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

Abstract

Aberrant nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is associated with chondrocalcinosis, osteoarthritis, and type 2 diabetes. The potential of NPP1 inhibitors as therapeutic agents, and the scarceness of their structure–activity relationship, encouraged us to develop new NPP1 inhibitors. Specifically, we synthesized ATP-α-thio-β,γ- CH2 (1), ATP-α-thio-β,γ-CCl2 (2), ATP-α-CH2-γ-thio (3), and 8-SH-ATP (4) and established their resistance to hydrolysis by NPP1,3 and NTPDase1,2,3,8 (<5% hydrolysis) (NTPDase = ectonucleoside triphosphate diphosphohydrolase). Analogues 1–3 at 100 μM inhibited thymidine 5′-monophosphate p-nitrophenyl ester hydrolysis by NPP1 and NPP3 by >90% and 23–43%, respectively, and only slightly affected (0–40%) hydrolysis of ATP by NTPDase1,2,3,8. Analogue 3 is the most potent NPP1 inhibitor currently known, Ki = 20 nM and IC50 = 0.39 μM. Analogue 2a is a selective NPP1 inhibitor with Ki = 685 nM and IC50 = 0.57 μM. Analogues 1–3 were found mostly to be nonagonists of P2Y1/P2Y2/P2Y11 receptors. Docking analogues 1–3 into the NPP1 model suggested that activity correlates with the number of H-bonds with binding site residues. In conclusion, we propose analogues 2a and 3 as highly promising NPP1 inhibitors.

A critical look at the function of the P2Y11 receptor

“…Many other nucleotide compounds target the P2Y 11 receptor, including diadenosine polyphosphate Ap2A and its isomers P18 and P24 [56,57]. Nicotinamide adenine dinucleotide (NAD + ) is also capable of activating P2Y 11 and causing an increase in intracellular Ca 2+ , IP 3 , and cAMP in P2Y 11 transfected 1321N1 cells [58,59].…”

ATP-γ-S-(α,β-CH2) protects against oxidative stress and amyloid beta toxicity in neuronal culture

“…ATP-g-S-(a,b-CH 2 ) was prepared according to Nadel et al [12]. Trolox was obtained from Acros Organics (Morris Plains, NJ).…”

“…Likewise, ATP-g-S-(a,b-CH 2 ) at 100 mM was not catabolized by NPP1 or NPP3 and was highly stable to tissue non-specific alkaline phosphatase (TNAP) hydrolysis. Furthermore, ATP-g-S-(a,b-CH 2 ) was found to be an potent NPP1 inhibitor, Ki 20 nM [12].…”

“…Recently we reported that ATP-g-S-(a,b-CH 2 ) was a 6.7-fold more potent P2Y11-R agonist than the endogenous agonist, ATP (EC 50 1 and 6.7 mM, respectively), and was selective to P2Y11-R exhibiting no activity at P2Y1 or P2Y2 receptors [12].…”

“…The highly promising properties of ATP-g-S-(a,b-CH 2 ) as a Fenton reaction inhibitor [10], an effective ion chelator [16], a powerful agent for in-vitro dissolution of Ab 40/42 -M(II) oligomers and aggregates [10], together with its enzymatic stability, and its high activity at P2Y11-R [12], encouraged us to explore the potential of ATP-g-S-(a,b-CH 2 ) for the rescue of primary neurons subjected to insults typical of Alzheimer's disease.…”