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

Abstract

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 μM 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 Ki,app values of the seven most potent and selective inhibitors were in the range of 0.5-56 μM, 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 Ki,app (0.5 μM) was also inactive toward P2Y receptors. Overall, analogue 2 is the most potent and selective NPP1 inhibitor described so far.

Highly Potent and Selective Ectonucleotide Pyrophosphatase/Phosphodiesterase I Inhibitors Based on an Adenosine 5′-(α or γ)-Thio-(α,β- or β,γ)-methylenetriphosphate Scaffold

“…As previously reported, 22 the preferred binding of all analogues to NPP1 rather than to NPP3 could be readily explained by comparing the binding sites of the two proteins. In NPP1, a unique arrangement of Lys residues (seven in total) which is absent in NPP3 (a single binding site Lys residue) results in a highly positive electrostatic potential which renders this site more suitable for binding negatively charged nucleotides.…”

“…On the basis of docking results, such coordination is not possible for the borano compound since in this compound, the nonchelating borano group at P α points toward the Zn1 ion. 22 …”

“…Recent reports suggested that quinazoline-4-piperidine-4-methylsulfamide is an NPP1 inhibitor lacking binding affinity for hERG 19 and that 1,3,4-oxadiazole(thiadiazole)-2(3 H )-thiones are noncompetitive human NPP1 inhibitors with K i values of 360 μ M. 20 Previously, we reported that bis(2′-deoxyadenosine) α , β:δ , ε -dimethylenepentaphosphonate (Figure 1A) is an NPP1 inhibitor with an IC 50 of 13 μ M and a K i of 9 μ M. 21 In addition, we recently reported that adenosine 5′- α -borano- β , γ -methylenetriphosphate (isomer A) (Figure 1B) is a selective inhibitor of NPP1 with a K i of 0.5 μ M. 22 …”

Zinc deficiency causes delayed ATP clearance and adenosine generation in rats and cell culture models

“…ENPP activity was measured as described elsewhere 51 with certain modifications. Briefly, 3 μg of membrane proteins or 5 μL of rat plasma was incubated at 37 °C for 10 min, and then 100 μL of 0.1 mM p -nitrophenyl thymidine 5′-monophosphate ( p NP-TMP; Sigma) solution was added and incubated at 37 °C for 15 min.…”

Structural basis for nucleotide recognition by the ectoenzyme CD203c

“…Despite these multiple possible roles, the biochemistry of NPP3 has not been extensively characterized, and the determinants of its substrate specificity are unknown. The 3D structures of all the other mammalian NPPs have been elucidated [11,[22][23][24][25][26][27][28], whereas NPP3 has been the subject of modeling studies to assist isozyme-specific inhibitor design [29][30][31]. Here, we report the crystal structure of human NPP3 as well as its complex with a substrate analog, and explore the dimerization properties of this enzyme.…”