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1.  Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening 
Abstract
Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z′ = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.
doi:10.1089/adt.2015.669
PMCID: PMC4605357  PMID: 26383544
2.  Altered cytotoxicity of ROS-inducing compounds by sodium pyruvate in cell culture medium depends on the location of ROS generation 
SpringerPlus  2015;4:269.
Induction of oxidative stress by drugs and other xenobiotics is an important mechanism of cytotoxicity. However, in vitro studies on the relationship between oxidative stress and cytotoxicity in cultured cells is frequently complicated by the fact that cell culture medium components affect reactive oxygen species (ROS) exposures in ways that vary with the mode of ROS production. The objectives of this study were to first determine the mode of ROS induction by certain model compounds when they are applied to cultured cells, and then to determine how ROS induction and cytotoxicity were affected by the ROS-quenching medium component pyruvate. Three compounds, eseroline, benserazide, and pyrogallol induced H2O2 in cell culture media independent of cells. However, another compound, menadione, induced H2O2 in a manner largely dependent on the MDA-MB-231 breast cancer cells used in this study, which is consistent with its known mechanism of inducing ROS through intracellular redox cycling. 1 mM pyruvate, as well as catalase, reduced the H2O2 in culture wells with each ROS inducer tested but it only reduced the cytotoxicity of cell-independent inducers. It reduced the cytotoxicity of benserazide and pyrogallol >10-fold and of eseroline about 2.5-fold, but had no effect on menadione cytotoxicity. From this data, it was concluded that depending on the mechanism of ROS induction, whether intra- or extracellular, a ROS-quenching medium component such as pyruvate will differentially affect the net ROS-induction and cytotoxicity of a test compound.
doi:10.1186/s40064-015-1063-y
PMCID: PMC4469600  PMID: 26090316
Cell culture; Hydrogen peroxide; Reactive oxygen species; Cytotoxicity
3.  A Novel Steroidal Inhibitor of Estrogen-Related Receptor α (ERRα) 
Biochemical pharmacology  2010;80(6):819-826.
The orphan nuclear receptor estrogen-related receptor α (ERRα) has been implicated in the development of various human malignancies, including breast, prostate, ovary, and colon cancer. ERRα, bound to a co-activator protein (e.g., peroxisome proliferator receptor γ co-activator-1α, PGC-1α), regulates cellular energy metabolism by activating transcription of genes involved in various metabolic processes, such as mitochondrial genesis, oxidative phosphorylation, and fatty acid oxidation. Accumulating evidence suggests that ERRα is a novel target for solid tumor therapy, conceivably through effects on the regulation of tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. This report describes a novel steroidal antiestrogen (SR16388) that binds selectively to ERRα, but not to ERRβ or ERRγ, as determined using a time-resolved fluorescence resonance energy transfer assay. SR16388 potently inhibits ERRα's transcriptional activity in reporter gene assays, and prevents endogenous PGC-1α and ERRα from being recruited to the promoters or enhancers of target genes. Representative in vivo results show that SR16388 inhibited the growth of human prostate tumor xenografts in nude mice as a single agent at 30 mg/kg given once daily and 100 mg/kg given once weekly. In a combination study, SR16388 (10 mg/kg, once daily) and paclitaxel (7.5 mg/kg, twice weekly) inhibited the growth of prostate tumor xenografts in nude mice by 61% compared to untreated xenograft tumors. SR16388 also inhibited the proliferation of diverse human tumor cell lines after a 24-h exposure to the compound. SR16388 thus has utility both as an experimental antitumor agent and as a chemical probe of ERRα biology.
doi:10.1016/j.bcp.2010.05.024
PMCID: PMC2918268  PMID: 20513360
Estrogen-related receptor α; steroid; antitumor agent; metabolism
4.  A High-throughput Fluorescence Polarization Assay for Inhibitors of Gyrase B 
Journal of biomolecular screening  2011;16(2):230-238.
DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A2B2 heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin–Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buffer additives on the interaction. The assay was developed into a 21-μL, 384-well assay format and has been validated for use in high-throughput screening against a collection of Food and Drug Administration–approved compounds. The assay performed with an average Z′ factor of 0.80 and was able to identify GyrB inhibitors from a screening library.
doi:10.1177/1087057110392038
PMCID: PMC3176662  PMID: 21245469
fluorescence polarization; gyrase; assay development; high-throughput screen; anthracycline
5.  Phosphorylation sites of Epstein-Barr virus EBNA1 regulate its function 
The Journal of general virology  2009;90(Pt 9):2251-2259.
Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and a risk factor for developing a variety of lymphomas and carcinomas. EBV nuclear antigen 1 (EBNA1) is the only viral protein found in all EBV-related malignancies. It plays a key role in establishing and maintaining the altered state of cells transformed with EBV. EBNA1 is required for a variety of functions, including gene regulation, replication, and maintenance of the viral genome, but the regulation of EBNA1's functions is poorly understood. We demonstrate that phosphorylation affects EBNA1's functions. Using electron transfer dissociation tandem mass spectrometry, ten specific phosphorylated EBNA1 residues were identified. A mutant derivative preventing the phosphorylation of all ten phosphosites retained the unusually long half-life and the ability to translocate into the nucleus of wildtype EBNA1. This phosphorylation-deficient mutant, however, was significantly reduced in its ability to activate transcription and maintain EBV's plasmids in cells.
doi:10.1099/vir.0.012260-0
PMCID: PMC2786306  PMID: 19439552
6.  Phosphorylation sites of Epstein–Barr virus EBNA1 regulate its function 
The Journal of General Virology  2009;90(Pt 9):2251-2259.
Epstein–Barr virus (EBV) is the causative agent of infectious mononucleosis and a risk factor for developing a variety of lymphomas and carcinomas. EBV nuclear antigen 1 (EBNA1) is the only viral protein found in all EBV-related malignancies. It plays a key role in establishing and maintaining the altered state of cells transformed with EBV. EBNA1 is required for a variety of functions, including gene regulation, replication and maintenance of the viral genome, but the regulation of EBNA1's functions is poorly understood. We demonstrate that phosphorylation affects the functions of EBNA1. By using electron-transfer dissociation tandem mass spectrometry, ten specific phosphorylated EBNA1 residues were identified. A mutant derivative preventing the phosphorylation of all ten phosphosites retained the unusually long half-life and the ability to translocate into the nucleus of wild-type EBNA1. This phosphorylation-deficient mutant, however, had a significantly reduced ability to activate transcription and to maintain EBV's plasmids in cells.
doi:10.1099/vir.0.012260-0
PMCID: PMC2786306  PMID: 19439552
7.  Large-scale Epstein-Barr virus EBNA1 protein purification 
The protein-DNA and protein-protein interactions of Epstein-Barr virus nuclear antigen 1 (EBNA1) are known to play an important role in the many functions of this viral protein. Large quantities of pure EBNA1 protein would be useful in biochemical assays to elucidate such interactions. In particular, the crystal structure of the full-length protein would be important to show possible regions of interaction and/or post-translational modification. Recently, we described a novel approach to overexpress and purify EBNA1 from Escherichia coli; however, it is not ideal for large-scale production of EBNA1. We were able to optimize this protocol by 1) adding a polyethyleneimine precipitation step prior to Ni-NTA chromatography to reduce complexity of the sample and remove nucleic acid, 2) optimizing the Ni-NTA gradient to further separate EBNA1 from impurities, and 3) concluding with a MonoS cation-exchange chromatography step to further purify and concentrate EBNA1. We were able to recover 10-mg quantities of pure EBNA1 protein.
doi:10.1016/j.pep.2008.09.012
PMCID: PMC2744152  PMID: 18848996
8.  Antigen-Binding Properties of Monoclonal Antibodies Reactive with EBNA1 and Use in Immunoaffinity Chromatography 
PLoS ONE  2009;4(2):e4614.
Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was overexpressed and purified from Escherichia coli. Mouse monoclonal antibodies (mAbs) were prepared that react with EBNA1. Eleven high affinity mAbs were recovered. Nine mAbs are isotype IgG (all subisotype IgG1) and two mAbs are isotype IgM. All mAbs react strongly with EBNA1 in an ELISA assay while only one mAb (designated 1EB6) fails to react in a Western blot assay. The epitopes for these mAbs were mapped to seven different regions, providing good coverage of the entire EBNA1 protein. The mAbs had differing affinity for an EBNA1/DNA complex with four mAbs able to supershift the complex completely. All mAbs can immunoprecipitate EBNA1 from E. coli overexpressing EBNA1. A modified ELISA assay, termed ELISA-elution assay, was used to screen for mAbs that release EBNA1 in the presence of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotropic salt. MAbs with this property, termed polyol-responsive (PR)-mAbs, allow gentle elution of labile proteins and protein complexes. Four mAbs are polyol-responsive with two showing usefulness in gentle immunoaffinity chromatography. Purification with these PR-mAbs may be useful in purifying EBNA1 complexes and elucidating EBNA1-associated proteins. This panel of anti-EBNA1 mAbs will advance the study of EBV by providing new tools to detect and purify EBNA1.
doi:10.1371/journal.pone.0004614
PMCID: PMC2644765  PMID: 19242546

Results 1-8 (8)