RESUMO
Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a Kb of 26 nM. The maximum response to DETQ alone was â¼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was â¼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3-20 mg/kg orally) caused a robust (â¼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30-240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists.
Assuntos
Comportamento Animal/efeitos dos fármacos , Técnicas de Introdução de Genes , Isoquinolinas/farmacologia , Locomoção/efeitos dos fármacos , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Taquifilaxia , Adamantano/análogos & derivados , Adamantano/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Benzopiranos/farmacologia , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Isoquinolinas/efeitos adversos , Masculino , Camundongos , Transporte Proteico/efeitos dos fármacos , Receptores de Dopamina D1/agonistasRESUMO
The Th17 pathway has been implicated in autoimmune diseases. The retinoic acid receptor-related orphan receptor C2 (RORγt) is a master regulator of Th17 cells and controls the expression of IL-17A. RORγt is expressed primarily in IL-17A-producing lymphoid cells. Here we describe a virtual screen of the ligand-binding pocket and subsequent screen in a binding assay that identified the 1-benzyl-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-2'-carboxamide scaffold as a starting point for optimization of binding affinity and functional activity guided by structure-based design. Compound 12 demonstrated activity in a mouse PK/PD model and efficacy in an inflammatory arthritis mouse model that were used to define the level and duration of target engagement required for efficacy in vivo. Further optimization to improve ADME and physicochemical properties with guidance from simulations and modeling provided compound 22, which is projected to achieve the level and duration of target engagement required for efficacy in the clinic.
Assuntos
Ligantes , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Tiofenos/química , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Artrite/patologia , Sítios de Ligação , Cristalografia por Raios X , Modelos Animais de Doenças , Desenho de Fármacos , Feminino , Meia-Vida , Humanos , Interleucina-17/genética , Interleucina-17/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/química , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Ligação Proteica , Relação Estrutura-Atividade , Tiofenos/metabolismo , Tiofenos/farmacologia , Tiofenos/uso terapêuticoRESUMO
Clinical development of catechol-based orthosteric agonists of the dopamine D1 receptor has thus far been unsuccessful due to multiple challenges. To address these issues, we identified LY3154207 (3) as a novel, potent, and subtype selective human D1 positive allosteric modulator (PAM) with minimal allosteric agonist activity. Conformational studies showed LY3154207 adopts an unusual boat conformation, and a binding pose with the human D1 receptor was proposed based on this observation. In contrast to orthosteric agonists, LY3154207 showed a distinct pharmacological profile without a bell-shaped dose-response relationship or tachyphylaxis in preclinical models. Identification of a crystalline form of free LY3154207 from the discovery lots was not successful. Instead, a novel cocrystal form with superior solubility was discovered and determined to be suitable for development. This cocrystal form was advanced to clinical development as a potential first-in-class D1 PAM and is now in phase 2 studies for Lewy body dementia.
Assuntos
Isoquinolinas/farmacologia , Receptores de Dopamina D1/agonistas , Acetilcolina/metabolismo , Administração Oral , Regulação Alostérica/efeitos dos fármacos , Animais , Sítios de Ligação , Cristalografia por Raios X , AMP Cíclico/metabolismo , Células HEK293 , Meia-Vida , Humanos , Isoquinolinas/química , Isoquinolinas/farmacocinética , Rim/efeitos dos fármacos , Rim/metabolismo , Locomoção/efeitos dos fármacos , Camundongos , Conformação Molecular , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/metabolismo , Ratos , Receptores de Dopamina D1/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
Multiple therapeutic opportunities have been suggested for compounds capable of selective activation of metabotropic glutamate 3 (mGlu3) receptors, but small molecule tools are lacking. As part of our ongoing efforts to identify potent, selective, and systemically bioavailable agonists for mGlu2 and mGlu3 receptor subtypes, a series of C4ß-N-linked variants of (1 S,2 S,5 R,6 S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 1 (LY354740) were prepared and evaluated for both mGlu2 and mGlu3 receptor binding affinity and functional cellular responses. From this investigation we identified (1 S,2 S,4 S,5 R,6 S)-2-amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 8p (LY2794193), a molecule that demonstrates remarkable mGlu3 receptor selectivity. Crystallization of 8p with the amino terminal domain of hmGlu3 revealed critical binding interactions for this ligand with residues adjacent to the glutamate binding site, while pharmacokinetic assessment of 8p combined with its effect in an mGlu2 receptor-dependent behavioral model provides estimates for doses of this compound that would be expected to selectively engage and activate central mGlu3 receptors in vivo.
Assuntos
Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/farmacologia , Agonistas de Aminoácidos Excitatórios/síntese química , Agonistas de Aminoácidos Excitatórios/farmacologia , Receptores de Glutamato Metabotrópico/agonistas , Animais , Compostos Bicíclicos com Pontes/farmacocinética , Cristalografia por Raios X , AMP Cíclico/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacocinética , Antagonistas de Aminoácidos Excitatórios/farmacologia , Humanos , Masculino , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenciclidina/antagonistas & inibidores , Fenciclidina/farmacologia , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
Identification of orthosteric mGlu(2/3) receptor agonists capable of discriminating between individual mGlu2 and mGlu3 subtypes has been highly challenging owing to the glutamate-site sequence homology between these proteins. Herein we detail the preparation and characterization of a series of molecules related to (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate 1 (LY354740) bearing C4-thiotriazole substituents. On the basis of second messenger responses in cells expressing other recombinant human mGlu2/3 subtypes, a number of high potency and efficacy mGlu2 receptor agonists exhibiting low potency mGlu3 partial agonist/antagonist activity were identified. From this, (1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 14a (LY2812223) was further characterized. Cocrystallization of 14a with the amino terminal domains of hmGlu2 and hmGlu3 combined with site-directed mutation studies has clarified the underlying molecular basis of this unique pharmacology. Evaluation of 14a in a rat model responsive to mGlu2 receptor activation coupled with a measure of central drug disposition provides evidence that this molecule engages and activates central mGlu2 receptors in vivo.
Assuntos
Compostos Bicíclicos com Pontes/química , Receptores de Glutamato Metabotrópico/agonistas , Triazóis/química , Regulação Alostérica , Animais , Ligação Competitiva , Compostos Bicíclicos com Pontes/farmacocinética , Compostos Bicíclicos com Pontes/farmacologia , Cálcio/metabolismo , AMP Cíclico/metabolismo , Cães , Agonismo Parcial de Drogas , Humanos , Masculino , Camundongos , Modelos Moleculares , Atividade Motora/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Estereoisomerismo , Triazóis/farmacocinética , Triazóis/farmacologiaRESUMO
The demonstrated functional interaction of metabotropic glutamate 5 (mGlu5) receptors with N-methyl-d-aspartate (NMDA) receptors has prompted speculation that their activation may offer a potential treatment for aspects of schizophrenia. Development of selective mGlu5 agonists has been difficult, but several different positive allosteric modulator (PAM) molecules have now been identified. This study describes two novel mGlu5 PAMs, LSN2463359 (N-(1-methylethyl)-5-(pyridin-4-ylethynyl)pyridine-2-carboxamide) and LSN2814617 [(7S)-3-tert-butyl-7-[3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-A]pyridine], which are useful tools for this field of research. Both compounds are potent and selective potentiators of human and rat mGlu5 receptors in vitro, displaying curve shift ratios of two to three fold in the concentration-response relationship to glutamate or the glutamate receptor agonist, DHPG, with no detectable intrinsic agonist properties. Both compounds displaced the mGlu5 receptor antagonist radioligand, [³H]MPEP in vitro and, following oral administration reached brain concentrations sufficient to occupy hippocampal mGlu5 receptors as measured in vivo by dose-dependent displacement from the hippocampus of intravenously administered MPEPy. In vivo EEG studies demonstrated that these mGlu5 PAMs have marked wake-promoting properties but little in the way of rebound hypersomnolence. In contrast, the previously described mGlu5 PAMs CDPPB and ADX47273 showed relatively poor evidence of in vivo target engagement in either receptor occupancy assays or EEG disturbance. Wake-promoting doses of LSN2463359 and LSN2814617 attenuated deficits in performance induced by the competitive NMDA receptor antagonist SDZ 220,581 in two tests of operant behaviour: the variable interval 30 s task and the DMTP task. These effects were lost if the dose of either compound extended into the range which disrupted performance in the baseline DMTP task. However, the improvements in response accuracy induced by the mGlu5 potentiators in SDZ 220,581-treated rats were not delay-dependent and, therefore, perhaps more likely reflected optimization of general arousal than specific beneficial effects on discrete cognitive processes. The systematic profiling of LSN2463359 and LSN2814617 alongside other previously described molecules will help determine more precisely how mGlu5 potentiator pharmacology might provide therapeutic benefit. This article is part of a Special Issue entitled 'Cognitive Enhancers'.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Drogas em Investigação/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Neurônios/efeitos dos fármacos , Nootrópicos/farmacologia , Receptores de Glutamato Metabotrópico/agonistas , Esquizofrenia/tratamento farmacológico , Regulação Alostérica , Animais , Nível de Alerta/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , Drogas em Investigação/efeitos adversos , Drogas em Investigação/metabolismo , Drogas em Investigação/uso terapêutico , Embrião de Mamíferos/citologia , Agonistas de Aminoácidos Excitatórios/efeitos adversos , Agonistas de Aminoácidos Excitatórios/metabolismo , Agonistas de Aminoácidos Excitatórios/uso terapêutico , Humanos , Masculino , Neurônios/citologia , Neurônios/metabolismo , Nootrópicos/efeitos adversos , Nootrópicos/metabolismo , Nootrópicos/uso terapêutico , Ratos , Ratos Sprague-Dawley , Receptor de Glutamato Metabotrópico 5 , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fases do Sono/efeitos dos fármacos , Distribuição TecidualRESUMO
As part of our ongoing interest in identifying novel agonists acting at metabotropic glutamate (mGlu) 2/3 receptors, we have explored the effect of structural modifications of 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740), a potent and pharmacologically balanced mGlu2/3 receptor agonist. Incorporation of relatively small substituents (e.g., F, O) at the C4 position of this molecule resulted in additional highly potent mGlu2/3 agonists that demonstrate excellent selectivity over the other mGlu receptor subtypes, while addition of larger C4-substituents (e.g., SPh) led to a loss of agonist potency and/or the appearance of weak mGlu2/3 receptor antagonist activity. Further characterization of the α-fluoro-substituted analogue (LY459477) in vivo revealed that this molecule possesses good oral bioavailability in rats and effectively suppresses phencyclidine-evoked locomotor activity at doses that do not impair neuromuscular coordination. This molecule therefore represents a valuable new addition to the arsenal of pharmacological tools competent to investigate mGlu2/3 receptor function both in vitro and in vivo.