ArticleAssay and drug development technologies2009
Characterization and optimization of a red-shifted fluorescence polarization ADP detection assay.
Article in Assay and drug development technologies, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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Who cites it
19 citing papers in PubMed, 45 citations in OpenAlex.
- ATP is dispensable for E. coli DNA replication and eukaryotic helicase activity.Nature communications · 2026Article
- Discovery and optimization of narrow spectrum inhibitors of Tousled like kinase 2 (TLK2) using quantitative structure activity relationships.European journal of medicinal chemistry · 2024Article
- Discovery and Optimization of Narrow Spectrum Inhibitors of Tousled Like Kinase 2 (TLK2) Using Quantitative Structure Activity Relationships.bioRxiv : the preprint server for biology · 2023Article
- Fluorescence Polarization-Based Bioassays: New Horizons.Sensors (Basel, Switzerland) · 2020Review
- Molecular Characterization of a Fungus Producing Membrane Active Metabolite and Analysis of the Produced Secondary MetaboliteIranian biomedical journal · 2019Article
- Bioluminescence Methods for Assaying Kinases in Quantitative High-Throughput Screening (qHTS) Format Applied to Yes1 Tyrosine Kinase, Glucokinase, and PI5P4Kα Lipid Kinase.Methods in molecular biology (Clifton, N.J.) · 2016Article
- Method for Assaying the Lipid Kinase Phosphatidylinositol-5-phosphate 4-kinase α in Quantitative High-Throughput Screening (qHTS) Bioluminescent Format.Methods in molecular biology (Clifton, N.J.) · 2016Article
- Development of a Sensitive Assay for SERCA Activity Using FRET Detection of ADP.Analytical methods : advancing methods and applications · 2014Article
- High-throughput screening for RecA inhibitors using a transcreener adenosine 5'-O-diphosphate assay.Assay and drug development technologies · 2012Article
- Development and validation of a generic fluorescent methyltransferase activity assay based on the transcreener AMP/GMP assay.Journal of biomolecular screening · 2012Article
- Regulators of G-protein signaling and their Gα substrates: promises and challenges in their use as drug discovery targets.Pharmacological reviews · 2011Review
- Analysis of protein-ligand interactions by fluorescence polarization.Nature protocols · 2011Article
- Phosphatase-coupled universal kinase assay and kinetics for first-order-rate coupling reaction.PloS one · 2011Article
- Fluorescence polarization assays in small molecule screening.Expert opinion on drug discovery · 2011Article
- Development and validation of a transcreener assay for detection of AMP- and GMP-producing enzymes.Assay and drug development technologies · 2010Article
- A fluorescent, reagentless biosensor for ADP based on tetramethylrhodamine-labeled ParM.ACS chemical biology · 2010Article
- Two Galpha(i1) rate-modifying mutations act in concert to allow receptor-independent, steady-state measurements of RGS protein activity.Journal of biomolecular screening · 2009Article
- Comparison of bioluminescent kinase assays using substrate depletion and product formation.Assay and drug development technologies · 2009Article
- A biosensor for fluorescent determination of ADP with high time resolution.The Journal of biological chemistry · 2009Article
Corrections and comments
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Authors and funding
12 authors at 2 institutions in 1 country.
Funding
Abstract
ATP depletion and ADP formation are generic detection methods used for the identification of kinase and other ATP-utilizing enzyme inhibitors in high-throughput screening campaigns. However, the most widely used nucleotide detection approaches require high ATP consumption rates or involve the use of coupling enzymes, which can complicate the selection of lead compounds. As an alternative, we have developed the Transcreener (BellBrook Labs, Madison, WI) platform, which relies on the direct immunodetection of nucleotides. Here we describe the development of antibodies with >100-fold selectivity for ADP versus ATP, which enable robust detection of initial velocity rates (Z' > 0.7 at 10% substrate consumption) at ATP concentrations ranging from 0.1 microM to 1,000 microM in a competitive fluorescence polarization (FP) immunoassay. Competitive binding experiments indicate similar affinities for other nucleotide diphosphates, including 2' -deoxy ADP, GDP, and UDP. The antibody-tracer complex and the red-shifted, ratiometric FP signal are stable for at least 24 h at room temperature, providing suitable conditions for high-throughput screening. A method for calculating a kinase ATP Km with this FP immunoassay is also presented. The Transcreener ADP assay provides a simple, generic assay platform for inhibitor screening and selectivity profiling that can be used for any ADP-generating enzyme.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.