RESUMEN
The delta opioid receptor (DOR) is a crucial receptor system that regulates pain, mood, anxiety, and similar mental states. DOR agonists, such as SNC80, and DOR-neutral antagonists, such as naltrindole, were developed to investigate the DOR in vivo and as potential therapeutics for pain and depression. However, few inverse agonists and non-competitive/irreversible antagonists have been developed, and none are widely available. This leaves a gap in our pharmacological toolbox and limits our ability to investigate the biology of this receptor. Thus, we designed and synthesized the novel compounds SRI-9342 as an irreversible antagonist and SRI-45128 as an inverse agonist. These compounds were then evaluated in vitro for their binding affinity by radioligand binding, their functional activity by 35S-GTPγS coupling, and their cAMP accumulation in cells expressing the human DOR. Both compounds demonstrated high binding affinity and selectivity at the DOR, and both displayed their hypothesized molecular pharmacology of irreversible antagonism (SRI-9342) or inverse agonism (SRI-45128). Together, these results demonstrate that we have successfully designed new inverse agonists and irreversible antagonists of the DOR based on a novel chemical scaffold. These new compounds will provide new tools to investigate the biology of the DOR or even new potential therapeutics.
Asunto(s)
Analgésicos Opioides/química , Unión Competitiva , Descubrimiento de Drogas , Receptores Opioides delta/química , Analgésicos Opioides/síntesis química , Analgésicos Opioides/farmacología , Técnicas de Química Sintética , Descubrimiento de Drogas/métodos , Humanos , Ligandos , Estructura Molecular , Unión Proteica , Receptores Opioides delta/agonistas , Relación Estructura-ActividadRESUMEN
NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission. Most native NMDA receptors are tetrameric assemblies of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. Co-assembly of the glycine-binding GluN1 with glycine-binding GluN3 subunits (GluN3A-B) creates glycine activated receptors that possess strikingly different functional and pharmacological properties compared to GluN1/GluN2 NMDA receptors. The role of GluN1/GluN3 receptors in neuronal function remains unknown, in part due to lack of pharmacological tools with which to explore their physiological roles. We have identified the negative allosteric modulator EU1180-438, which is selective for GluN1/GluN3 receptors over GluN1/GluN2 NMDA receptors, AMPA, and kainate receptors. EU1180-438 is also inactive at GABA, glycine, and P2X receptors, but displays inhibition of some nicotinic acetylcholine receptors. Furthermore, we demonstrate that EU1180-438 produces robust inhibition of glycine-activated current responses mediated by native GluN1/GluN3A receptors in hippocampal CA1 pyramidal neurons. EU1180-438 is a non-competitive antagonist with activity that is independent of membrane potential (i.e. voltage-independent), glycine concentration, and extracellular pH. Non-stationary fluctuation analysis of neuronal current responses provided an estimated weighted mean unitary conductance of 6.1 pS for GluN1/GluN3A channels, and showed that EU1180-438 has no effect on conductance. Site-directed mutagenesis suggests that structural determinants of EU1180-438 activity reside near a short pre-M1 helix that lies parallel to the plane of the membrane below the agonist binding domain. These findings demonstrate that structural differences between GluN3 and other glutamate receptor subunits can be exploited to generate subunit-selective ligands with utility in exploring the roles GluN3 in neuronal function.
Asunto(s)
Antagonistas de Aminoácidos Excitadores/farmacología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Relación Dosis-Respuesta a Droga , Agonistas de Aminoácidos Excitadores/farmacología , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Técnicas de Cultivo de Órganos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Xenopus laevisRESUMEN
Zolvix® is a recently introduced anthelmintic drench containing monepantel as the active ingredient. Monepantel is a positive allosteric modulator of DEG-3/DES-2 type nicotinic acetylcholine receptors (nAChRs) in several nematode species. The drug has been reported to produce hypercontraction of Caenorhabditis elegans and Haemonchus contortus somatic muscle. We investigated the effects of monepantel on nAChRs from Ascaris suum and Oesophagostomum dentatum heterologously expressed in Xenopus laevis oocytes. Using two-electrode voltage-clamp electrophysiology, we studied the effects of monepantel on a nicotine preferring homomeric nAChR subtype from A. suum comprising of ACR-16; a pyrantel/tribendimidine preferring heteromeric subtype from O. dentatum comprising UNC-29, UNC-38 and UNC-63 subunits; and a levamisole preferring subtype (O. dentatum) comprising UNC-29, UNC-38, UNC-63 and ACR-8 subunits. For each subtype tested, monepantel applied in isolation produced no measurable currents thereby ruling out an agonist action. When monepantel was continuously applied, it reduced the amplitude of acetylcholine induced currents in a concentration-dependent manner. In all three subtypes, monepantel acted as a non-competitive antagonist on the expressed receptors. ACR-16 from A. suum was particularly sensitive to monepantel inhibition (IC50 values: 1.6⯱â¯3.1â¯nM and 0.2⯱â¯2.3⯵M). We also investigated the effects of monepantel on muscle flaps isolated from adult A. suum. The drug did not significantly increase baseline tension when applied on its own. As with acetylcholine induced currents in the heterologously expressed receptors, contractions induced by acetylcholine were antagonized by monepantel. Further investigation revealed that the inhibition was a mixture of competitive and non-competitive antagonism. Our findings suggest that monepantel is active on multiple nAChR subtypes.
Asunto(s)
Aminoacetonitrilo/análogos & derivados , Ascaris suum/efectos de los fármacos , Antagonistas Nicotínicos/farmacología , Oesophagostomum/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Acetilcolina/farmacología , Aminoacetonitrilo/farmacología , Animales , Antihelmínticos/farmacología , Ascaris suum/citología , Electrofisiología/métodos , Músculos/efectos de los fármacos , Músculos/fisiología , Oesophagostomum/citología , Oocitos , Receptores Nicotínicos/genética , Xenopus laevis/genéticaRESUMEN
BACKGROUND: The isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and, to a lesser extent, of inhibitory glutamate-gated chloride channels (GluCls). Lotilaner (Credelio™), a novel representative of this chemical class, is currently evaluated for its excellent ectoparasiticide properties. METHODS: In this study, we investigated the molecular mode of action and pharmacology of lotilaner. We report the successful gene identification, cDNA cloning and functional expression in Xenopus oocytes of Drosohpila melanogaster (wild type and dieldrin/fipronil-resistant forms), Lepeophtheirus salmonis (an ectoparasite copepod crustacean of salmon), Rhipicephalus microplus and Canis lupus familiaris GABACls. Automated Xenopus oocyte two-electrode voltage clamp electrophysiology was used to assess GABACls functionality and to compare ion channel inhibition by lotilaner with that of established insecticides addressing GABACls as targets. RESULTS: In these assays, we demonstrated that lotilaner is a potent non-competitive antagonist of insects (fly) GABACls. No cross-resistance with dieldrin or fipronil resistance mutations was detected, suggesting that lotilaner might bind to a site at least partly different from the one bound by known GABACl blockers. Using co-application experiments, we observed that lotilaner antagonism differs significantly from the classical open channel blocker fipronil. We finally confirmed for the first time that isoxazoline compounds are not only powerful antagonists of GABACls of acari (ticks) but also of crustaceans (sea lice), while no activity on a dog GABAA receptor was observed up to a concentration of 10 µM. CONCLUSIONS: Together, these results demonstrate that lotilaner is a non-competitive antagonist specific to invertebrate's γ-aminobutyric acid-gated chloride channels (GABACls). They contribute to our understanding of the mode of action of this new ectoparasiticide compound.
Asunto(s)
Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/química , Insecticidas/farmacología , Invertebrados/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismo , Animales , Canales de Cloruro/genética , Clonación Molecular , Copépodos/efectos de los fármacos , Copépodos/fisiología , ADN Complementario , Drosophila melanogaster/fisiología , Insectos , Insecticidas/química , Insecticidas/metabolismo , Invertebrados/genética , Invertebrados/fisiología , Oocitos , Técnicas de Placa-Clamp , Pirazoles/farmacología , Rhipicephalus/efectos de los fármacos , Rhipicephalus/fisiología , XenopusRESUMEN
The interaction of (-)-reboxetine, a non-tricyclic norepinephrine selective reuptake inhibitor, with muscle-type nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that (-)-reboxetine: (a) inhibits (±)-epibatidine-induced Ca(2+) influx in human (h) muscle embryonic (hα1ß1γδ) and adult (hα1ß1εδ) AChRs in a non-competitive manner and with potencies IC50=3.86±0.49 and 1.92±0.48 µM, respectively, (b) binds to the [(3)H]TCP site with ~13-fold higher affinity when the Torpedo AChR is in the desensitized state compared to the resting state, (c) enhances [(3)H]cytisine binding to the resting but activatableTorpedo AChR but not to the desensitized AChR, suggesting desensitizing properties, (d) overlaps the PCP luminal site located between rings 6' and 13' in the Torpedo but not human muscle AChRs. In silico mutation results indicate that ring 9' is the minimum structural component for (-)-reboxetine binding, and (e) interacts to non-luminal sites located within the transmembrane segments from the Torpedo AChR γ subunit, and at the α1/ε transmembrane interface from the adult muscle AChR. In conclusion, (-)-reboxetine non-competitively inhibits muscle AChRs by binding to the TCP luminal site and by inducing receptor desensitization (maybe by interacting with non-luminal sites), a mechanism that is shared by tricyclic antidepressants.