Anales RANF

P.23 DEVELOPMENT OF A HIGH-THROUGHPUT SCREENING ASSAY FOR THE IDENTIFICATION AND CHARACTERIZATION OF NUCLEOTIDE PYROPHOSPHATASE / PHOSPHODIESTERASE 4 (NPP4) INHIBITORS V. Lopez, a S-Y. Lee, a S. Holger b and C.E. Müller a a University of Bonn, Bonn, Germany; b Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany. Nucleotide pyrophosphatase/phosphodiesterase 4 (NPP4) is a membrane-bound enzyme that hydrolyzes extracellular diadenosine polyphosphates such as Ap 3 A and Ap 4 A. The NPP4, which is present on the surface of endothelial cells, was reported to promote platelet aggregation by hydrolyzing Ap 3 A to AMP and ADP, since ADP activates pro-thrombotic G protein-coupled P2Y 1 and P2Y 12 receptors. Thus, inhibitors of NPP4 have potential as novel anticoagulant drugs. To discover NPP4 inhibitors, a high-throughput screening method is required. In the present study, we developed a luminescence-based assay using recombinant human soluble NPP4 expressed in Sf9 insect cells, and 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 method is highly sensitive with a limit of detection (LOD) and a limit of quantification (LOQ) of 16.6 nM, and 44.3 nM, respectively. The determined Z’-factor was 0.68 indicating that the developed assay is suitable for high-throughput screening. Subsequently, we applied it to studying the enzyme kinetics of NPP4 and for inhibitor screening. This led to the discovery of the first NPP4 inhibitors, which are required for target validation studies.