PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-8 (8)
 

Clipboard (0)
None
Journals
Authors
more »
Year of Publication
Document Types
1.  A Robotic Platform for Quantitative High-Throughput Screening 
Abstract
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 1,536-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 HTS (qHTS) paradigm, which tests each library compound at multiple concentrations to construct concentration-response curves (CRCs) generating a comprehensive data set for each assay. The practical implementation of qHTS for cell-based and biochemical assays across libraries of > 100,000 compounds (e.g., between 700,000 and 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 National Institutes of Health's 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 led to the generation of over 6 million CRCs from > 120 assays in the last 3 years and is a technology that can be widely implemented to increase efficiency of screening and lead generation.
doi:10.1089/adt.2008.150
PMCID: PMC2651822  PMID: 19035846
2.  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.
doi:10.1089/adt.2008.151
PMCID: PMC2752819  PMID: 18699726
3.  A 1,536-Well-Based Kinetic HTS Assay for Inhibitors of Schistosoma mansoni Thioredoxin Glutathione Reductase 
Abstract
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 in part because of a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principal 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, glutathione 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 a highly miniaturized and robust screening assay for TGR. The 5-μl final volume assay is based on the Ellman 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 that utilize DTNB as a model substrate.
doi:10.1089/adt.2008.149
PMCID: PMC2669305  PMID: 18665782
4.  Development of a 384-Well Colorimetric Assay to Quantify Hydrogen Peroxide Generated by the Redox Cycling of Compounds in the Presence of Reducing Agents 
Abstract
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 μ 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.
doi:10.1089/adt.2008.151
PMCID: PMC2752819  PMID: 18699726
5.  A Comparative Analysis of Standard Microtiter Plate Reading Versus Imaging in Cellular Assays 
Abstract
We evaluated the performance of two plate readers (the Beckman Coulter [Fullerton, CA] DTX and the PerkinElmer [Wellesley, MA] EnVision™) and a plate imager (the General Electric [Fairfield, CT] IN Cell 1000 Analyzer™) in a primary fluorescent cellular screen of 10,000 Molecular Libraries Screening Center Network library compounds for up- and down-regulation of vascular cell adhesion molecule (VCAM)-1, which has been shown to be up-regulated in artherothrombotic vascular disease and is a general indicator of chronic inflammatory disease. Prior to screening, imaging of a twofold, six-step titration of fluorescent cells in a 384-well test plate showed greater consistency, sensitivity, and dynamic range of signal detection curves throughout the detection range, as compared to the plate readers. With the same 384-well test plate, the detection limits for fluorescent protein-labeled cells on the DTX and EnVision instruments were 2,250 and 560 fluorescent cells per well, respectively, as compared to 280 on the IN Cell 1000. During VCAM screening, sensitivity was critical for detection of antagonists, which reduced brightness of the primary immunofluorescence readout; inhibitor controls yielded Z′ values of 0.41 and 0.16 for the IN Cell 1000 and EnVision instruments, respectively. The best 1% of small molecule inhibitors from all platforms were visually confirmed using images from the IN Cell 1000. The EnVision and DTX plate readers mutually identified approximately 57% and 21%, respectively, of the VCAM-1 inhibitors visually confirmed in the IN Cell best 1% of inhibitors. Furthermore, the plate reader hits were largely exclusive, with only 6% agreement across all platforms (three hits out of 47). Taken together, the imager outperformed the plate readers at hit detection in this bimodal assay because of superior sensitivity and had the advantage of speeding hit confirmation during post-acquisition analysis.
doi:10.1089/adt.2008.139
PMCID: PMC2956680  PMID: 18795873
6.  Evaluation of an Orthogonal Pooling Strategy for Rapid High-Throughput Screening of Proteases 
Abstract
Orthogonal pooling was evaluated as a strategy for the rapid screening of multiple cysteine and serine proteases against large compound libraries. To validate the method the human cysteine protease cathepsin B was screened against a library of 64,000 individual compounds and also against the same library mixed 10 compounds per well. The orthogonal pooling method used resulted in each compound being present in two wells, mixed with a different set of nine other compounds in each location. Thus hits were identified based on activity in both locations, avoiding the need for retesting of each component of active mixtures. Hits were tested in dose–response both in the dithiothreitol (DTT)-containing buffer used in the primary HTS and in buffer containing cysteine in place of DTT to rule out artifacts due to oxidative inactivation of the enzyme. Comparison of the confirmed actives from single-compound and mixture screening showed that mixture screening identified all of the actives from single-compound HTS. Based on these results the orthogonal pooling strategy has been used successfully to rapidly screen several cysteine and serine proteases.
doi:10.1089/adt.2007.110
PMCID: PMC2971631  PMID: 18593377
7.  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.
doi:10.1089/adt.2008.149
PMCID: PMC2669305  PMID: 18665782
8.  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.
doi:10.1089/adt.2008.150
PMCID: PMC2651822  PMID: 19035846
quantitative high-throughput screening; screening; qHTS; 1536-well plate; laboratory automation; interleaved screens; laser cytometer

Results 1-8 (8)