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1.  Development and Validation of a High-density Fluorescence Polarization-based Assay for the Trypanosoma RNA Triphosphatase TbCet1 
RNA triphosphatases are attractive and mostly unexplored therapeutic targets for the development of broad spectrum antiprotozoal, antiviral and antifungal agents. The use of malachite green as a readout for phosphatases is well characterized and widely employed. However, the reaction depends on high quantities of inorganic phosphate to be generated, which makes this assay not easily amenable to screening in 1536-well format. The overly long reading times required also prohibit its use to screen large chemical libraries. To overcome these limitations, we sought to develop a fluorescence polarization (FP) -based assay for triphosphatases, compatible with miniaturization and fast readouts. For this purpose, we took advantage of the nucleoside triphosphatase activity of this class of enzyme to successfully adapt the Transcreener™ ADP assay based on the detection of generated ADP by immunocompetition fluorescence polarization to the RNA triphosphatase TbCet1 in 1536-well format. We also tested the performance of this newly developed assay in a pilot screen of 3,000 compounds and we confirmed the activity of the obtained hits. We present and discuss our findings and their importance for the discovery of novel drugs by high-throughput screening.
PMCID: PMC3626118  PMID: 19275531
triphosphatase; drug discovery; high-throughput screening; fluorescence polarization
2.  Revisiting Old Drugs as Novel Agents for Retinoblastoma: In vitro and In vivo Antitumor Activity of Cardenolides 
PURPOSE
Intraarterial delivery of chemotherapeutic agents offers a new and exciting opportunity for the treatment of advanced intraocular retinoblastoma. It allows local delivery of relatively high doses of chemo agents while bypassing general blood circulation. For this reason we sought to revisit some of the FDA approved drugs for the treatment of retinoblastoma.
METHODS
High throughput screening (HTS) of 2,640 approved drugs and bioactive compounds resulted in the identification of cytotoxic agents with potent activity toward both the Y79 and RB355 human retinoblastoma cell lines. Subsequent profiling of the drug candidates was performed in a panel of ocular cancer cell lines. Induction of apoptosis in Y79 cells was assessed by immunofluorescence detection of activated Caspase-3. Therapeutic effect was evaluated in a xenograft model of retinoblastoma.
RESULTS
We have identified several FDA approved drugs with potent cytotoxic activity toward retinoblastoma cell lines in vitro. Among them were several cardiac glycosides, a class of cardenolides historically associated with the prevention and treatment of congestive heart failure. Caspase-3 activation studies provided an insight into the mechanism of action of cardenolides in retinoblastoma cells. When tested in a xenograft model of retinoblastoma, the cardenolide ouabain induced complete tumor regression in the treated mice.
CONCLUSIONS
We have identified cardenolides as a new class of antitumor agents for the treatment of retinoblastoma. We propose that members of this class of cardiotonic drugs could be repositioned for retinoblastoma if administered locally via direct intraarterial infusion.
doi:10.1167/iovs.08-3158
PMCID: PMC3617409  PMID: 19151399
3.  Live Cell Imaging of Caspase Activation for High Content Screening 
Journal of biomolecular screening  2009;14(8):956-969.
Caspases are central to the execution of programmed cell death and their activation constitutes the biochemical hallmark of apoptosis. In this article, we report the successful adaptation of a high content assay method utilizing the DEVD-NucView488™ fluorogenic substrate, and for the first time, we show caspase activation in live cells induced either by drugs or siRNA. The fluorogenic substrate was found to be non-toxic over an exposure period of several days; during which we demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the anti-apoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to Doxorubicin, Etoposide or cell death siRNA. Our method was further validated against two well characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to Erlotinib; where we show a differential time dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, our results demonstrate the feasibility of using this newly adapted and validated high content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells.
doi:10.1177/1087057109343207
PMCID: PMC3613133  PMID: 19726787
High content assay; RNAi HT screening; Chemical HT screening; caspase; apoptosis; cancer; live cells
4.  A Miniaturized 1536-Well Format γ-Secretase Assay 
γ-Secretase is an aspartyl protease that cleaves multiple substrates including the amyloid precursor protein (APP) and the Notch proteins. Abnormal proteolysis of APP is involved in the pathogenesis of Alzheimer’s disease (AD) and overactive Notch signaling plays an oncogenic role in a variety of cancers. γ-Secretase has emerged as a promising target for drug development in the treatment of AD and cancer. Assays with increased capacity for high-throughput screening would allow for quicker screening of chemical libraries and facilitate inhibitor development. We have developed a homogeneous time-resolved fluorescence (HTRF)-based assay that makes use of a novel biotinylated recombinant APP substrate and solubilized membrane preparation as the source of the γ-secretase enzyme. The assay was miniaturized to a 1536-well format and validated in a pilot screen against a library of ∼3,000 compounds. The overall assay performance was robust due to a calculated Z′ factor of 0.74 and its demonstrated ability to identify known γ-secretase inhibitors such as pepstatin A. This validated assay can readily be used for primary screening against large chemical libraries searching for novel inhibitors of γ-secretase activity that may represent potential therapeutics for AD and a variety of neoplasms.
doi:10.1089/adt.2009.0202
PMCID: PMC3096545  PMID: 19715456

Results 1-4 (4)