Development of a cell-based assay for high-throughput screening of inhibitors against HCV genotypes 1a and 1b in a single well

2009 ◽  
Vol 82 (1) ◽  
pp. 82-88 ◽  
Author(s):  
Rubina Mondal ◽  
Gennadiy Koev ◽  
Tami Pilot-Matias ◽  
Yupeng He ◽  
Teresa Ng ◽  
...  
2008 ◽  
Vol 78 (2) ◽  
pp. A48
Author(s):  
William Severson ◽  
Joseph Maddry ◽  
Xi Chen ◽  
Subramaniam Ananthan ◽  
Adrian Poffenberger ◽  
...  

2011 ◽  
Vol 16 (8) ◽  
pp. 925-931 ◽  
Author(s):  
Amy Emery ◽  
David A. Sorrell ◽  
Stacy Lawrence ◽  
Emma Easthope ◽  
Mark Stockdale ◽  
...  

Aurora A kinase is a key regulator of mitosis, which is upregulated in several human cancers, making it a potential target for anticancer therapeutics. Consequently, robust medium- to high-throughput cell-based assays to measure Aurora A kinase activity are critical for the development of small-molecule inhibitors. Here the authors compare measurement of the phosphorylation of two Aurora A substrates previously used in high-content screening Aurora A assays, Aurora A itself and TACC3, with a novel substrate Lats2. Using antibodies directed against phosphorylated forms of Aurora A (pThr288), P-TACC3 (pSer558), and P-Lats2 (pSer83), the authors investigate their suitability in parallel for development of a cell-based assay using several reference Aurora inhibitors: MLN8054, VX680, and AZD1152-HQPA. They validate a combined assay of target-specific phosphorylation of Lats2 at the centrosome and an increase in mitotic index as a measure of Aurora A activity. The assay is both sensitive and robust and has acceptable assay performance for high-throughput screening or potency estimation from concentration–response assays. It has the advantage that it can be carried out using a commercially available monoclonal antibody against phospho-Lats2 and the widely available Cellomics ArrayScan HCS reader and thus represents a significant addition to the tools available for the identification of Aurora A specific inhibitors.


2019 ◽  
Vol 294 (29) ◽  
pp. 11259-11275 ◽  
Author(s):  
Mitsuharu Ueda ◽  
Masamitsu Okada ◽  
Mineyuki Mizuguchi ◽  
Barbara Kluve-Beckerman ◽  
Kyosuke Kanenawa ◽  
...  

2019 ◽  
Vol 20 (12) ◽  
pp. 3112 ◽  
Author(s):  
Viviana Gatta ◽  
Polina Ilina ◽  
Alison Porter ◽  
Stuart McElroy ◽  
Päivi Tammela

Since quorum sensing (QS) is linked to the establishment of bacterial infection, its inactivation represents one of the newest strategies to fight bacterial pathogens. LsrK is a kinase playing a key role in the processing of autoinducer-2 (AI-2), a quorum-sensing mediator in gut enteric bacteria. Inhibition of LsrK might thus impair the quorum-sensing cascade and consequently reduce bacterial pathogenicity. Aiming for the development of a target-based assay for the discovery of LsrK inhibitors, we evaluated different assay set-ups based on ATP detection and optimized an automation-compatible method for the high-throughput screening of chemical libraries. The assay was then used to perform the screening of a 2000-compound library, which provided 12 active compounds with an IC50 ≤ 10 µM confirming the effectiveness and sensitivity of our assay. Follow-up studies on the positive hits led to the identification of two compounds, harpagoside and rosolic acid, active in a cell-based AI-2 QS interference assay, which are at the moment the most promising candidates for the development of a new class of antivirulence agents based on LsrK inhibition.


2005 ◽  
Vol 49 (12) ◽  
pp. 5185-5188 ◽  
Author(s):  
Sofiya Micheva-Viteva ◽  
Annmarie L. Pacchia ◽  
Yacov Ron ◽  
Stuart W. Peltz ◽  
Joseph P. Dougherty

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is not eliminated from patients even after years of antiretroviral therapy, apparently due to the presence of latently infected cells. Here we describe the development of a cell-based system of latency that can be used for high-throughput screening aimed at novel drug discovery to eradicate HIV-1 infection.


2019 ◽  
Vol 25 (1) ◽  
pp. 79-86
Author(s):  
Tara Walhart ◽  
Erin Isaacson-Wechsler ◽  
Kean-Hooi Ang ◽  
Michelle Arkin ◽  
Sharof Tugizov ◽  
...  

Like cervical cancer, anal cancer is caused by human papillomavirus (HPV). HPV is the most common sexually transmitted agent and is found in the anal canal of almost all HIV-positive men who have sex with men (MSM). Rates of HPV anal cancer are disproportionately higher in this population. Although the nanovalent HPV vaccine is efficacious in protecting against oncogenic HPV types, a substantial proportion of MSM remains unvaccinated and anal HPV infection continues to be an important public health burden. Therefore, it is important to identify strategies to prevent HPV infection. We report on two promising and interlinked strategies: (1) the development of a cell-based Renilla luminescence reporter assay using HPV-16 pseudovirions that encapsidate SV40-driven Renilla luminescence reporter expression plasmid and (2) use of this assay for high-throughput screening (HTS) of FDA- and internationally approved drugs to identify those that could be repurposed to prevent HPV infection. We conducted a screen of 1906 drugs. The assay was valid with a Z′ of 0.67 ± 0.04, percent coefficient of variance of 10.0, and signal-to-background noise window of 424.0 ± 8.0. Five drugs were chosen for further analyses based on selection parameters of ≥77.0% infection of HPV-16 pseudovirion-driven Renilla expression with <20.0% cytotoxicity. Of these, the antifungal pentamidine and a gamma-amino butyric acid receptor agonist securinine exhibited ≥90.0% infection with <10.0% cytotoxicity. This luminescent cell-based reporter expression plasmid assay for HTS is a valid method to identify FDA- and internationally approved drugs with the potential to be repurposed into prevention modalities for HPV infection.


2015 ◽  
Vol 10 (1) ◽  
pp. 55 ◽  
Author(s):  
Jasmina Hodzic ◽  
Ilse Dingjan ◽  
Mariëlle Maas ◽  
Ida H van der Meulen-Muileman ◽  
Renee X de Menezes ◽  
...  

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