RESUMEN
Quantitative microscopy is a powerful method for performing phenotypic screens from which image-based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image-based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments. Here, we examine the performance of the Cell Painting assay across multiple high-throughput microscope systems and find that all are compatible with this assay. Furthermore, we determine independently for each microscope system the best performing settings, providing those who wish to adopt this assay an ideal starting point for their own assays. We also explore the impact of microscopy setting changes in the Cell Painting assay and find that few dramatically reduce the quality of a Cell Painting profile, regardless of the microscope used.
Asunto(s)
Bioensayo , Microscopía , Humanos , Microscopía/métodos , Bioensayo/métodosRESUMEN
Quantitative microscopy is a powerful method for performing phenotypic screens from which image-based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image-based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments. Here, we examine the performance of the Cell Painting assay across multiple high-throughput microscope systems and find that all are compatible with this assay. Furthermore, we determine independently for each microscope system the best performing settings, providing those who wish to adopt this assay an ideal starting point for their own assays. We also explore the impact of microscopy setting changes in the Cell Painting assay and find that few dramatically reduce the quality of a Cell Painting profile, regardless of the microscope used.
RESUMEN
Cystic fibrosis (CF) is a lethal genetic disorder caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Despite recent groundbreaking approval of genotype-specific small-molecule drugs, a significant portion of CF patients still lack effective therapeutic options that address the underlying cause of the disease. Through a phenotypic high-throughput screen of approximately 54,000 small molecules, we identified a novel class of CFTR modulators called amplifiers. The identified compound, the characteristics of which are represented here by PTI-CH, selectively increases the expression of immature CFTR protein across different CFTR mutations, including F508del-CFTR, by targeting the inefficiencies of early CFTR biosynthesis. PTI-CH also augments the activity of other CFTR modulators and was found to possess novel characteristics that distinguish it from CFTR potentiator and corrector moieties. The PTI-CH-mediated increase in F508del-CFTR did not elicit cytosolic or endoplasmic reticulum-associated cellular stress responses. Based on these data, amplifiers represent a promising new class of CFTR modulators for the treatment of CF that can be used synergistically with other CFTR modulators.
Asunto(s)
Fibrosis Quística/tratamiento farmacológico , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Línea Celular , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/genética , Genotipo , Humanos , Mutación/genética , FenotipoRESUMEN
Immunoreagents formed the basis of early fixed end point high content screening (HCS) assays and their use in HCS applications in drug discovery will continue to increase. One important application of immunoreagents is their incorporation into multiplexed HCS assays in which multiple physiological features are simultaneously measured and related in the same cells. However, creating multiplexed HCS assays that incorporate multiple immunoreagents presents issues such as reagent compatibility, spectral signal overlap, and reproducibility that must be addressed. Here, an example multiplexed fixed end point HCS assay is used to guide potential assay developers on how to optimize complex, yet cellular information rich, multiplexed HCS assays although avoiding some common pitfalls.
Asunto(s)
Anticuerpos/análisis , Colorantes Fluorescentes/análisis , Análisis de Matrices Tisulares/métodos , Humanos , Células Tumorales CultivadasRESUMEN
Reagents that are used as part of a discovery platform for the measurement and manipulation of cell functions are at the heart of single and multiplexed high content screening assays. Measurement reagents include physiological indicators, immunoreagents, fluorescent analogs of macromolecules, positional biosensors, and fluorescent protein biosensors. Recent developments in reagents that manipulate specific cell functions including small inhibitory RNAs, caged peptides, proteins, and RNAs, and gene switches complement measurement reagents, especially when both classes of reagents are used in the same living cells. The use measurement and manipulation reagents in multiplexed high content screening assays promises to enable a systems cell biology approach to drug discovery and biomedical research.
Asunto(s)
Investigación Biomédica/métodos , Células/metabolismo , Sondas Moleculares/análisis , Sondas Moleculares/metabolismo , Animales , Técnicas Biosensibles , Supervivencia Celular , Evaluación Preclínica de Medicamentos/métodos , Determinación de Punto Final , Células HeLa , Histocitoquímica , Humanos , Indicadores y Reactivos , Proteínas/metabolismoRESUMEN
Cystic fibrosis (CF), an inherited genetic disease, is caused by mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes an ion channel involved in hydration maintenance by anion homeostasis. Ninety percent of CF patients possess one or more copies of the F508del CFTR mutation. This mutation disrupts trafficking of the protein to the plasma membrane and diminishes function of mature CFTR. Identifying small molecule modulators of mutant CFTR activity or biosynthesis may yield new tools for discovering novel CF treatments. One strategy utilizes a 384-well, cell-based fluorescence-quenching assay, which requires extensive wash steps, but reports sensitive changes in fluorescence-quenching kinetic rates. In this study, we describe the methods of adapting the protocol to a homogeneous, miniaturized 1,536-well format and further optimization of this functional F508del CFTR assay. The assay utilizes a cystic fibrosis bronchial epithelial (CFBE41o-) cell line, which was engineered to report CFTR-mediated intracellular flux of iodide by a halide-sensitive yellow fluorescence protein (YFP) reporter. We also describe the limitations of quench rate analysis and the subsequent incorporation of a novel, kinetic data analysis modality to quickly and efficiently find active CFTR modulators. This format yields a Z' value interval of 0.61 ± 0.05. As further evidence of high-throughput screen suitability, we subsequently completed a screening campaign of >645,000 compounds, identifying 2,811 initial hits. After completing secondary and tertiary follow-up assays, we identified 187 potential CFTR modulators, which EC50's < 5 µM. Thus, the assay has integrated the advantages of a phenotypic screen with high-throughput scalability to discover new small-molecule CFTR modulators.
Asunto(s)
Proteínas Bacterianas/análisis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Proteínas Luminiscentes/análisis , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Bacterianas/metabolismo , Células Cultivadas , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/agonistas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Proteínas Luminiscentes/metabolismo , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
A new discipline of biology has emerged since 2004, which we call "systems cell biology" (SCB). Systems cell biology is the study of the living cell, the basic unit of life, an integrated and interacting network of genes, proteins, and myriad metabolic reactions that give rise to function. SCB takes advantage of high-content screening platforms, but delivers more detailed profiles of cellular systemic function, including the application of advanced reagents and informatics tools to sophisticated cellular models. Therefore, an SCB profile is a cellular systemic response as measured by a panel of reagents that quantify a specific set of biomarkers.
Asunto(s)
Biología de Sistemas/instrumentación , Biología de Sistemas/métodos , Animales , Biomarcadores/metabolismo , Línea Celular Tumoral , Química Farmacéutica/métodos , Biología Computacional/métodos , Diseño de Fármacos , Regulación de la Expresión Génica , Humanos , Tecnología Farmacéutica/métodosRESUMEN
High content screening (HCS) has emerged as an important platform technology for early drug discovery from target identification through in vitro ADME/Tox. The focus is now on implementing multiplexed assays, developing and using advanced reagents and developing and harnessing more sophisticated informatics tools. Multiplexed HCS assays have the potential to dramatically improve the early drug discovery process by creating systems cell biology profiles on the activities of compounds. It is predicted that multiplexed HCS assays will accelerate the overall workflow and produce deeper functional knowledge, thereby permitting better decisions on what compounds to pursue.
Asunto(s)
Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento/métodos , Biología de Sistemas/métodos , Animales , Biología Celular , Humanos , Interferencia de ARNRESUMEN
High content screening (HCS), the large-scale automated analysis of the temporal and spatial changes in cells and cell constituents in arrays of cells, has the potential to create enormous systems cell biology knowledge bases. HCS is being employed along with the continuum of the early drug discovery process, including lead optimization where new knowledge is being used to facilitate the decision-making process. We demonstrate methodology to build new systems cell biology knowledge using a multiplexed HCS assay, designed with the aid of knowledge-mining tools, to measure the phenotypic response of a panel of human tumor cell types to a panel of natural product-derived microtubule-targeted anticancer agents and their synthetic analogs. We show how this new systems cell biology knowledge can be used to design a lead compound optimization strategy for at least two members of the panel, (-)-laulimalide and (+)-discodermolide, that exploits cell killing activity while minimally perturbing the regulation of the cell cycle and the stability of microtubules. Furthermore, this methodology can also be applied to basic biomedical research on cells.
Asunto(s)
Antineoplásicos/administración & dosificación , Bioensayo/métodos , Técnicas de Cultivo de Célula/métodos , Evaluación Preclínica de Medicamentos/métodos , Biología de Sistemas/métodos , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/fisiología , Inteligencia Artificial , Bioensayo/instrumentación , Técnicas de Cultivo de Célula/instrumentación , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/instrumentación , Humanos , Robótica/métodos , Biología de Sistemas/instrumentación , Células Tumorales Cultivadas/citologíaRESUMEN
High content screening (HCS) has emerged as an important platform technology for early drug discovery from target identification through in vitro ADME/Tox. The focus is now on implementing multiplexed assays, developing and using advanced reagents and developing and harnessing more sophisticated informatics tools. Multiplexed HCS assays have the potential to dramatically improve the early drug discovery process by creating systems cell biology profiles on the activities of compounds. It is predicted that multiplexed HCS assays will accelerate the overall workflow and produce deeper functional knowledge, thereby permitting better decisions on what compounds to pursue.:
RESUMEN
(+)-Discodermolide, a C24:4, trihydroxylated, octamethyl, carbamate-bearing fatty acid lactone originally isolated from a Caribbean sponge, has proven to be the most potent of the microtubule-stabilizing agents. Recent studies suggest that it or its analogues may have advantages over other classes of microtubule-stabilizing agents. (+)-Discodermolide's complex molecular architecture has made structure-activity relationship analysis in this class of compounds a formidable task. The goal of this study was to prepare simplified analogues of (+)-discodermolide and to analyze their biological activities to expand structure-activity relationships. A small library of analogues was prepared wherein the (+)-discodermolide methyl groups at C-14 and C-16 and the C-7 hydroxyl were removed, and the lactone was replaced by simple esters. The library components were analyzed for microtubule-stabilizing actions in vitro, antiproliferative activity against a small panel of human carcinoma cells, and cell signaling, microtubule architecture and mitotic spindle alterations by a multiparameter fluorescence cell-based screening technique. The results show that even drastic structural simplification can lead to analogues with actions related to microtubule targeting and signal transduction, but that these subtle effects were illuminated only through the high information content cell-based screen.
Asunto(s)
Alcanos , Antineoplásicos/farmacología , Carbamatos , Carcinoma/patología , Lactonas/farmacología , Microtúbulos/efectos de los fármacos , Sitios de Unión , Carbono/química , División Celular , Relación Dosis-Respuesta a Droga , Biblioteca de Genes , Células HeLa , Humanos , Microtúbulos/metabolismo , Modelos Químicos , Paclitaxel/farmacología , Fosforilación , Pironas , Transducción de Señal , Huso Acromático , Tubulina (Proteína)/metabolismo , Células Tumorales CultivadasRESUMEN
Drug-drug interactions play an important role in the discovery and development of therapeutic agents. High-content profiling was developed to unravel the complexity of these interactions by providing multiparameter measurements of target activity at the cellular and subcellular levels. Two microtubule drugs, vinblastine and curacin A, were shown to modulate multiple cellular processes, including nuclear condensation, the activation of the extracellular signal-regulated kinase pathway as measured by RSK90 phosphorylation, and the regulation of the microtubule cytoskeleton as measured in detergent-extracted cells. The heterogeneity of the response, addressed through population analysis and multiparameter comparisons within single cells, was consistent with vinblastine and curacin A having similar effects on nuclear morphology and 90 kDa ribosomal s6 kinase (RSK90) phosphorylation despite having distinct effects on the microtubule cytoskeleton. Ketoconazole, originally developed as an antifungal agent, exhibited concentration-dependent inhibitory and potentiating effects on both drugs in HeLa and PC-3 cells at concentration ranges near the plasma levels of ketoconazole attained in human subjects. Thus, high-content profiling was used to dissect the cellular and molecular responses to interacting drugs and is therefore a potentially important tool in the selection, characterization, and optimization of lead therapeutic compounds.
Asunto(s)
Antifúngicos/farmacología , Ciclopropanos/farmacología , Interacciones Farmacológicas , Cetoconazol/farmacología , Tiazoles/farmacología , Vinblastina/farmacología , Animales , Antifúngicos/metabolismo , Antineoplásicos Fitogénicos/metabolismo , Línea Celular Tumoral , Tamaño de la Célula , Ciclopropanos/metabolismo , Humanos , Cetoconazol/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Transducción de Señal , Tiazoles/metabolismo , Vinblastina/metabolismoRESUMEN
Deciphering the effects of compounds on molecular events within living cells is becoming an increasingly important component of drug discovery. In a model application of the industrial drug discovery process, the authors profiled a panel of 22 compounds using hierarchical cluster analysis of multiparameter high-content screening measurements from nearly 500,000 cells per microplate. RNAi protein knockdown methodology was used with high-content screening to dissect the effects of 2 anticancer drugs on multiple target activities. Camptothecin activated p53 in A549 lung carcinoma cells pretreated with scrambled siRNA, exhibited concentration-dependent cell cycle blocks, and induced moderate microtubule stabilization. Knockdown of camptothecin-induced p53 protein expression with p53 siRNA inhibited the G1/S blocking activity of the drug and diminished its microtubule-stabilizing activity. Paclitaxel activated p53 protein at low concentrations but exhibited G2/M cell cycle blocking activity at higher concentrations where microtubules were stabilized. In cells treated with p53 siRNA, paclitaxel failed to activate p53 protein, but the knockdown did not have a significant effect on the ability of paclitaxel to stabilize microtubules or induce a G2/M cell cycle block. Thus, this model application of the use of RNAi technology within the context of high-content screening shows the potential to provide massive amounts of combinatorial cell biological information on the temporal and spatial responses that cells mount to treatment by promising therapeutic candidates.
Asunto(s)
Ensayos de Selección de Medicamentos Antitumorales/métodos , ARN Interferente Pequeño/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Antineoplásicos Fitogénicos/farmacología , Camptotecina/farmacología , Ciclo Celular/efectos de los fármacos , Análisis por Conglomerados , ADN/análisis , Técnica del Anticuerpo Fluorescente , Humanos , Modelos Biológicos , Paclitaxel/farmacología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/efectos de los fármacosRESUMEN
Cell-based target validation, secondary screening, lead optimization, and structure-activity relationships have been recast with the advent of HCS. Prior to HCS, a computational approach to the characterization of the functions of specific target proteins and other cellular constituents, along with whole-cell functions employing fluorescence cell-based assays and microscopy, required extensive interaction among the researcher, instrumentation, and software tools. Early HCS platforms were instrument-centric and addressed the need to interface fully automated fluorescence microscopy, plate-handling automation, and seamless image analysis. HCS has since evolved into an integrated solution for accelerated drug discovery by encompassing the workflow components of assay and reagent design, robust instrumentation for automated fixed-end-point and live cell kinetic analysis, generalized and specific BioApplication software (Cellomics, Pittsburgh, PA) modules that produce information on drug responses from cell image data, and informatics/bioinformatics solutions that build knowledge from this information while providing a means to globalize HCS throughout an entire organization. This review communicates how these recent advances are incorporated into the drug discovery workflow by presenting a real-world use case.
Asunto(s)
Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Tecnología Farmacéutica/métodos , Tecnología Farmacéutica/tendencias , Modelos BiológicosRESUMEN
The integration of high-content screening (HCS) readers with organ-specific cell models, panels of functional biomarkers, and advanced informatics is a powerful approach to identifying the toxic liabilities of compounds early in the development process and forms the basis of "early safety assessment." This cellular systems biology (CSB) approach (CellCiphr profile) has been used to integrate rodent and human cellular hepatic models with panels of functional biomarkers measured at multiple time points to profile both the potency and specificity of the cellular toxicological response. These profiles also provide initial insights on the mechanism of the toxic response. The authors describe here mechanistic assay profiles designed to further dissect the toxic mechanisms of action and elucidate subtle effects apparent in subpopulations of cells. They measured 8 key mechanisms of toxicity with multiple biomarker feature measurements made simultaneously in populations of living primary hepatocytes and HepG2 cells. Mining the cell population response from these mechanistic profiles revealed the concentration dependence and nature of the heterogeneity of the response, as well as relationships between the functional responses. These more detailed mechanistic profiles define differences in compound activities that are not apparent in the average population response. Because cells and tissues encounter wide ranges of drug doses in space and time, these mechanistic profiles build on the CellCiphr profile and better reflect the complexity of the response in vivo.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Bibliotecas de Moléculas Pequeñas/toxicidad , Biología de Sistemas/métodos , Animales , Bioensayo , Biomarcadores/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Hep G2 , Hepatocitos/citología , Humanos , Lisosomas/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/análisis , Factores de TiempoRESUMEN
We present here the characterization and optimization of a novel imaging-based positional biosensor high-content screening (HCS) assay to identify disruptors of p53-hDM2 protein-protein interactions (PPIs). The chimeric proteins of the biosensor incorporated the N-terminal PPI domains of p53 and hDM2, protein targeting sequences (nuclear localization and nuclear export sequence), and fluorescent reporters, which when expressed in cells could be used to monitor p53-hDM2 PPIs through changes in the subcellular localization of the hDM2 component of the biosensor. Coinfection with the recombinant adenovirus biosensors was used to express the NH-terminal domains of p53 and hDM2, fused to green fluorescent protein and red fluorescent protein, respectively, in U-2 OS cells. We validated the p53-hDM2 PPI biosensor (PPIB) HCS assay with Nutlin-3, a compound that occupies the hydrophobic pocket on the surface of the N-terminus of hDM2 and blocks the binding interactions with the N-terminus of p53. Nutlin-3 disrupted the p53-hDM2 PPIB in a concentration-dependent manner and provided a robust, reproducible, and stable assay signal window that was compatible with HCS. The p53-hDM2 PPIB assay was readily implemented in HCS and we identified four (4) compounds in the 1,280-compound Library of Pharmacologically Active Compounds that activated the p53 signaling pathway and elicited biosensor signals that were clearly distinct from the responses of inactive compounds. Anthracycline (topoisomerase II inhibitors such as mitoxantrone and ellipticine) and camptothecin (topoisomerase I inhibitor) derivatives including topotecan induce DNA double strand breaks, which activate the p53 pathway through the ataxia telangiectasia mutated-checkpoint kinase 2 (ATM-CHK2) DNA damage response pathway. Although mitoxantrone, ellipticine, camptothecin, and topotecan all exhibited concentration-dependent disruption of the p53-hDM2 PPIB, they were much less potent than Nutlin-3. Further, their corresponding cellular images and quantitative HCS data did not completely match the Nutlin-3 phenotypic profile.
Asunto(s)
Antineoplásicos/farmacología , Técnicas Biosensibles , Ensayos Analíticos de Alto Rendimiento , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Camptotecina/farmacología , Línea Celular Tumoral , Quinasa de Punto de Control 2 , Elipticinas/farmacología , Genes p53 , Humanos , Imidazoles/farmacología , Mitoxantrona/farmacología , Piperazinas/farmacología , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Bibliotecas de Moléculas Pequeñas , Espectrometría de Fluorescencia , Topotecan/farmacologíaRESUMEN
In recent years, advances in structure-based drug design and the development of an impressive variety of high-throughput screening (HTS) assay formats have yielded an expanding list of protein-protein interaction inhibitors. Despite these advances, protein-protein interaction targets are still widely considered difficult to disrupt with small molecules. The authors present here the results from screening 220,017 compounds from the National Institute of Health's small-molecule library in a novel p53-hDM2 protein-protein interaction biosensor (PPIB) assay. The p53-hDM2 positional biosensor performed robustly and reproducibly throughout the high-content screening (HCS) campaign, and analysis of the multiparameter data from images of the 3 fluorescent channels enabled the authors to identify and eliminate compounds that were cytotoxic or fluorescent artifacts. The HCS campaign yielded 3 structurally related methylbenzo-naphthyridin-5-amine (MBNA) hits with IC(50)s between 30 and 50 microM in the p53-hDM2 PPIB. In HCT116 cells with wild-type (WT) p53, the MBNAs enhanced p53 protein levels, increased the expression of p53 target genes, caused a cell cycle arrest in G1, induced apoptosis, and inhibited cell proliferation with an IC(50) ~4 microM. The prototype disruptor of p53-hDM2 interactions Nutlin-3 was more potent than the MBNAs in the p53-hDM2 PPIB assay but produced equivalent biological results in HCT116 cells WT for p53. Unlike Nutlin-3, however, MBNAs also increased the percentage of apoptosis in p53 null cells and exhibited similar potencies for growth inhibition in isogenic cell lines null for p53 or p21. Neither the MBNAs nor Nutin-3 caused cell cycle arrest in p53 null HCT116 cells. Despite the relatively modest size of the screening library, the combination of a novel p53-hDM2 PPIB assay together with an automated imaging HCS platform and image analysis methods enabled the discovery of a novel chemotype series that disrupts p53-hDM2 interactions in cells.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/efectos de los fármacos , Técnicas Biosensibles , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Daño del ADN , Humanos , Naftiridinas/farmacología , Unión Proteica/efectos de los fármacos , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Building cellular models of disease based on the approach of Cellular Systems Biology (CSB) has the potential to improve the process of creating drugs as part of the continuum from early drug discovery through drug development and clinical trials and diagnostics. This paper focuses on the application of CSB to early drug discovery. We discuss the integration of protein-protein interaction biosensors with other multiplexed, functional biomarkers as an example in using CSB to optimize the identification of quality lead series compounds.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Modelos Biológicos , Neoplasias , Biología de Sistemas/métodos , Biomarcadores de Tumor/análisis , Descubrimiento de Drogas , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Mitogen-activated protein kinase phosphatase-1 (MKP-1) is a dual specificity phosphatase that is overexpressed in many human tumors and can protect cells from apoptosis caused by DNA-damaging agents or cellular stress. Small molecule inhibitors of MKP-1 have not been reported, in part because of the lack of structural guidance for inhibitor design and definitive assays for MKP-1 inhibition in intact cells. Herein we have exploited a high content chemical complementation assay to analyze a diverse collection of pure natural products for cellular MKP-1 inhibition. Using two-dimensional Kolmogorov-Smirnov statistics, we identified sanguinarine, a plant alkaloid with known antibiotic and antitumor activity but no primary cellular target, as a potent and selective inhibitor of MKP-1. Sanguinarine inhibited cellular MKP-1 with an IC50 of 10 microM and showed selectivity for MKP-1 over MKP-3. Sanguinarine also inhibited MKP-1 and the MKP-1 like phosphatase, MKP-L, in vitro with IC50 values of 17.3 and 12.5 microM, respectively, and showed 5-10-fold selectivity for MKP-3 and MKP-1 over VH-1-related phosphatase, Cdc25B2, or protein-tyrosine phosphatase 1B. In a human tumor cell line with high MKP-1 levels, sanguinarine caused enhanced ERK and JNK/SAPK phosphorylation. A close congener of sanguinarine, chelerythrine, also inhibited MKP-1 in vitro and in whole cells, and activated ERK and JNK/SAPK. In contrast, sanguinarine analogs lacking the benzophenanthridine scaffold did not inhibit MKP-1 in vitro or in cells nor did they cause ERK or JNK/SAPK phosphorylation. These data illustrate the utility of a chemical complementation assay linked with multiparameter high content cellular screening.
Asunto(s)
Alcaloides/química , Alcaloides/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/química , Inhibidores Enzimáticos/farmacología , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Proteínas Inmediatas-Precoces/química , Neoplasias/tratamiento farmacológico , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/química , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/química , Benzofenantridinas , Dominio Catalítico , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Daño del ADN , Relación Dosis-Respuesta a Droga , Fosfatasa 1 de Especificidad Dual , Fosfatasa 6 de Especificidad Dual , Electroforesis en Gel Bidimensional , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Biblioteca de Genes , Células HeLa , Humanos , Concentración 50 Inhibidora , Isoquinolinas , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 4 , Microscopía Fluorescente , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Químicos , Modelos Estadísticos , Fosforilación , Extractos Vegetales/farmacología , Proteína Fosfatasa 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas/metabolismo , Factores de Tiempo , Transfección , Fosfatasas cdc25/metabolismoRESUMEN
(-)-Dictyostatin is a sponge-derived, 22-member macrolactone natural product shown to cause cells to accumulate in the G2/M phase of the cell cycle, with changes in intracellular microtubules analogous to those observed with paclitaxel treatment. Dictyostatin also induces assembly of purified tubulin more rapidly than does paclitaxel, and nearly as vigorously as does dictyostatin's close structural congener, (+)-discodermolide (Isbrucker et al. (2003), Biochem. Pharmacol. 65, 75-82). We used synthetic (-)-dictyostatin to study its biochemical and cytological activities in greater detail. The antiproliferative activity of dictyostatin did not differ greatly from that of paclitaxel or discodermolide. Like discodermolide, dictyostatin retained antiproliferative activity against human ovarian carcinoma cells resistant to paclitaxel due to beta-tubulin mutations and caused conversion of cellular soluble tubulin pools to microtubules. Detailed comparison of the abilities of dictyostatin and discodermolide to induce tubulin assembly demonstrated that the compounds had similar potencies. Dictyostatin inhibited the binding of radiolabeled discodermolide to microtubules more potently than any other compound examined, and dictyostatin and discodermolide had equivalent activity as inhibitors of the binding of both radiolabeled epothilone B and paclitaxel to microtubules. These results are consistent with the idea that the macrocyclic structure of dictyostatin represents the template for the bioactive conformation of discodermolide.