RESUMEN
RATIONALE: Associated with frank neuropathology, patients with Alzheimer's disease suffer from a host of neuropsychiatric symptoms that include depression, apathy, agitation, and aggression. Negative allosteric modulators (NAMs) of α5-containing GABAA receptors have been suggested to be a novel target for antidepressant action. We hypothesized that pharmacological modulation of this target would engender increased motivation in stressful environments. METHODS: We utilized electrophysiological recordings from Xenopus oocytes and behavioral measures in mice to address this hypothesis. RESULTS: In the forced-swim assay in mice that detects antidepressant drugs, the α5ß3γ2 GABAΑ receptor NAM, RY-080 produced a marked antidepressant phenotype. Another compound, PWZ-029, was characterized as an α5ß3γ2 receptor NAM of lower intrinsic efficacy in electrophysiological studies in Xenopus oocytes. In contrast to RY-080, PWZ-029 was only moderately active in the forced-swim assay and the α5ß3γ2 receptor antagonist, Xli-093, was not active at all. The effects of RY-080 were prevented by the non-selective benzodiazepine receptor antagonist flumazenil as well as by the selective ligands, PWZ-029 and Xli-093. These findings demonstrate that this effect of RY-080 is driven by negative allosteric modulation of α5ßγ2 GABAA receptors. RY-080 was not active in the tail-suspension test. We also demonstrated a reduction in the age-dependent hyperactivity exhibited by transgenic mice that accumulate pathological tau (rTg4510 mice) by RY-080. The decrease in hyperactivity by RY-080 was selective for the hyperactivity of the rTg4510 mice since the locomotion of control strains of mice were not significantly affected by RY-080. CONCLUSIONS: α5ßγ2 GABAA receptor NAMs might function as a pharmacological treatment for mood, amotivational syndromes, and psychomotor agitation in patients with Alzheimer's and other neurodegenerative disorders.
Asunto(s)
Envejecimiento/efectos de los fármacos , Antidepresivos/farmacología , Agitación Psicomotora/tratamiento farmacológico , Receptores de GABA-A/fisiología , Proteínas tau/antagonistas & inhibidores , Envejecimiento/fisiología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Benzodiazepinas/farmacología , Benzodiazepinas/uso terapéutico , Trastorno Depresivo/tratamiento farmacológico , Trastorno Depresivo/psicología , Relación Dosis-Respuesta a Droga , Femenino , Flumazenil/farmacología , Flumazenil/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Agitación Psicomotora/genética , Xenopus laevis , Proteínas tau/genéticaRESUMEN
The 'neurotrophic sesquiterpenes' refer to a group of molecules derived from the Illicium genus of flowering plant. They display neurotrophic effects in cultured neuron preparations and have been suggested to be cognitive enhancers and potential therapeutics for neurodegenerative disorders and dementias. Recent synthetic advances generated sufficient quantities of jiadifenolide for in vivo investigation into its biological effects. Jiadifenolide did not induce convulsions in mice nor did it enhance or diminish convulsions induced by pentylenetetrazole. Other negative allosteric modulators of GABAA receptors, picrotoxin, tetramethylenedisulfotetramine (TETS), and bilobalide all induced convulsions. Either i.p. or i.c.v. dosing generated micromolar plasma and brain levels of jiadifenolide but only small effects on locomotion of mice. However, jiadifenolide decreased d-amphetamine-induced hyperlocomotion in mice, an antipsychotic-like drug effect. Jiadifenolide did not significantly alter body temperature or behavior in the forced-swim test in mice. Molecular simulation data suggested a potential site in the pore/M2 helix region that is at an overlapping, yet lower position than those observed for other 'cage convulsant' compounds such as TETS and picrotoxin. We hypothesize that a position nearer to the entrance of the pore channel may allow for easier displacement of jiadifenolide from its blocking location leading to lower potency and lower side-effect liability. Like jiadifenolide, memantine (Namenda), one of the few drugs used in the symptomatic treatment of dementias, occupies a unique site on the NMDA receptor complex that creates low binding affinity that is associated with its reduced side-effect profile. Given the potential therapeutic applications of jiadifenolide and its relatively inert effects on overt behavior, the possibility of clinical utility for jiadifenolide and related compounds becomes intriguing.
Asunto(s)
Convulsivantes/metabolismo , Convulsivantes/farmacología , Progresión de la Enfermedad , Enfermedades Neurodegenerativas/metabolismo , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacología , Animales , Convulsivantes/química , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Locomoción/efectos de los fármacos , Locomoción/fisiología , Masculino , Ratones , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/farmacología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Agitación Psicomotora/metabolismo , Sesquiterpenos/químicaRESUMEN
Voltage-gated calcium channels represent important drug targets. The implementation of higher throughput electrophysiology assays is necessary to characterize the interaction of test compounds with several conformational states of the channel, but has presented significant challenges. We report on the development of a high-throughput, automated electrophysiology assay for Cav2.2 on the IonWorks Barracuda™ platform. The assay provides an assessment of the potency of the test compound on the resting/closed and inactivated states of the channel in the same assay run. Inclusion of the heavy metal chelator 2,3-bis(sulfanyl)propane-1-sulfonate in the assay solutions improved the data quality by reversing a loss of current seen in wells directly above the ground electrodes. We hypothesize that the loss of current is caused by block of Cav2.2 currents by silver ions originating from the electrodes.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/metabolismo , Quelantes/farmacología , Ensayos Analíticos de Alto Rendimiento , Plata/farmacología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electrodos , Células HEK293 , Humanos , Técnicas de Placa-Clamp , Ratas , Relación Estructura-ActividadRESUMEN
Voltage-gated sodium channels represent important drug targets. The implementation of higher throughput electrophysiology assays is necessary to characterize the interaction of test compounds with several conformational states of the channel, but has presented significant challenges. We describe a novel high throughput approach to assess the effects of test agents on voltage-gated sodium currents. The multiple protocol mode of the automated electrophysiology instrument IonWorks Barracuda was used to control the level of inactivation and monitor current stability. Good temporal stability of currents and spatial uniformity of inactivation were obtained by optimizing the experimental conditions. The resulting assay allowed for robust assessment of state-dependent effects of test agents and enabled direct comparison of compound potency across several sodium channel subtypes at equivalent levels of inactivation.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Amitriptilina/farmacología , Animales , Células CHO , Células Cultivadas , Cricetulus , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Lidocaína/farmacología , Técnicas de Placa-Clamp , Fenitoína/farmacología , Relación Estructura-Actividad , Tetracaína/farmacologíaRESUMEN
N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an important role in synaptic plasticity and learning and memory formation. Malfunctioning of NMDARs, in particular the reduction in NMDAR activity, is thought to be implicated in major neurological disorders. NMDAR positive allosteric modulators (PAMs) represent potential therapeutic interventions for restoring normal NMDAR function. We report a novel screening approach for identification and characterization of NMDAR-PAMs. The approach combines high-throughput fluorescence imaging with automated electrophysiological recording of glutamate-evoked responses in HEK-293 cells expressing NR1/NR2A NMDAR subunits. Initial high-throughput screening (HTS) of a chemical library containing >810,000 compounds using a calcium flux assay in 1536-well plate format identified a total of 864 NMDAR-PAMs. Concentration response determination in both calcium flux and automated electrophysiological assays found several novel chemical series with EC50 values between 0.49 and 10 µM. A small subset (six series) was selected and analyzed for pharmacological properties, subtype selectivity, mode of action, and activity at native NMDARs. Our approach demonstrates the successful application of HTS functional assays that led to identification of NMDAR-PAMs providing the foundation for further medicinal chemistry work that may lead to novel therapies for treatment of cognitive impairment associated with Alzheimer's disease and schizophrenia.
Asunto(s)
Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Receptores de N-Metil-D-Aspartato/metabolismo , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Regulación Alostérica/efectos de los fármacos , Enfermedad de Alzheimer/tratamiento farmacológico , Calcio/química , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Células HEK293 , Humanos , Plasticidad Neuronal/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Esquizofrenia/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
With the aim of improving HCV protease inhibitors reported in our previous manuscripts, we synthesized and evaluated a series of 1a-based tetrapeptidyl alpha-ketoamides with additional P4 modification. The promising analog discovered through this SAR, 5a, was further derivatized at P1' or P1 position. As a result of these efforts, we found that replacement of the P4 valine as seen in 1a with cyclohexylglycine (Chg) resulted in the discovery of 5a, 5c, and 5e endowed with improved cellular activity in comparison to 1a.
Asunto(s)
Amidas/síntesis química , Antivirales/síntesis química , Inhibidores de Serina Proteinasa/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Amidas/farmacología , Antivirales/farmacología , Replicón/efectos de los fármacos , Inhibidores de Serina Proteinasa/farmacología , Relación Estructura-ActividadRESUMEN
We describe herein the synthesis and biological evaluation of a series of tripeptidyl alpha-ketoamides as human rhinovirus (HRV) 3C protease inhibitors. The most potent inhibitor discussed in this manuscript, 4I, exhibited impressive enzyme inhibitory activity as well as antiviral activity against HRV-14.
Asunto(s)
Amidas/síntesis química , Amidas/farmacología , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Proteínas Virales/antagonistas & inhibidores , Proteasas Virales 3C , Amidas/química , Cisteína Endopeptidasas , Evaluación Preclínica de Medicamentos , Humanos , Inhibidores de Proteasas/química , Relación Estructura-ActividadRESUMEN
We describe herein the design, syntheses and evaluation of a number of bicycloproline P2 bearing HCV protease inhibitors endowed with impressive enzyme potency, enzyme selectivity, cellular activity and favorable ADME profiles.