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ABSTRACT: The human pathogen Giardia lamblia is an anaerobic protozoan parasite that causes giardiasis, one of the most common diarrheal diseases worldwide. Although several drugs are available for the treatment of giardisis, resistance to these drugs has been reported and is likely to increase. The Giardia carbamate kinase (glCK) plays an essential role in Giardia metabolism and has no homologs in humans, making it an attractive candidate for anti-Giardia drug development. We have developed a luminescent enzyme coupled assay to measure the activity of glCK by quantitating the amount of ATP produced by the enzyme. This assay is homogeneous and has been miniaturized into a 1536-well plate format. A pilot screen against 4,096 known compounds using this assay yielded a signal-to-basal ratio of 11.5 fold and Z' factor of 0.8 with a hit rate of 0.9 % of inhibitors of glCK. Therefore, this Giardia lamblia carbamate kinase assay is useful for high throughput screening of large compound collection for identification of the inhibitors for drug development.
Current Chemical Genomics 01/2012; 6:93-102.
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Ruili Huang,
Menghang Xia,
Ming-Hsuang Cho,
Srilatha Sakamuru, Paul Shinn,
Keith A Houck,
David J Dix,
Richard S Judson,
Kristine L Witt,
Robert J Kavlock,
Raymond R Tice,
Christopher P Austin
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ABSTRACT: The large and increasing number of chemicals released into the environment demands more efficient and cost-effective approaches for assessing environmental chemical toxicity. The U.S. Tox21 program has responded to this challenge by proposing alternative strategies for toxicity testing, among which the quantitative high-throughput screening (qHTS) paradigm has been adopted as the primary tool for generating data from screening large chemical libraries using a wide spectrum of assays.
The goal of this study was to develop methods to evaluate the data generated from these assays to guide future assay selection and prioritization for the Tox21 program.
We examined the data from the Tox21 pilot-phase collection of approximately 3,000 environmental chemicals profiled in qHTS format against a panel of 10 human nuclear receptors (AR, ERα, FXR, GR, LXRβ, PPARγ, PPARδ, RXRα, TRβ, and VDR) for reproducibility, concordance of biological activity profiles with sequence homology of the receptor ligand binding domains, and structure-activity relationships.
We determined the assays to be appropriate in terms of biological relevance. We found better concordance for replicate compounds for the agonist-mode than for the antagonist-mode assays, likely due to interference of cytotoxicity in the latter assays. This exercise also enabled us to formulate data-driven strategies for discriminating true signals from artifacts, and to prioritize assays based on data quality.
The results demonstrate the feasibility of qHTS to identify the potential for environmentally relevant chemicals to interact with key toxicity pathways related to human disease induction.
Environmental Health Perspectives 05/2011; 119(8):1142-8. · 7.04 Impact Factor
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ABSTRACT: Small-molecule compounds approved for use as drugs may be "repurposed" for new indications and studied to determine the mechanisms of their beneficial and adverse effects. A comprehensive collection of all small-molecule drugs approved for human use would be invaluable for systematic repurposing across human diseases, particularly for rare and neglected diseases, for which the cost and time required for development of a new chemical entity are often prohibitive. Previous efforts to build such a comprehensive collection have been limited by the complexities, redundancies, and semantic inconsistencies of drug naming within and among regulatory agencies worldwide; a lack of clear conceptualization of what constitutes a drug; and a lack of access to physical samples. We report here the creation of a definitive, complete, and nonredundant list of all approved molecular entities as a freely available electronic resource and a physical collection of small molecules amenable to high-throughput screening.
Science translational medicine 04/2011; 3(80):80ps16. · 7.80 Impact Factor
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ABSTRACT: The pregnane X receptor (PXR) binds xenobiotics and regulates the expression of several drug-metabolizing enzymes and transporters. Human PXR (hPXR) activation and CYP3A4 induction can be involved in drug-drug interactions, resulting in reduced efficacy or increased toxicity. However, there are known species-specific differences with regard to PXR activation that should be taken into account when animal PXR data are extrapolated to humans. We profiled 2816 clinically used drugs from the National Institutes of Health Chemical Genomics Center Pharmaceutical Collection for their ability to activate hPXR and rat PXR (rPXR) at the cellular level, induce human CYP3A4 at the cellular level, and bind human PXR at the protein level. From 6 to 11% of drugs were identified as active across the four assays, which included assay-specific and pan-active compounds. The lowest concordance was observed between the hPXR and rPXR assays, and many compounds active in both assays nonetheless demonstrated significant potency differences between species. Analysis based on clustering potency values demonstrated the greatest activity correlation between the hPXR activation and CYP3A4 induction assays. Structure-activity relationship analysis identified chemical scaffolds that were pan-active (e.g., dihydropyridine calcium channel blockers) and others that were uniquely active in individual assays (e.g., steroids and fatty acids). These results provide important information on PXR activation by clinically used drugs, highlight the species specificity of PXR activation by xenobiotics, and provide a means of prioritizing compounds for follow-up studies and optimization efforts.
Drug metabolism and disposition: the biological fate of chemicals 10/2010; 39(1):151-9. · 3.74 Impact Factor
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Richard S Judson,
Matthew T Martin,
David M Reif,
Keith A Houck,
Thomas B Knudsen,
Daniel M Rotroff,
Menghang Xia,
Srilatha Sakamuru,
Ruili Huang, Paul Shinn,
Christopher P Austin,
Robert J Kavlock,
David J Dix
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ABSTRACT: The Deepwater Horizon oil spill has led to the use of >1 M gallons of oil spill dispersants, which are mixtures of surfactants and solvents. Because of this large scale use there is a critical need to understand the potential for toxicity of the currently used dispersant and potential alternatives, especially given the limited toxicity testing information that is available. In particular, some dispersants contain nonylphenol ethoxylates (NPEs), which can degrade to nonylphenol (NP), a known endocrine disruptor. Given the urgent need to generate toxicity data, we carried out a series of in vitro high-throughput assays on eight commercial dispersants. These assays focused on the estrogen and androgen receptors (ER and AR), but also included a larger battery of assays probing other biological pathways. Cytotoxicity in mammalian cells was also quantified. No activity was seen in any AR assay. Two dispersants showed a weak ER signal in one assay (EC50 of 16 ppm for Nokomis 3-F4 and 25 ppm for ZI-400). NPs and NPEs also had a weak signal in this same ER assay. Note that Corexit 9500, the currently used product, does not contain NPEs and did not show any ER activity. Cytotoxicity values for six of the dispersants were statistically indistinguishable, with median LC50 values approximately 100 ppm. Two dispersants, JD 2000 and SAF-RON GOLD, were significantly less cytotoxic than the others with LC50 values approaching or exceeding 1000 ppm.
Environmental Science & Technology 08/2010; 44(15):5979-85. · 4.80 Impact Factor
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ABSTRACT: Nuclear factor-kappa B (NF-kappaB) is a transcription factor that plays a critical role across many cellular processes including embryonic and neuronal development, cell proliferation, apoptosis, and immune responses to infection and inflammation. Dysregulation of NF-kappaB signaling is associated with inflammatory diseases and certain cancers. Constitutive activation of NF-kappaB signaling has been found in some types of tumors including breast, colon, prostate, skin and lymphoid, hence therapeutic blockade of NF-kappaB signaling in cancer cells provides an attractive strategy for the development of anticancer drugs. To identify small molecule inhibitors of NF-kappaB signaling, we screened approximately 2800 clinically approved drugs and bioactive compounds from the NIH Chemical Genomics Center Pharmaceutical Collection (NPC) in a NF-kappaB mediated beta-lactamase reporter gene assay. Each compound was tested at fifteen different concentrations in a quantitative high throughput screening format. We identified nineteen drugs that inhibited NF-kappaB signaling, with potencies as low as 20 nM. Many of these drugs, including emetine, fluorosalan, sunitinib malate, bithionol, narasin, tribromsalan, and lestaurtinib, inhibited NF-kappaB signaling via inhibition of IkappaBalpha phosphorylation. Others, such as ectinascidin 743, chromomycin A3 and bortezomib utilized other mechanisms. Furthermore, many of these drugs induced caspase 3/7 activity and had an inhibitory effect on cervical cancer cell growth. Our results indicate that many currently approved pharmaceuticals have previously unappreciated effects on NF-kappaB signaling, which may contribute to anticancer therapeutic effects. Comprehensive profiling of approved drugs provides insight into their molecular mechanisms, thus providing a basis for drug repurposing.
Biochemical pharmacology 05/2010; 79(9):1272-80. · 4.25 Impact Factor
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ABSTRACT: Activation of G(q) 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 1,536-well plate format for high-throughput screening (HTS). Here, we have applied the latest generation of fluorescence kinetic plate readers to multiplex the agonist and antagonist screens of a G protein-coupled receptor (GPCR). This multiplexed assay format provides an efficient and cost-effective method for HTS of G(q)-coupled GPCR targets.
Assay and Drug Development Technologies 03/2010; 8(3):367-79. · 1.73 Impact Factor
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Bryan T Mott,
Cordelle Tanega,
Min Shen,
David J Maloney, Paul Shinn,
William Leister,
Juan J Marugan,
James Inglese,
Christopher P Austin,
Tom Misteli,
Douglas S Auld,
Craig J Thomas
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ABSTRACT: A series of substituted 6-arylquinazolin-4-amines were prepared and analyzed as inhibitors of Clk4. Synthesis, structure-activity relationships and the selectivity of a potent analogue against a panel of 402 kinases are presented. Inhibition of Clk4 by these agents at varied concentrations of assay substrates (ATP and receptor peptide) highly suggests that this chemotype is an ATP competitive inhibitor. Molecular docking provides further evidence that inhibition is the result of binding at the kinase hinge region. Selected compounds represent novel tools capable of potent and selective inhibition of Clk1, Clk4, and Dyrk1A.
Bioorganic & medicinal chemistry letters 12/2009; 19(23):6700-5. · 2.65 Impact Factor
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ABSTRACT: The authors describe how room temperature storage of a 1120-member compound library prepared in either DMSO or in a hydrated-DMSO/water (67/33) mixture affects the reproducibility of potency values as monitored using cytochrome P450 1A2 and 2D6 isozyme assays. The bioluminescent assays showed Z' factors of 0.71 and 0.62, with 17% and 32% of the library found as active against the CYP 1A2 and 2D6 isozymes, respectively. The authors tested the library using quantitative high-throughput screening to generate potency values for every library member, which was measured at 7 time intervals spanning 37 weeks. They calculated the minimum significant ratio (MSR) from these potency values at each time interval and found that for the library stored in DMSO, the CYP 1A2 and 2D6 assay MSRs progressed from approximately 2.0 to 5.0. The hydrated conditions showed similar performance in both MSR progression and analytical quality control results. Based on this study, the authors recommend that DMSO samples be stored in 1536-well plates for <4 months at room temperature. Furthermore, the study illustrates the degree and time scale of apparent compound potency changes due to sample storage.
Journal of Biomolecular Screening 06/2009; 14(5):538-46. · 2.05 Impact Factor
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Anton Simeonov,
Ajit Jadhav,
Craig J Thomas,
Yuhong Wang,
Ruili Huang,
Noel T Southall, Paul Shinn,
Jeremy Smith,
Christopher P Austin,
Douglas S Auld,
James Inglese
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ABSTRACT: Chromo/fluorophoric properties often accompany the heterocyclic scaffolds and impurities that comprise libraries used for high-throughput screening (HTS). These properties affect assay outputs obtained with optical detection, thus complicating analysis and leading to false positives and negatives. Here, we report the fluorescence profile of more than 70,000 samples across spectral regions commonly utilized in HTS. The quantitative HTS paradigm was utilized to test each sample at seven or more concentrations over a 4-log range in 1,536-well format. Raw fluorescence was compared with fluorophore standards to compute a normalized response as a function of concentration and spectral region. More than 5% of library members were brighter than the equivalent of 10 nM 4-methyl umbelliferone, a common UV-active probe. Red-shifting the spectral window by as little as 100 nm was accompanied by a dramatic decrease in autofluorescence. Native compound fluorescence, fluorescent impurities, novel fluorescent compounds, and the utilization of fluorescence profiling data are discussed.
Journal of Medicinal Chemistry 05/2008; 51(8):2363-71. · 5.25 Impact Factor
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ABSTRACT: An efficient and versatile Compound Management operation is essential for the success of all downstream processes in high-throughput screening (HTS) and small molecule lead development. Staff, equipment, and processes need to be not only reliable, but remain flexible and prepared to incorporate paradigm changes. In the present report, we describe a system and associated processes which enable handling of compounds for both screening and follow-up purposes at the NIH Chemical Genomics Center (NCGC), a recently-established HTS and probe development center within the Molecular Libraries Initiative of the NIH Roadmap. Our screening process, termed quantitative HTS (qHTS), involves assaying the complete compound library, currently containing >200,000 members, at a series of dilutions to construct a full concentration-response profile. As such, Compound Management at the NCGC has been uniquely tasked to prepare, store, register, and track a vertically-developed plate dilution series (i.e., inter-plate titrations) in the 384-well format. These are compressed into a series of 1,536-well plates and are registered to track all subsequent plate storage. Here, we present details on the selection of equipment to enable automated, reliable and parallel compound manipulation in 384- and 1,536-well formats, protocols for preparation of inter-plate dilution series for qHTS, as well as qHTS-specific processes and issues.
Journal of the Association for Laboratory Automation 04/2008; 13(2):79-89. · 1.42 Impact Factor
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Steve Titus,
Susanne Neumann,
Wei Zheng,
Noel Southall,
Sam Michael,
Carleen Klumpp,
Adam Yasgar, Paul Shinn,
Craig J Thomas,
James Inglese,
Marvin C Gershengorn,
Christopher P Austin
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ABSTRACT: The thyroid-stimulating hormone (TSH; thyrotropin) receptor belongs to the glycoprotein hormone receptor subfamily of 7-transmembrane spanning receptors. TSH receptor (TSHR) is expressed mainly in thyroid follicular cells and is activated by TSH, which regulates the growth and function of thyroid follicular cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small-molecule agonists of the TSHR are available. To screen for novel TSHR agonists, the authors miniaturized a commercially available cell-based cyclic adenosine 3',5' monophosphate (cAMP) assay into a 1536-well plate format. This assay uses an HEK293 cell line stably transfected with the TSHR coupled to a cyclic nucleotide gated ion channel as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal homogeneous time-resolved fluorescence cAMP-based assay. Forty-nine compounds in several structural classes have been confirmed as the small-molecule TSHR agonists that will serve as a starting point for chemical optimization and studies of thyroid physiology in health and disease.
Journal of Biomolecular Screening 03/2008; 13(2):120-7. · 2.05 Impact Factor
