Nonhydrolyzable ATP Analogues as Selective Inhibitors of Human NPP1: A Combined Computational/Experimental Study

Elevated nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is implicated in health disorders including pathological calcification. Specific NPP1 inhibitors would therefore be valuable for studying this enzyme and as potential therapeutic agents. Here we present a combined computational/experimental study characterizing 13 nonhydrolyzable ATP analogues as selective human NPP1 inhibitors. All analogues at 100 mu M inhibited (66-99%) the hydrolysis of pnp-TMP by both recombinant NPP1 and cell surface NPP1 activity of osteocarcinoma (HTB-85) cells. These analogues only slightly altered the activity of other ectonucleotidases, NPP3 and NTPDases. The K-i,K-app values of the seven most potent and selective inhibitors were in the range of 0.5-56 mu M, all with mixed type inhibition, predominantly competitive. Those molecules were docked into a newly developed homology model of human NPP1. All adopted ATP-like binding modes, suggesting competitive inhibition with the endogenous ligand. NPP1 selectivity versus NPP3 could be explained in terms of the electrostatic potential of the two proteins that of NPP1 favoring negatively charged ligands. Inhibitor 2 that had the lowest K-i,K-app (0.5 mu M) was also inactive toward P2Y receptors. Overall, analogue 2 is the most potent and selective NPP1 inhibitor described so far.