RESUMEN
Polycomb repressive complex 2 (PRC2), a histone H3 lysine 27 methyltransferase, plays a key role in gene regulation and is a known epigenetics drug target for cancer therapy. The WD40 domain-containing protein EED is the regulatory subunit of PRC2. It binds to the tri-methylated lysine 27 of the histone H3 (H3K27me3), and through which stimulates the activity of PRC2 allosterically. Recently, we disclosed a novel PRC2 inhibitor EED226 which binds to the K27me3-pocket on EED and showed strong antitumor activity in xenograft mice model. Here, we further report the identification and validation of four other EED binders along with EED162, the parental compound of EED226. The crystal structures for all these five compounds in complex with EED revealed a common deep pocket induced by the binding of this diverse set of compounds. This pocket was created after significant conformational rearrangement of the aromatic cage residues (Y365, Y148 and F97) in the H3K27me3 binding pocket of EED, the width of which was delineated by the side chains of these rearranged residues. In addition, all five compounds interact with the Arg367 at the bottom of the pocket. Each compound also displays unique features in its interaction with EED, suggesting the dynamics of the H3K27me3 pocket in accommodating the binding of different compounds. Our results provide structural insights for rational design of novel EED binder for the inhibition of PRC2 complex activity.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Simulación del Acoplamiento Molecular , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Sulfonas/farmacología , Triazoles/farmacología , Animales , Sitios de Unión , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Ratones , Complejo Represivo Polycomb 2/química , Complejo Represivo Polycomb 2/metabolismo , Relación Estructura-Actividad Cuantitativa , Sulfonas/química , Triazoles/químicaRESUMEN
Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1-4 kinase inhibitors, the optimization of which enabled cocrystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1-4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Descubrimiento de Drogas , Células HEK293 , Células HT29 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Antígenos de Histocompatibilidad Menor/química , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
One of the central questions in the characterization of enzyme inhibitors is determining the mode of inhibition (MOI). Classically, this is done with a number of low-throughput methods in which inhibition models are fitted to the data. The ability to rapidly characterize the MOI for inhibitors arising from high-throughput screening in which hundreds to thousands of primary inhibitors may need to be characterized would greatly help in lead selection efforts. Here we describe a novel method for determining the MOI of a compound without the need for curve fitting of the enzyme inhibition data. We provide experimental data to demonstrate the utility of this new high-throughput MOI classification method based on nonparametric analysis of the activity derived from a small matrix of substrate and inhibitor concentrations (e.g., from a 4S × 4I matrix). Lists of inhibitors from four different enzyme assays are studied, and the results are compared with the previously described IC50-shift method for MOI classification. The MOI results from this method are in good agreement with the known MOI and compare favorably with those from the IC50-shift method. In addition, we discuss some advantages and limitations of the method and provide recommendations for utilization of this MOI classification method.
Asunto(s)
Inhibidores Enzimáticos/química , Enzimas/química , Ensayos Analíticos de Alto Rendimiento/métodos , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/clasificación , HumanosRESUMEN
The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RASERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 µM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RASERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.
Asunto(s)
Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Piperidinas/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Pirimidinas/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Concentración 50 Inhibidora , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Desnudos , Modelos Moleculares , Neoplasias/patología , Proteína Oncogénica p21(ras)/metabolismo , Piperidinas/química , Piperidinas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Estabilidad Proteica/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Pirimidinas/química , Pirimidinas/uso terapéutico , Reproducibilidad de los Resultados , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein-ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.
Asunto(s)
Antineoplásicos/química , Piperidinas/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Pirazinas/química , Pirimidinas/química , Administración Oral , Regulación Alostérica , Sitio Alostérico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Diseño de Fármacos , Femenino , Xenoinjertos , Ensayos Analíticos de Alto Rendimiento , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Desnudos , Modelos Moleculares , Trasplante de Neoplasias , Piperidinas/síntesis química , Piperidinas/farmacocinética , Piperidinas/farmacología , Conformación Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Pirazinas/síntesis química , Pirazinas/farmacocinética , Pirazinas/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
Pilot testing of an assay intended for high-throughput screening (HTS) with small compound sets is a necessary but often time-consuming step in the validation of an assay protocol. When the initial testing concentration is less than optimal, this can involve iterative testing at different concentrations to further evaluate the pilot outcome, which can be even more time-consuming. Quantitative HTS (qHTS) enables flexible and rapid collection of assay performance statistics, hits at different concentrations, and concentration-response curves in a single experiment. Here we describe the qHTS process for pilot testing in which eight-point concentration-response curves are produced using an interplate asymmetric dilution protocol in which the first four concentrations are used to represent the range of typical HTS screening concentrations and the last four concentrations are added for robust curve fitting to determine potency/efficacy values. We also describe how these data can be analyzed to predict the frequency of false-positives, false-negatives, hit rates, and confirmation rates for the HTS process as a function of screening concentration. By taking into account the compound pharmacology, this pilot-testing paradigm enables rapid assessment of the assay performance and choosing the optimal concentration for the large-scale HTS in one experiment.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Bioensayo , Línea Celular , Relación Dosis-Respuesta a Droga , Reacciones Falso Positivas , Genes Reporteros , Humanos , Proyectos Piloto , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
Ezh2 (Enhancer of zeste homolog 2) protein is the enzymatic component of the Polycomb repressive complex 2 (PRC2), which represses gene expression by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation and differentiation during embryonic development. Recently, hot-spot mutations of Ezh2 were identified in diffused large B-cell lymphomas and follicular lymphomas. To investigate if tumor growth is dependent on the enzymatic activity of Ezh2, we developed a potent and selective small molecule inhibitor, EI1, which inhibits the enzymatic activity of Ezh2 through direct binding to the enzyme and competing with the methyl group donor S-Adenosyl methionine. EI1-treated cells exhibit genome-wide loss of H3K27 methylation and activation of PRC2 target genes. Furthermore, inhibition of Ezh2 by EI1 in diffused large B-cell lymphomas cells carrying the Y641 mutations results in decreased proliferation, cell cycle arrest, and apoptosis. These results provide strong validation of Ezh2 as a potential therapeutic target for the treatment of cancer.
Asunto(s)
Linfoma de Células B Grandes Difuso/patología , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Embrión de Mamíferos/citología , Proteína Potenciadora del Homólogo Zeste 2 , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Linfoma de Células B Grandes Difuso/genética , Metilación/efectos de los fármacos , Ratones , Mutación/genética , Fenotipo , Complejo Represivo Polycomb 2/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Ensayo de Tumor de Célula Madre , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Histone methylation is a regulated feature of nucleosomes that can have an impact on gene expression. The methylation state of histone residues has also been found in recent years to be associated with various disorders. Tools for detecting methylation state changes are very useful for dissecting the function of these epigenetic marks. In this work, a sensitive homogeneous assay for histone demethylase activity at the H3K4 site has been developed in a time-resolved fluorescent resonance energy transfer assay format. The assay is based on the detection of the unmethylated H3 peptide by a fluorescent europium-chelate labeled monoclonal antibody binding specifically to the H3K4 site. The assay was validated for histone lysine-specific demethylase 1 and was demonstrated to be a suitable assay for inhibitor profiling and high-throughput screening.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Histona Desacetilasas/farmacología , Histona Demetilasas/antagonistas & inhibidores , Anticuerpos Monoclonales/química , Sitios de Unión , Quelantes/química , Interpretación Estadística de Datos , Europio , Transferencia Resonante de Energía de Fluorescencia , Histona Demetilasas/química , Peroxidasa de Rábano Silvestre/química , Humanos , Peróxido de Hidrógeno/química , Indicadores y Reactivos , Especificidad por SustratoRESUMEN
The synthesis and preliminary studies of the SAR of novel 3,5-diarylazole inhibitors of Protein Kinase D (PKD) are reported. Notably, optimized compounds in this class have been found to be active in cellular assays of phosphorylation-dependant HDAC5 nuclear export, orally bioavailable, and highly selective versus a panel of additional putative histone deacetylase (HDAC) kinases. Therefore these compounds could provide attractive tools for the further study of PKD/HDAC5 signaling.
Asunto(s)
Azoles/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Animales , Azoles/síntesis química , Azoles/química , Azoles/farmacocinética , Disponibilidad Biológica , Histona Desacetilasas/metabolismo , Concentración 50 Inhibidora , Estructura Molecular , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Relación Estructura-ActividadRESUMEN
The main goal of high-throughput screening (HTS) is to identify active chemical series rather than just individual active compounds. In light of this goal, a new method (called compound set enrichment) to identify active chemical series from primary screening data is proposed. The method employs the scaffold tree compound classification in conjunction with the Kolmogorov-Smirnov statistic to assess the overall activity of a compound scaffold. The application of this method to seven PubChem data sets (containing between 9389 and 263679 molecules) is presented, and the ability of this method to identify compound classes with only weakly active compounds (potentially latent hits) is demonstrated. The analysis presented here shows how methods based on an activity cutoff can distort activity information, leading to the incorrect activity assignment of compound series. These results suggest that this method might have utility in the rational selection of active classes of compounds (and not just individual active compounds) for followup and validation.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Bioensayo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de MedicamentosRESUMEN
A novel 2,6-naphthyridine was identified by high throughput screen (HTS) as a dual protein kinase C/D (PKC/PKD) inhibitor. PKD inhibition in the heart was proposed as a potential antihypertrophic mechanism with application as a heart failure therapy. As PKC was previously identified as the immediate upstream activator of PKD, PKD vs PKC selectivity was essential to understand the effect of PKD inhibition in models of cardiac hypertrophy and heart failure. The present study describes the modification of the HTS hit to a series of prototype pan-PKD inhibitors with routine 1000-fold PKD vs PKC selectivity. Example compounds inhibited PKD activity in vitro, in cells, and in vivo following oral administration. Their effects on heart morphology and function are discussed herein.
Asunto(s)
Aminopiridinas/síntesis química , Naftiridinas/síntesis química , Proteína Quinasa C/antagonistas & inhibidores , Transporte Activo de Núcleo Celular , Administración Oral , Aminopiridinas/farmacocinética , Aminopiridinas/farmacología , Animales , Antihipertensivos/síntesis química , Antihipertensivos/química , Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Cardiomegalia/tratamiento farmacológico , Cardiomegalia/patología , Núcleo Celular/metabolismo , Histona Desacetilasas/metabolismo , Isoenzimas/antagonistas & inhibidores , Masculino , Modelos Moleculares , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Células Musculares/patología , Miocardio/metabolismo , Miocardio/patología , Naftiridinas/farmacocinética , Naftiridinas/farmacología , Fosforilación , Unión Proteica , Ratas , Ratas Endogámicas Dahl , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Many attractive targets for therapeutic intervention are enzymes that catalyze biological reactions involving small molecules such as lipids, fatty acids, amino acid derivatives, nucleic acid derivatives, and cofactors. Some of the reactions are difficult to detect by methods commonly used in high-throughput screening (HTS) without specific radioactive or fluorescent labeling of substrates. In addition, there are instances when labeling has a detrimental effect on the biological response. Generally, applicable assay methodologies for detection of such reactions are thus required. Mass spectrometry (MS), being a label-free detection tool, has been actively pursued for assay detection in HTS in the past several years. The authors have explored the use of multiparallel liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for high-throughput detection of biochemical reactions. In this report, we describe in detail the assay development and screening with a LC/MS-based system for inhibitors of human diacylglycerol acyltransferase (DGAT1) with a chemical library of approximately 800,000 compounds. Several strategies and process improvements have been investigated to overcome technical challenges such as data variation and throughput. Results indicated that, through these innovative approaches, the LC/MS-based screening method is both feasible and suitable for high-throughput primary screening.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos/métodos , Pruebas de Enzimas/métodos , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/farmacología , Espectrometría de Masas/métodos , Cromatografía Liquida , Diacilglicerol O-Acetiltransferasa/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Estándares de Referencia , Reproducibilidad de los Resultados , Solventes/química , Factores de Tiempo , VolumetríaRESUMEN
High-throughput screening (HTS) is an important tool for finding active compounds to initiate medicinal chemistry programs in pharmaceutical discovery research. Traditional HTS methods rely on fluorescent or radiolabeled reagents and/or coupling assays to permit quantitation of enzymatic target inhibition or activation. Mass spectrometry-based high-throughput screening (MS-HTS) is an alternative that is not susceptible to the limitations imposed by labeling and coupling enzymes. MS-HTS offers a selective and sensitive analytical method for unlabeled substrates and products. Furthermore, method development times are reduced without the need to incorporate labels or coupling assays. MS-HTS also permits screening of targets that are difficult or impossible to screen by other techniques. For example, enzymes that are challenging to purify can lead to the nonspecific detection of structurally similar components of the impure enzyme or matrix of membraneous enzymes. The high selectivity of tandem mass spectrometry (MS/MS) enables these screens to proceed with low levels of background noise to sensitively discover interesting hits even with relatively weak activity. In this article, we describe three techniques that we have adapted for large-scale (approximately 175,000 sample) compound library screening, including four-way parallel multiplexed electrospray liquid chromatography tandem mass spectrometry (MUX-LC/MS/MS), four-way parallel staggered gradient liquid chromatography tandem mass spectrometry (LC/MS/MS), and eight-way staggered flow injection MS/MS following 384-well plate solid-phase extraction (SPE). These methods are capable of analyzing a 384-well plate in 37 min, with typical analysis times of less than 2 h. The quality of the MS-HTS approach is demonstrated herein with screening data from two large-scale screens.
Asunto(s)
Técnicas Químicas Combinatorias , Preparaciones Farmacéuticas/análisis , Espectrometría de Masas en Tándem/métodos , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Evaluación Preclínica de Medicamentos/instrumentación , Evaluación Preclínica de Medicamentos/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Extracción en Fase Sólida/instrumentación , Extracción en Fase Sólida/métodos , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
Despite a large body of references on assay development, assay optimization, strategies, and methodologies for high-throughput screening (HTS), there have been few reports on investigations of the efficiency of primary screening in a systematic and quantitative manner for a typical HTS process. Recently, the authors investigated the primary hit comparison and the effect of measurement variability by screening a library of approximately 25,000 random compounds in multiple replicate tests in a nuclear receptor recruitment assay with 2 different assay detection technologies. In this report, we utilized these sets of multiple replicate screening data from a different perspective and conducted a systematic data analysis in order to gain some insights into the hit-finding efficiency of a typical primary screening process. Specifically, hit confirmation, false-positive (declaration) rates, and false-negative rates at different hit cutoff limits were explored and calculated from the 2 different assay formats. Results and analyses provided some quantitative estimation regarding the reliability and efficiency of the primary screening process. For the 2 assay formats tested in this report, the confirmation rate (activity repeated at or above a certain hit limit) was found to be 65% or above. It was also suggested that, at least in this case, applying some hit-selection strategies, it is possible to decrease the number of false-negative or false-positive hits without significantly increasing the efforts in primary screening.
Asunto(s)
Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Reacciones Falso Negativas , Reacciones Falso Positivas , Transferencia Resonante de Energía de Fluorescencia/métodos , Ligandos , Receptores Citoplasmáticos y Nucleares/análisis , Receptores Citoplasmáticos y Nucleares/química , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Just-in-time cell supply for cell-based high-throughput screening (HTS) is frequently problematic. In addition to scheduling and logistical issues, quality issues and variability due to passage effect, cell cycle, or confluency contribute to day-to-day signal variability in the course of cell-based HTS campaigns. Cell division-arrest and cryopreservation technologies permit the use of cells as assay-ready reagents for HTS and other cell-based profiling and structure-activity studies. In this report, the authors compare division-arrested and dividing cells in 2 assay types that are dependent on movement of proteins within or through cell membranes: a receptor tyrosine kinase assay involving A431 cells responsive to epidermal growth factor, and a secretion reporter assay, which measures secretion of a reporter gene, secreted alkaline phosphatase. In both assays, dividing and division-arrested cells yielded similar basal and maximal signals at a given cell density. Similar IC50s were obtained for reference inhibitors in each assay, type in both dividing and division-arrested cells. In addition, for the secretion reporter assay, when comparing IC50s obtained from 44 compounds randomly chosen from a primary screening hit list, the rank order of potency obtained from dividing cells and division-arrested cells was essentially identical. Furthermore, the results show that, under certain assay conditions, data generated using division-arrested cells are less variable than those generated using dividing cells. In summary, the results suggest that, in many cases, division-arrested cells can substitute for dividing cells and offer certain advantages for cell-based assays.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Industria Farmacéutica/métodos , Fosfatasa Alcalina/metabolismo , División Celular , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Química Farmacéutica/métodos , Dimetilsulfóxido/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Genes Reporteros , Humanos , Concentración 50 Inhibidora , Fosforilación , Proteínas Tirosina Quinasas Receptoras/metabolismo , Reproducibilidad de los ResultadosRESUMEN
High-throughput screening (HTS) has grown rapidly in the past decade, with many advances in new assay formats, detection technologies, and laboratory automation. Recently, several studies have shown that the choice of assay technology used for the screening process is particularly important and can yield quite different primary screening outcomes. However, because the screening assays in these previous studies were performed in a single-point determination, it is not clear to what extent the difference observed in the screening results between different assay technologies is attributable to inherent assay variability and day-to-day measurement variation. To address this question, a nuclear receptor coactivator recruitment assay was carried out in 2 different assay formats, namely, AlphaScreen and time-resolved fluorescence resonance energy transfer, which probed the same biochemical binding events but with different detection technologies. For each assay format, 4 independent screening runs in a typical HTS setting were completed to evaluate the run-to-run screening variability. These multiple tests with 2 assay formats allow an unambiguous comparison between the discrepancies of different assay formats and the effects of the variability of assay and screening measurements on the screening outcomes. The results provide further support that the choice of assay format or technology is a critical factor in HTS assay development.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos/métodos , Factores de Transcripción/antagonistas & inhibidores , Animales , Automatización , Biotina/química , Proteínas de Unión al ADN/metabolismo , Industria Farmacéutica/instrumentación , Industria Farmacéutica/métodos , Transferencia Resonante de Energía de Fluorescencia , Ligandos , Miniaturización , Estructura Terciaria de Proteína , Ratas , Receptores Citoplasmáticos y Nucleares , Reproducibilidad de los Resultados , Proyectos de Investigación , Espectroscopía Infrarroja por Transformada de Fourier , Factores de Transcripción/metabolismoAsunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Anticuerpos/metabolismo , Proteína de Unión a CREB , Histona Acetiltransferasas , Ligandos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Coactivador 1 de Receptor Nuclear , Péptidos/química , Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-myb/inmunología , Receptores Citoplasmáticos y Nucleares/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transactivadores/genética , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
The mechanism by which ligands of nuclear receptors show differential effects on gene transcription is not fully understood, but is believed to result in part from the preferential recruitment and/or displacement of coactivators and corepressors. We have explored the interaction of several known ligands and the nuclear receptor (peroxisome proliferator activated receptor alpha, PPARalpha) using scintillation proximity assay (SPA) and the interaction of LXXLL containing peptides derived from three coactivators (SRC-1, CBP and PGC-1) with PPARalpha in the presence of PPARalpha agonist ligands using fluorescence resonance energy transfer (FRET). The EC(50)s of the individual ligands for recruitment showed the same rank order regardless of the coactivator peptide used, with GW2331Asunto(s)
Receptores Citoplasmáticos y Nucleares/metabolismo
, Factores de Transcripción/metabolismo
, Línea Celular
, Núcleo Celular/metabolismo
, ADN Complementario/metabolismo
, Transferencia de Energía
, Escherichia coli/metabolismo
, Histona Acetiltransferasas
, Humanos
, Cinética
, Ligandos
, Coactivador 1 de Receptor Nuclear
, Péptidos/química
, Péptidos/metabolismo
, Plásmidos/metabolismo
, Unión Proteica
, Proteínas Proto-Oncogénicas c-myc/metabolismo
, Espectrofotometría
, Transfección
RESUMEN
The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors activated by fatty acids and their metabolites. The PPARdelta subtype is believed to be involved in lipoprotein regulation and may have a role in reverse cholesterol transport. While the range of biological roles of PPARdelta still remains unclear, it is of therapeutic interest in cardiovascular diseases. Here we report a homogeneous in vitro assay for studying ligand activation of PPARdelta. We surveyed a panel of peptides containing the LXXLL motifs derived from coactivator protein sequences. Peptides with the best response were used to develop a sensitive and homogeneous recruitment assay for PPARdelta. The optimized assay has a signal-to-background ratio of about 8:1 and an assay quality parameter Z'-factor value of 0.8. The assay signal generated is stable for hours to even overnight. This simple recruitment assay can provide agonist and/or antagonist information that cannot be assessed by receptor-binding assay, and can be used for characterization and screening of ligands that modulate the activation of PPARdelta.