A High-Throughput Assay for Cyclic Nucleotide Phosphodiesterases

Schilling, Roger J.; Morgan, David R.; Kilpatrick, Brian F.

doi:10.1006/abio.1994.1019

Cited by 23 publications

(17 citation statements)

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“…This assay was chosen because it can potentially be upgraded to high throughput screening and because it produces less liquid radioactive waste than other procedures. The assay was validated and found to provide results essentially identical to those with other assay formats (40). The reaction contained 50 mM HEPES, pH 7.5, 0.5 mM EDTA, 10 mM MgCl 2 , and…”

Section: Tacgccggtattccaatgtagg-3ј; Openmentioning

confidence: 90%

Characterization of TbPDE2A, a Novel Cyclic Nucleotide-specific Phosphodiesterase from the Protozoan Parasite Trypanosoma brucei

Zoraghi¹,

Kunz

Gong³

et al. 2001

Journal of Biological Chemistry

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This study reports the identification and characterization of a cAMP-specific phosphodiesterase from the parasitic hemoflagellate Trypanosoma brucei. TbPDE2A is a class I phosphodiesterase. Its catalytic domain exhibits 30 -40% sequence identity with those of all 11 mammalian phosphodiesterase (PDE) families, as well as with PDE2 from Saccharomyces cerevisiae, dunce from Drosophila melanogaster, and regA from Dictyostelium discoideum. The overall structure of TbPDE2A resembles that of human PDE11A in that its N-terminal region contains a single GAF domain. This domain is very similar to those of the mammalian PDE2, -5, -6, -10, and -11, where it constitutes a potential cGMP binding site. TbPDE2A can be expressed in S. cerevisiae, and it complements an S. cerevisiae PDE deletion strain. Recombinant TbPDE2A is specific for cAMP, with a K m of ϳ2 M. It is entirely resistant to the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine, but it is sensitive to trequinsin, dipyridamole, sildenafil, and ethaverine with IC 50 values of 5.4, 5.9, 9.4, and 14.2 M, respectively. All four compounds inhibit proliferation of bloodstream form trypanosomes in culture, indicating that TbPDE2A is an essential enzyme.

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Section: Tacgccggtattccaatgtagg-3ј; Openmentioning

confidence: 90%

Characterization of TbPDE2A, a Novel Cyclic Nucleotide-specific Phosphodiesterase from the Protozoan Parasite Trypanosoma brucei

Zoraghi¹,

Kunz

Gong³

et al. 2001

Journal of Biological Chemistry

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“…[5][6][7] The most widely used assays are radiometric and employ 32 P-, 14 C-, or 3 H-labeled purine nucleotide substrate. 8,9 Traditionally, many PDE inhibitors originate from natural product sources, [10][11][12] and about one-third of all current drugs are from natural origin or derived from natural products. 13,14 Pharmacologically active compounds have been isolated from a wide variety of natural sources, ranging from marine organisms 15 to microbes.…”

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confidence: 99%

Screening of Natural Products Extracts for the Presence of Phosphodiesterase Inhibitors Using Liquid Chromatography Coupled Online to Parallel Biochemical Detection and Chemical Characterization

et al. 2003

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The ability to rapidly identify active compounds in a complex mixture (e.g., natural products extract) is still one of the major problems in natural products screening programs. An elegant way to overcome this problem is to separate the complex mixture by gradient liquid chromatography followed by online biochemical detection parallel with chemical characterization, referred to as high-resolution screening (HRS). To find and identify phosphodiesterase (PDE) inhibitors in natural products extracts using the HRS technology, the authors developed a continuous-flow PDE enzymatic assay. The suitability of the continuous-flow PDE enzymatic assay for natural products screening was demonstrated. After optimization of the continuousflow PDE assay, the limit of detection for 3-isobutyl-1-methyl-xanthine (IBMX) was 1 µM, with a dynamic range from 1 to 100 µM IBMX. The applicability of the HRS technology for the detection of PDE inhibitors in natural products extracts was demonstrated by the analysis of a plant extract spiked with 2 naturally occurring PDE inhibitors. The plant extract was analyzed with 2 assay lines in parallel, enabling background fluorescence correction of the sample. The simultaneous quantification of the active compounds using evaporative light-scattering detection allowed the estimation of the IC 50 value of the active compounds directly in the crude extract. (Journal of Biomolecular Screening 2003:421-429)

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“…Recent adaptation of the methodology has allowed the use of this assay in a 96-well format [3]. Another approach that has emerged uses selective precipitation, where either [ 3 H]cAMP in the supernatant or precipitated [ 3 H]AMP is measured [4]. The PDE assay has been adapted to the scintillation proximity assay (SPA) format, and this represents one of the most widely used contemporary methods [5,6].…”

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confidence: 99%

A reversed-phase HPLC-based method for the assay of cyclic nucleotide phosphodiesterase activity

Abbott

Thompson

2005

Analytical Biochemistry

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A High-Throughput Assay for Cyclic Nucleotide Phosphodiesterases

Cited by 23 publications

References 0 publications

Characterization of TbPDE2A, a Novel Cyclic Nucleotide-specific Phosphodiesterase from the Protozoan Parasite Trypanosoma brucei

Characterization of TbPDE2A, a Novel Cyclic Nucleotide-specific Phosphodiesterase from the Protozoan Parasite Trypanosoma brucei

Screening of Natural Products Extracts for the Presence of Phosphodiesterase Inhibitors Using Liquid Chromatography Coupled Online to Parallel Biochemical Detection and Chemical Characterization

A reversed-phase HPLC-based method for the assay of cyclic nucleotide phosphodiesterase activity

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