Convolamine, a tropane alkaloid extracted from Convolvulus plauricalis, is a potent sigma-1 receptor-positive modulator with cognitive and neuroprotective properties.

BACKGROUND AND AIM: By using an in vivo phenotypic screening assay in zebrafish, we identified Convolamine, a tropane alkaloid from Convulvus plauricalis, as a positive modulator of the sigma-1 receptor (S1R). The wfs1abKO zebrafish larva, a model of Wolfram syndrome, exhibits an increased visual-motor response due to a mutation in Wolframin, a protein involved in endoplasmic reticulum-mitochondria communication. We previously reported that ligand activating S1R, restored the cellular and behavioral deficits in patient fibroblasts and zebrafish and mouse models. EXPERIMENTAL PROCEDURES: We screened a library of 108 repurposing and natural compounds on zebrafish motor response. KEY RESULTS: One hit, the tropane alkaloid Convolamine, restored normal mobility in wfs1abKO larvae without affecting wfs1abWT controls. They did not bind to the S1R agonist/antagonist binding site nor dissociated S1R from BiP, an S1R activity assay in vitro, but behaved as a positive modulator by shifting the IC50 value of the reference agonist PRE-084 to lower values. Convolamine restored learning in Wfs1∆Exon8 , Dizocilpine-treated, and Aß25-35 -treated mice. These effects were observed at low ~1 mg/kg doses, not shared by Convolvine, the desmethyl metabolite, and blocked by an S1R antagonist. CONCLUSION AND IMPLICATIONS: Convolamine therefore acts as an S1R positive modulator and this pharmacological action is relevant to the traditional use of Shankhpushpi in memory and cognitive protection.

Coumarin Derivatives Exert Anti-Lung Cancer Activity by Inhibition of Epithelial-Mesenchymal Transition and Migration in A549 Cells.

A series of coumarin derivatives and isosteres were synthesized from the reaction of triflic intermediates with phenylboronic acids, terminal alkynes, and organozinc compounds through palladium-catalyzed cross-coupling reactions. The in vitro cytotoxic effect of the compounds was evaluated against two non-small cell lung carcinoma (NSCLC) cell lines (A-549 and H2170) and a normal cell line (NIH-3T3) using cisplatin as a reference drug. Additionally, the effects of the most promising coumarin derivative (9f) in reversing the epithelial-to-mesenchymal transition (EMT) in IL-1ß-stimulated A549 cells and in inhibiting the EMT-associated migratory ability in A549 cells were also evaluated. 9f had the greatest cytotoxic effect (CC50 = 7.1 ± 0.8 and 3.3 ± 0.5 µM, respectively against A549 and H2170 cells) and CC50 value of 25.8 µM for NIH-3T3 cells. 9f inhibited the IL-1ß-induced EMT in epithelial cells by inhibiting the F-actin reorganization, attenuating changes in the actin cytoskeleton reorganization, and downregulating vimentin in A549 cells stimulated by IL-1ß. Treatment of A549 cells with 9f at 7 µM for 24 h significantly reduced the migration of IL-1ß-stimulated cells, which is a phenomenon confirmed by qualitative assessment of the wound closure. Taken together, our findings suggest that coumarin derivatives, especially compound 9f, may become a promising candidate for lung cancer therapy, especially in lung cancer promoted by NSCLC cell lines.

Antiallodynic action of phosphodiesterase inhibitors in a mouse model of peripheral nerve injury.

Neuropathic pain arises as a consequence of a lesion or disease affecting the somatosensory nervous system. It is accompanied by neuronal and non-neuronal alterations, including alterations in intracellular second messenger pathways. Cellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) are regulated by phosphodiesterase (PDE) enzymes. Here, we studied the impact of PDE inhibitors (PDEi) in a mouse model of peripheral nerve injury induced by placing a cuff around the main branch of the sciatic nerve. Mechanical hypersensitivity, evaluated using von Frey filaments, was relieved by sustained treatment with the non-selective PDEi theophylline and ibudilast (AV-411), with PDE4i rolipram, etazolate and YM-976, and with PDE5i sildenafil, zaprinast and MY-5445, but not by treatments with PDE1i vinpocetine, PDE2i EHNA or PDE3i milrinone. Using pharmacological and knock-out approaches, we show a preferential implication of delta opioid receptors in the action of the PDE4i rolipram and of both mu and delta opioid receptors in the action of the PDE5i sildenafil. Calcium imaging highlighted a preferential action of rolipram on dorsal root ganglia non-neuronal cells, through PDE4B and PDE4D inhibition. Rolipram had anti-neuroimmune action, as shown by its impact on levels of the pro-inflammatory cytokine tumor necrosis factor-α (TNFα) in the dorsal root ganglia of mice with peripheral nerve injury, as well as in human peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharides. This study suggests that PDEs, especially PDE4 and 5, may be targets of interest in the treatment of neuropathic pain.

Synthesis, Antileishmanial Activity and in silico Studies of Aminoguanidine Hydrazones (AGH) and Thiosemicarbazones (TSC) Against Leishmania chagasi Amastigotes.

BACKGROUND: Leishmaniasis is a worldwide health problem, highly endemic in developing countries. Among the four main clinical forms of the disease, visceral leishmaniasis is the most severe, fatal in 95% of cases. The undesired side-effects from first-line chemotherapy and the reported drug resistance search for effective drugs that can replace or supplement those currently used in an urgent need. Aminoguanidine hydrazones (AGH's) have been explored for exhibiting a diverse spectrum of biological activities, in particular the antileishmanial activity of MGBG. The bioisosteres thiosemicarbazones (TSC's) offer a similar biological activity diversity, including antiprotozoal effects against Leishmania species and Trypanosoma cruzi. OBJECTIVES: Considering the impact of leishmaniasis worldwide, this work aimed to design, synthesize, and perform a screening upon L. chagasi amastigotes and for the cytotoxicity of the small "inhouse" library of both AGH and TSC derivatives and their structurally-related compounds. METHODS: A set of AGH's (3-7), TSC's (9, 10), and semicarbazones (11) were initially synthesized. Subsequently, different semi-constrained analogs were designed and also prepared, including thiazolidines (12), dihydrothiazines (13), imidazolines (15), pyrimidines (16, 18) azines (19, 20), and benzotriazepinones (23-25). All intermediates and target compounds were obtained with satisfactory yields and exhibited spectral data consistent with their structures. All final compounds were evaluated against L. chagasi amastigotes and J774.A1 cell line. Molecular docking was performed towards trypanothione reductase using GOLD® software. RESULTS: The AGH's 3i, 4a, and 5d, and the TSC's 9i, 9k, and 9o were selected as valuable hits. These compounds presented antileishmanial activity compared with pentamidine, showing IC50 values ranged from 0.6 to 7.27 µM, maximal effects up to 55.3%, and satisfactory SI values (ranged from 11 to 87). On the other hand, most of the resulting semi-constrained analogs were found cytotoxic or presented reduced antileishmanial activity. In general, TSC class is more promising than its isosteric AGH analogs, and the beneficial aromatic substituent effects are not similar in both series. In silico studies have suggested that these hits are capable of inhibiting the trypanothione reductase from the amastigote forms. CONCLUSION: The promising antileishmanial activity of three AGH's and three TSC's was characterized. These compounds presented antileishmanial activity compared with PTD, showing IC50 values ranged from 0.6 to 7.27 µM, and satisfactory SI values. Further pharmacological assays involving other Leishmania strains are in progress, which will help choose the best hits for in vivo experiments.

Design, Synthesis and Biological Evaluation of Arylpyridin-2-yl Guanidine Derivatives and Cyclic Mimetics as Novel MSK1 Inhibitors. An Application in an Asthma Model.

Mitogen- and Stress-Activated Kinase 1 (MSK1) is a nuclear kinase, taking part in the activation pathway of the pro-inflammatory transcription factor NF-kB and is demonstrating a therapeutic target potential in inflammatory diseases such as asthma, psoriasis and atherosclerosis. To date, few MSK1 inhibitors were reported. In order to identify new MSK1 inhibitors, a screening of a library of low molecular weight compounds was performed, and the results highlighted the 6-phenylpyridin-2-yl guanidine (compound 1a, IC50~18 µM) as a starting hit for structure-activity relationship study. Derivatives, homologues and rigid mimetics of 1a were designed, and all synthesized compounds were evaluated for their inhibitory activity towards MSK1. Among them, the non-cytotoxic 2-aminobenzimidazole 49d was the most potent at inhibiting significantly: (i) MSK1 activity, (ii) the release of IL-6 in inflammatory conditions in vitro (IC50~2 µM) and (iii) the inflammatory cell recruitment to the airways in a mouse model of asthma.

Indole-3-guanylhydrazone hydrochloride mitigates long-term cognitive impairment in a neonatal sepsis model with involvement of MAPK and NFκB pathways.

BACKGROUND: Neonatal sepsis is defined as a systemic inflammatory response caused by a suspected or proven infection, occurring in the first month of life, and remains one of the main causes of morbidity and mortality in newborn and preterm infants. Frequently, survivors of neonatal sepsis have serious long-term cognitive impairment and adverse neurologic outcomes. There is currently no specific drug treatment for sepsis. Indole-3-guanylhydrazone hydrochloride (LQM01) is an aminoguanidine derivative that has been described as an anti-inflammatory, antihypertensive and antioxidant with potential applicability in inflammatory diseases. METHODS: We used a LPS-challenged neonatal sepsis rodent model to investigate the effect of LQM01 on cognitive impairment and anxiety-like behavior in sepsis mice survivors, and examined the possible molecular mechanisms involved. RESULTS: It was found that LQM01 exposure during the neonatal period reduces anxiety-like behavior and cognitive impairment caused by lipopolysaccharides (LPS) in adult life. Additionally, treatment with LQM01 decreased pro-inflammatory cytokine levels and reduced NFκB, COX-2, MAPK and microglia activation in hippocampus of neonatal mice. Furthermore, LQM01 was also able to prevent oxidative damage in hippocampus of neonatal mice and preserve brain barrier integrity. CONCLUSIONS: LQM01 attenuated inflammatory reactions in an LPS-challenged neonatal sepsis mice model through the MAPK and NFκB signaling pathways and microglia activation suppression. All these findings are associated with mitigated cognitive impairment in 70 days-old LQM01 treated-mice. GENERAL SIGNIFICANCE: We revealed the effect of LQM01 as an anti-septic agent, and the role of crucial molecular pathways in mitigating the potential damage caused by neonatal sepsis.

TSPO Ligands Boost Mitochondrial Function and Pregnenolone Synthesis.

Translocator protein 18 kDa (TSPO) is located in the mitochondrial outer membrane and plays an important role in steroidogenesis and cell survival. In the central nervous system (CNS), its expression is upregulated in neuropathologies such as Alzheimer's disease (AD). Previously, we demonstrated that two new TSPO ligands based on an imidazoquinazolinone termed 2a and 2b, stimulated pregnenolone synthesis and ATP production in vitro. In the present study, we compared their effects to those of TSPO ligands described in the literature (XBD173, SSR-180,575, and Ro5-4864) by profiling the mitochondrial bioenergetic phenotype before and after treatment and investigating the protective effects of these ligands after oxidative injury in a cellular model of AD overexpressing amyloid-ß (Aß). Of note, ATP levels increased with rising pregnenolone levels suggesting that the energetic performance of mitochondria is linked to an increased production of this neurosteroid via TSPO modulation. Our results further demonstrate that the TSPO ligands 2a and 2b exerted neuroprotective effects by improving mitochondrial respiration, reducing reactive oxygen species and thereby decreasing oxidative stress-induced cell death as well as lowering Aß levels. The compounds 2a and 2b show similar or even better functional effects than those obtained with the reference TSPO ligands XBD173 and SSR-180.575. These findings indicate that the new TSPO ligands modulate mitochondrial bioenergetic phenotype and protect against oxidative injury probably through the de novo synthesis of neurosteroids, suggesting that these compounds could be potential new therapeutic tools for the treatment of neurodegenerative disease.

Design and synthesis of 3-aminophthalazine derivatives and structural analogues as PDE5 inhibitors: anti-allodynic effect against neuropathic pain in a mouse model.

Neuropathic pain is a chronic pain caused by a lesion or disease affecting the somatosensory nervous system. To date, no specific treatment has been developed to cure this pain. Antidepressants and anticonvulsant drugs are used, but they do not demonstrate universal efficacy, and they often cause detrimental adverse effects. Some studies highlighted the efficacy of sildenafil, a well-known inhibitor of phosphodiesterase 5 (PDE5, (IC50 = 3.3 nM)), in models of pain. Based on these results, we focused our attention on MY 5445, another known PDE5 inhibitor. Homologues, isosteres and structural analogues of MY 5445 were designed and all synthesized compounds were evaluated for their inhibitory activity toward PDE5. Selectivity profiles towards other PDE1-4 isoenzymes, water solubility and stability in acidic medium of the most potent PDE5 inhibitors were determined and the aminophthalazine 16h and its mimetic 41n (3-aminoindazole) were evaluated in comparison to MY 5445 (4b) in vivo in a model of neuropathic pain induced by sciatic nerve cuffing in mice (3 and 0.5 mg/kg, ip twice a day). Both compounds showed the same efficacy on neuropathic allodynia as MY 5445, and thus produced a significant relief of mechanical hypersensitivity after 12 days of treatment.

Aminoguanidine Hydrazone Derivatives as Nonpeptide NPFF1 Receptor Antagonists Reverse Opioid Induced Hyperalgesia.

Neuropeptide FF receptors (NPFF1R and NPFF2R) and their endogenous ligand neuropeptide FF have been shown previously to display antiopioid properties and to play a critical role in the adverse effects associated with chronic administrations of opiates including the development of opioid-induced hyperalgesia and analgesic tolerance. In this work, we sought to identify novel NPFF receptors ligands by focusing our interest in a series of heterocycles as rigidified nonpeptide NPFF receptor ligands, starting from already described aminoguanidine hydrazones (AGHs). Binding experiments and functional assays highlighted AGH 1n and its rigidified analogue 2-amino-dihydropyrimidine 22e for in vivo experiments. As shown earlier with the prototypical dipeptide antagonist RF9, both 1n and 22e reduced significantly the long lasting fentanyl-induced hyperalgesia in rodents. Altogether these data indicate that AGH rigidification maintains nanomolar affinities for both NPFF receptors, while improving antagonist character toward NPFF1R.

Evidence for the involvement of TNF-α, IL-1ß and IL-10 in the antinociceptive and anti-inflammatory effects of indole-3-guanylhydrazone hydrochloride, an aromatic aminoguanidine, in rodents.

BACKGROUND: Indole-3-guanylhydrazone hydrochloride (LQM01) is a new derivative of aminoguanidine hydrochloride, an aromatic aminoguanidine. METHODS: Mice were treated with LQM01 (5, 10, 25 or 50 mg/kg, i.p.), vehicle (0.9% saline i.p.) or a standard drug. The mice were subjected to carrageenan-induced pleurisy, abdominal writhing induced by acetic acid, the formalin test and the hot-plate test. The model of non-inflammatory chronic muscle pain induced by saline acid was also used. Mice from the chronic protocol were assessed for withdrawal threshold, muscle strength and motor coordination. LQM01 or vehicle treated mice were evaluated for Fos protein. RESULTS: LQM01 inhibits TNF-α and IL-1ß production, as well as leukocyte recruitment during inflammation process. The level of IL-10 in LQM01-treated mice increased in pleural fluid. In addition, LQM01 decreased the nociceptive behavior in the acetic acid induced writhing test, the formalin test (both phases) and increased latency time on the hot-plate. LQM01 treatment also decreased mechanical hyperalgesia in mice with chronic muscle pain, with no changes in muscle strength and motor coordination. LQM01 reduced the number of Fos positive cells in the superficial dorsal horn. This compound exhibited antioxidant properties in in vitro assays. CONCLUSIONS: LQM01 has an outstanding anti-inflammatory and analgesic profile, probably mediated through a reduction in proinflammatory cytokines release, increase in IL-10 production and reduction in neuron activity in the dorsal horn of the spinal cord in mice. GENERAL SIGNIFICANCE: Beneficial effects of LQM01 suggest that it has some important clinical features and can play a role in the management of 'dysfunctional pain' and inflammatory diseases.

Phenylpyridine-2-ylguanidines and rigid mimetics as novel inhibitors of TNFα overproduction: Beneficial action in models of neuropathic pain and of acute lung inflammation.

4-phenylpyridin-2-yl-guanidine (5b): a new inhibitor of the overproduction of pro-inflammatory cytokines (TNFα and Il1ß) was identified from a high-throughput screening of a chemical library on human peripheral blood mononuclear cells (PBMCs) after LPS stimulation. Derivatives, homologues and rigid mimetics of 5b were designed and synthesized, and their cytotoxicity and ability to inhibit TNFα overproduction were evaluated. Among them, compound 5b and its mimetic 12 (2-aminodihydroquinazoline) showed similar inhibitory activities, and were evaluated in vivo in models of lung inflammation and neuropathic pain in mice. In particular, compound 12 proved to be active (5 mg/kg, ip) in both models.

Efficient and Mild Ullmann-Type N-Arylation of Amides, Carbamates, and Azoles in Water.

A simple, sustainable, efficient, mild, and low-cost protocol was developed for d-glucose-assisted Cu-catalyzed Ullmann reactions in water for amides, carbamates, and nitrogen-containing heterocycles. The reaction was compatible with diverse aryl/heteroaryl iodides, giving highly substituted pyridine, indole, or indazole rings. This method offers an attractive alternative to existing protocols, because the reaction proceeds in aqueous media, occurs at or near ambient temperature, and provides the N-arylated products in good to high yields.

RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes effects of RF-amide-related peptide-3 and opioid-induced hyperalgesia in rodents.

Although opiates represent the most effective analgesics, their use in chronic treatments is associated with numerous side effects including the development of pain hypersensitivity and analgesic tolerance. We recently identified a novel orally active neuropeptide FF (NPFF) receptor antagonist, RF313, which efficiently prevents the development of fentanyl-induced hyperalgesia in rats. In this study, we investigated the properties of this compound into more details. We show that RF313 exhibited a pronounced selectivity for NPFF receptors, antagonist activity at NPFF1 receptor (NPFF1R) subtype both in vitro and in vivo and no major side effects when administered in mice up to 30 mg/kg. When co-administered with opiates in rats and mice, it improved their analgesic efficacy and prevented the development of long lasting opioid-induced hyperalgesia. Moreover, and in marked contrast with the dipeptidic NPFF receptor antagonist RF9, RF313 displayed negligible affinity and no agonist activity (up to 100 µM) toward the kisspeptin receptor. Finally, in male hamster, RF313 had no effect when administered alone but fully blocked the increase in LH induced by RFRP-3, while RF9 per se induced a significant increase in LH levels which is consistent with its ability to activate kisspeptin receptors. Altogether, our data indicate that RF313 represents an interesting compound for the development of therapeutic tools aiming at improving analgesic action of opiates and reducing adverse side effects associated with their chronic administration. Moreover, its lack of agonist activity at the kisspeptin receptor indicates that RF313 might be considered a better pharmacological tool, when compared to RF9, to examine the regulatory roles of RF-amide-related peptides and NPFF1R in reproduction.

d-Glucose: An Efficient Reducing Agent for a Copper(II)-Mediated Arylation of Primary Amines in Water.

A copper-catalyzed Ullmann-type amination with primary amines in water with a combination of copper(II) triflate [Cu(OTf)2 ], dipivaloylmethane, and d-glucose is reported. The mild conditions and the use of an inexpensive catalyst as well as a renewable feedstock (d-glucose and the surfactant TPGS-750-M, which is derived from vitamin E) make this protocol a safe and convenient strategy for efficient C-N bond formation. This easy-to-handle procedure is extremely competitive compared to palladium-based reactions and may be used to synthesize N-containing molecules, such as drugs or organic light-emitting diodes (OLEDs).

Development of Dipeptidic hGPR54 Agonists.

A series of dipeptides were designed as potential agonists of the human KiSS1-derived peptide receptor (hGPR54). While the sequence Arg-Trp-NH2 was the most efficient in terms of affinity, we established a convergent synthetic strategy to optimize the N terminus. Using two successive Sonogashira cross-coupling reactions on a solid-supported peptide, we were able to introduce various alkynes at the N terminus to afford compounds with sub-micromolar affinities for hGPR54. However, functional assays indicated the benzoylated dipeptide Bz-Arg-Trp-NH2 as the most promising compound in terms of agonistic properties. Interestingly, this compound appeared much more stable than the endogenous neuropeptide kisspeptin, both in serum and in liver microsomes of rats. This compound was also found to be able to induce a significant in vivo increase in testosterone levels in male rats.

Functionalization of 2H-1,2,3-Triazole C-Nucleoside Template via N(2) Selective Arylation.

C-Nucleosides are an underexplored and important class of nucleosides with antiviral and anticancer activity. In addition, triazole heterocycles are well employed as a strategy to modify nucleobase in nucleoside analogues, although rare examples were described for triazoyl C-nucleosides. N(2)-Aryl-1,2,3-triazole C-nucleoside compounds that could be obtained by selective 1,2,3-triazole heterocycle N(2) arylation in 1-ß-d-ribofuranosyl-2H-1,2,3-triazole substrate were designed in this study. The optimized condition used AdBrettPhos/[PdCl(allyl)]2 as the catalyst system. This transformation was accomplished by aryl halides bearing an electron donor and withdrawing groups, as well as by heterocyclic halides in good to excellent yields. The transformation developed in this study represents a significant contribution to the nucleoside field, once it allows for the synthesis of unexplored scaffolds through selective functionalization of triazole nucleosides.

Effects of systematic N-terminus deletions and benzoylations of endogenous RF-amide peptides on NPFF1R, NPFF2R, GPR10, GPR54 and GPR103.

Mammalian RF-amide peptides including RF-amide-related peptides-1 and -3, neuropeptides AF and FF, Prolactin releasing peptides, Kisspeptins and RFa peptides are currently considered endogenous peptides for the GPCRs NPFF1R, NPFF2R, GPR10, GPR54 and GPR103, respectively. While NPFF1R and NPFF2R displayed high affinity for all the RF-amide peptides, GPR10, GPR54 and GPR103 only bind their cognate ligands. Through a systematic and sequential N-terminus deletion and benzoylation of either RF-amide neuropeptide (RFRP-3, NPFF, Kp-10, PrRP20, and 26RFa), we report the corresponding impact on affinity and activity towards all the RF-amide receptors (NPFF1R, NPFF2R, GPR10, GPR54 and GPR103). Our results highlight the difficulty to develop selective peptide ligands for GPR10, GPR54 or GPR103 without a modification of the C-terminus RF-amide signature, but open the door to the design of new RF-amide peptides acting as agonist for one receptor and antagonist for another one.

Neuropeptide FF receptors as novel targets for limbic seizure attenuation.

Neuropeptide Y (NPY) is a well established anticonvulsant and first-in-class antiepileptic neuropeptide. In this study, the controversial role of NPY1 receptors in epilepsy was reassessed by testing two highly selective NPY1 receptor ligands and a mixed NPY1/NPFF receptor antagonist BIBP3226 in a rat model for limbic seizures. While BIBP3226 significantly attenuated the pilocarpine-induced seizures, neither of the highly selective NPY1 receptor ligands altered the seizure severity. Administration of the NPFF1/NPFF2 receptor antagonist RF9 also significantly attenuated limbic seizure activity. To further prove the involvement of NPFF receptors in these seizure-modulating effects, low and high affinity antagonists for the NPFF receptors were tested. We observed that the low affinity ligand failed to exhibit anticonvulsant properties while the two high affinity ligands significantly attenuated the seizures. Continuous NPFF1 receptor agonist administration also inhibited limbic seizures whereas bolus administration of the NPFF1 receptor agonist was without effect. This suggests that continuous agonist perfusion could result in NPFF1 receptor desensitization and mimic NPFF1 receptor antagonist administration. Our data unveil for the first time the involvement of the NPFF system in the management of limbic seizures.

Design and validation of a homogeneous time-resolved fluorescence cell-based assay targeting the ligand-gated ion channel 5-HT3A.

Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy involves the capability to screen large chemical libraries. We present a new Tag-lite ligand binding assay targeting LGICs on living cells. This technology combines the use of suicide enzyme tags fused to channels of interest with homogeneous time-resolved fluorescence (HTRF) as the detection readout. Using the 5-HT3 receptor as system model, we showed that the pharmacology of the HALO-5HT3 receptor was identical to that of the native receptor. After validation of the assay by using 5-HT3 agonists and antagonists of reference, a pilot screen enabled us to identify azelastine, a well-known histamine H1 antagonist, as a potent 5-HT3 antagonist. This interesting result was confirmed with electrophysiological experiments. The method described here is easy to implement and could be applicable for other LGICs, opening new ways for the screening of chemical libraries.

Neuropeptide FF and prolactin-releasing peptide decrease cortical excitability through activation of NPFF receptors.

OBJECTIVE: Drugs with a novel mechanism of action are needed to reduce the number of people with epilepsy that are refractory to treatment. Increasing attention is paid to neuropeptide systems and several anticonvulsant neuropeptides have already been described, such as galanin, ghrelin, and neuropeptide Y (NPY). Many others, however, have not been investigated for their ability to affect epileptic seizures. In this study, the potential anticonvulsant activities of three members of the RF-amide neuropeptide family, neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP), and kisspeptin (Kp) and other receptor ligands (NPFF1/2 R, GPR10, and GRP54, respectively) were tested in the motor cortex stimulation model. METHODS: A train of pulses with increasing intensity (0-10 mA over 150 s, 50 Hz, pulse width 2 msec) was delivered to the motor cortex of rats. The threshold intensity for eliciting a motor response (i.e., motor threshold) was determined through behavioral observation and used as a measure for cortical excitability. The threshold was determined before, during, and after the intracerebroventricular (i.c.v.) administration of various NPFF1/2 R, GPR10, and GPR54 receptor ligands. RESULTS: NPFF and PrRP significantly increased the motor threshold by a maximum of 143 ± 27 and 83 ± 13 µA, respectively, for the doses of 1 nmol/h (p < 0.05). The increase of motor threshold by NPFF and PrRP was prevented by pretreatment and co-treatment with the NPFF1/2 R antagonist RF9. Pretreatment with a selective NPFF1 R antagonist also prevented the threshold increase induced by NPFF. Kp did not increase motor threshold. SIGNIFICANCE: Intracerebroventricular infusion of NPFF or PrRP decreases cortical excitability in rats through activation of NPFFRs. Furthermore, the NPFF1 R is required for the NPFF-induced decrease in cortical excitability.