Development and Validation of a Transcreener Assay for Detection of AMP- and GMP-Producing Enzymes

Staeben, Matt; Kleman-Leyer, Karen; Kopp, Andrew L.; Westermeyer, Thane A.; Lowery, Robert G.

doi:10.1089/adt.2009.0254

Cited by 31 publications

(41 citation statements)

References 28 publications

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“…Successful off-label use of the PDE3 inhibitor enoximone was described in the context of status asthmaticus (32). Next to its PDE3 inhibitory properties (IC50 = 5.9 μM), enoximone also exhibits some PDE4 inhibitory effect, more specifically a myocardial PDE4A inhibitory effect (IC50 = 21.1 μM) (57,58). Cardiomyocytes express mainly PDE4A1A (59).…”

Section: Pde3a -/-And Pde3b -/-Mice Show Reduced Albumin Leakage In Amentioning

confidence: 99%

A pathophysiological role of PDE3 in allergic airway inflammation

et al. 2018

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Section: Pde3a -/-And Pde3b -/-Mice Show Reduced Albumin Leakage In Amentioning

confidence: 99%

A pathophysiological role of PDE3 in allergic airway inflammation

et al. 2018

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“…To circumvent the problems associated with peptide substrate-based kinase assays, the Transcreener ™ FPIA platform (BellBrook Labs, Madison, WI) detects the invariant nucleotide product through high-affinity nucleotide-specific antibodies, obviating the need for kinase-specific antibody. The Transcreener ™ assay is comprised of a red-shifted fluorophore labeled nucleotide, an antibody that binds to the nucleotide with high affinity and specificity [55, 56], and quench buffer used to stop the enzymatic reaction. Similar to competitive FPIA assays that utilize phosphospecific antibodies (Figure 3C), Transcreener ™ FPIA assays follow a reduction in FP value as the nucleotides generated by reaction displace tracer bound by the antibodies (Figure 3D).…”

Section: Fp Applicationsmentioning

confidence: 99%

Fluorescence polarization assays in small molecule screening

Lea

Simeonov²

2010

Expert Opinion on Drug Discovery

307

225

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Importance of the field Fluorescence polarization (FP) is a homogeneous method that allows rapid and quantitative analysis of diverse molecular interactions and enzyme activities. This technique has been widely utilized in clinical and biomedical settings, including the diagnosis of certain diseases and monitoring therapeutic drug levels in body fluids. Recent developments in the field has been symbolized by the facile adoption of FP in high-throughput screening (HTS) and small molecule drug discovery of an increasing range of target classes. Areas covered in this review The article provides a brief overview on the theoretical foundation of FP, followed by updates on recent advancements in its application for various drug target classes, including G-protein coupled receptors (GPCRs), enzymes and protein-protein interactions (PPIs). The strengths and weaknesses of this method, practical considerations in assay design, novel applications, and future directions are also discussed. What the reader will gain The reader will be informed of the most recent advancements and future directions of FP application to small molecule screening. Take home message In addition to its continued utilization in high-throughput screening, FP has expanded into new disease and target areas and has been marked by increased use of labeled small molecule ligands for receptor binding studies.

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“…BellBrook has developed a line of HTS assays, called Transcreener, that use highly specific immunodetection of nucleotide reaction products with homogenous fluorescent readouts including FP, TRF and FI 15; 19; 24 . Direct immunodetection of SAH would be advantageous as it would eliminate the potential for compound interference with coupling enzymes, however it requires an antibody that specifically binds SAH in the presence of excess SAM; ie, that differentiates on the basis of a single methyl group.…”

Section: Introductionmentioning

confidence: 99%

Development and Validation of a Generic Fluorescent Methyltransferase Activity Assay Based on the Transcreener AMP/GMP Assay

et al. 2012

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Methylation is a ubiquitous covalent modification used to control the function of diverse biomolecules including hormones, neurotransmitters, xenobiotics, proteins, nucleic acids and lipids. Histone methyltransferases (HMTs) are currently of high interest as drug targets because of their role in epigenetic regulation, however most HMT assay methods are either not amenable to an HTS environment or are applicable to a limited number of enzymes. We developed a generic methyltransferase assay method using fluorescent immunodetection of AMP, which is formed from the MT reaction product S-adenosylhomocysteine in a dual enzyme coupling step. The detection range of the assay, its suitability for HTS, including stability of reagents following dispensing and after addition to reactions as well as the potential for interference from drug like molecules was investigated. In addition, the use of the assay for measuring inhibitor potencies with peptide or intact protein substrates was examined through pilot screening with selected reference enzymes including HMT G9a. By combining a novel enzymatic coupling step with the well characterized Transcreener® AMP/GMP assay, we have developed a robust HTS assay for HMTs which should be broadly applicable to other types of methyltransferases as well.

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Development and Validation of a Transcreener Assay for Detection of AMP- and GMP-Producing Enzymes

Abstract: Screening of AMP-and GMP-producing enzymes such as phosphodiesterases (PDEs), ligases, and synthetases would be

Cited by 31 publications

References 28 publications

A pathophysiological role of PDE3 in allergic airway inflammation

A pathophysiological role of PDE3 in allergic airway inflammation

Fluorescence polarization assays in small molecule screening

Development and Validation of a Generic Fluorescent Methyltransferase Activity Assay Based on the Transcreener AMP/GMP Assay

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