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1.
J Pharmacol Exp Ther ; 369(3): 345-363, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30910921

RESUMEN

Nonselective glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists are efficacious in chronic pain but have significant tolerability issues, likely arising from the ubiquitous expression of AMPA receptors in the central nervous system (CNS). Recently, LY3130481 has been shown to selectively block AMPA receptors coassembled with the auxiliary protein, transmembrane AMPA receptor regulatory protein (TARP) γ8, which is highly expressed in the hippocampus but also in pain pathways, including anterior cingulate (ACC) and somatosensory cortices and the spinal cord, suggesting that selective blockade of γ8/AMPA receptors may suppress nociceptive signaling with fewer CNS side effects. The potency of LY3130481 on recombinant γ8-containing AMPA receptors was modulated by coexpression with other TARPs; γ2 subunits affected activity more than γ3 subunits. Consistent with these findings, LY3130481 had decreasing potency on receptors from rat hippocampal, cortical, spinal cord, and cerebellar neurons that was replicated in tissue from human brain. LY3130481 partially suppressed, whereas the nonselective AMPA antagonist GYKI53784 completely blocked, AMPA receptor-dependent excitatory postsynaptic potentials in ACC and spinal neurons in vitro. Similarly, LY3130481 attenuated short-term synaptic plasticity in spinal sensory neurons in vivo in response to stimulation of peripheral afferents. LY3130481 also significantly reduced nocifensive behaviors after intraplantar formalin that was correlated with occupancy of CNS γ8-containing AMPA receptors. In addition, LY3130481 dose-dependently attenuated established gait impairment after joint damage and tactile allodynia after spinal nerve ligation, all in the absence of motor side effects. Collectively, these data demonstrate that LY3130481 can suppress excitatory synaptic transmission and plasticity in pain pathways containing γ8/AMPA receptors and significantly reduce nocifensive behaviors, suggesting a novel, effective, and safer therapy for chronic pain conditions.


Asunto(s)
Canales de Calcio/metabolismo , Dolor Crónico/tratamiento farmacológico , Dolor Crónico/metabolismo , Terapia Molecular Dirigida , Receptores AMPA/metabolismo , Animales , Benzotiazoles/farmacología , Benzotiazoles/uso terapéutico , Dolor Crónico/fisiopatología , Masculino , Plasticidad Neuronal/efectos de los fármacos , Nocicepción/efectos de los fármacos , Pirazoles/farmacología , Pirazoles/uso terapéutico , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/efectos de los fármacos , Distribución Tisular
2.
Br J Pharmacol ; 176(1): 110-126, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30276808

RESUMEN

BACKGROUND AND PURPOSE: We aimed to identify and develop novel, selective muscarinic M1 receptor agonists as potential therapeutic agents for the symptomatic treatment of Alzheimer's disease. EXPERIMENTAL APPROACH: We developed and utilized a novel M1 receptor occupancy assay to drive a structure activity relationship in a relevant brain region while simultaneously tracking drug levels in plasma and brain to optimize for central penetration. Functional activity was tracked in relevant native in vitro assays allowing translational (rat-human) benchmarking of structure-activity relationship molecules to clinical comparators. KEY RESULTS: Using this paradigm, we identified a series of M1 receptor selective molecules displaying desirable in vitro and in vivo properties and optimized key features, such as central penetration while maintaining selectivity and a partial agonist profile. From these compounds, we selected spiropiperidine 1 (SPP1). In vitro, SPP1 is a potent, partial agonist of cortical and hippocampal M1 receptors with activity conserved across species. SPP1 displays high functional selectivity for M1 receptors over native M2 and M3 receptor anti-targets and over a panel of other targets. Assessment of central target engagement by receptor occupancy reveals SPP1 significantly and dose-dependently occupies rodent cortical M1 receptors. CONCLUSIONS AND IMPLICATIONS: We report the discovery of SPP1, a novel, functionally selective, brain penetrant partial orthosteric agonist at M1 receptors, identified by a novel receptor occupancy assay. SPP1 is amenable to in vitro and in vivo study and provides a valuable research tool to further probe the role of M1 receptors in physiology and disease.


Asunto(s)
Osteopontina/agonistas , Piperidinas/farmacología , Receptor Muscarínico M1/agonistas , Compuestos de Espiro/farmacología , Animales , Células CHO , Células Cultivadas , Cricetulus , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Piperidinas/química , Ratas , Ratas Sprague-Dawley , Compuestos de Espiro/química , Relación Estructura-Actividad , Xenopus
3.
Drug Discov Today ; 24(2): 533-543, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30395928

RESUMEN

It is important in drug discovery to demonstrate that activity of novel drugs found by screening on recombinant receptors translates to activity on native human receptors in brain areas affected by disease. In this review, we summarise the development and use of the microtransplantation technique. Native receptors are reconstituted from human brain tissues into oocytes from the frog Xenopus laevis where they can be functionally assessed. Oocytes microtransplanted with hippocampal tissue from an epileptic patient were used to demonstrate that new antiepileptic agents act on receptors in diseased tissue. Furthermore, frozen post-mortem human tissues were used to show that drugs are active on receptors in brain areas associated with a disease; but not in areas associated with side effects.


Asunto(s)
Encéfalo/metabolismo , Oocitos/metabolismo , Receptores de Superficie Celular/fisiología , Trasplante Heterólogo/métodos , Animales , Descubrimiento de Drogas , Humanos
4.
Nat Neurosci ; 21(1): 130-138, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29203896

RESUMEN

The postsynaptic proteome of excitatory synapses comprises ~1,000 highly conserved proteins that control the behavioral repertoire, and mutations disrupting their function cause >130 brain diseases. Here, we document the composition of postsynaptic proteomes in human neocortical regions and integrate it with genetic, functional and structural magnetic resonance imaging, positron emission tomography imaging, and behavioral data. Neocortical regions show signatures of expression of individual proteins, protein complexes, biochemical and metabolic pathways. We characterized the compositional signatures in brain regions involved with language, emotion and memory functions. Integrating large-scale GWAS with regional proteome data identifies the same cortical region for smoking behavior as found with fMRI data. The neocortical postsynaptic proteome data resource can be used to link genetics to brain imaging and behavior, and to study the role of postsynaptic proteins in localization of brain functions.


Asunto(s)
Neocórtex/patología , Proteínas del Tejido Nervioso/metabolismo , Sinapsis/metabolismo , Sinaptosomas/metabolismo , Animales , Biología Computacional , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Potenciales de la Membrana/genética , Microinyecciones , Neocórtex/diagnóstico por imagen , Proteínas del Tejido Nervioso/genética , Oocitos , Oxígeno/sangre , Técnicas de Placa-Clamp , Tomografía de Emisión de Positrones , Proteómica , Accidente Cerebrovascular/patología , Sinapsis/ultraestructura , Xenopus laevis , Ácido gamma-Aminobutírico/farmacología
5.
Biochem Biophys Res Commun ; 495(1): 481-486, 2018 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-29127015

RESUMEN

Muscarinic acetylcholine M1 receptors play an important role in synaptic plasticity in the hippocampus and cortex. Potentiation of NMDA receptors as a consequence of muscarinic acetylcholine M1 receptor activation is a crucial event mediating the cholinergic modulation of synaptic plasticity, which is a cellular mechanism for learning and memory. In Alzheimer's disease, the cholinergic input to the hippocampus and cortex is severely degenerated, and agonists or positive allosteric modulators of M1 receptors are therefore thought to be of potential use to treat the deficits in cognitive functions in Alzheimer's disease. In this study we developed a simple system in which muscarinic modulation of NMDA receptors can be studied in vitro. Human M1 receptors and NR1/2B NMDA receptors were co-expressed in Xenopus oocytes and various muscarinic agonists were assessed for their modulatory effects on NMDA receptor-mediated responses. As expected, NMDA receptor-mediated responses were potentiated by oxotremorine-M, oxotremorine or xanomeline when the drugs were applied between subsequent NMDA responses, an effect which was fully blocked by the muscarinic receptor antagonist atropine. However, in oocytes expressing NR1/2B NMDA receptors but not muscarinic M1 receptors, oxotremorine-M co-applied with NMDA also resulted in a potentiation of NMDA currents and this effect was not blocked by atropine, demonstrating that oxotremorine-M is able to directly potentiate NMDA receptors. Oxotremorine, which is a close analogue of oxotremorine-M, and xanomeline, a chemically distinct muscarinic agonist, did not potentiate NMDA receptors by this direct mechanism. Comparing the chemical structures of the three different muscarinic agonists used in this study suggests that the tri-methyl ammonium moiety present in oxotremorine-M is important for the compound's interaction with NMDA receptors.


Asunto(s)
Agonistas Muscarínicos/farmacología , Oxotremorina/análogos & derivados , Receptores de N-Metil-D-Aspartato/agonistas , Animales , Humanos , Agonistas Muscarínicos/química , Oxotremorina/química , Oxotremorina/farmacología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Xenopus
6.
Nat Med ; 22(12): 1496-1501, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27820603

RESUMEN

Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.


Asunto(s)
Anticonvulsivantes/farmacología , Benzotiazoles/farmacología , Cerebelo/efectos de los fármacos , Epilepsia/tratamiento farmacológico , Prosencéfalo/efectos de los fármacos , Pirazoles/farmacología , Piridonas/farmacología , Receptores AMPA/antagonistas & inhibidores , Animales , Anticonvulsivantes/efectos adversos , Canales de Calcio/metabolismo , Cerebelo/metabolismo , Convulsivantes/toxicidad , Modelos Animales de Enfermedad , Mareo/inducido químicamente , Epilepsia/inducido químicamente , Ratones , Nitrilos , Pentilenotetrazol/toxicidad , Prosencéfalo/metabolismo , Piridonas/efectos adversos , Ratas , Receptores AMPA/metabolismo , Convulsiones/inducido químicamente , Convulsiones/tratamiento farmacológico
7.
Eur J Pharmacol ; 791: 221-228, 2016 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-27590358

RESUMEN

Inhibition of KCNQ (Kv7) potassium channels by activation of muscarinic acetylcholine receptors has been well established, and the ion currents through these channels have been long known as M-currents. We found that this cross-talk can be reconstituted in Xenopus oocytes by co-transfection of human recombinant muscarinic M1 receptors and KCNQ2/3 potassium channels. Application of the muscarinic acetylcholine receptor agonist Oxotremorine-methiodide (Oxo-M) between voltage pulses to activate KCNQ2/3 channels caused inhibition of the subsequent KCNQ2/3 responses. This effect of Oxo-M was blocked by the muscarinic acetylcholine receptor antagonist atropine. We also found that KCNQ2/3 currents were inhibited when Oxo-M was applied during an ongoing KCNQ2/3 response, an effect that was not blocked by atropine, suggesting that Oxo-M inhibits KCNQ2/3 channels directly. Indeed, also in oocytes that were transfected with only KCNQ2/3 channels, but not with muscarinic M1 receptors, Oxo-M inhibited the KCNQ2/3 response. These results show that besides the usual muscarinic acetylcholine receptor-mediated inhibition, Oxo-M also inhibits KCNQ2/3 channels by a direct mechanism. We subsequently tested xanomeline, which is a chemically distinct muscarinic acetylcholine receptor agonist, and oxotremorine, which is a close analogue of Oxo-M. Both compounds inhibited KCNQ2/3 currents via activation of M1 muscarinic acetylcholine receptors but, in contrast to Oxo-M, they did not directly inhibit KCNQ2/3 channels. Xanomeline and oxotremorine do not contain a positively charged trimethylammonium moiety that is present in Oxo-M, suggesting that such a charged moiety could be a crucial component mediating this newly described direct inhibition of KCNQ2/3 channels.


Asunto(s)
Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ3/antagonistas & inhibidores , Oxotremorina/análogos & derivados , Bloqueadores de los Canales de Potasio/farmacología , Animales , Humanos , Oxotremorina/farmacología , Piridinas/farmacología , Receptor Muscarínico M1/metabolismo , Tiadiazoles/farmacología , Xenopus
8.
J Neurochem ; 138(3): 384-96, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27216696

RESUMEN

Disruption in the expression and function of synaptic proteins, and ion channels in particular, is critical in the pathophysiology of human neuropsychiatric and neurodegenerative diseases. However, very little is known regarding the functional and pharmacological properties of native synaptic human ion channels, and their potential changes in pathological conditions. Recently, an electrophysiological technique has been enabled for studying the functional and pharmacological properties of ion channels present in crude membrane preparation obtained from post-mortem frozen brains. We here extend these studies by showing that human synaptic ion channels also can be studied in this way. Synaptosomes purified from different regions of rodent and human brain (control and Alzheimer's) were characterized biochemically for enrichment of synaptic proteins, and expression of ion channel subunits. The same synaptosomes were also reconstituted in Xenopus oocytes, in which the functional and pharmacological properties of the native synaptic ion channels were characterized using the voltage clamp technique. We show that we can detect GABA, (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and NMDA receptors, and modulate them pharmacologically with selective agonists, antagonists, and allosteric modulators. Furthermore, changes in ion channel expression and function were detected in synaptic membranes from Alzheimer's brains. Our present results demonstrate the possibility to investigate synaptic ion channels from healthy and pathological brains. This method of synaptosomes preparation and injection into oocytes is a significant improvement over the earlier method. It opens the way to directly testing, on native ion channels, the effects of novel drugs aimed at modulating important classes of synaptic targets. Disruption in the expression and function of synaptic ion channels is critical in the pathophysiology of human neurodegenerative diseases. We here show that synaptosomes purified from rodent and human frozen brain (control and Alzheimer disease) can be studied both biochemically and functionally. This method opens the way to directly testing the effects of novel drugs on native ion channels.


Asunto(s)
Encéfalo/metabolismo , Canales Iónicos/metabolismo , Oocitos/metabolismo , Sinaptosomas/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Fenómenos Electrofisiológicos/efectos de los fármacos , Fenómenos Electrofisiológicos/fisiología , Femenino , Humanos , Técnicas de Placa-Clamp/métodos , Ratas Wistar , Receptores de GABA-A/metabolismo , Xenopus laevis , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacología
9.
J Pharmacol Exp Ther ; 355(2): 247-54, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26324167

RESUMEN

The citric acid cycle intermediate citrate plays a crucial role in metabolic processes such as fatty acid synthesis, glucose metabolism, and ß-oxidation. Citrate is imported from the circulation across the plasma membrane into liver cells mainly by the sodium-dependent citrate transporter (NaCT; SLC13A5). Deletion of NaCT from mice led to metabolic changes similar to caloric restriction; therefore, NaCT has been proposed as an attractive therapeutic target for the treatment of obesity and type 2 diabetes. In this study, we expressed mouse and human NaCT into Xenopus oocytes and examined some basic functional properties of those transporters. Interestingly, striking differences were found between mouse and human NaCT with respect to their sensitivities to citric acid cycle intermediates as substrates for these transporters. Mouse NaCT had at least 20- to 800-fold higher affinity for these intermediates than human NaCT. Mouse NaCT is fully active at physiologic plasma levels of citrate, but its human counterpart is not. Replacement of extracellular sodium by other monovalent cations revealed that human NaCT was markedly less dependent on extracellular sodium than mouse NaCT. The low sensitivity of human NaCT for citrate raises questions about the translatability of this target from the mouse to the human situation and raises doubts about the validity of this transporter as a therapeutic target for the treatment of metabolic diseases in humans.


Asunto(s)
Ciclo del Ácido Cítrico , Transportadores de Ácidos Dicarboxílicos/fisiología , Simportadores/fisiología , Animales , Cationes Monovalentes , Colina/metabolismo , Transportadores de Ácidos Dicarboxílicos/genética , Femenino , Humanos , Litio/metabolismo , Oocitos/metabolismo , Técnicas de Placa-Clamp , Potasio/metabolismo , Sodio/metabolismo , Especificidad por Sustrato , Simportadores/genética , Xenopus laevis
10.
PLoS One ; 10(6): e0130572, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26086615

RESUMEN

The existence of α7ß2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer's disease, it is critical to determine whether α7ß2 nAChRs are present in the human brain, in which brain areas, and whether they differ functionally from α7 nAChR homomers. We used α-bungarotoxin to affinity purify α7-containing nAChRs from surgically excised human temporal cortex, and found that α7 subunits co-purify with ß2 subunits, indicating the presence of α7ß2 nAChRs in the human brain. We validated these results by demonstrating co-purification of ß2 from wild-type, but not α7 or ß2 knock-out mice. The pharmacology and kinetics of human α7ß2 nAChRs differed significantly from that of α7 homomers in response to nAChR agonists when expressed in Xenopus oocytes and HEK293 cells. Notably, α7ß2 heteromers expressed in HEK293 cells display markedly slower rise and decay phases. These results demonstrate that α7 subunits in the human brain form heteromeric complexes with ß2 subunits, and that human α7ß2 nAChR heteromers respond to nAChR agonists with a unique pharmacology and kinetic profile. α7ß2 nAChRs thus represent an alternative mechanism for the reported clinical efficacy of α7 nAChR ligands.


Asunto(s)
Corteza Cerebral/metabolismo , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Corteza Cerebral/patología , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Receptores Nicotínicos/deficiencia , Receptores Nicotínicos/genética , Xenopus laevis/crecimiento & desarrollo , Xenopus laevis/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/deficiencia , Receptor Nicotínico de Acetilcolina alfa 7/genética
11.
Eur J Pharmacol ; 726: 77-86, 2014 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-24485886

RESUMEN

α7ß2 is a novel type of nicotinic acetylcholine receptor shown to be uniquely expressed in cholinergic neurons of the basal forebrain and in hippocampal interneurons. We have compared the pharmacological properties of recombinant homomeric α7 and heteromeric α7ß2 nicotinic acetylcholine receptors in order to reveal the pharmacological consequences of ß2 subunit incorporation into the pentamer. The non-selective agonist epibatidine did not distinguish α7ß2 from α7 nicotinic acetylcholine receptors, but three other non-selective agonists (nicotine, cytisine and varenicline) were less efficacious on α7ß2 than on α7. A more dramatic change in efficacy was seen with eight different selective α7 agonists. Because of their very low intrinsic efficacy, some compounds became very efficacious functional antagonists at α7ß2 receptors. Three α4ß2 nicotinic receptor selective agonists that were not active on α7, were also inactive on α7ß2, and dihydro-ß-erythroidine, an α4ß2 receptor-preferring antagonist, inhibited α7 and α7ß2 in a similar manner. These results reveal significant effects of ß2 incorporation in determining the relative efficacy of several non-selective and α7 selective agonists, and also show that incorporation of ß2 subunits does not cause a shift to a more "ß2-like" pharmacology of α7 nicotinic acetylcholine receptors.


Asunto(s)
Oocitos/metabolismo , Multimerización de Proteína , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Xenopus laevis/genética , Receptor Nicotínico de Acetilcolina alfa 7/química , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Expresión Génica , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Estructura Cuaternaria de Proteína , Ratas , Receptores Nicotínicos/genética , Receptor Nicotínico de Acetilcolina alfa 7/genética
12.
Br J Pharmacol ; 165(4): 1006-16, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21838750

RESUMEN

BACKGROUND AND PURPOSE: Partial agonists selective for α4ß2 nicotinic ACh receptors have been developed for smoking cessation as they induce weak activation of native α4ß2* receptors and inhibit effect of nicotine. However, it is unclear whether at brain functions there is an existence of receptor reserve that allows weak receptor activation to induce maximum physiological effects. We assessed the extent of α4ß2 partial agonist-induced increase of firing rate in dopaminergic neurons and evaluated the influence of receptor reserve. EXPERIMENTAL APPROACH: The relative maximal effects and potencies of six nicotinic agonists were assessed on recombinant human α4ß2 and α7 receptors expressed in mammalian cell lines by measuring calcium influx. Agonist-induced increase of the spontaneous firing rate of dopaminergic neurons was recorded using microelectrodes in the ventral tegmental area of rat brain slices. KEY RESULTS: All α4ß2 partial and full agonists increased the firing rate concentration-dependently. Their sensitivity to subtype-selective antagonists showed predominant activation of native α4ß2* receptors. However, partial agonists with relative maximal effects as low as 33% on α4ß2 receptors maximally increased the firing rate and induced additional depolarization block of firing, demonstrating that partial activation of receptors caused the maximum increase in firing rate in the presence of a receptor reserve. CONCLUSIONS AND IMPLICATIONS: Partial α4ß2 agonists induced relatively enhanced effects on the firing rate of dopaminergic neurons, and the effect was mainly attributed to the existence of native α4ß2* receptor reserve. The results have implications in the understanding of physiological effects and therapeutic efficacies of α4ß2 partial agonists.


Asunto(s)
Neuronas Dopaminérgicas/efectos de los fármacos , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Calcio/fisiología , Neuronas Dopaminérgicas/fisiología , Agonismo Parcial de Drogas , Células HEK293 , Humanos , Masculino , Mesencéfalo/efectos de los fármacos , Mesencéfalo/fisiología , Nicotina/agonistas , Nicotina/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley
13.
Proc Natl Acad Sci U S A ; 108(14): 5867-72, 2011 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-21436053

RESUMEN

Conventional nicotinic acetylcholine receptor (nAChR) agonists, such as acetylcholine, act at an extracellular "orthosteric" binding site located at the interface between two adjacent subunits. Here, we present evidence of potent activation of α7 nAChRs via an allosteric transmembrane site. Previous studies have identified a series of nAChR-positive allosteric modulators (PAMs) that lack agonist activity but are able to potentiate responses to orthosteric agonists, such as acetylcholine. It has been shown, for example, that TQS acts as a conventional α7 nAChR PAM. In contrast, we have found that a compound with close chemical similarity to TQS (4BP-TQS) is a potent allosteric agonist of α7 nAChRs. Whereas the α7 nAChR antagonist metyllycaconitine acts competitively with conventional nicotinic agonists, metyllycaconitine is a noncompetitive antagonist of 4BP-TQS. Mutation of an amino acid (M253L), located in a transmembrane cavity that has been proposed as being the binding site for PAMs, completely blocks agonist activation by 4BP-TQS. In contrast, this mutation had no significant effect on agonist activation by acetylcholine. Conversely, mutation of an amino acid located within the known orthosteric binding site (W148F) has a profound effect on agonist potency of acetylcholine (resulting in a shift of ∼200-fold in the acetylcholine dose-response curve), but had little effect on the agonist dose-response curve for 4BP-TQS. Computer docking studies with an α7 homology model provides evidence that both TQS and 4BP-TQS bind within an intrasubunit transmembrane cavity. Taken together, these findings provide evidence that agonist activation of nAChRs can occur via an allosteric transmembrane site.


Asunto(s)
Moduladores del Transporte de Membrana/farmacología , Modelos Moleculares , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/metabolismo , Acetilcolina , Aconitina/análogos & derivados , Regulación Alostérica/fisiología , Animales , Sitios de Unión/genética , Sitios de Unión/fisiología , Simulación por Computador , Electrofisiología , Humanos , Ratones , Estructura Molecular , Mutación Missense/genética , Naftalenos/química , Técnicas de Placa-Clamp , Quinolinas/química , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Sulfonamidas/química , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7
14.
Eur J Pharmacol ; 643(2-3): 218-24, 2010 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-20624387

RESUMEN

Positive allosteric modulators of centrally expressed nicotinic acetylcholine receptors have therapeutic potentials in areas of cognition, motor function and reward. Several chemical classes of allosteric modulators that are selective for alpha7 nicotinic receptors have been characterised, but potentiators for the most widely expressed alpha4beta2 nicotinic receptor subtype are few and less defined, owing probably to the difficulty to achieve selectivity over other heteromeric receptor subtypes. 2087101 (2-amino-5-keto)thiazole) is a potent potentiator of both alpha7 and alpha4beta2 receptors and it has selectivity against the alpha3beta4 subtype, which may be responsible for the undesirable peripheral side effects. To further characterise its ability to differentiate between native nicotinic receptors, we examined the effects of 2087101 on alpha7, alpha4beta2* and alpha3beta4* receptor-mediated responses in the rat brain in electrophysiological and neurochemical experiments. 2087101 significantly potentiated agonist-induced, alpha7 and non-alpha7 receptor-mediated, GABAergic postsynaptic currents in cultured hippocampal neurones, but not the nicotine-stimulated [(3)H]noradrenaline release from hippocampal slices, which was primarily mediated by alpha3beta4* receptors, confirming its selectivity for alpha7 and alpha4beta2* receptors in native systems. 2087101 also significantly enhanced nicotine-stimulated firing increase in dopamine neurones of the ventral tegmental area, an effect that was dihydro-beta-erythroidine-sensitive and thereby mediated by alpha4beta2* nicotinic receptors. 2087101 can therefore enhance native nicotinic activities mediated by alpha7 and alpha4beta2*, but not alpha3beta4* receptors, showing its unique ability to discriminate between native heteromeric nicotinic receptor subtypes and its therapeutic potential for treating brain disorders by concurrent modulation of both alpha7 and alpha4beta2* nicotinic receptors.


Asunto(s)
Hipocampo/efectos de los fármacos , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/metabolismo , Transmisión Sináptica/efectos de los fármacos , Tiazoles/farmacología , Área Tegmental Ventral/efectos de los fármacos , Regulación Alostérica , Animales , Animales Recién Nacidos , Células Cultivadas , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipocampo/citología , Hipocampo/metabolismo , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Norepinefrina/metabolismo , Concentración Osmolar , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Factores de Tiempo , Área Tegmental Ventral/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7
15.
Proc Natl Acad Sci U S A ; 105(38): 14686-91, 2008 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-18791069

RESUMEN

Positive allosteric modulators of alpha7 nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as potential tools for the treatment of neurological and psychiatric disorders such as Alzheimer's disease and schizophrenia. However, despite the potential therapeutic usefulness of these compounds, little is known about their mechanism of action. Here, we have examined two allosteric potentiators of alpha7 nAChRs (PNU-120596 and LY-2087101). From studies with a series of subunit chimeras, we have identified the transmembrane regions of alpha7 as being critical in facilitating potentiation of agonist-evoked responses. Furthermore, we have identified five transmembrane amino acids that, when mutated, significantly reduce potentiation of alpha7 nAChRs. The amino acids we have identified are located within the alpha-helical transmembrane domains TM1 (S222 and A225), TM2 (M253), and TM4 (F455 and C459). Mutation of either A225 or M253 individually have particularly profound effects, reducing potentiation of EC(20) concentrations of acetylcholine to a tenth of the level seen with wild-type alpha7. Reference to homology models of the alpha7 nAChR, based on the 4A structure of the Torpedo nAChR, indicates that the side chains of all five amino acids point toward an intrasubunit cavity located between the four alpha-helical transmembrane domains. Computer docking simulations predict that the allosteric compounds such as PNU-120596 and LY-2087101 may bind within this intrasubunit cavity, much as neurosteroids and volatile anesthetics are thought to interact with GABA(A) and glycine receptors. Our findings suggest that this is a conserved modulatory allosteric site within neurotransmitter-gated ion channels.


Asunto(s)
Sitio Alostérico , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Regulación Alostérica/efectos de los fármacos , Aminoácidos/genética , Animales , Sitios de Unión , Pollos , Simulación por Computador , Relación Dosis-Respuesta a Droga , Humanos , Isoxazoles/farmacología , Ratones , Modelos Moleculares , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Mutación , Oocitos/metabolismo , Compuestos de Fenilurea/farmacología , Estructura Terciaria de Proteína , Ratas , Receptores Nicotínicos/genética , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7
16.
J Neurosci ; 28(27): 6884-94, 2008 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-18596163

RESUMEN

The alpha4beta2 subtype is the most abundant nicotinic acetylcholine receptor (nAChR) in the brain and possesses the high-affinity binding site for nicotine. The alpha4 and beta2 nAChR subunits assemble into two alternate stoichiometries, (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2), which differ in their functional properties and sensitivity to chronic exposure to nicotine. Here, we investigated the sensitivity of both receptor stoichiometries to modulation by Zn2+. We show that Zn2+ exerts an inhibitory modulatory effect on (alpha4)(2)(beta2)(3) receptors, whereas it potentiates or inhibits, depending on its concentration, the function of (alpha4)(3)(beta2)(2) receptors. Furthermore, Zn2+ inhibition on (alpha4)(2)(beta2)(3) nAChRs is voltage-dependent, whereas it is not on (alpha4)(3)(beta2)(2) receptors. We used molecular modeling in conjunction with alanine substitution and functional studies to identify two distinct sets of residues that determine these effects and may coordinate Zn(2+). Zn(2+) inhibition is mediated by a site located on the beta2(+)/alpha4(-) subunit interfaces on both receptor stoichiometries. alpha4(H195) and beta2(D218) are key determinants of this site. Zn2+ potentiation on (alpha4)(3)(beta2)(2) nAChRs is exerted by a site that resides on the alpha4(+)/alpha4(-) of this receptor stoichiometry. alpha4(H195) on the (-) side of the ACh-binding alpha4 subunit and alpha4(E224) on the (+) side of the non-ACh-binding alpha4 subunit critically contribute to this site. We also identified residues within the beta2 subunit that confer voltage dependency to Zn2+ inhibition on (alpha4)(2)(beta2)(3), but not on (alpha4)(3)(beta2)(2) nAChRs.


Asunto(s)
Membrana Celular/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Zinc/farmacología , Acetilcolina/química , Acetilcolina/metabolismo , Alanina/química , Alanina/metabolismo , Secuencia de Aminoácidos/fisiología , Sustitución de Aminoácidos/fisiología , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Membrana Celular/efectos de los fármacos , Femenino , Humanos , Ligandos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Modelos Moleculares , Oocitos , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/efectos de los fármacos , Subunidades de Proteína/metabolismo , Agregación de Receptores/efectos de los fármacos , Agregación de Receptores/fisiología , Receptor Cross-Talk/efectos de los fármacos , Receptor Cross-Talk/fisiología , Receptores Nicotínicos/química , Transmisión Sináptica/fisiología , Xenopus laevis , Zinc/química
17.
Mol Pharmacol ; 73(6): 1838-43, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18367540

RESUMEN

Sazetidine-A has been recently proposed to be a "silent desensitizer" of alpha4beta2 nicotinic acetylcholine receptors (nAChRs), implying that it desensitizes alpha4beta2 nAChRs without first activating them. This unusual pharmacological property of sazetidine-A makes it, potentially, an excellent research tool to distinguish between the role of activation and desensitization of alpha4beta2 nAChRs in mediating the central nervous system effects of nicotine itself, as well as those of new nicotinic drugs. We were surprised to find that sazetidine-A potently and efficaciously stimulated nAChR-mediated dopamine release from rat striatal slices, which is mediated by alpha4beta2(*) and alpha6beta2(*) subtypes of nAChR. The agonist effects on native striatal nAChRs prompted us to re-examine the effects of sazetidine-A on recombinant alpha4beta2 nAChRs in more detail. We expressed the two alternative stoichiometries of alpha4beta2 nAChR in Xenopus laevis oocytes and investigated the agonist properties of sazetidine-A on both alpha4(2)beta2(3) and alpha4(3)beta2(2) nAChRs. We found that sazetidine-A potently activated both stoichiometries of alpha4beta2 nAChR: it was a full agonist on alpha4(2)beta2(3) nAChRs, whereas it had an efficacy of only 6% on alpha4(3)beta2(2) nAChRs. In contrast to what has been published before, we therefore conclude that sazetidine-A is an agonist of native and recombinant alpha4beta2 nAChRs but shows differential efficacy on alpha4beta2 nAChRs subtypes.


Asunto(s)
Azetidinas/metabolismo , Agonistas Nicotínicos/metabolismo , Piridinas/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Animales , Azetidinas/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Agonistas Nicotínicos/farmacología , Piridinas/farmacología , Ratas , Xenopus laevis
18.
Mol Pharmacol ; 71(2): 389-97, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17065235

RESUMEN

5-(Trifluoromethyl)-6-(1-methyl-azepan-4-yl)methyl-1H-quinolin-2-one (TMAQ) is a novel nicotinic acetylcholine receptor (nAChR) agonist with strong selectivity for beta4-containing receptors. TMAQ also exhibits remarkable species selectivity, being a potent agonist of nAChRs containing the human beta4 subunit but having no detectable agonist activity on nAChRs containing the rat beta4 subunit. With the aim of identifying subunit domains and individual amino acids, which contribute to the species selectivity of TMAQ, a series of chimeric and mutated beta4 subunits has been constructed. Recombinant receptors containing wild-type, chimeric, or mutated beta4 subunits have been examined by radioligand binding, intracellular calcium assays, and electrophysiological recording. Two adjacent amino acids located within the extracellular loop D domain of the beta4 subunit (amino acids 55 and 56) have been identified as playing a critical role in determining the agonist potency of TMAQ. Mutagenesis of these two residues within the rat beta4 subunit to the corresponding amino acids in the human beta4 subunit (S55N and I56V mutations) confers sensitivity to TMAQ. The converse mutations in the human beta4 subunit (N55S and V56I) largely abolish sensitivity to TMAQ. In contrast, these mutations have little or no effect on sensitivity to the nonselective nicotinic agonist epibatidine. Despite acting as a potent agonist of human beta4-containing nAChRs, TMAQ acts as an antagonist of rat beta4-containing receptors. Our experimental data, together with homology models of the rat and human alpha3beta4 nAChRs, suggest that amino acids 55 and 56 may be involved in the coupling of agonist binding and channel gating.


Asunto(s)
Receptores Nicotínicos/efectos de los fármacos , Aminoácidos , Animales , Antraquinonas , Humanos , Activación del Canal Iónico , Mutación , Agonistas Nicotínicos , Unión Proteica , Ingeniería de Proteínas , Subunidades de Proteína , Ratas , Receptores Nicotínicos/genética , Especificidad de la Especie , Relación Estructura-Actividad
19.
J Neurochem ; 98(3): 876-85, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16787419

RESUMEN

Alpha4 and beta2 nicotinic acetylcholine (nACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixture of receptors with high and low agonist sensitivity whose relative abundance is influenced by the heteropentamer subunit ratio. We have found that inhibition of protein kinase A by KT5720 decreased maximal [3H]cytisine binding and acetylcholine (ACh)-induced current responses, and increased the relative proportion of alpha4beta2 receptors with high agonist sensitivity. Mutation of serine 467, a putative protein kinase A substrate in a chaperone protein binding motif within the large cytoplasmic domain of the alpha4 subunit, to alanine or asparate decreased or increased, respectively, maximal [3H]cytisine binding and ACh response amplitude. Expression of alpha4S467A mutant subunits decreased steady levels of alpha4 and the relative proportion of alpha4beta2 receptors with low agonist sensitivity, whilst expression of alpha4S467D increased steady levels of alpha4 and alpha4beta2 receptors with low agonist sensitivity. Difopein, an inhibitor of chaperone 14-3-3 proteins, decreased [3H]cytisine binding and ACh responses and increased the proportion of alpha4beta2 with high sensitivity to activation by ACh. Thus, post-translational modification affecting steady-state levels of alpha4 subunits provides a possible means for physiologically relevant, chaperone-mediated variation in the relative proportion of high and low agonist sensitivity alpha4beta2 nACh receptors.


Asunto(s)
Proteínas 14-3-3/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Chaperonas Moleculares/fisiología , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/biosíntesis , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Sensibilidad y Especificidad , Xenopus laevis
20.
J Pharmacol Exp Ther ; 318(3): 1108-17, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16738207

RESUMEN

Here we report the discovery, by high-throughput screening, of three novel (2-amino-5-keto)thiazole compounds that act as selective potentiators of nicotinic acetylcholine receptors. Compound selectivity was assessed at seven human nicotinic acetylcholine receptors (alpha1beta1gammadelta, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7) expressed in mammalian cells or Xenopus oocytes. At alpha2beta4, alpha4beta2, alpha4beta4, and alpha7, but not alpha1beta1gammadelta, alpha3beta2, or alpha3beta4, submaximal responses to nicotinic agonists were potentiated in a concentration-dependent manner by all compounds. At similar concentrations, no potentiation of 5-hydroxytryptamine, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, GABA(A), and N-methyl-d-aspartate receptors or voltage-gated Na(+) and Ca(2+) channels was observed. Furthermore, these compounds did not inhibit acetylcholine esterase. Further profiling revealed that these compounds enhanced the potency and maximal efficacy of a range of nicotinic agonists at alpha4beta2 nicotinic acetylcholine receptors, a profile typical of allosteric potentiators. At concentrations required for potentiation, the compounds did not displace [(3)H]epibatidine from the agonist-binding site, and potentiation was observed at all agonist concentrations, suggesting a noncompetitive mechanism of action. Blockade of common second messenger systems did not affect potentiation. At concentrations higher then required for potentiation the compounds also displayed intrinsic agonist activity, which was blocked by competitive and noncompetitive nicotinic acetylcholine receptor (nAChR) antagonists. These novel selective nicotinic receptor potentiators should help in clarifying the potential therapeutic utility of selective nAChR modulation for the treatment of central nervous system disorders.


Asunto(s)
Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/efectos de los fármacos , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular , Sinergismo Farmacológico , Galantamina/farmacología , Humanos , Subunidades de Proteína , Piridinas/farmacología , Receptores Nicotínicos/química , Proteínas Recombinantes/efectos de los fármacos , Xenopus laevis
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