Abstract 18748: High Resolution Structure Determination and Small Molecule Inhibitor Identification of NPP4, a Procoagulant Enzyme Localized to Brain Vascular Endothelium

Abstract

We recently identified the enzyme NPP4, an uncharacterized member of the nucleotide pyrophosphatase/phosphodiesterase family, as present at high concentrations on the vascular surface of the brain and promotes platelet aggregation in a dose dependent manner at physiologic concentrations of AP3A. AP3A is a small molecule stored at high concentrations in platelet dense core granules and is released upon platelet activation at high concentrations in the vicinity of the growing thrombus. Ap3A has a significantly longer lifespan in whole blood than ADP and has long been thought to promote stable thrombus formation by serving as a ‘chemically masked’ source of ADP. The goal of the present study was to determine the high-resolution three-dimensional structure of NPP4, and to identify small molecule inhibitors of NPP4 useful for the in vitro and in vivo validation of the physiologic role of the enzyme. We used X-ray crystallography to determine the high-resolution structure of the enzyme, and high-throughput small molecule screening to identify lead small molecules as lead inhibitors. The three-dimensional structure of the enzyme was determined to1.5Å resolution in the presence and absence of its enzymatic product (AMP), and high-affinity (nM) small molecule inhibitors of NPP4 were identified as lead inhibitors in high throughput small molecule screens. Structure-activity relationships (SAR) of the lead compound were explored via chemical derivatization, and three-dimensional structures of the lead compounds were obtained to assist a structure-guided approach to lead optimization. Our combined studies lay the foundation for the validation of the biological role of NPP4 in hemostasis and establishes the necessary tools and reagents for the translational application of our preliminary findings into the clinic.