RESUMEN
N-methyl-D-aspartate-receptors (NMDARs) are ionotropic glutamate receptors that function in synaptic transmission, plasticity and cognition. Malfunction of NMDARs has been implicated in a variety of nervous system disorders, making them attractive therapeutic targets. Overexpression of functional NMDAR in non-neuronal cells results in cell death by excitotoxicity, hindering the development of cell-based assays for NMDAR drug discovery. Here we report a plate-based, high-throughput approach to study NMDAR function. Our assay enables the functional study of NMDARs with different subunit composition after activation by glycine/D-serine or glutamate and hence presents the first plate-based, high throughput assay that allows for the measurement of NMDAR function in glycine/D-serine and/or glutamate sensitive modes. This allows to investigate the effect of small molecule modulators on the activation of NMDARs at different concentrations or combinations of the co-ligands. The reported assay system faithfully replicates the pharmacology of the receptor in response to known agonists, antagonists, positive and negative allosteric modulators, as well as the receptor's sensitivity to magnesium and zinc. We believe that the ability to study the biology of NMDARs rapidly and in large scale screens will enable the identification of novel therapeutics whose discovery has otherwise been hindered by the limitations of existing cell based approaches.
Asunto(s)
Calcio/metabolismo , Ácido Glutámico/metabolismo , Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/metabolismo , Expresión Génica , Células HEK293 , Humanos , Unión Proteica , Multimerización de Proteína , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Proteínas RecombinantesRESUMEN
Vast amounts of bioactivity data have been generated for small molecules across public and corporate domains. Biological signatures, either derived from systematic profiling efforts or from existing historical assay data, have been successfully employed for small molecule mechanism-of-action elucidation, drug repositioning, hit expansion and screening subset design. This article reviews different types of biological descriptors and applications, and we demonstrate how biological data can outlive the original purpose or project for which it was generated. By comparing 150 HTS campaigns run at Novartis over the past decade on the basis of their active and inactive chemical matter, we highlight the opportunities and challenges associated with cross-project learning in drug discovery.
Asunto(s)
Minería de Datos , Bases de Datos de Compuestos Químicos , Bases de Datos Farmacéuticas , Descubrimiento de Drogas/métodos , Preparaciones Farmacéuticas/química , Animales , Simulación por Computador , Minería de Datos/historia , Bases de Datos de Compuestos Químicos/historia , Bases de Datos Farmacéuticas/historia , Descubrimiento de Drogas/historia , Historia del Siglo XXI , Humanos , Modelos Moleculares , Estructura Molecular , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The disrupted in schizophrenia 1 (DISC1) gene is a candidate susceptibility factor for schizophrenia, but its mechanistic role in the disorder is unknown. Here we report that the gene encoding phosphodiesterase 4B (PDE4B) is disrupted by a balanced translocation in a subject diagnosed with schizophrenia and a relative with chronic psychiatric illness. The PDEs inactivate adenosine 3',5'-monophosphate (cAMP), a second messenger implicated in learning, memory, and mood. We show that DISC1 interacts with the UCR2 domain of PDE4B and that elevation of cellular cAMP leads to dissociation of PDE4B from DISC1 and an increase in PDE4B activity. We propose a mechanistic model whereby DISC1 sequesters PDE4B in resting cells and releases it in an activated state in response to elevated cAMP.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/genética , AMP Cíclico/metabolismo , Proteínas del Tejido Nervioso/genética , Esquizofrenia/genética , Transducción de Señal , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Adulto , Trastornos Psicóticos Afectivos/genética , Trastornos Psicóticos Afectivos/metabolismo , Animales , Cadherinas/genética , Línea Celular , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 16 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Activación Enzimática , Predisposición Genética a la Enfermedad , Humanos , Masculino , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Ratas , Esquizofrenia/enzimología , Esquizofrenia/metabolismo , Translocación GenéticaRESUMEN
Recently, nuclear distribution element-like (NUDEL) has been implicated to play a role in lissencephaly and schizophrenia through interactions with the lissencephaly gene 1 (Lis1) and disrupted-in-schizophrenia 1 (DISC1) products, respectively. Interestingly, NUDEL is the same protein as endooligopeptidase A (EOPA), a thiol-activated peptidase involved in conversion and inactivation of a number of bioactive peptides. In this study, we have cloned EOPA from the human brain and have confirmed that it is equivalent to NUDEL, leading us to suggest a single name, NUDEL-oligopeptidase. In the brain, the monomeric form of NUDEL-oligopeptidase is responsible for the peptidase activity whose catalytic mechanism is likely to involve a reactive cysteine, because mutation of Cys-273 fully abolished NUDEL-oligopeptidase activity without disrupting the protein's secondary structure. Cys-273 is very close to the DISC1-binding site on NUDEL-oligopeptidase. Intriguingly, DISC1 inhibits NUDEL-oligopeptidase activity in a competitive fashion. We suggest that the activity of NUDEL-oligopeptidase is under tight regulation through protein-protein interactions and that disruption of these interactions, as postulated in a Scottish DISC1 translocation schizophrenia cohort, may lead to aberrant regulation of NUDEL-oligopeptidase, perhaps providing a substrate for the pathology of schizophrenia.