Overview on High‐Throughput Screening

Entzeroth, Michael

doi:10.1002/0471141755.ph0904s22

Cited by 15 publications

(16 citation statements)

References 84 publications

(81 reference statements)

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“…In order to evaluate assay performance and robustness, we applied a high-throughput screen format to the assay using a small library of 1600 bioactive compounds. The average Z factor (>0.72) among all the plates screened met the acceptance criteria for assay validation [23]. Assay signal-to-noise was consistently above 9.5 (Supplementary Table S1 at http://www.biochemj.org/bj/461/bj4610323add.htm), a satisfactory window for distinguishing between active and inactive compounds.…”

Section: Inhibition Assay and Compound Library Screenmentioning

confidence: 88%

Characterization of the histone methyltransferase PRDM9 using biochemical, biophysical and chemical biology techniques

Koh-Stenta

Joy

Poulsen

et al. 2014

Biochemical Journal

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PRDM proteins have emerged as important regulators of disease and developmental processes. To gain insight into the mechanistic actions of the PRDM family, we have performed comprehensive characterization of a prototype member protein, the histone methyltransferase PRDM9, using biochemical, biophysical and chemical biology techniques. In the present paper we report the first known molecular characterization of a PRDM9-methylated recombinant histone octamer and the identification of new histone substrates for the enzyme. A single C321P mutant of the PR/SET domain was demonstrated to significantly weaken PRDM9 activity. Additionally, we have optimized a robust biochemical assay amenable to high-throughput screening to facilitate the generation of small-molecule chemical probes for this protein family. The present study has provided valuable insight into the enzymology of an intrinsically active PRDM protein.

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Section: Inhibition Assay and Compound Library Screenmentioning

confidence: 88%

Characterization of the histone methyltransferase PRDM9 using biochemical, biophysical and chemical biology techniques

Koh-Stenta

Joy

Poulsen

et al. 2014

Biochemical Journal

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“…These include in silico HTS, in vitro enzymatic assays, cell-based reporter assays, image-based whole infection assays, among others [19]. In the work reported here, we describe the development of an image-based high-throughput/high-content assay (HT/HCA) screening method for anti-dengue compounds using an infectious virus system and an innovative approach in image analysis.…”

Section: Introductionmentioning

confidence: 99%

High Content Screening of a Kinase-Focused Library Reveals Compounds Broadly-Active against Dengue Viruses

et al. 2013

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Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates.

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“…To enter into a study that produces large volumes of data without a clear idea of the question being asked and a framework for how the study will be interpreted is both foolish and irresponsible. If experimental design was important in the days of a single radioligand binding study being run in a test tube, how much more so is it important in current high‐throughput experiments that use 1536‐well plates and other formats that extend to 8000‐plus reagent wells/assay (Entzeroth, Flotow, & Condon, ; Macarron et al., )? Although laboratory work at times may be routine, the questions being asked often are not.…”

Section: Experimental Designmentioning

confidence: 99%

Logical Experimental Design and Execution in the Biomedical Sciences

Holder

Marino²

2017

CP Pharmacology

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Lack of reproducibility has been highlighted as a significant problem in biomedical research. The present unit is devoted to describing ways to help ensure that research findings can be replicated by others, with a focus on the design and execution of laboratory experiments. Essential components for this include clearly defining the question being asked, using available information or information from pilot studies to aid in the design the experiment, and choosing manipulations under a logical framework based on Mill's "methods of knowing" to build confidence in putative causal links. Final experimental design requires systematic attention to detail, including the choice of controls, sample selection, blinding to avoid bias, and the use of power analysis to determine the sample size. Execution of the experiment is done with care to ensure that the independent variables are controlled and the measurements of the dependent variables are accurate. While there are always differences among laboratories with respect to technical expertise, equipment, and suppliers, execution of the steps itemized in this unit will ensure well-designed and well-executed experiments to answer any question in biomedical research. © 2017 by John Wiley & Sons, Inc.

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Overview on High‐Throughput Screening

Cited by 15 publications

References 84 publications

Characterization of the histone methyltransferase PRDM9 using biochemical, biophysical and chemical biology techniques

Characterization of the histone methyltransferase PRDM9 using biochemical, biophysical and chemical biology techniques

High Content Screening of a Kinase-Focused Library Reveals Compounds Broadly-Active against Dengue Viruses

Logical Experimental Design and Execution in the Biomedical Sciences

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