RESUMO
M(1) muscarinic acetylcholine receptors (mAChRs) represent a viable target for treatment of multiple disorders of the central nervous system (CNS) including Alzheimer's disease and schizophrenia. The recent discovery of highly selective allosteric agonists of M(1) receptors has provided a major breakthrough in developing a viable approach for the discovery of novel therapeutic agents that target these receptors. Here we describe the characterization of two novel M(1) allosteric agonists, VU0357017 and VU0364572, that display profound differences in their efficacy in activating M(1) coupling to different signaling pathways including Ca(2+) and ß-arrestin responses. Interestingly, the ability of these agents to differentially activate coupling of M(1) to specific signaling pathways leads to selective actions on some but not all M(1)-mediated responses in brain circuits. These novel M(1) allosteric agonists induced robust electrophysiological effects in rat hippocampal slices, but showed lower efficacy in striatum and no measureable effects on M(1)-mediated responses in medial prefrontal cortical pyramidal cells in mice. Consistent with these actions, both M(1) agonists enhanced acquisition of hippocampal-dependent cognitive function but did not reverse amphetamine-induced hyperlocomotion in rats. Together, these data reveal that M(1) allosteric agonists can differentially regulate coupling of M(1) to different signaling pathways, and this can dramatically alter the actions of these compounds on specific brain circuits important for learning and memory and psychosis.
Assuntos
Comportamento Animal/efeitos dos fármacos , Benzamidas/farmacologia , Compostos de Bifenilo/farmacologia , Encéfalo/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Receptor Muscarínico M1/agonistas , Animais , Arrestinas/metabolismo , Células CHO , Cálcio/metabolismo , Linhagem Celular , Corpo Estriado/fisiologia , Cricetinae , Cricetulus , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Espaço Extracelular/fisiologia , Medo/psicologia , Perfilação da Expressão Gênica , Hipocampo/fisiologia , Humanos , Masculino , Aprendizagem em Labirinto , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Córtex Pré-Frontal/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Parkinson's disease (PD) is a debilitating neurodegenerative disorder associated with severe motor impairments caused by the loss of dopaminergic innervation of the striatum. Previous studies have demonstrated that positive allosteric modulators (PAMs) of metabotropic glutamate receptor 4 (mGlu4), including N-phenyl-7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxamide, can produce antiparkinsonian-like effects in preclinical models of PD. However, these early mGlu4 PAMsexhibited unsuitable physiochemical properties for systemic dosing, requiring intracerebroventricular administration and limiting their broader utility as in vivo tools to further understand the role of mGlu4 in the modulation of basal ganglia function relevant to PD. In the present study, we describe the pharmacologic characterization of a systemically active mGlu4 PAM, N-(3-chlorophenyl)picolinamide (VU0364770), in several rodent PD models. VU0364770 showed efficacy alone or when administered in combination with L-DOPA or an adenosine 2A (A2A) receptor antagonist currently in clinical development (preladenant). When administered alone, VU0364770 exhibited efficacy in reversing haloperidol-induced catalepsy, forelimb asymmetry-induced by unilateral 6-hydroxydopamine (6-OHDA) lesions of the median forebrain bundle, and attentional deficits induced by bilateral 6-OHDA nigrostriatal lesions in rats. In addition, VU0364770 enhanced the efficacy of preladenant to reverse haloperidol-induced catalepsy when given in combination. The effects of VU0364770 to reverse forelimb asymmetry were also potentiated when the compound was coadministered with an inactive dose of L-DOPA, suggesting that mGlu4 PAMs may provide L-DOPA-sparing activity. The present findings provide exciting support for the potential role of selective mGlu4 PAMs as a novel approach for the symptomatic treatment of PD and a possible augmentation strategy with either L-DOPA or A2A antagonists.
Assuntos
Antagonistas do Receptor A2 de Adenosina/uso terapêutico , Levodopa/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Ácidos Picolínicos/uso terapêutico , Receptores de Glutamato Metabotrópico/agonistas , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Antagonistas do Receptor A2 de Adenosina/sangue , Antagonistas do Receptor A2 de Adenosina/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Sinalização do Cálcio/efeitos dos fármacos , Catalepsia/induzido quimicamente , Catalepsia/tratamento farmacológico , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Corpo Estriado/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Ácido Glutâmico/farmacologia , Células HEK293 , Haloperidol/farmacologia , Humanos , Levodopa/metabolismo , Masculino , Monoaminoxidase/metabolismo , Doença dos Neurônios Motores/induzido quimicamente , Doença dos Neurônios Motores/tratamento farmacológico , Doença dos Neurônios Motores/metabolismo , Doença dos Neurônios Motores/patologia , Doença dos Neurônios Motores/fisiopatologia , Oxidopamina/farmacologia , Ácidos Picolínicos/sangue , Ácidos Picolínicos/metabolismo , Ácidos Picolínicos/farmacocinética , Ácidos Picolínicos/farmacologia , Ligação Proteica , Desempenho Psicomotor/efeitos dos fármacos , Pirimidinas/sangue , Pirimidinas/metabolismo , Pirimidinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Tempo de Reação/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glutamato Metabotrópico/genéticaRESUMO
Nicotinic acetylcholine receptors (nAChRs) form ligand-gated ion channels that mediate fast signal transmission at synapses. These receptors are members of a large family of pentameric ion channels that are of active medical interest. An expression system utilizing a chimerical construct of the N-terminal extracellular ligand binding domain of alpha7 type nAChR and the C-terminal transmembrane portion of 5HT3 type receptor resulted high level of expressions. Two ligand affinity chromatography purification methods for this receptor have been developed. One method relies on the covalent immobilization of a high affinity small molecule alpha7 nAChR agonist, (R)-5-(4-aminophenyl)-N-(quinuclidin-3-yl) furan-2-carboxamide, and the other uses mono biotinylated alpha-bungarotoxin, an antagonist, that forms a quasi-irreversible complex with alpha7 nAChR. Detergent solubilized alpha7/5HT(3) chimeric receptors were selectively retained on the affinity resins and could be eluted with free ligand or biotin. The proteins purified by both methods were characterized by gel electrophoresis, mass spectra, amino acid composition analysis, and N-terminal sequence determination. These analyses confirmed the isolation of a mature alpha7/5HT(3) receptor with the signal peptide removed. These results suggest a scalable path forward to generate multi-milligram amounts of purified complexes for additional studies including protein crystallization.
Assuntos
Receptores Nicotínicos/genética , Receptores Nicotínicos/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Agonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/metabolismo , Ligação Proteica , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ultracentrifugação , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Profiling of putative lead compounds against a representative panel of relevant enzymes, receptors, ion channels, and transporters is a pragmatic approach to establish a preliminary view of potential issues that might later hamper development. An early idea of which off-target activities must be minimized can save valuable time and money during the preclinical lead optimization phase if pivotal questions are asked beyond the usual profiling at hERG. The best data for critical evaluation of activity at ion channels is obtained using functional assays, since binding assays cannot detect all interactions and do not provide information on whether the interaction is that of an agonist, antagonist, or allosteric modulator. For ion channels present in human cardiac muscle, depending on the required throughput, manual-, or automated-patch-clamp methodologies can be easily used to evaluate compounds individually to accurately reveal any potential liabilities. The issue of expanding screening capacity against a cardiac panel has recently been addressed by developing a series of robust, high-throughput, cell-based counter-screening assays employing fluorescence-based readouts. Similar assay development approaches can be used to configure panels of efficacy assays that can be used to assess selectivity within a family of related ion channels, such as Nav1.X channels. This overview discusses the benefits of in vitro assays, specific decision points where profiling can be of immediate benefit, and highlights the development and validation of patch-clamp and fluorescence-based profiling assays for ion channels (for examples of fluorescence-based assays, see Bhave et al., 2010; and for high-throughput patch-clamp assays see Mathes, 2006; Schrøder et al., 2008).