libraries, structure/function analysis of receptors and analysis of receptor antagonists or agonists.     

Serotonin (5HT), fluoxetine, imipramine and dopamine target distinct 5HT receptor signaling to modulate Caenorhabditis elegans egg-laying behavior

Drugs that target the serotonergic system are the most commonly prescribed therapeutic agents and are used for treatment of a wide range of behavioral and neurological disorders. However, the mechanism of the drug action remain a conjecture. Here, we dissect the genetic targets of serotonin (5HT), the selective 5HT reuptake inhibitor (SSRI) fluoxetine (Prozac), the tricyclic antidepressant imipramine, and dopamine. Using the well-established serotonergic response in C. elegans egg-laying behavior as a paradigm, we show that action of fluoxetine and imipramine at the 5HT reuptake transporter (SERT) and at 5HT receptors are separable mechanisms. Even mutants completely lacking 5HT or SERT can partially respond to fluoxetine and imipramine. Furthermore, distinct mechanisms for each drug can be recognized to mediate these responses. Deletion of SER-1, a 5HT1 receptor, abolishes the response to 5HT but has only a minor effect on the response to imipramine and no effect on the response to fluoxetine. In contrast, deletion of SER-4, a 5HT2 receptor, confers significant resistance to imipramine while leaving the responses to 5HT or fluoxetine intact. Further, fluoxetine can stimulate egg laying via the Gq protein EGL-30, independent of SER-1, SER-4, or 5HT. We also show that dopamine antagonizes the 5HT action via the 5HT-gated ion channel MOD-1 signaling, suggesting that this channel activity couples 5HT and dopamine signaling. These results suggest that the actions of these drugs at specific receptor subtypes could determine their therapeutic efficacy. SSRIs and tricyclic antidepressants may regulate 5HT outputs independently of synaptic levels of 5HT.     

Desensitization of the human V2 vasopressin receptor. Homologous effects in the absence of heterologous desensitization.

Three main pathways have been implicated in desensitization of receptors that stimulate adenylylcyclase (AC): cAMP-mediated phosphorylation; cAMP-independent phosphorylation, and receptor internalization. Cell lines derived from the murine Ltk- cell were found useful in exploring the contribution of cAMP-dependent phosphorylation in V2 vasopressin receptor desensitization. The HTB-2 cell expresses the human V2 vasopressin receptor, introduced by transfection of human genomic DNA, and the prostaglandin E1 (PGE1) receptor, endogenous to the Ltk- cell. The A7 cell expresses the hamster beta 2-adrenoceptor, which undergoes the above-mentioned desensitization processes. Treatment of HTB-2 cells with arginine-vasopressin (AVP) had no effect on AC responsiveness to PGE1, but promoted desensitization of the AVP response. This was seen as a 5-6-fold right shift in the dose-response curves for AVP action (cAMP accumulation in intact cells and AC stimulation in homogenates and isolated membranes) and in a decrease in the maximum effect of AVP on these parameters. AVP treatment caused a decrease in cell surface receptors to approximately 75% of control without changes in KD, as determined by Scatchard analysis. When cAMP was increased by treatment with 10 microM PGE1 and isobutylmethylxanthine, desensitization of the PGE1 receptor was observed but not of the AVP receptor. In A7 cells the same treatment caused, as expected, a 3-fold right shift in the dose-response curve for AC stimulation by isoproterenol, indicating that L cells can mediate heterologous desensitization. These data demonstrate that the V2 vasopressin and the PGE1 receptors undergo homologous desensitization in the absence of cAMP-mediated phosphorylation and that this component is not required for vasopressin receptor internalization.     

Pharmacological profile of a model for central serotonin receptor activation

The head shake response in rats after systemic administration of the serotonin (5HT) precursor 5-hydroxytryptophan (5HTP) was pharmacologically characterized and shown to be a useful animal model to quantify brain 5HT receptor activation. The behavior occurred in a dose-dependent manner after injection of 5HTP and the 5HT agonist quipazine. Head shakes were also observed after injection of L-tryptophan, 5-methoxydimethyltryptamine and fenfluramine. The 5HT antagonists cyproheptadine and metergoline were potent blockers of the response. Xylamidine, a peripheral 5HT antagonist, had no effect on head shaking. Inhibition of 5HT uptake with fluoxetine potentiated the head shake response after 5HTP. Manipulation of central cholinergic or GABAergic mechanisms did not alter 5HTP-induced shakes. Alpha-noradrenergic receptor blockade had no significant effect on head shakes. However, desmethylimipramine was equipotent with methysergide as an antagonist of the behavior. Beta-noradrenergic receptor blockade had no specific effect on 5HTP head shakes. Concomitant dopamine receptor activation with SK&;F 38393 did not affect head shakes but the neuroleptics chlorpromazine and pimozide reduced the number of head shakes after 5HTP. The H₁ receptor antagonist pyrilamine had no effect on head shakes. It is concluded that 5HTP-induced head shakes in rats is a quantitative model of brain 5HT receptor activation which is particularly sensitive to 5HT antagonists.     

Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor

UTP activates P2Y₂ receptors in both 1321N1 cell transfectants expressing the P2Y₂ receptor and human HT-29 epithelial cells expressing endogenous P2Y₂ receptors with an EC₅₀ of 0.2- 1.0 M. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC₅₀ for UTP-induced receptor desensitization was 0.3 - 1.0 M for both systems). Desensitization and down-regulation of the P2Y₂ nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y₂ receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y,₂ receptor. In these cells, potent P2Y₂ receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca²⁺]_i, after addition of desensitizing concentrations of UTP, indicating that P2Y₂ receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca²⁺]_i induced by UTP in 1321N1 cell transfectants expressing the P2Y₂ receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y₂ receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), reduced the subsequent response to UTP by about 50% whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases - 1 and -2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y₂ receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y₂ receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y₂ nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y₂ receptor agonists.     

Design and synthesis of orally-active and selective azaindane 5HT2c agonist for the treatment of obesity

Based on our original pyrazine hit, CP-0809101, novel conformationally-restricted 5HT2c receptor agonists with 2-piperazin-azaindane scaffold were designed. Synthesis and structure–activity relationship (SAR) studies are described with emphasis on optimization of the selectivity against 5HT2a and 5HT2b receptors with excellent 2c potency. Orally-active and selective compounds were identified with dose–responsive in vivo efficacy in our pre-clinical food intake model.