Synthesis of novel 5-substituted-2-aminotetralin analogs: 5-HT1A and 5-HT7 G protein-coupled receptor affinity, 3D-QSAR and molecular modeling

The serotonin 5-HT₇ G protein-coupled receptor (GPCR) is a proposed pharmacotherapeutic target for a variety of central and peripheral indications, albeit, there are no approved drugs selective for binding 5-HT₇. We previously reported that a lead analog based on the 5-substituted-N,N-disubstituted-1,2,3,4-tetrahydronaphthalen-2-amine (5-substituted-2-aminotetralin, 5-SAT) scaffold binds with high affinity at the 5-HT₇ GPCR, and can treat symptoms of autism in mouse models; subsequently, the lead was found to have high affinity at the 5-HT_1A GPCR. Herein, we report the synthesis of novel 5-SAT analogs to develop a 3-dimensional quantitative structure—affinity relationship (3D-QSAR) at the human 5-HT₇ receptor for comparison with similar studies at the highly homologous 5-HT_1A receptor. We report 35 new 5-SAT ligands, some with very high affinity (K_i ≤ 1 nM) and stereoselectivity at 5-HT₇ + or 5-HT_1A receptors, several with modest selectivity (up to 12-fold) for binding at 5-HT₇, and, several ligands with high selectivity (up to 40-fold) at the 5-HT_1A receptor. 3D-QSAR results indicate that steric extensions at the C(5)-position improve selectivity for the 5-HT₇ over 5-HT_1A receptor, while steric and hydrophobic extensions at the chiral C(2)-amino position impart 5-HT_1A selectivity. In silico receptor homology modeling studies, supplemented with molecular dynamics simulations and binding free energy calculations, were used to rationalize experimentally-determined receptor selectivity and stereoselective affinity results. The data from these studies indicate that the 5-SAT chemotype, previously shown to be safe and efficacious in rodent paradigms of neurodevelopmental and neuropsychiatric disorders, is amenable to structural modification to optimize affinity at serotonin 5-HT₇ vs. 5-HT_1A GPCRs, as may be required for successful clinical translation.     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.     

Molecular basis for cis-urocanic acid as a 5-HT2A receptor agonist

The underlying mechanisms of urocanic acid (UA) to induce immune suppression remain elusive until the recent finding that cis-UA acts via the serotonin, 5-hydroxytryptamine (5-HT) receptor subtype 5-HT_2A. In the present study, the interactions of cis-UA to 5-HT_2A receptor were explored and compared with those of 5-HT to the same receptor using computational docking. Similar binding modes were observed for cis-UA and 5-HT with 5-HT_2A receptor and the former possessed relatively higher binding affinity, which may account for cis-UA being a serotonin receptor agonist. Moreover, the molecular basis for the distinct binding affinities between the trans- and cis-UA with 5-HT_2A receptor was also provided.     

The Cardiac Ventricular 5-HT4 Receptor Is Functional in Late Foetal Development and Is Reactivated in Heart Failure

A positive inotropic responsiveness to serotonin, mediated by 5-HT₄ and 5-HT_2A receptors, appears in the ventricle of rats with post-infarction congestive heart failure (HF) and pressure overload-induced hypertrophy. A hallmark of HF is a transition towards a foetal genotype which correlates with loss of cardiac functions. Thus, we wanted to investigate whether the foetal and neonatal cardiac ventricle displays serotonin responsiveness. Wistar rat hearts were collected day 3 and 1 before expected birth (days -3 and -1), as well as day 1, 3, 5 and 113 (age matched with Sham and HF) after birth. Hearts from post-infarction HF and sham-operated animals (Sham) were also collected. Heart tissue was examined for mRNA expression of 5-HT₄, 5-HT_2A and 5-HT_2B serotonin receptors, 5-HT transporter, atrial natriuretic peptide (ANP) and myosin heavy chain (MHC)-α and MHC-β (real-time quantitative RT-PCR) as well as 5-HT-receptor-mediated increase in contractile function ex vivo (electrical field stimulation of ventricular strips from foetal and neonatal rats and left ventricular papillary muscle from adult rats in organ bath). Both 5-HT₄ mRNA expression and functional responses were highest at day -3 and decreased gradually to day 5, with a further decrease to adult levels. In HF, receptor mRNA levels and functional responses reappeared, but to lower levels than in the foetal ventricle. The 5-HT_2A and 5-HT_2B receptor mRNA levels increased to a maximum immediately after birth, but of these, only the 5-HT_2A receptor mediated a positive inotropic response. We suggest that the 5-HT₄ receptor is a representative of a foetal cardiac gene program, functional in late foetal development and reactivated in heart failure.     

Prefrontal Serotonergic Denervation Induces Increase in the Density of 5-HT<Subscript>2A</Subscript> Receptors in Adult Rat Prefrontal Cortex

The 5-HTergic system and particularly 5-HT_2A receptors have been involved in prefrontal cognitive functions, but the underlying mechanisms by which the serotonin (5-HT) system modulates these processes are still unclear. In this work, the effects of prefrontal 5-HTergic denervation on the density and expression levels of 5-HT_2A receptors were evaluated by immunohistochemical and molecular biology studies in the prefrontal cortex (PFC). The [³H]-Ketanserin binding study revealed an increase in the B_max, along with no change in the binding affinity (K_D) for 5-HT_2A receptors. The increase in PFC of 5-HT_2A receptor density in response to denervation was accompanied by increase in 5-HT_2A receptor mRNA and protein levels. This increase in the number of 5-HT_2A receptors may be interpreted as an adaptive plastic change, i.e., hypersensitivity; resulting from the selective pharmacological lesion of the raphe-proceeding 5-HTergic fibers to the PFC. Based on previous evidence, this could be strongly related to the abnormal expression of short-term memory.     

Regulation of GABAergic inhibition by serotonin signaling in prefrontal cortex: molecular mechanisms and functional implications

Serotonergic neurotransmission in prefrontal cortex (PFC) plays a key role in regulating emotion and cognition under normal and pathological conditios. Increasing evidence suggests that serotonin receptors are involved in the complex regulation of GABAergic inhibitory transmission in PFC. Activation of postsynaptic 5-HT₂ receptors in PFC pyramidal neurons inhibits GABA_A-receptor currents via phosphorylation of GABA_A receptor γ2 subunits by RACK1-anchored PKC. In contrast, activation of postsynaptic 5-HT₄ receptors produces an activity-dependent bi-directional regulation of GABA-evoked currents in PFC pyramidal neurons, which is mediated through phosphorylation of GABA_A-receptor β subunits by anchored PKA. On the presynaptic side, GABAergic inhibition is regulated by 5-HT through the activation of 5-HT₂, 5-HT₁, and 5-HT₃ receptors on GABAergic intereneurons. These data provide a molecular and cellular mechanism for serotonin to dynamically regulate synaptic transmission and neuronal excitability in the PFC network, which may underlie the actions of many antidepressant and antipsychotic drugs.     

Relationship between 5-HT2A receptor mRNA density and contractility in trachea and aorta from guinea pig and rat

The present studies document marked differences in contractile responsiveness to serotonin in trachea and aorta between guinea pig and rat. For example, the guinea pig trachea and rat aorta markedly contract in response to serotonin via activation of 5-HT_2A receptors. In contrast, the rat and guinea pig aorta only modestly contract to serotonin. The availability of 5-HT_2A receptor selective cDNA clones from brain of both guinea pig and rat permitted molecular probes to be designed and PCR amplification studies initiated to identify and quantify 5-HT_2A receptor specific mRNA in these tissues. For trachea, 3-fold higher concentrations of 5-HT_2A receptor specific mRNA were found in guinea pig relative to rat trachea. These data are consistent with the more profound contractile response to serotonin in guinea pig versus rat trachea and suggest that differences in tracheal contractility to serotonin correlate with the density of 5-HT_2A receptor mRNA. In contrast, although rat aorta contracted more dramatically to serotonin than guinea pig aorta, rat aorta possessed a similar concentration of 5-HT_2A receptor specific mRNA as compared to guinea pig aorta. Thus, for the aorta, differences in the concentration of 5-HT_2A receptor mRNA are not sufficient to explain the observed differences in contractility between tissues from guinea pig and rat. These studies documenting 5-HT_2A receptor mRNA in rat trachea and guinea pig aorta, two tissues that do not markedly contract in response to serotonin indicate that 5-HT_2A receptor mRNA although present, has not resulted in a receptor capable of mediating a contractile response in these tissues.     

Brainstem 5-Hydroxytrytamine1A Binding Sites are not Down-Regulated by Agonists which Induce Tolerance in the Rat: Myoclonus and other Serotonergic Behaviors

Abstract

To study the regulation of 5-HT_1A receptors in the brainstem, the region most relevant to the serotonin syndrome and to serotonin-responsive human myoclonic disorders, we chronically treated rats with various 5-HT_1A agonists and labeled 5-HT_1A sites with [³H]8-OH-DPAT. Daily injection for 30 consecutive days of 10 mg/kg ip 8-OH-DPAT (pre- and post-synaptic 5-HT_1A agonist) significantly decreased 8-OH-DPAT-evoked flat body posture, forelimb myoclonus, and hypothermia compared to chronic vehicle injection. There was no cross tolerance to 8-OH-DPAT in rats chronically injected with ipsapirone or buspirone (presynaptic 5-HT_1A agonists). However, none of the 5HT_1A agonists significantly altered B_max of brainstem 5-HT_1A binding sites. Chronic injection with other drugs such as 1-propranolol, (±) pindolol and spiperone (5-HT_1A and 5-HT₂ antagonists), methysergide (5-HT₁ and 5-HT₂ antagonist), and agonists and antagonists at various other 5-HT receptors also had no effect on binding parameters. These data demonstrate lack of cross-tolerance between pre- and post-synaptically acting 5-HT_1A agonists and absence of down-regulation of presynaptic 5-HT_1A sites at doses which induced tolerance of 5-HT_1A-mediated behaviors of the serotonin syndrome. They suggest changes in the post-synaptic cell rather than the receptor recognition site as the mechanism of tolerance.     

Polymorphism of the Serotonin System Genes in Some Finno-Ugric Populations

Polymorphic sites in the genes encoding monoamine oxidase A (MAO-A), serotonin transporter (hSERT) and 5-HT_2A receptor were typed in Khant and Komi ethnic groups with the purpose of revealing possible inerpopulation differences in genotype and allele frequencies. No statistically significant differences in the hSERT and 5-HT_2A gene frequencies were detected. At the same time, the populations examined had statistically significantly different MAO-A genotype and allele frequencies. These results obtained indicate the prevalence of the site gain alleles of theEcoRV and Fnu4HI RFLP loci at the MAO-A gene in Komis and the of the corresponding site loss alleles in Khants.     

Discovery of SMP-304, a novel benzylpiperidine derivative with serotonin transporter inhibitory activity and 5-HT1A weak partial agonistic activity showing the antidepressant-like effect

We report the discovery of a novel benzylpiperidine derivative with serotonin transporter (SERT) inhibitory activity and 5-HT_1A receptor weak partial agonistic activity showing the antidepressant-like effect. The 3-methoxyphenyl group and the phenethyl group of compound 1, which has weak SERT binding activity, but potent 5-HT_1A binding activity, were optimized, leading to compound 35 with potent and balanced dual SERT and 5-HT_1A binding activity, but also potent CYP2D6 inhibitory activity. Replacement of the methoxy group in the left part of compound 35 with a larger alkoxy group, such as ethoxy, isopropoxy or methoxy-ethoxy group ameliorated CYP2D6 inhibition, giving SMP-304 as a candidate. SMP-304 with serotonin uptake inhibitory activity and 5-HT_1A weak partial agonistic activity, which could work as a 5-HT_1A antagonist, displayed faster onset of antidepressant-like effect than a representative SSRI paroxetine in an animal model.     

Phosphatidylinositol 4-phosphate 5-kinase Iγ_v6, a new splice variant found in rodents and humans

It has been shown that anti-cancer drug induces secretion of serotonin (5-HT) from small intestine which activates serotonin type 3 (5-HT₃) receptor to cause nausea and vomiting. In general, antagonist for 5-HT₃ receptor is used as anti-emetics during chemotherapy. However, we found that anti-cancer drug irinotecan itself inhibits 5-HT-gated current through the homomeric 5-HT_3A and heteromeric 5-HT_3AB receptor in a concentration-dependent manner. The inhibitory effect of irinotecan on 5-HT_3A receptor was more potent than that on 5-HT_3AB receptor. On the other hand, SN-38, a metabolite of irinotecan, had no effect on the responsiveness. Our findings suggest that irinotecan itself could have anti-emetic activities through inhibition of the 5-HT_3A and 5-HT_3AB receptor.     

5-HT2 Receptors Facilitate JC Polyomavirus Entry

The human JC polyomavirus (JCPyV) causes the rapidly progressing demyelinating disease progressive multifocal leukoencephalopathy (PML). The disease occurs most often in individuals with AIDS but also occurs in individuals receiving immunomodulatory therapies for immune-related diseases such as multiple sclerosis. JCPyV infection of host cells requires the pentasaccharide lactoseries tetrasaccharide c (LSTc) and the serotonin receptor 5-hydroxytryptamine (5-HT) receptor 5-HT_2AR. While LSTc is involved in the initial attachment of virus to cells via interactions with VP1, the mechanism by which 5-HT_2AR contributes to infection is not clear. To further define the roles of serotonin receptors in infection, HEK293A cells, which are poorly permissive to JCPyV, were transfected with 14 different isoforms of serotonin receptor. Only 5-HT₂ receptors were found to support infection by JCPyV. None of the other 11 isoforms of serotonin receptor supported JCPyV infection. Expression of 5-HT₂ receptors did not increase binding of JCPyV to cells, but this was not unexpected, given that the cells uniformly expressed the major attachment receptor, LSTc. Infection of these cells remained sensitive to inhibition with soluble LSTc, confirming that LSTc recognition is required for JCPyV infection. Virus internalization into HEK293A cells was significantly and specifically enhanced when 5HT₂ receptors were expressed. Taken together, these data confirm that the carbohydrate LSTc is the attachment receptor for JCPyV and that the type 2 serotonin receptors contribute to JCPyV infection by facilitating entry.     

Synthesis and evaluation of a series of 2-substituted-5-thiopropylpiperazine (piperidine)-1,3,4-oxadiazoles derivatives as atypical antipsychotics

Background

It is important to develop novel antipsychotics that can effectively treat schizophrenia with minor side-effects. The aim of our work is to develop novel antipsychotics that act on dopamine D₂ and D₃, serotonin 5-HT_1A and 5-HT_2A receptors with low affinity for the serotonin 5-HT_2C and H₁ receptors, which can effectively cure positive symptoms, negative symptoms and cognitive impairment without the weight gain side-effect.

Methodology/Principal Findings

A series of 2-substituted-5-thiopropylpiperazine (piperidine) -1,3,4-oxadiazoles derivatives have been synthesized and the target compounds were evaluated for binding affinities to D₂, 5-HT_1A and 5-HT_2A receptors. Preliminary results indicated that compounds 14, 16 and 22 exhibited high affinities to D₂, 5-HT_1A and 5-HT_2A receptors among these compounds. Further binding tests showed that compound 22 had high affinity for D₃ receptor, and low affinity for serotonin 5-HT_2C and H₁ receptors. In addition, compound 22 inhibited apomorphine-induced climbing behavior and MK-801-induced hyperactivity with no extrapyramidal symptoms liability in mice. Moreover, compound 22 exhibited acceptable pharmacokinetic properties.

Conclusions/Significance

Compound 22 showed an atypical antipsychotic activity without liability for extrapyramidal symptoms. We anticipate compound 22 to be useful for developing a novel class of drug for the treatment of schizophrenia.     

5-HT<Subscript>1A</Subscript> receptor expression in pyramidal neurons of cortical and limbic brain regions

We studied expression of the 5-HT_1A receptor in cortical and limbic areas of the brain of the tree shrew. In situ hybridization with a receptor-specific probe and immunocytochemistry with various antibodies was used to identify distinct neurons expressing the receptor. In vitro receptor autoradiography with ³H-8-OH-DPAT (³H-8-hydroxy-2-[di-n-propylamino]tetralin) was performed to visualize receptor-binding sites. In the prefrontal, insular, and occipital cortex, 5-HT_1A receptor mRNA was expressed in pyramidal neurons of layer 2, whereas ³H-8-OH-DPAT labeled layers 1 and 2 generating a columnar-like pattern in the prefrontal and occipital cortex. In the striate and ventral occipital cortex, receptor mRNA was present within layers 5 and 6 in pyramidal neurons and Meynert cells. Pyramid-like neurons in the claustrum and anterior olfactory nucleus also expressed the receptor. Principal neurons in hippocampal region CA1 expressed 5-HT_1A receptor mRNA, and ³H-8-OH-DPAT labeled both the stratum oriens and stratum radiatum. CA3 pyramidal neurons displayed low 5-HT_1A receptor expression, whereas granule neurons in the dentate gyrus revealed moderate expression of this receptor. In the amygdala, large pyramid-like neurons in the basal magnocellular nucleus strongly expressed the receptor. Immunocytochemistry with antibodies against parvalbumin, calbindin, and gamma aminobutyric acid (GABA) provided no evidence for 5-HT_1A receptor expression in GABAergic neurons in cortical and limbic brain areas. Our data agree with previous findings showing that the 5-HT_1A receptor mediates the modulation of glutamatergic neurons. Expression in the limbic and cortical areas suggested an involvement of 5-HT_1A receptors in emotional and cognitive processes.This work was supported by the German Science Foundation (SFB 406; C4 to G.F.).     

Synthesis and antidepressant-like activity of novel alkoxy-piperidine derivatives targeting SSRI/5-HT1A/5-HT7

A series of novel alkoxy-piperidine derivatives were synthesized and evaluated for their serotonin reuptake inhibitory and binding affinities for 5-HT_1A/5-HT₇ receptors. In vivo antidepressant activities of the selective compounds were explored using the forced swimming test (FST) and tail suspension test (TST) in mice. The results showed that compounds 7a (reuptake inhibition (RUI), IC₅₀ = 177 nM; 5-HT_1A, K_i = 12 nM; 5-HT₇, K_i = 25 nM) and 15g (RUI, IC₅₀ = 85 nM; 5-HT_1A, K_i = 17 nM; 5-HT₇, K_i = 35 nM) were potential antidepressant agents in animal behavioral models with high 5-HT_1A/5-HT₇ receptor affinities and moderate serotonin reuptake inhibition, and good metabolic stability in vitro.     

PKCδ Knockout Mice Are Protected from Dextromethorphan-Induced Serotonergic Behaviors in Mice: Involvements of Downregulation of 5-HT<Subscript>1A</Subscript> Receptor and Upregulation of Nrf2-Dependent GSH Synthesis

We investigated whether a specific serotonin (5-HT) receptor-mediated mechanism was involved in dextromethorphan (DM)-induced serotonergic behaviors. We firstly observed that the activation of 5-HT_1A receptor, but not 5-HT_2A receptor, contributed to DM-induced serotonergic behaviors in mice. We aimed to determine whether the upregulation of 5-HT_1A receptor induced by DM facilitates the specific induction of certain PKC isoform, because previous reports suggested that 5-HT_1A receptor activates protein kinase C (PKC). A high dose of DM (80 mg/kg, i.p.) induced a selective induction of PKCδ out of PKCα, PKCβI, PKCβII, PKCξ, and PKCδ in the hypothalamus of wild-type (WT) mice. More importantly, 5-HT_1A receptor co-immunoprecipitated PKCδ in the presence of DM. Consistently, rottlerin, a pharmacological inhibitor of PKCδ, or PKCδ knockout significantly protected against increases in 5-HT_1A receptor gene expression, 5-HT turnover rate, and serotonergic behaviors induced by DM. Treatment with DM resulted in an initial increase in nuclear factor erythroid-2-related factor 2 (Nrf2) nuclear translocation and DNA-binding activity, γ-glutamylcysteine (GCL) mRNA expression, and glutathione (GSH) level. This compensative induction was further potentiated by rottlerin or PKCδ knockout. However, GCL mRNA and GSH/GSSG levels were decreased 6 and 12 h post-DM. These decreases were attenuated by PKCδ inhibition. Our results suggest that interaction between 5-HT_1A receptor and PKCδ is critical for inducing DM-induced serotonergic behaviors and that inhibition of PKCδ attenuates the serotonergic behaviors via downregulation of 5-HT_1A receptor and upregulation of Nrf2-dependent GSH synthesis.     

Involvement of 5-HT<Subscript>2A</Subscript> receptors in genetic mechanisms of autoregulation of brain 5-HT system

The brain serotonin (5-HT) system has been implicated in the pathophysiology of anxiety, depression, drug addiction, and schizophrenia. 5-HT_2A receptors are involved in the mechanisms of stressinduced psychopathology and impulsive behavior. In this work, we investigated the role of 5-HT_2A receptors in the autoregulation of the brain 5-HT system. Chronic treatment with DOI, a 5-HT_2A receptor agonist (1.0 mg/kg, i.p./14 days), produced a considerable decrease in the number of 5-HT_2A receptor-mediated head twitches in AKR/J mice, indicating the desensitization of 5-HT_2A receptors. Chronic DOI treatment did not affect the expression of the 5-HT_2A receptor gene in the midbrain, hippocampus and frontal cortex. At the same time, an increase in the expression of the gene encoding a key enzyme of 5-HT synthesis, tryptophan hydroxylase-2 (TPH-2), accompanied with an increase in TPH-2 activity and 5-HT levels, and decreased expression of the serotonin transporter (5-HTT) gene were observed in the midbrain of DOI-treated mice. These results provide new evidence of receptor-gene cross-talk in the brain 5-HT system and implication 5-HT_2A receptors in the autoregulation of the brain 5-HT system.