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1.  Identification of repurposed small molecule drugs for chordoma therapy 
Cancer Biology & Therapy  2013;14(7):638-647.
Chordoma is a rare, slow growing malignant tumor arising from remnants of the fetal notochord. Surgery is the first choice for chordoma treatment, followed by radiotherapy, although postoperative complications remain significant. Recurrence of the disease occurs frequently due to the anatomy of the tumor location and violation of the tumor margins at the initial surgery. Currently, there are no effective drugs available for patients with chordoma. Due to the rarity of the disease, there is limited opportunity to test agents in clinical trials and no concerted effort to develop agents for chordoma in the pharmaceutical industry. To rapidly and efficiently identify small molecules that inhibit chordoma cell growth, we screened the NCGC Pharmaceutical Collection (NPC) containing approximately 2800 clinically approved and investigational drugs at 15 different concentrations in chordoma cell lines, U-CH1 and U-CH2. We identified a group of drugs including bortezomib, 17-AAG, digitoxin, staurosporine, digoxin, rubitecan, and trimetrexate that inhibited chordoma cell growth, with potencies from 10 to 370 nM in U-CH1 cells, but less potently in U-CH2 cells. Most of these drugs also induced caspase 3/7 activity with a similar rank order as the cytotoxic effect on U-CH1 cells. Cantharidin, digoxin, digitoxin, staurosporine, and bortezomib showed similar inhibitory effect on cell lines and 3 primary chordoma cell cultures. The combination treatment of bortezomib with topoisomerase I and II inhibitors increased the therapeutic potency in U-CH2 and patient-derived primary cultures. Our results provide information useful for repurposing currently approved drugs for chordoma and potential approach of combination therapy.
doi:10.4161/cbt.24596
PMCID: PMC3742493  PMID: 23792643
chordoma; NCGC Pharmaceutical Collection; cell viability; caspase 3/7; U-CH1; U-CH2; qHTS
2.  Chemical Genomics Profiling of Environmental Chemical Modulation of Human Nuclear Receptors 
Environmental Health Perspectives  2011;119(8):1142-1148.
Background: 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.
Objectives: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
doi:10.1289/ehp.1002952
PMCID: PMC3237348  PMID: 21543282
assay performance; chemical genomics; cytotoxicity; nuclear receptors; qHTS; Tox21
3.  Weighted Feature Significance: A Simple, Interpretable Model of Compound Toxicity Based on the Statistical Enrichment of Structural Features 
Toxicological Sciences  2009;112(2):385-393.
In support of the U.S. Tox21 program, we have developed a simple and chemically intuitive model we call weighted feature significance (WFS) to predict the toxicological activity of compounds, based on the statistical enrichment of structural features in toxic compounds. We trained and tested the model on the following: (1) data from quantitative high–throughput screening cytotoxicity and caspase activation assays conducted at the National Institutes of Health Chemical Genomics Center, (2) data from Salmonella typhimurium reverse mutagenicity assays conducted by the U.S. National Toxicology Program, and (3) hepatotoxicity data published in the Registry of Toxic Effects of Chemical Substances. Enrichments of structural features in toxic compounds are evaluated for their statistical significance and compiled into a simple additive model of toxicity and then used to score new compounds for potential toxicity. The predictive power of the model for cytotoxicity was validated using an independent set of compounds from the U.S. Environmental Protection Agency tested also at the National Institutes of Health Chemical Genomics Center. We compared the performance of our WFS approach with classical classification methods such as Naive Bayesian clustering and support vector machines. In most test cases, WFS showed similar or slightly better predictive power, especially in the prediction of hepatotoxic compounds, where WFS appeared to have the best performance among the three methods. The new algorithm has the important advantages of simplicity, power, interpretability, and ease of implementation.
doi:10.1093/toxsci/kfp231
PMCID: PMC2777082  PMID: 19805409
modeling; toxicity prediction; structural features; cell viability; caspase-3,7 activation; in vivo toxicity
4.  Identification of small molecule compounds that inhibit the HIF-1 signaling pathway 
Molecular Cancer  2009;8:117.
Background
Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that regulates cellular responses to low oxygen environments. HIF-1 is composed of two subunits: hypoxia-inducible HIF-1α and constitutively-expressed HIF-1β. During hypoxic conditions, HIF-1α heterodimerizes with HIF-1β and translocates to the nucleus where the HIF-1 complex binds to the hypoxia-response element (HRE) and activates expression of target genes implicated in cell growth and survival. HIF-1α protein expression is elevated in many solid tumors, including those of the cervix and brain, where cells that are the greatest distance from blood vessels, and therefore the most hypoxic, express the highest levels of HIF-1α. Therapeutic blockade of the HIF-1 signaling pathway in cancer cells therefore provides an attractive strategy for development of anticancer drugs. To identify small molecule inhibitors of the HIF-1 pathway, we have developed a cell-based reporter gene assay and screened a large compound library by using a quantitative high-throughput screening (qHTS) approach.
Results
The assay is based upon a β-lactamase reporter under the control of a HRE. We have screened approximate 73,000 compounds by qHTS, with each compound tested over a range of seven to fifteen concentrations. After qHTS we have rapidly identified three novel structural series of HIF-1 pathway Inhibitors. Selected compounds in these series were also confirmed as inhibitors in a HRE β-lactamase reporter gene assay induced by low oxygen and in a VEGF secretion assay. Three of the four selected compounds tested showed significant inhibition of hypoxia-induced HIF-1α accumulation by western blot analysis.
Conclusion
The use of β-lactamase reporter gene assays, in combination with qHTS, enabled the rapid identification and prioritization of inhibitors specific to the hypoxia induced signaling pathway.
doi:10.1186/1476-4598-8-117
PMCID: PMC2797767  PMID: 20003191
5.  A Bioluminescent Cytotoxicity Assay for Assessment of Membrane Integrity Using a Proteolytic Biomarker 
Measurement of cell membrane integrity has been widely used to assess chemical cytotoxity. Several assays are available for determining cell membrane integrity including differential labeling techniques using neutral red and trypan blue dyes or fluorescent compounds such as propidium iodide. Other common methods for assessing cytotoxicity are enzymatic “release” assays which measure the extracellular activities of lactate dehydrogenase (LDH), adenylate kinase (AK), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in culture medium. However, all these assays suffer from several practical limitations, including multiple reagent additions, scalability, low sensitivity, poor linearity, or requisite washes and medium exchanges. We have developed a new cytotoxicity assay which measures the activity of released intracellular proteases as a result of cell membrane impairment. It allows for a homogenous, one-step addition assay with a luminescent readout. We have optimized and miniaturized this assay into a 1536-well format, and validated it by screening a library of known toxins from the National Toxicology Program (NTP) using HEK 293 and human renal mesangial cells by quantitative high-throughput screening (qHTS). Several known and novel membrane disrupters were identified from the library, which indicates that the assay is robust and suitable for large scale library screening. This cytotoxicity assay, combined with the qHTS platform, allowed us to quickly and efficiently evaluate compound toxicities related to cell membrane integrity.
doi:10.1016/j.tiv.2008.02.013
PMCID: PMC2386563  PMID: 18400464
1536-well; NTP 1408 compound library; membrane integrity; cytotoxicity assay; protease release assay; qHTS; renal mesangial cells; HEK293 cells
6.  Characterization of Diversity in Toxicity Mechanism Using In Vitro Cytotoxicity Assays in Quantitative High Throughput Screening 
Chemical research in toxicology  2008;21(3):659-667.
Assessing the potential health risks of environmental chemical compounds is an expensive undertaking which has motivated the development of new alternatives to traditional in vivo toxicological testing. One approach is to stage the evaluation, beginning with less expensive and higher throughput in vitro testing before progressing to more definitive trials. In vitro testing can be used to generate a hypothesis about a compound's mechanism of action, which can then be used to design an appropriate in vivo experiment. Here we begin to address the question of how to design such a battery of in vitro cell-based assays by combining data from two different types of assays, cell viability and caspase activation, with the aim of elucidating mechanism of action. Because caspase activation is a transient event during apoptosis, it is not possible to design a single end-point assay protocol that would identify all instances of compound-induced caspase activation. Nevertheless, useful information about compound mechanism of action can be obtained from these assays in combination with cell viability data. Unsupervised clustering in combination with Dunn's cluster validity index is a robust method for identifying mechanisms of action without requiring any a priori knowledge about mechanisms of toxicity. The performance of this clustering method is evaluated by comparing the clustering results against literature annotations of compound mechanisms.
doi:10.1021/tx700365e
PMCID: PMC2668196  PMID: 18281954
cell-based assay; cell viability; caspase-3/7; apoptosis; qHTS; mechanism of action
7.  Identification of a Potent New Chemotype for the Selective Inhibition of PDE4 
A series of substituted 3,6-diphenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines were prepared and analyzed as inhibitors of phosphodiesterase 4 (PDE4). Synthesis, structure activity relationships and the selectivity of a highly potent analogue against related phosphodiesterase isoforms are presented.
doi:10.1016/j.bmcl.2008.01.028
PMCID: PMC2268978  PMID: 18243697
8.  Compound Cytotoxicity Profiling Using Quantitative High-Throughput Screening 
Environmental Health Perspectives  2007;116(3):284-291.
Background
The propensity of compounds to produce adverse health effects in humans is generally evaluated using animal-based test methods. Such methods can be relatively expensive, low-throughput, and associated with pain suffered by the treated animals. In addition, differences in species biology may confound extrapolation to human health effects.
Objective
The National Toxicology Program and the National Institutes of Health Chemical Genomics Center are collaborating to identify a battery of cell-based screens to prioritize compounds for further toxicologic evaluation.
Methods
A collection of 1,408 compounds previously tested in one or more traditional toxicologic assays were profiled for cytotoxicity using quantitative high-throughput screening (qHTS) in 13 human and rodent cell types derived from six common targets of xenobiotic toxicity (liver, blood, kidney, nerve, lung, skin). Selected cytotoxicants were further tested to define response kinetics.
Results
qHTS of these compounds produced robust and reproducible results, which allowed cross-compound, cross-cell type, and cross-species comparisons. Some compounds were cytotoxic to all cell types at similar concentrations, whereas others exhibited species- or cell type–specific cytotoxicity. Closely related cell types and analogous cell types in human and rodent frequently showed different patterns of cytotoxicity. Some compounds inducing similar levels of cytotoxicity showed distinct time dependence in kinetic studies, consistent with known mechanisms of toxicity.
Conclusions
The generation of high-quality cytotoxicity data on this large library of known compounds using qHTS demonstrates the potential of this methodology to profile a much broader array of assays and compounds, which, in aggregate, may be valuable for prioritizing compounds for further toxicologic evaluation, identifying compounds with particular mechanisms of action, and potentially predicting in vivo biological response.
doi:10.1289/ehp.10727
PMCID: PMC2265061  PMID: 18335092
1,536-well; cell viability; NTP 1,408 compound library; PubChem; qHTS; RT-CES

Results 1-8 (8)