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1.  A High-throughput 1536-well Luminescence Assay for Glutathione S-Transferase Activity 
Glutathione S-transferases (GSTs) constitute a family of detoxification enzymes that catalyze the conjugation of glutathione with a variety of hydrophobic compounds, including drugs and their metabolites, to yield water soluble derivatives which are excreted in urine or bile. Profiling the effect of small molecules on GST activity is an important component in the characterization of drug candidates and compound libraries. Additionally, specific GST isozymes have been implicated in drug resistance, especially in cancer, and thus represent potential targets for intervention. To date, there are no sensitive miniaturized high-throughput assays available for GST activity detection. A series of GST substrates containing a masked luciferin moiety have been described recently, offering the potential for configuring a sensitive screening assay via coupled luciferase reaction and standard luminescence detection. We report on the optimization and miniaturization of this homogeneous method to 1,536-well format using GSTs from three different species: mouse isozyme A4-4, human isozymes A1-1, M1-1, and P1-1, and the major GST from the parasitic worm Schistosoma japonicum.
PMCID: PMC2864799  PMID: 20085484
2.  A Multiplex Calcium Assay for Identification of GPCR Agonists and Antagonists 
Activation of Gq protein-coupled receptors can be monitored by measuring the increase in intracellular calcium with fluorescent dyes. Recent advances in fluorescent kinetic plate readers and liquid-handling technology have made it possible to follow these transient changes in intracellular calcium in a 1536-well plate format for high-throughput screening. Here, we have applied the latest generation of fluorescence kinetic plate readers to multiplex the agonist and antagonist screens of a GPCR. This multiplexed assay format provides an efficient and cost-effective method for high-throughput screening of Gq-coupled GPCR targets.
PMCID: PMC2893246  PMID: 20230302
GPCR; calcium assay; FDSS; multiplex; high-throughput screening
3.  Quantification of lipid droplets and associated proteins in cellular models of obesity via high content/high throughput microscopy and automated image analysis 
Intracellular lipid droplets are associated with a myriad of afflictions including obesity, fatty liver disease, coronary artery disease and infectious diseases (e.g., HCV and tuberculosis). To develop high content assay (HCA) techniques to analyze lipid droplets and associated proteins, primary human pre-adipocytes, were plated in 96-well dishes in the presence of rosiglitazone (rosi), a PPARγ agonist which promotes adipogenesis. The cells were then labeled for nuclei, lipid droplets, and proteins such as perilipin, protein kinase C (PKC), and hormone sensitive lipase (HSL). The cells were imaged via automated digital microscopy and algorithms were developed to quantify lipid droplet (Lipid Droplet algorithm) and protein expression and colocalization (Colocalization algorithm). The algorithms, which were incorporated into Vala Science Inc’s CyteSeer® image cytometry program, quantified the rosi-induced increases in lipid droplet number, size, and intensity, and the expression of perilipin with exceptional consistency (Z’ values of 0.54 to 0.71). Regarding colocalization with lipid droplets, Pearson’s Correlation coefficients of 0.38 (highly colocalized), 0.16 (moderate), and − 0.0010 (random) were found for perilipin, PKC, and HSL, respectively. For hepatocytes (AML12, Huh7, and primary cells), the algorithms also quantified the stimulatory and inhibitory effect of oleic acid and triacsin c on lipid droplets (Z’s > 0.50) and ADFP expression/colocalization. Oleic-acid induced lipid droplets in HeLa cells and macrophages (THP-1) were also well quantified. The results suggest that HCA techniques can be utilized to quantify lipid droplets and associated proteins in many cell models relevant to a variety of diseases.
PMCID: PMC2872546  PMID: 19895345
4.  A dual-fluorescence high-throughput cell line system for probing multidrug resistance 
The efflux pump P-glycoprotein (ABCB1, MDR1, P-gp) has long been known to contribute to multidrug resistance (MDR) against cancer chemotherapeutics. We describe the development of a dual-fluorescent cell line system to allow multiplexing of drug-sensitive and P-gp-mediated MDR cell lines. The parental OVCAR-8 human ovarian carcinoma cell line and the isogenic MDR NCI/ADR-RES subline, which stably expresses high levels of endogenous P-gp, were transfected to express the fluorescent proteins DsRed2 and EGFP respectively. Co-culture conditions were defined and fluorescent barcoding of each cell line allowed for the direct, simultaneous comparison of resistance to cytotoxic compounds in sensitive and MDR cell lines. We show that this assay system retains the phenotypes of the original lines and is suitable for multiplexing using confocal microscopy, flow cytometry or laser-scanning microplate cytometry in 1,536-well plates, enabling the high-throughput screening (HTS) of large chemical libraries.
PMCID: PMC2814070  PMID: 19548831
5.  Development of a 384-Well Colorimetric Assay to Quantify Hydrogen Peroxide Generated by the Redox Cycling of Compounds in the Presence of Reducing Agents 
We report here the development and optimization of a simple 384-well colorimetric assay to measure H2O2 generated by the redox cycling of compounds incubated with reducing agents in high-throughput screening (HTS) assay buffers. The phenol red-horseradish peroxidase (HRP) assay readily detected H2O2 either added exogenously or generated by the redox cycling of compounds in dithiothreitol (DTT). The generation of H2O2 was dependent on the concentration of both the compound and DTT and was abolished by catalase. Although both DTT and tris(2-carboxyethyl)-phosphine sustain the redox cycling generation of H2O2 by a model quinolinedione, 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione (NSC 663284; DA3003-1), other reducing agents such as β-mercaptoethanol, glutathione, and cysteine do not. The assay is compatible with HTS. Once terminated, the assay signal was stable for at least 5 h, allowing for a reasonable throughput. The assay tolerated up to 20% dimethyl sulfoxide, allowing a wide range of compound concentrations to be tested. The assay signal window was robust and reproducible with average Z-factors of ≥0.8, and the redox cycling generation of H2O2 by DA3003-1 in DTT exhibited an average 50% effective concentration of 0.830 ± 0.068 μM. Five of the mitogen-activated protein kinase phosphatase (MKP) 1 inhibitors identified in an HTS were shown to generate H2O2 in the presence of DTT, and their inhibition of MKP-1 activity was shown to be time dependent and was abolished or significantly reduced by either 100 U of catalase or by higher DTT levels. A cross-target query of the PubChem database with three structurally related pyrimidotriazinediones revealed active flags in 36–39% of the primary screening assays. Activity was confirmed against a number of targets containing active site cysteines, including protein tyrosine phosphatases, cathepsins, and caspases, as well as a number of cellular cytotoxicity assays. Rather than utilize resources to conduct a hit characterization effort involving several secondary assays, the phenol red-HRP assay provides a simple, rapid, sensitive, and inexpensive method to identify compounds that redox cycle in DTT or tris(2-carboxyethyl)phosphine to produce H2O2 that may indirectly modulate target activity and represent promiscuous false-positives from a primary screen.
PMCID: PMC2752819  PMID: 18699726
6.  A 1536-well Based Kinetic HTS Assay for Inhibitors of Schistosoma mansoni Thioredoxin Glutathione Reductase 
Schistosomiasis is a major neglected tropical disease that currently affects over 200 million people and leads to over 200,000 annual deaths. Schistosoma mansoni parasites survive in humans due in part to a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principle component of this defense system has been recently identified as thioredoxin glutathione reductase (TGR), a parasite-specific enzyme that combines the functions of two human counterparts, gluotathione reductase and thioredoxin reductase, and as such this enzyme presents an attractive new target for anti-schistosomiasis drug development. Herein, we present the development of highly-miniaturized and robust screening assay for TGR. The 5 μL final volume assay is based on the Ellman’s reagent (5,5′ dithiobis(2-nitrobenzoic acid), DTNB) and utilizes a high-speed absorbance kinetic read to minimize the effect of dust, absorbance interference, and meniscus variation. This assay is further applicable to the testing of other redox enzymes which utilize DTNB as a model substrate.
PMCID: PMC2669305  PMID: 18665782
7.  A Robotic Platform for Quantitative High Throughput Screening 
High-throughput screening (HTS) is increasingly being adopted in academic institutions, where the decoupling of screening and drug development has led to unique challenges, as well as novel uses of instrumentation, assay formulations, and software tools. Advances in technology have made automated unattended screening in the 1536-well plate format broadly accessible and have further facilitated the exploration of new technologies and approaches to screening. A case in point is our recently-developed quantitative high-throughput screening (qHTS) paradigm which tests each library compound at multiple concentrations to construct concentration-response curves (CRCs) generating a comprehensive data set for each assay (Inglese et al, Proc. Natl. Acad. Sci USA 103, 11473–11478). The practical implementation of qHTS for cell-based and biochemical assays across libraries of >100,000 compounds (e.g. between 700,000 –2,000,000 sample wells tested) requires maximal efficiency and miniaturization, and the ability to easily accommodate many different assay formats and screening protocols. Here, we describe the design and utilization of a fully-integrated and automated screening system for qHTS at the NIH Chemical Genomics Center. We report system productivity, reliability, and flexibility, as well as modifications made to increase throughput, add additional capabilities, and address limitations. The combination of this system and qHTS has lead to the generation of over 6 million CRCs from >120 assays in the last three years, and is a technology that can be widely implemented to increase efficiency of screening and lead generation.
PMCID: PMC2651822  PMID: 19035846
quantitative high-throughput screening; screening; qHTS; 1536-well plate; laboratory automation; interleaved screens; laser cytometer
8.  High yield expression of serine/threonine protein phosphatase type 5, and a fluorescent assay suitable for use in the detection of catalytic inhibitors 
Protein phosphatase type 5 (PP5) belongs to the PPP-family of serine/threonine protein phosphatases and is expressed in most, if not all, human tissues. Although the physiological roles played by PP5 are not yet clear, PP5 is found in association with several proteins that influence intracellular signaling networks initiated by hormones (i.e. glucocorticoids) or cellular stress (i.e. hypoxia, oxidative stress). Recently, studies conducted with siRNA and antisense oligonucleotides indicate that PP5 plays an important role in the regulation of stress-induced signaling cascades that influence both cell growth and the onset of apoptosis. Therefore, the identification of small molecule inhibitors of PP5 is desired for use in studies to further define the biological/pathological roles of PP5. Such inhibitors may also prove useful for development into novel antitumor agents. Here we describe methods to express and purify large amounts of biologically active PP5c, an inhibitor-titration based assay to determine the amount of PP5 in solution, and a fluorescent phosphatase assay that can be used to screen chemical libraries and natural extracts for the presence of catalytic inhibitors.
PMCID: PMC2493290  PMID: 17939754
phosphatase; PP5; PPP5; DiFMUP; 6; 8-difluoro-4-methylumbelliferyl phosphate; umbelliferone; fluorescent; assay

Results 1-8 (8)