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Paul A Johnston

Publications and source records attributed to Paul A Johnston.

8 recordsLinked to original sources
PubMed2007

Requirements, features, and performance of high content screening platforms.

High content screening (HCS) platforms integrate fluorescence microscopy with image analysis algorithms and informatics to automate cell analysis. The initial applications of HCS to secondary screening in drug discovery have spread throughout the discovery pipeline, and now into the expanding research field of systems cell biology, in which new manipulation tools enable the use of large scale screens to understand cellular pathways, and cell functions. In this chapter we discuss the requirements for HCS and the systems that have been designed to meet these application needs. The number of HCS systems available in the market place, and the range of features available, has grown considerably in the past 2 yr. Of the two general optical designs, the confocal systems have dominated the high-throughput HCS market, whereas the more cost effective wide-field systems have dominated all other market segments, and have a much larger market share. The majority of available systems have been optimized for fixed cell applications; however, there is growing interest in live cell kinetic assays, and four systems have successfully penetrated this application area. The breadth of applications for these systems continues to expand, especially with the integration of new technologies. New applications, improved software, better data visualization tools, and new detection methods such as multispectral imaging and fluorescence lifetime are predicted to drive the development of future HCS platforms.

Animals↗
PubMed2006

Generation and characterization of a stable MK2-EGFP cell line and subsequent development of a high-content imaging assay on the Cellomics ArrayScan platform to screen for p38 mitogen-activated protein kinase inhibitors.

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.

Automation↗
PubMed2006

Development and implementation of three mitogen-activated protein kinase (MAPK) signaling pathway imaging assays to provide MAPK module selectivity profiling for kinase inhibitors: MK2-EGFP translocation, c-Jun, and ERK activation.

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.

Biochemistry↗
PubMed2006

Assay development and case history of a 32K-biased library high-content MK2-EGFP translocation screen to identify p38 mitogen-activated protein kinase inhibitors on the ArrayScan 3.1 imaging platform.

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.

Biological Assay↗
PubMed2005

Secretion of proinflammatory cytokines and chemokines during amphotericin B exposure is mediated by coactivation of toll-like receptors 1 and 2.

Amphotericin B (AmB) is a ligand of toll-like receptor 2 (TLR2). Here, we demonstrate the participation of TLR1 in AmB-induced cell activation that led to the secretion of tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-8. Hence, TLR2-TLR1 coactivation serves as the underlying mechanism for the proinflammatory toxicities associated with AmB.

Amphotericin B↗
PubMed2005

The multidrug resistance protein 5 (ABCC5) confers resistance to 5-fluorouracil and transports its monophosphorylated metabolites.

5'-Fluorouracil (5-FU), used in the treatment of colon and breast cancers, is converted intracellularly to 5'-fluoro-2'-deoxyuridine (5-FUdR) by thymidine phosphorylase and is subsequently phosphorylated by thymidine kinase to 5'-fluoro-2'-dUMP (5-FdUMP). This active metabolite, along with the reduced folate cofactor, 5,10-methylenetetrahydrofolate, forms a stable inhibitory complex with thymidylate synthase that blocks cellular growth. The present study shows that the ATP-dependent multidrug resistance protein-5 (MRP5, ABCC5) confers resistance to 5-FU by transporting the monophosphate metabolites. MRP5- and vector-transfected human embryonic kidney (HEK) cells were employed in these studies. In 3-day cytotoxicity assays, MRP5-transfected cells were approximately 9-fold resistant to 5-FU and 6-thioguanine. Studies with inside-out membrane vesicles prepared from transfected cells showed that MRP5 mediates ATP-dependent transport of 5 micromol/L [(3)H]5-FdUMP, [(3)H]5-FUMP, [(3)H]dUMP, and not [(3)H]5-FUdR, or [(3)H]5-FU. The ATP-dependent transport of 5-FdUMP showed saturation with increasing concentrations and had a K(m) of 1.1 mmol/L and V(max) of 439 pmol/min/mg protein. Uptake of 250 micromol/L 5-FdUMP was inhibited by dUMP, cyclic nucleotide, cyclic guanosine 3',5'-monophosphate, amphiphilic anions such as probenecid, MK571, the phosphodiesterase inhibitors, trequinsin, zaprinast, and sildenafil, and by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid and glybenclamide. Furthermore, the 5-FU drug sensitivity of HEK-MRP5 cells was partially modulated to that of the HEK-vector by the presence of 40 micromol/L 5-nitro-2-(3-phenylpropylamino)-benzoic acid but not by 2 mmol/L probenecid. Thus, MRP5 transports the monophosphorylated metabolite of this nucleoside and when MRP5 is overexpressed in colorectal and breast tumors, it may contribute to 5-FU drug resistance.

Adenosine Triphosphate↗
PubMed2002

Cellular platforms for HTS: three case studies.

The field of cell-based screening is expanding rapidly as innovations in target selection and instrumentation increase the number of targets that can be efficiently screened in cellular formats. Cell-based screens can be configured to provide a broad range of data on chemical compound activity, mechanism of action and drugability. However, the decision to pursue a cell-based approach should not be made lightly, as cell-based assays can be challenging to implement in the high-throughput screening (HTS) laboratory. In this review, we describe three case studies in which targets were successfully interrogated in cell-based HTS, and highlight the necessary steps to ensure the validity of these screens.

Animals↗