Journal
ANALYTICAL BIOCHEMISTRY
Volume 603, Issue -, Pages -Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2020.113774
Keywords
Antithrombotic drug; Assay development; Ectonucleotidase; Recombinant enzyme expression; High-throughput screening; Luminescence detection; NPP4 inhibitors
Funding
- Federal Ministry of Education and Research (BMBF, BIGS DrugS project)
- Deutsche Forschungsgemeinschaft (DFG) [SFB1328]
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Nucleotide pyrophosphatase/phosphodiesterase 4 (NPP4) is a membrane-bound enzyme that hydrolyzes extracellular diadenosine polyphosphates such as diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap(4)A) yielding mononucleotides. NPP4 on the surface of endothelial cells was reported to promote platelet aggregation by hydrolyzing Ap(3)A to ADP, which activates pro-thrombotic G protein-coupled P2Y(1) and P2Y(12) receptors. Thus, NPP4 inhibitors have potential as novel antithrombotic drugs. In the present study we expressed soluble human NPP4 in Sf9 insect cells and established an enzyme assay using diadenosine tetraphosphate (Ap(4)A) as a substrate. The reaction product ATP was quantified by luciferin-luciferase reaction in a 96-well plate format. The sensitive method displayed a limit of detection (LOD) of 14.6 nM, and a Z'-factor of 0.68 indicating its suitability for high-throughput screening. The new assay was applied for studying enzyme kinetics and led to the identification of the first NPP4 inhibitors.
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