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Family Based Mental Health Services (FBMHS) with an embedded clinical model, Ecosystemic Structural Family Therapy, is an intervention designed for youth with a serious emotional disturbance (SED) who are at risk of out-of-home placement. The current evaluation examines the association between receipt of FBMHS and rates of out-of-home and community-based care during and after an episode of FBMHS. We identified 25,016 Medicaid-enrolled youth ages 3 to 17 years with receipt of a new FBMHS episode from 1/1/2015 to 6/30/2021. 14% of youth received out-of-home services. Rates of out-of-home service decreased during receipt of FBMHS (14.25-6.98%, p < .0001) and remained lower 6 months following discharge (to 6.95%, p < .0001). Short and longer doses of service were both associated with decreased rates of out-of-home services. FBMHS has been scaled across a large geographic area and is associated with lower rates of out-of-home placement for youth with SED.
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Terapia Familiar , Medicaid , Readmisión del Paciente , Humanos , Adolescente , Femenino , Masculino , Niño , Preescolar , Readmisión del Paciente/estadística & datos numéricos , Terapia Familiar/métodos , Estados Unidos , Servicios Comunitarios de Salud Mental , Síntomas Afectivos/terapia , Síntomas Afectivos/psicologíaRESUMEN
Plant-based diets, defined here as including both vegan and lacto-ovo-vegetarian diets, are growing in popularity throughout the Western world for various reasons, including concerns for human health and the health of the planet. Plant-based diets are more environmentally sustainable than meat-based diets and have a reduced environmental impact, including producing lower levels of greenhouse gas emissions. Dietary guidelines are normally formulated to enhance the health of society, reduce the risk of chronic diseases, and prevent nutritional deficiencies. We reviewed the scientific data on plant-based diets to summarize their preventative and therapeutic role in cardiovascular disease, cancer, diabetes, obesity, and osteoporosis. Consuming plant-based diets is safe and effective for all stages of the life cycle, from pregnancy and lactation, to childhood, to old age. Plant-based diets, which are high in fiber and polyphenolics, are also associated with a diverse gut microbiota, producing metabolites that have anti-inflammatory functions that may help manage disease processes. Concerns about the adequate intake of a number of nutrients, including vitamin B12, calcium, vitamin D, iron, zinc, and omega-3 fats, are discussed. The use of fortified foods and/or supplements as well as appropriate food choices are outlined for each nutrient. Finally, guidelines are suggested for health professionals working with clients consuming plant-based diets.
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Enfermedad Crónica/prevención & control , Dieta Vegana/normas , Dieta Vegetariana/normas , Dietética/normas , Política Nutricional , Adolescente , Adulto , Anciano , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Guías de Práctica Clínica como Asunto , Embarazo , Adulto JovenRESUMEN
To plan for a proactive approach to support patients traveling for their treatment during the COVID-19 pandemic, a network of oncology hospitals worked within existing collaborative agreements to define policies and procedures to transition care for patients living in communities in close proximity to a member institution. Nurse leaders were instrumental in collaborating with and leading interprofessional partners to achieve these outcomes. These efforts led to patients' abilities to continue treatment in their local community, ensuring continuity of cancer care.
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Infecciones por Coronavirus/epidemiología , Atención a la Salud/organización & administración , Neoplasias/terapia , Pandemias , Neumonía Viral/epidemiología , COVID-19 , Continuidad de la Atención al Paciente/organización & administración , Conducta Cooperativa , Práctica Clínica Basada en la Evidencia , Humanos , Enfermería OncológicaRESUMEN
Membership in professional organizations is a way for professionals with similar objectives to join together to have the voice of many, versus the voice of one. Joining an organization can lead to personal growth, enhanced professional prospective and knowledge development, and support for creating change.
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Personal de Enfermería , Sociedades , Humanos , Relaciones Interprofesionales , Estudios ProspectivosRESUMEN
BACKGROUND: The management of patients with cancer and concurrent heart failure (HF) is challenging. The increased complexity of treatment and the occurrence of multiple overlapping symptoms may lead to frequent hospital admissions, which may result in cancer treatment delays, a diminished quality of life, and an increased financial burden for the patient's family. To provide holistic care to oncology patients with HF, we implemented the Heart Success Program (HSP), a patient-centered, interprofessional collaborative practice, which decreased the 30-day hospital readmission rate for HF diagnosis from 40 to 27%. However, this rate remains higher than that reported for Medicare beneficiaries. AIM: To identify the factors contributing to frequent readmissions, the HSP committee participated in the institution's Clinical Safety and Effectiveness and utilize quality improvement methodologies and tools to decrease hospital readmission for HF. METHODS: The DMAIC (Define, Measure, Analyze, Improve and Control) method was used to guide this quality improvement. Areas considered as having high impact and requiring low effort to address were patient education barriers, lack of documentation clarity, and care provider knowledge gaps about the HSP. We implemented workflow changes, improved clarity with documentation of HF diagnosis, and increase provider knowledge about the HSP. FINDINGS: After 6 months of implementing quality improvement techniques, the 30-day hospital readmission rate for HF patients fell by 23.43% (from 31.7% for the baseline period to 8.27%), exceeding the target project goal of 10%. Our quality improvement method may also be effective in improving the management of patients with cancer and other comorbid conditions.
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BACKGROUND: Emerging immunotherapies are associated with numerous toxicities. Although traditional health records allow nurses to document system-based assessments, few offer immunotherapy-based documentation templates to assess and grade toxicities.â©. OBJECTIVES: The aim of this article is to present the development of a standardized template for documenting genetically modified cellular product-related toxicities in an electronic health record (EHR).â©. METHODS: Through interprofessional collaboration, a documentation template for genetically modified cellular product-related toxicities was developed in an EHR, allowing for standardized documentation, data reporting, and tracking of immune-related toxicities. â©. FINDINGS: The documentation template has enhanced the quality and safety of practice at the authors' institution and provides a framework for other nursing units when initiating immunotherapy care.â©.
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Tratamiento Basado en Trasplante de Células y Tejidos/efectos adversos , Documentación/normas , Registros Electrónicos de Salud/normas , Inmunoterapia , Neoplasias/inmunología , Neoplasias/terapia , Atención al Paciente/normas , Adulto , Anciano , Anciano de 80 o más Años , Educación Continua en Enfermería/organización & administración , Femenino , Humanos , Masculino , Persona de Mediana Edad , Registros de Enfermería/normasRESUMEN
An interprofessional team developed a symptom word documentation tool based on surveillance definitions for specific types of infections published by the Centers for Disease Control and Prevention. Nursing documentation was monitored pre- and postimplementation of the tool, revealing increased compliance with symptom documentation. Although symptom word documentation does not reduce the number of infections, it may impact the central line-associated bloodstream infection (CLABSI) rate by better differentiating CLABSI from other infection sources, as was observed in this institution.
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Infección Hospitalaria/diagnóstico , Infección Hospitalaria/patología , Manejo de la Enfermedad , Métodos Epidemiológicos , Técnicas de Apoyo para la Decisión , HumanosRESUMEN
This quality improvement project evaluated the accuracy of harm scores entered into an event reporting system by inpatient nursing staff at a National Cancer Institute-designated comprehensive cancer center. Nurses scored 10 safety scenarios using 2 versions of the Agency for Healthcare Research and Quality scale to determine interrater reliability. Results indicated inconsistency in the way nurses scored the scenarios, suggesting that the event reporting system may not accurately portray the severity of harm in patient safety events. Nurse executives can use this information to guide the development and implementation of incident reporting systems.
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Reducción del Daño , Errores Médicos/prevención & control , Seguridad del Paciente , Mejoramiento de la Calidad , Adolescente , Adulto , Femenino , Humanos , Liderazgo , Masculino , Errores Médicos/estadística & datos numéricos , Persona de Mediana Edad , Neoplasias/terapia , Reproducibilidad de los ResultadosRESUMEN
Chemotherapy administration in the home setting poses risks to patients, caregivers, and the environment, particularly in the event of spills. Although the response to chemotherapy spills in the hospital setting is vigorous and includes standard disposal practices for contaminated items, the management of spills in the home setting may vary. A standardized method for managing chemotherapy spills at home that includes education and distribution of spill cleanup materials is imperative to reduce these risks.
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Antineoplásicos/uso terapéutico , Relaciones Interprofesionales , Eliminación de Residuos Sanitarios , Neoplasias/tratamiento farmacológico , Guías de Práctica Clínica como Asunto , Administración de la Seguridad , Antineoplásicos/efectos adversos , Educación Continua en Enfermería , Humanos , Neoplasias/enfermería , Educación del Paciente como AsuntoRESUMEN
PURPOSE: The purpose of this study was to examine the impact of a consumer-led equipment and device program [Equipment and Assistive Technology Initiative (EATI) in British Columbia, Canada] from the perspective of program participants. The importance of collaborative assessments for obtaining the right assistive technology (AT) for meeting an individual's needs is discussed in light of the program's participant-centered "Participation Model", or philosophy by which the program is structured. METHOD: A cross-sectional survey with participants and semi-structured interviews were conducted with participants (≥ 18 years) who held a range of disabilities. The survey asked participants to rank their AT and to identify the method by which they obtained the technology [by self, prescribed by a health professional or collaborative (self and professional)]. Interviews addressed participants' opinions about obtaining and using AT. RESULTS: In total, 357 people responded to the survey (17% response rate) and 16 people participated in the interviews. The highest ranking AT was assigned to devices assessed via a collaborative method (self = 31%, practitioner = 26%, collaborative = 43%; χ(2) (16,180) = 39.604, p < 0.001). CONCLUSIONS: Shared decision-making between health professionals and people with disabilities within the assessment process for assistive technology leads to what participants perceive as the right AT. IMPLICATIONS FOR REHABILITATION: Collaborative decision-making can lead to the selection of assistive technology that is considered needed and right for the individual. Person-centered philosophy associated with assistive technology assessment is contributing to attaining "the right" AT.
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Conducta Cooperativa , Toma de Decisiones , Personas con Discapacidad/rehabilitación , Relaciones Profesional-Paciente , Dispositivos de Autoayuda , Adolescente , Adulto , Anciano , Colombia Británica , Estudios Transversales , Femenino , Humanos , Entrevistas como Asunto , Masculino , Persona de Mediana Edad , Evaluación de NecesidadesRESUMEN
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.
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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
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.
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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.
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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
Registered nurse (RN) job satisfaction is a major predictor of intent to stay and job turnover, serious concerns to health care leaders. Predictors of job satisfaction include autonomy, control over daily practice, nurse-physician collaboration, transformational leadership, group cohesion, job stress, structural empowerment, and psychological empowerment. In the model of psychological empowerment, stress resiliency is the product of persons' interpretive styles and influences psychological empowerment. This study has evaluated the influence of stress resiliency on job stress, psychological empowerment, job satisfaction, and intent to stay using causal modeling. Participants are 464 RNs employed in five acute care hospitals in West Virginia. The final model has provided a very good fit to the data. Stress resiliency is a predictor of psychological empowerment, situational stress, and job satisfaction. This study provides the first evidence of the influence of stress resiliency on job stress, psychological empowerment, job satisfaction, and intent to stay in a sample of RNs.
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Agotamiento Profesional/psicología , Intención , Satisfacción en el Trabajo , Personal de Enfermería en Hospital/psicología , Reorganización del Personal , Resiliencia Psicológica , Adulto , Análisis de Varianza , Actitud del Personal de Salud , Agotamiento Profesional/diagnóstico , Agotamiento Profesional/epidemiología , Agotamiento Profesional/prevención & control , Distribución de Chi-Cuadrado , Femenino , Humanos , Relaciones Interprofesionales , Liderazgo , Masculino , Modelos Psicológicos , Rol de la Enfermera/psicología , Investigación Metodológica en Enfermería , Personal de Enfermería en Hospital/educación , Personal de Enfermería en Hospital/organización & administración , Reorganización del Personal/estadística & datos numéricos , Poder Psicológico , Autonomía Profesional , Análisis de Regresión , Encuestas y Cuestionarios , West Virginia/epidemiologíaRESUMEN
The authors review the literature and describe the potential impact of electronic medical records on nursing efficiency, as measured by a reduction in the amount of time nurses spend on activities that are not part of direct patient care. They also identify factors leading to success or failure in achieving electronic medical record-enabled nursing efficiency and illustrate their impact using a case study.
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Eficiencia , Sistemas de Registros Médicos Computarizados , Modelos Organizacionales , Estudios de Casos Organizacionales , TexasRESUMEN
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.
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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
The integration of fluorescent microscopy imaging technologies and image analysis into high-content screening (HCS) has been applied throughout the drug discovery pipeline to identify, evaluate, and advance compounds from early lead generation through preclinical candidate selection. In this chapter we describe the development, validation, and implementation of an HCS assay to screen compounds from a kinase-focused small-molecule library to identify inhibitors of the p38 pathway using the GE InCell 3000 automated imaging platform. The assay utilized a genetically modified HeLa cell line stably expressing mitogen-activated, protein-activating protein kinase-2 fused to enhanced green fluorescent protein (MK2-EGFP) and measured the subcellular distribution of the MK2-EGFP as a direct readout of p38 activation. The MK2-EGFP translocation assay performed in 384-well glass bottom microtiter plates exhibited a robust Z-factor of 0.46 and reproducible EC50 and IC50 determinations for activators and inhibitors, respectively. A total of 32,891 compounds were screened in singlicate at 50 microM and 156 were confirmed as inhibitors of p38-mediated MK2-EGFP translocation in follow-up IC50 concentration response curves. Thirty-one compounds exhibited IC50s less than 1 microM, and at least one novel structural class of p38 inhibitor was identified using this HCA/HCS chemical biology screening approach.
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Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/análisis , Microscopía Confocal/métodos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Calcio/metabolismo , Células HeLa , HumanosRESUMEN
This chapter describes the generation and characterization of a stable MK2-EGFP expressing HeLa cell line and the subsequent development of a high-content imaging assay on the Cellomics ArrayScan platform to screen for p38 MAPK inhibitors. Mitogen-activated protein kinase activating protein kinase-2 (MK2) is a substrate of p38 MAPK kinase, and p38-induced phosphorylation of MK-2 induces a nucleus to cytoplasm translocation (Engel et al., 1998; Neininger et al., 2001; Zu et al., 1995). Through a process of heterologous expression of a MK2-EGFP fusion protein in HeLa cells using retroviral infection, antibiotic selection, and flow sorting, we were able to isolate a cell line in which the MK2-EGFP translocation response could be robustly quantified on the Cellomics ArrayScan platform using the nuclear translocation algorithm. A series of assay development experiments using the A4-MK2-EGFP-HeLa cell line are described to optimize the assay with respect to cell seeding density, length of anisomycin stimulation, dimethyl sulfoxide tolerance, assay signal window, and reproducibility. The resulting MK2-EGFP translocation assay is compatible with high-throughput screening and was shown to be capable of identifying p38 inhibitors. The MK2-EGF translocation response is susceptible to other classes of inhibitors, including nonselective kinase inhibitors, kinase inhibitors that inhibit upstream kinases in the p38 MAPK signaling pathway, and kinases involved in cross talk between different modules (ERKs, JNKs, and p38s) of the MAPK signaling pathways. An example of mining "high-content" image-based multiparameter data to extract additional information on the effects of compound treatment of cells is presented.
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Bioensayo/instrumentación , Bioensayo/métodos , Técnicas de Cultivo de Célula/métodos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Automatización , Línea Celular , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Sistema de Señalización de MAP Quinasas , Microscopía Fluorescente , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Proteínas Serina-Treonina Quinasas , Reproducibilidad de los Resultados , Transducción de SeñalRESUMEN
This chapter describes the development and implementation of three independent imaging assays for the major mitogen-activated protein kinase (MAPK) signaling modules: p38, JNK, and ERK. There are more than 500 protein kinases encoded in the human genome that share an ATP-binding site and catalytic domain conserved in both sequence and structure. The majority of kinase inhibitors have been found to be competitive with ATP, raising concerns regarding kinase selectivity and potency in an environment of millimolar intracellular concentrations of ATP, as well as the potential for off-target effects via the many other cellular proteins that bind and/or utilize ATP. The apparent redundancy of the kinase isoforms and functions in the MAPK signaling modules present additional challenges for kinase inhibitor selectivity and potency. Imaging assays provide a method to address many of these concerns. Cellular imaging approaches facilitate analysis of the targets expressed in the context of their endogenous substrates and scaffolding proteins and in a complex environment for which subcellular localization, cross talk between pathways, phosphatase regulatory control, and intracellular ATP concentrations are relevant to the functions of the kinase. The assays described herein provide a strategy to profile kinase inhibitors for MAPK pathway selectivity while simultaneously providing information on cell morphology or toxicity. Results suggest that the MAPK pathways are indeed susceptible to nonselective kinase inhibitors such as staurosporin and inhibitors that inhibit upstream MAPK Kinase Kinases (MKKKs) and MAPK Kinases (MKKs) in the MAPK signaling pathway, especially those involved in cross talk between the pathways. However, selective MAPK inhibitors were identified that exhibited pathway selectivity as evidenced by significantly lower IC(50) values for their respective p38, JNK, or ERK signaling pathway assays.
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Bioquímica/métodos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Dominio Catalítico , Diferenciación Celular , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Isoformas de Proteínas , Proteínas Serina-Treonina Quinasas , Transporte de Proteínas , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
This chapter describes the conversion and assay development of a 96-well MK2-EGFP translocation assay into a higher density 384-well format high-content assay to be screened on the ArrayScan 3.1 imaging platform. The assay takes advantage of the well-substantiated hypothesis that mitogen-activated protein kinase-activating protein kinase-2 (MK2) is a substrate of p38 MAPK kinase and that p38-induced phosphorylation of MK-2 induces a nucleus-to-cytoplasm translocation. This chapter also presents a case history of the performance of the MK2-EGFP translocation assay, run as a "high-content" screen of a 32K kinase-biased library to identify p38 inhibitors. The assay performed very well and a number of putative p38 inhibitor hits were identified. Through the use of multiparameter data provided by the nuclear translocation algorithm and by checking images, a number of compounds were identified that were potential artifacts due to interference with the imaging format. These included fluorescent compounds, or compounds that dramatically reduced cell numbers due to cytotoxicity or by disrupting cell adherence. A total of 145 compounds produced IC(50) values <50.0 muM in the MK2-EGFP translocation assay, and a cross target query of the Lilly-RTP HTS database confirmed their inhibitory activity against in vitro kinase targets, including p38a. Compounds were confirmed structurally by LCMS analysis and profiled in cell-based imaging assays for MAPK signaling pathway selectivity. Three of the hit scaffolds identified in the MK2-EGFP translocation HCS run on the ArrayScan were selected for a p38a inhibitor hit-to-lead structure activity relationship (SAR) chemistry effort.