RESUMEN
Glutamate signalling through the N-methyl-d-aspartate receptor (NMDAR) activates the enzyme neuronal nitric oxide synthase (nNOS) to produce the signalling molecule nitric oxide (NO). We hypothesized that disruption of the protein-protein interaction between nNOS and the scaffolding protein postsynaptic density 95 kDa (PSD95) would block NMDAR-dependent NO signalling and represent a viable therapeutic route to decrease opioid reward and relapse-like behaviour without the unwanted side effects of NMDAR antagonists. We used a conditioned place preference (CPP) paradigm to evaluate the impact of two small-molecule PSD95-nNOS inhibitors, IC87201 and ZL006, on the rewarding effects of morphine. Both IC87201 and ZL006 blocked morphine-induced CPP at doses that lacked intrinsic rewarding or aversive properties. Furthermore, in vivo fast-scan cyclic voltammetry (FSCV) was used to ascertain the impact of ZL006 on morphine-induced increases in dopamine (DA) efflux in the nucleus accumbens shell (NAc shell) evoked by electrical stimulation of the medial forebrain bundle (MFB). ZL006 attenuated morphine-induced increases in DA efflux at a dose that did not have intrinsic effects on DA transmission. We also employed multiple intravenous drug self-administration approaches to examine the impact of ZL006 on the reinforcing effects of morphine. Interestingly, ZL006 did not alter acquisition or maintenance of morphine self-administration, but reduced lever pressing in a morphine relapse test after forced abstinence. Our results provide behavioural and neurochemical support for the hypothesis that inhibition of PSD95-nNOS protein-protein interactions decreases morphine reward and relapse-like behaviour, highlighting a previously unreported application for these novel therapeutics in the treatment of opioid addiction.
Asunto(s)
Morfina , Recompensa , Animales , Homólogo 4 de la Proteína Discs Large , Morfina/farmacología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Núcleo Accumbens/metabolismo , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , RecurrenciaRESUMEN
G-protein-gated inwardly-rectifying K+ (GIRK) channels are targets of Gi/o-protein-signaling systems that inhibit cell excitability. GIRK channels exist as homotetramers (GIRK2 and GIRK4) or heterotetramers with nonfunctional homomeric subunits (GIRK1 and GIRK3). Although they have been implicated in multiple conditions, the lack of selective GIRK drugs that discriminate among the different GIRK channel subtypes has hampered investigations into their precise physiological relevance and therapeutic potential. Here, we report on a highly-specific, potent, and efficacious activator of brain GIRK1/2 channels. Using a chemical screen and electrophysiological assays, we found that this activator, the bromothiophene-substituted small molecule GAT1508, is specific for brain-expressed GIRK1/2 channels rather than for cardiac GIRK1/4 channels. Computational models predicted a GAT1508-binding site validated by experimental mutagenesis experiments, providing insights into how urea-based compounds engage distant GIRK1 residues required for channel activation. Furthermore, we provide computational and experimental evidence that GAT1508 is an allosteric modulator of channel-phosphatidylinositol 4,5-bisphosphate interactions. Through brain-slice electrophysiology, we show that subthreshold GAT1508 concentrations directly stimulate GIRK currents in the basolateral amygdala (BLA) and potentiate baclofen-induced currents. Of note, GAT1508 effectively extinguished conditioned fear in rodents and lacked cardiac and behavioral side effects, suggesting its potential for use in pharmacotherapy for post-traumatic stress disorder. In summary, our findings indicate that the small molecule GAT1508 has high specificity for brain GIRK1/2 channel subunits, directly or allosterically activates GIRK1/2 channels in the BLA, and facilitates fear extinction in a rodent model.
Asunto(s)
Encéfalo/metabolismo , Extinción Psicológica/efectos de los fármacos , Miedo/efectos de los fármacos , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Regulación Alostérica/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Sitios de Unión , Cognición/efectos de los fármacos , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/agonistas , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Células HEK293 , Atrios Cardíacos/diagnóstico por imagen , Humanos , Ligandos , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Mutación/genética , Miocardio/metabolismo , Especificidad de Órganos , Compuestos de Fenilurea/farmacología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosforilación/efectos de los fármacos , Estructura Secundaria de Proteína , Subunidades de Proteína/metabolismo , Pirazoles/farmacología , XenopusRESUMEN
Allosteric modulators of cannabinoid 1 receptor (CB1R) show translational promise over orthosteric ligands due to their potential to elicit therapeutic benefit without cannabimimetic side effects. The prototypic 2-phenylindole CB1R allosteric modulator, GAT211 (1), demonstrates preclinical efficacy in various disease models. The limited systematic structure-activity relationship (SAR) data at the C2 position of the indole ring within GAT211 invites the opportunity for further modifications to improve GAT211's pharmacological profile while serving to amplify and variegate this library of therapeutically attractive agents. These considerations prompted this focused SAR study in which we substituted the GAT211 C2-phenyl ring with heteroaromatic substituents. The synthesized GAT211 analogs were then evaluated in vitro as CB1R allosteric modulators in cAMP and ß-arrestin2 assays with CP55,940 as the orthosteric ligand. Furan and thiophene rings (15c-f and 15m) were the best-tolerated substituents at the C2 position of GAT211 for engagement with human CB1R (hCB1R). The SAR around the novel ligands reported allowed direct experimental characterization of the interaction profile of that pharmacophore with its binding domain in functional, human CB1R, thus offering guidance for accessing subsequent-generation hCB1R allosteric modulators as potential therapeutics. The most potent analog, 15d, markedly promoted orthosteric ligand binding to hCB1R. Pharmacological profiling in the GTPγS and mouse vas deferens assays demonstrated that 15d behaves as a CB1R agonist-positive allosteric modulator (ago-PAM), as confirmed electrophysiologically in autoptic neurons. In vivo, 15d was efficacious as a topical agent that significantly reduced intraocular pressure (IOP) in the ocular normotensive murine model of glaucoma. Since elevated IOP is a decisive risk factor for glaucoma and attendant vision loss, our data support the proposition that the 2-phenylindole class of CB1R ago-PAMs has therapeutic potential for glaucoma and other diseases where potentiation of CB1R signaling may be therapeutic.
Asunto(s)
Agonistas de Receptores de Cannabinoides/farmacología , Diseño de Fármacos , Indoles/farmacología , Receptor Cannabinoide CB1/agonistas , Regulación Alostérica/efectos de los fármacos , Agonistas de Receptores de Cannabinoides/síntesis química , Agonistas de Receptores de Cannabinoides/química , Relación Dosis-Respuesta a Droga , Humanos , Indoles/síntesis química , Indoles/química , Presión Intraocular/efectos de los fármacos , Estructura Molecular , Receptor Cannabinoide CB1/metabolismo , Relación Estructura-ActividadRESUMEN
The currents of α7 nicotinic acetylcholine receptors activated by acetylcholine (ACh) are brief. The channel has high permeability to calcium relative to monovalent cations and shows inward rectification. It has been previously noted that in the presence of positive allosteric modulators (PAMs), currents through the channels of α7 receptors differ from normal α7 currents both in sensitivity to specific channel blockers and their current-voltage (I-V) relationships, no longer showing inward rectification. Linear I-V functions are often associated with channels lacking calcium permeability, so we measured the I-V functions of α7 receptors activated by ACh when PAMs were bound to the allosteric binding site in the transmembrane domain. Currents were recorded in chloride-free Ringer's solution with low or high concentrations of extracellular calcium to determine the magnitude of the reversal potential shift in the two conditions as well as the I-V relationships. ACh-evoked currents potentiated by the allosteric agonist-PAMs (ago-PAMs) (3aR,4S,9bS)-4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (GAT107) and 3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propenamide (B-973B) showed reduced inward rectification and calcium-dependent reversal potential shifts decreased by 80%, and 50%, respectively, compared with currents activated by ACh alone, indicative of reduced calcium permeability. Currents potentiated by 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide were also linear and showed no calcium-dependent reversal potential shifts. The ago-PAMs GAT-107 and B-973B stimulated increases in intracellular calcium in stably transfected HEK293 cells. However, these calcium signals were delayed relative to channel activation produced by these agents and were insensitive to the channel blocker mecamylamine. Our results indicate that, although allosterically activated α7 nicotinic ACh receptor may affect intracellular calcium levels, such effects are not likely due to large channel-dependent calcium influx. SIGNIFICANCE STATEMENT: Positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptor can increase channel activation by two or more orders of magnitude, raising the concern that, due to the relatively high calcium permeability of α7 receptors activated by acetylcholine alone, such efficacious PAMs may have cytotoxic side effects. We show that PAMs alter the ion conduction pathway and, in general, reduce the calcium permeability of the channels. This supports the hypothesis that α7 effects on intracellular calcium may be independent of channel-mediated calcium influx.
Asunto(s)
Calcio/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Acetilcolina/metabolismo , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células HEK293 , Humanos , Oocitos , Técnicas de Placa-Clamp , Fenilpropionatos/farmacología , Piperazinas/farmacología , Quinolinas/farmacología , Sulfonamidas/farmacología , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7/agonistasRESUMEN
Many synthetic compounds to which we attribute specific activities are produced as racemic mixtures of stereoisomers, and it may be that all the desired activity comes from a single enantiomer. We have previously shown this to be the case with the α7 nicotinic acetylcholine receptor positive allosteric modulator (PAM) 3a,4,5,9b-Tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) and the α7 ago-PAM 4BP-TQS. Cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-te-trahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (2,3,5,6TMP-TQS), previously published as a "silent allosteric modulator" and an antagonist of α7 allosteric activation, shares the same scaffold with three chiral centers as the aforementioned compounds. We isolated the enantiomers of 2,3,5,6TMP-TQS and determined that the (-) isomer was a significantly better antagonist than the (+) isomer of the allosteric activation of both wild-type α7 and the nonorthosterically activatible C190A α7 mutant by the ago-PAM GAT107 (the active isomer of 4BP-TQS). In contrast, (+)2,3,5,6TMP-TQS proved to be an α7 PAM. (-)2,3,5,6TMP-TQS was shown to antagonize the allosteric activation of α7 by the structurally unrelated ago-PAM B-973B as well as the allosteric activation of the TQS-sensitive α4ß2L15'M mutant. In silico docking of 2,3,5,6TMP-TQS in the putative allosteric activation binding site suggested a specific interaction of the (-) enantiomer with α7T106, and allosteric activation of α7T106 mutants was not inhibited by (-)2,3,5,6TMP-TQS, confirming the importance of this interaction and supporting the model of the allosteric binding site. Comparisons and contrasts between 2,3,5,6TMP-TQS isomers and active and inactive enantiomers of other TQS-related compounds identify the orientation of the cyclopentenyl ring to the plane of the core quinoline to be a crucial determinate of PAM activity. SIGNIFICANCE STATEMENT: Many synthetic ligands are in use as racemic preparations. We show that one enantiomer of the TQS analog Cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-te-trahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, originally reported to lack activity when used as a racemic preparation, is an α7 nicotinic acetylcholine receptor positive allosteric modulator (PAM). The other enantiomer is not a PAM, but it is an effective allosteric antagonist. In silico studies and structural comparisons identify essential elements of both the allosteric ligands and receptor binding sites important for these allosteric activities.
Asunto(s)
Sulfonamidas/síntesis química , Sulfonamidas/farmacología , Xenopus laevis/genética , Receptor Nicotínico de Acetilcolina alfa 7/química , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Sitios de Unión , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Mutación , Estereoisomerismo , Sulfonamidas/química , Xenopus laevis/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of ligand-gated ion channels. Typically, channel activation follows the binding of agonists to the orthosteric binding sites of the receptor. α7 nAChRs have a very low probability of channel activation, which can be reversed by the binding of α7 selective positive allosteric modulators (PAMs) to putative sites within the transmembrane domains. Although typical PAMs, like PNU-120596, require coapplication of an orthosteric agonist to produce large channel activations, some, like GAT107 and B-973B [(S)-3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propanamide], are characterized as allosteric activating PAMs, which also bind to an allosteric activation (AA) site in the extracellular domain and activate the α7 ion channel by themselves. We had previously characterized N,N-diethyl-N'-phenylpiperazine analogs with various functions. In this work, we docked members of this family to a homology model of the α7 receptor extracellular domain. The compound 1,1-diethyl-4(naphthalene-2-yl)piperazin-1-ium (2NDEP) a weak partial agonist, showed particularly favorable docking and binding energies at the putative AA site of the receptor. We hypothesized that 2NDEP could couple with PAMs through the AA site. This hypothesis was tested with the α7 mutant C190A, which is not activated by orthosteric agonists but is effectively activated by GAT107. The results showed that 2NDEP acts as an allosteric agonist of α7C190A when coapplied with the PAM PNU-120596. Also, the allosteric activity was nearly abolished upon coapplication with the AA site-selective antagonist 2,3,5,6MP-TQS (cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), consistent with AA site involvement. Overall, our findings show a novel mode of agonism through an allosteric site in the extracellular domain of α7 nAChR.
Asunto(s)
Mutación , Agonistas Nicotínicos/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/química , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Animales , Sitios de Unión , Línea Celular , Humanos , Isoxazoles/química , Isoxazoles/farmacología , Masculino , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Agonistas Nicotínicos/química , Compuestos de Fenilurea/química , Compuestos de Fenilurea/farmacología , Piperazinas/química , Piperazinas/farmacología , Quinolinas/química , Quinolinas/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
B-973 is an efficacious type II positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptors that, like 4BP-TQS and its active isomer GAT107, can produce direct allosteric activation in addition to potentiation of orthosteric agonist activity, which identifies it as an allosteric activating (ago)-PAM. We compared the properties of B-973B, the active enantiomer of B-973, with those of GAT107 regarding the separation of allosteric potentiation and activation. Both ago-PAMs can strongly activate mutants of α7 that are insensitive to standard orthosteric agonists like acetylcholine. Likewise, the activity of both ago-PAMs is largely eliminated by the M254L mutation in the putative transmembrane PAM-binding site. Allosteric activation by B-973B appeared more protracted than that produced by GAT107, and B-973B responses were relatively insensitive to the noncompetitive antagonist mecamylamine compared with GAT107 responses. Similar differences are also seen in the single-channel currents. The two agents generate unique profiles of full-conductance and subconductance states, with B-973B producing protracted bursts, even in the presence of mecamylamine. Modeling and docking studies suggest that the molecular basis for these effects depends on specific interactions in both the extracellular and transmembrane domains of the receptor.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Agonistas Nicotínicos/farmacología , Fenilpropionatos/farmacología , Piperazinas/farmacología , Quinolinas/farmacología , Sulfonamidas/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Femenino , Humanos , Mecamilamina/farmacología , Proteínas de la Membrana/metabolismo , Dominios Proteicos/efectos de los fármacos , Xenopus laevisRESUMEN
Inwardly rectifying potassium (Kir) channels establish and regulate the resting membrane potential of excitable cells in the heart, brain, and other peripheral tissues. Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key direct activator of ion channels, including Kir channels. The gasotransmitter carbon monoxide has been shown to regulate Kir channel activity by altering channel-PIP2 interactions. Here, we tested in two cellular models the effects and mechanism of action of another gasotransmitter, hydrogen sulfide (H2S), thought to play a key role in cellular responses under ischemic conditions. Direct administration of sodium hydrogen sulfide as an exogenous H2S source and expression of cystathionine γ-lyase, a key enzyme that produces endogenous H2S in specific brain tissues, resulted in comparable current inhibition of several Kir2 and Kir3 channels. This effect resulted from changes in channel-gating kinetics rather than in conductance or cell-surface localization. The extent of H2S regulation depended on the strength of the channel-PIP2 interactions. H2S regulation was attenuated when channel-PIP2 interactions were strengthened and was increased when channel-PIP2 interactions were weakened by depleting PIP2 levels. These H2S effects required specific cytoplasmic cysteine residues in Kir3.2 channels. Mutation of these residues abolished H2S inhibition, and reintroduction of specific cysteine residues back into the background of the cytoplasmic cysteine-lacking mutant rescued H2S inhibition. Molecular dynamics simulation experiments provided mechanistic insights into how potential sulfhydration of specific cysteine residues could lead to changes in channel-PIP2 interactions and channel gating.
Asunto(s)
Canales de Potasio Rectificados Internamente Asociados a la Proteína G/antagonistas & inhibidores , Sulfuro de Hidrógeno/farmacología , Modelos Moleculares , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Sulfuros/farmacología , Regulación Alostérica/efectos de los fármacos , Sustitución de Aminoácidos , Animales , Células CHO , Cricetulus , Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Sulfuro de Hidrógeno/química , Sulfuro de Hidrógeno/metabolismo , Ratones , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mutación , Oocitos/citología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Fosfatidilinositol 4,5-Difosfato/química , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sulfuros/química , Sulfuros/metabolismo , Xenopus laevisRESUMEN
Homomeric α7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by α7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine that is unique to α7 at the 15' position of TM2 to leucine, the residue in most other nAChR subunits, largely eliminates the activity of such PAMs. We tested the effect of the reverse mutation (L15'M) in heteromeric nAChR receptors containing α4 and ß2, which are the nAChR subunits that are most abundant in the brain. Receptors containing these mutations were found to be strongly potentiated by the α7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) but insensitive to the alternative PAM 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea. The presence of the mutation in the ß2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the α4 subunit was to reduce responses to acetylcholine applied alone. Sensitivity to TQS required only a single mutant ß subunit, regardless of the position of the mutant ß subunit within the pentameric complex. Similar results were obtained when ß2L15'M was coexpressed with α2 or α3 and when the L15'M mutation was placed in ß4 and coexpressed with α2, α3, or α4. Functional receptors were not observed when ß1L15'M subunits were coexpressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the α4ß2L15'M receptor compared with α7 and the availability of high-resolution α4ß2 structures may provide new insights into the fundamental mechanisms of nAChR allosteric potentiation. SIGNIFICANCE STATEMENT: Heteromeric neuronal nAChRs have a relatively high initial probability of channel activation compared to receptors that are homomers of α7 subunits but are insensitive to PAMs, which greatly increase the open probability of α7 receptors. These features of heteromeric nAChR can be reversed by mutation of a single residue present in all neuronal heteromeric nAChR subunits to the sequence found in α7. Specifically, the mutation of the TM2 15' leucine to methionine in α subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal ß subunits allows heteromeric receptors to respond to select α7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChRs.
Asunto(s)
Mutación , Neuronas/metabolismo , Multimerización de Proteína , Subunidades de Proteína/genética , Receptor Nicotínico de Acetilcolina alfa 7/química , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Regulación Alostérica/genética , Secuencia de Aminoácidos , Animales , Humanos , Modelos Moleculares , Estructura Cuaternaria de Proteína , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
We report a facile, microwave-accelerated, one-pot tandem synthesis of unsymmetrical ureas via a Curtius rearrangement. In this method, one-pot microwave irradiation of commercially available (hetero)aromatic acids and amines in the presence of diphenylphosphoryl azide enabled extremely rapid (1-5 min) construction of an array of unsymmetrical ureas in good to excellent yields. We demonstrate the utility of our method in the efficient, gram-scale synthesis of key biologically active compounds targeting the cannabinoid 1 and α7 nicotinic acetylcholine receptors.
Asunto(s)
Química Orgánica/métodos , Microondas , Urea , Azidas/química , Estructura Molecular , Urea/análogos & derivados , Urea/síntesis química , Urea/químicaRESUMEN
Dravet syndrome is a catastrophic childhood epilepsy with multiple seizure types that are refractory to treatment. The endocannabinoid system regulates neuronal excitability so a deficit in endocannabinoid signaling could lead to hyperexcitability and seizures. Thus, we sought to determine whether a deficiency in the endocannabinoid system might contribute to seizure phenotypes in a mouse model of Dravet syndrome and whether enhancing endocannabinoid tone is anticonvulsant. Scn1a+/- mice model the clinical features of Dravet syndrome: hyperthermia-induced seizures, spontaneous seizures and reduced survival. We examined whether Scn1a+/- mice exhibit deficits in the endocannabinoid system by measuring brain cannabinoid receptor expression and endocannabinoid concentrations. Next, we determined whether pharmacologically enhanced endocannabinoid tone was anticonvulsant in Scn1a+/- mice. We used GAT229, a positive allosteric modulator of the cannabinoid (CB1) receptor, and ABX-1431, a compound that inhibits the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). The Scn1a+/- phenotype is strain-dependent with mice on a 129S6/SvEvTac (129) genetic background having no overt phenotype and those on an F1 (129S6/SvEvTac x C57BL/6J) background exhibiting a severe epilepsy phenotype. We observed lower brain cannabinoid CB1 receptor expression in the seizure-susceptible F1 compared to seizure-resistant 129 strain, suggesting an endocannabinoid deficiency might contribute to seizure susceptibility. GAT229 and ABX-1431 were anticonvulsant against hyperthermia-induced seizures. However, subchronic ABX1431 treatment increased spontaneous seizure frequency despite reducing seizure severity. Cnr1 is a putative genetic modifier of epilepsy in the Scn1a+/- mouse model of Dravet syndrome. Compounds that increase endocannabinoid tone could be developed as novel treatments for Dravet syndrome.
Asunto(s)
Anticonvulsivantes/farmacología , Agonistas de Receptores de Cannabinoides/farmacología , Endocannabinoides/antagonistas & inhibidores , Endocannabinoides/metabolismo , Epilepsias Mioclónicas/tratamiento farmacológico , Epilepsias Mioclónicas/metabolismo , Receptor Cannabinoide CB1/agonistas , Animales , Modelos Animales de Enfermedad , Endocannabinoides/deficiencia , Indoles/farmacología , Ratones , Ratones de la Cepa 129 , Ratones Transgénicos , Piperazinas/farmacología , Pirrolidinas/farmacologíaRESUMEN
Positive allosteric modulation of the type 1 cannabinoid receptor (CB1R) has substantial potential to treat both neurological and immune disorders. To date, a few studies have evaluated the structure-activity relationship (SAR) for CB1R positive allosteric modulators (PAMs). In this study, we separated the enantiomers of the previously characterized two potent CB1R ago-PAMs GAT591 and GAT593 to determine their biochemical activity at CB1R. Separating the enantiomers showed that the R-enantiomers (GAT1665 and GAT1667) displayed mixed allosteric agonist-PAM activity at CB1R while the S-enantiomers (GAT1664 and GAT1666) showed moderate activity. Furthermore, we observed that the R and S-enantiomers had distinct binding sites on CB1R, which led to their distinct behavior both in vitro and in vivo. The R-enantiomers (GAT1665 and GAT1667) produced ago-PAM effects in vitro, and PAM effects in the in vivo behavioral triad, indicating that the in vivo activity of these ligands may occur via PAM rather than agonist-based mechanisms. Overall, this study provides mechanistic insight into enantiospecific interaction of 2-phenylindole class of CB1R allosteric modulators, which have shown therapeutic potential in the treatment of pain, epilepsy, glaucoma, and Huntington's disease.
RESUMEN
Childhood absence epilepsy (CAE) is a non-convulsive seizure disorder primarily in children characterized by absence seizures. Absence seizures consist of 2.5-5 Hz spike-and-wave discharges (SWDs) detectable using electroencephalography (EEG). Current drug treatments are only partially effective and adverse side effects have spurred research into alternative treatment approaches. Recent research shows that positive allosteric modulation of the type-1 cannabinoid receptor (CB1R) reduces the frequency and duration of SWDs in Genetic Absence Epilepsy Rats from Strasbourg (GAERS), a model that recapitulates the SWDs in CAE. Here, we tested additional CB1R ago-PAMs, GAT591 and GAT593, for their potential in alleviating SWD activity in GAERS. In vitro experiments confirm that GAT591 and GAT593 exhibit increased potency and selectivity in cell cultures and behave as CB1R allosteric agonists and PAMs. To assess drug effects on SWDs, bilateral electrodes were surgically implanted in the somatosensory cortices of male GAERS and EEGs recorded for 4 h following systemic administration of GAT591 or GAT593 (1.0, 3.0 and 10.0 mg/kg). Both GAT591 and GAT593 dose-dependently reduced total SWD duration during the recording period. The greatest effect on SWD activity was observed at 10.0 mg/kg doses, with GAT591 and GAT593 reducing seizure duration by 36% and 34% respectively. Taken together, these results support the continued investigation of CB1R PAMs as a potential therapeutic to alleviate SWDs in absence epilepsy.
RESUMEN
The low sensitivity (α4)3(ß2)2 (LS) and high sensitivity (α4)2(ß2)3 (HS) nAChR isoforms may contribute to a variety of brain functions, pathophysiological processes, and pharmacological effects associated with nicotine use. In this study, we examined the contributions of the LS and HS α4ß2 nAChR isoforms in nicotine self-administration, withdrawal symptoms, antinociceptive and hypothermic effects. We utilized two nAChR positive allosteric modulators (PAMs): desformylflustrabromine (dFBr), a PAM of both the LS and HS α4ß2 nAChRs, and CMPI, a PAM selective for the LS nAChR. We found that dFBr, but not CMPI, decreased intravenous nicotine self-administration in male mice in a dose-dependent manner. Unlike dFBr, which fully reverses somatic and affective symptoms of nicotine withdrawal, CMPI at doses up to 15 mg/kg in male mice only partially reduced nicotine withdrawal-induced somatic signs, anxiety-like behavior and sucrose preference, but had no effects on nicotine withdrawal-induced hyperalgesia. These results indicate that potentiation of HS α4ß2 nAChRs is necessary to modulate nicotine's reinforcing properties that underlie nicotine intake and to reverse nicotine withdrawal symptoms that influence nicotine abstinence. In contrast, both dFBr and CMPI enhanced nicotine's hypothermic effect and reduced nicotine's antinociceptive effects in male mice. Therefore, these results indicate a more prevalent role of HS α4ß2 nAChR isoforms in mediating various behavioral effects associated with nicotine, whereas the LS α4ß2 nAChR isoform has a limited role in mediating body temperature and nociceptive responses. These findings will facilitate the development of more selective, efficacious, and safe nAChR-based therapeutics for nicotine addiction treatment.
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Conducta Animal/efectos de los fármacos , Hidrocarburos Bromados/farmacología , Alcaloides Indólicos/farmacología , Isoxazoles/farmacología , Nicotina/efectos adversos , Agonistas Nicotínicos/efectos adversos , Pirazoles/farmacología , Receptores Nicotínicos/efectos de los fármacos , Síndrome de Abstinencia a Sustancias/metabolismo , Tabaquismo/metabolismo , Regulación Alostérica , Animales , Ratones , Nicotina/administración & dosificación , Agonistas Nicotínicos/administración & dosificación , Isoformas de Proteínas , Receptores Nicotínicos/metabolismo , Autoadministración , Síndrome de Abstinencia a Sustancias/etiologíaRESUMEN
Altered interactions between endocannabinoid and glutamate signaling may be involved in the pathophysiology of schizophrenia and acute psychosis. As cognitive disturbances are involved in schizophrenia, increased understanding of the roles of these neurotransmitter systems in cognition may lead to the development of novel therapeutics for disorder. In the present study, we examined the effects of a recently synthesized cannabinoid receptor 1 (CB1R) positive allosteric modulator GAT211 in a rodent model of acute psychosis induced by systemic treatment with MK-801. To assess cognitive function, we used the Five-Choice Serial Reaction Time (5CSRT) task, conducted in touchscreen-equipped operant conditioning chambers. Our measures of primary interest were accuracy - indicative of visual attentional capacity - and the number of premature responses - indicative of impulsivity. We also measured latencies, omissions, and perseverative responding during all test sessions. Thirteen adult male Long Evans rats were trained on the 5CSRT and were then tested using a repeated measures design with acute MK-801 (0 or 0.15 mg/kg, i.p.) and GAT211 (0, 3, or 10 mg/kg, i.p.) administration. Acute MK-801 severely impaired accuracy, increased omissions, and increased the number of premature responses. MK-801 also significantly increased correct response latencies, without significant effects on incorrect or reward correction latencies. GAT211 had no significant effects when administered alone, or in combination with acute MK-801. These data confirm the dramatic effects of acute MK-801 treatment on behavioral measures of attention and impulsivity. Continued investigation of CB1R positive allosteric modulators as potential treatments for the cognitive symptoms of schizophrenia and related disorders should be pursued in other rodent models.
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Atención/efectos de los fármacos , Agonistas de Receptores de Cannabinoides/farmacología , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Conducta Impulsiva/efectos de los fármacos , Indoles/farmacología , Percepción Visual/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Conducta de Elección/efectos de los fármacos , Masculino , Ratas , Ratas Long-Evans , Tiempo de Reacción/efectos de los fármacosRESUMEN
RATIONALE: Antipsychotics help alleviate the positive symptoms associated with schizophrenia; however, their debilitating side effects have spurred the search for better treatment options. Novel compounds can be screened for antipsychotic potential in neuronal cell cultures and following acute N-methyl-D-aspartate (NMDA) receptor blockade with non-competitive antagonists such as MK-801 in rodent behavioral models. Given the known interactions between NMDA receptors and type 1 cannabinoid receptors (CB1R), compounds that modulate CB1Rs may have therapeutic potential for schizophrenia. OBJECTIVES: This study assessed whether the CB1R positive allosteric modulator GAT211, when compared to ∆9-tetrahydrocannabinol (THC), has potential to reduce psychiatric behavioral phenotypes following acute MK-801 treatment in rats, and block hyperdopaminergic signalling associated with those behaviors. METHODS: The effects of GAT211 and THC on cellular signaling were compared in Neuro2a cells, and behavioral effects of GAT211 and THC on altered locomotor activity and prepulse inhibition of the acoustic startle response caused by acute MK-801 treatment were assessed in male, Long Evans rats. RESULTS: GAT211 limited dopamine D2 receptor-mediated extracellular regulated kinase (ERK) phosphorylation in Neuro2a cells, whereas THC did not. As expected, acute MK-801 (0.15 mg/kg) produced a significant increase in locomotor activity and impaired PPI. GAT211 treatment alone (0.3-3.0 mg/kg) dose-dependently reduced locomotor activity and the acoustic startle response. GAT211 (3.0 mg/kg) also prevented hyperlocomotion caused by MK-801 but did not significantly affect PPI impairments. CONCLUSION: Taken together, these findings support continued preclinical research regarding the usefulness of CB1R positive allosteric modulators as antipsychotics.
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Antipsicóticos/farmacología , Indoles/farmacología , Receptor Cannabinoide CB1/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Línea Celular , Maleato de Dizocilpina , Relación Dosis-Respuesta a Droga , Dronabinol/farmacología , Antagonistas de Aminoácidos Excitadores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Masculino , Actividad Motora/efectos de los fármacos , Fosforilación , Inhibición Prepulso/efectos de los fármacos , Psicosis Inducidas por Sustancias/tratamiento farmacológico , Psicosis Inducidas por Sustancias/psicología , Ratas , Ratas Long-EvansRESUMEN
Childhood Absence Epilepsy (CAE) accounts for approximately 10% of all pediatric epilepsies. Current treatments for CAE are ineffective in approximately 1/3 of patients and can be associated with severe side effects such as hepatotoxicity. Certain cannabinoids, such as cannabidiol (CBD), have shown promise in the treatment of pediatric epilepsies. However, CBD remains limited or prohibited in many jurisdictions, and has not been shown to have efficacy in CAE. Modulation of the type 1 cannabinoid receptor (CB1R) may provide more desirable pharmacological treatments. Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model many aspects of CAE, including cortical spike and wave discharges (SWDs). We have recently demonstrated that Δ9-tetrahydrocannabinol (THC) increases SWDs in GAERS whereas CBD decreases these events. Here, we characterized aspects of the endocannabinoid system in brain areas relevant to seizures in GAERS and tested whether positive allosteric modulators (PAMs) of CB1R reduced SWDs. Both female and male GAERS had reduced (>50%) expression of CB1R and elevated levels of the endocannabinoid 2-AG in cortex compared to non-epileptic controls (NEC). We then administered the CB1R PAMs GAT211 and GAT229 to GAERS implanted with cortical electrodes. Systemic administration of GAT211 to male GAERS reduced SWDs by 40%. Systemic GAT229 administration reduced SWDs in female and male GAERS. Intracerebral infusion of GAT229 into the cortex of male GAERS reduced SWDs by >60% in a CB1R-dependent manner that was blocked by SR141716A. Together, these experiments identify altered endocannabinoid tone in GAERS and suggest that CB1R PAMs should be explored for treatment of absence seizures.
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Ondas Encefálicas/efectos de los fármacos , Agonistas de Receptores de Cannabinoides/farmacología , Epilepsia Tipo Ausencia/fisiopatología , Indoles/farmacología , Receptor Cannabinoide CB1/efectos de los fármacos , Regulación Alostérica , Animales , Ácidos Araquidónicos/metabolismo , Ondas Encefálicas/fisiología , Corteza Cerebral/metabolismo , Modelos Animales de Enfermedad , Endocannabinoides/metabolismo , Epilepsia Tipo Ausencia/genética , Femenino , Glicéridos/metabolismo , Masculino , Ratas , Receptor Cannabinoide CB1/metabolismoRESUMEN
We apply the magic methyl effect to improve the potency/efficacy of GAT211, the prototypic 2-phenylindole-based cannabinoid type-1 receptor (CB1R) agonist-positive allosteric modulator (ago-PAM). Introducing a methyl group at the α-position of nitro group generated two diastereomers, the greater potency and efficacy of erythro, (±)-9 vs threo, (±)-10 constitutes the first demonstration of diastereoselective CB1R-allosteric modulator interaction. Of the (±)-9 enantiomers, (-)-(S,R)-13 evidenced improved potency over GAT211 as a CB1R ago-PAM, whereas (+)-(R,S)-14 was a CB1R allosteric agonist biased toward G protein- vs ß-arrestin1/2-dependent signaling. (-)-(S,R)-13 and (+)-(R,S)-14 were devoid of undesirable side effects (triad test), and (+)-(R,S)-14 reduced intraocular pressure with an unprecedentedly long duration of action in a murine glaucoma model. (-)-(S,R)-13 docked into both a CB1R extracellular PAM and intracellular allosteric-agonist site(s), whereas (+)-(R,S)-14 preferentially engaged only the latter. Exploiting G-protein biased CB1R-allosteric modulation can offer safer therapeutic candidates for glaucoma and, potentially, other diseases.
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Agonistas de Receptores de Cannabinoides/uso terapéutico , Glaucoma/tratamiento farmacológico , Indoles/uso terapéutico , Receptor Cannabinoide CB1/agonistas , Sitio Alostérico , Animales , Células CHO , Agonistas de Receptores de Cannabinoides/síntesis química , Agonistas de Receptores de Cannabinoides/metabolismo , Cricetulus , Células HEK293 , Hipocampo/citología , Humanos , Indoles/síntesis química , Indoles/metabolismo , Presión Intraocular/efectos de los fármacos , Ligandos , Masculino , Ratones Endogámicos C57BL , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Neuronas/efectos de los fármacos , Receptor Cannabinoide CB1/química , Receptor Cannabinoide CB1/metabolismo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Opioid analgesics represent a critical treatment for chronic pain in the analgesic ladder of the World Health Organization. However, their use can result in a number of unwanted side-effects including incomplete efficacy, constipation, physical dependence, and overdose liability. Cannabinoids enhance the pain-relieving effects of opioids in preclinical studies and dampen unwanted side-effects resulting from excessive opioid intake. We recently reported that a CB1 positive allosteric modulator (PAM) exhibits antinociceptive efficacy in models of pathological pain and lacks the adverse side effects of direct CB1 receptor activation. In the present study, we evaluated whether a CB1 PAM would enhance morphine's therapeutic efficacy in an animal model of chemotherapy-induced neuropathic pain and characterized its impact on unwanted side-effects associated with chronic opioid administration. In paclitaxel-treated mice, both the CB1 PAM GAT211 and the opioid analgesic morphine reduced paclitaxel-induced behavioral hypersensitivities to mechanical and cold stimulation in a dose-dependent manner. Isobolographic analysis revealed that combinations of GAT211 and morphine resulted in anti-allodynic synergism. In paclitaxel-treated mice, a sub-threshold dose of GAT211 prevented the development of tolerance to the anti-allodynic effects of morphine over 20 days of once daily dosing. However, GAT211 did not reliably alter somatic withdrawal signs (i.e., jumps, paw tremors) in morphine-dependent neuropathic mice challenged with naloxone. In otherwise naïve mice, GAT211 also prolonged antinociceptive efficacy of morphine in the tail-flick test and reduced the overall right-ward shift in the ED50 for morphine to produce antinociception in the tail-flick test, consistent with attenuation of morphine tolerance. Pretreatment with GAT211 did not alter somatic signs of µ opioid receptor dependence in mice rendered dependent upon morphine via subcutaneous implantation of a morphine pellet. Moreover, GAT211 did not reliably alter µ-opioid receptor-mediated reward as measured by conditioned place preference to morphine. Our results suggest that a CB1 PAM may be beneficial in enhancing and prolonging the therapeutic properties of opioids while potentially sparing unwanted side-effects (e.g., tolerance) that occur with repeated opioid treatment.
RESUMEN
Cannabinoid 1 receptor (CB1R) allosteric ligands hold a far-reaching therapeutic promise. We report the application of fluoro- and nitrogen-walk approaches to enhance the drug-like properties of GAT211, a prototype CB1R allosteric agonist-positive allosteric modulator (ago-PAM). Several analogs exhibited improved functional potency (cAMP, ß-arrestin 2), metabolic stability, and aqueous solubility. Two key analogs, GAT591 (6r) and GAT593 (6s), exhibited augmented allosteric-agonist and PAM activities in neuronal cultures, improved metabolic stability, and enhanced orthosteric agonist binding (CP55,940). Both analogs also exhibited good analgesic potency in the CFA inflammatory-pain model with longer duration of action over GAT211 while being devoid of adverse cannabimimetic effects. Another analog, GAT592 (9j), exhibited moderate ago-PAM potency and improved aqueous solubility with therapeutic reduction of intraocular pressure in murine glaucoma models. The SAR findings and the enhanced allosteric activity in this class of allosteric modulators were accounted for in our recently developed computational model for CB1R allosteric activation and positive allosteric modulation.