8‐BuS‐ATP derivatives as specific NTPDase1 inhibitors

Abstract

Background and Purpose Ectonucleotidases control extracellular nucleotide levels and consequently, their (patho)physiological responses. Among these enzymes, nucleoside triphosphate diphosphohydrolase‐1 (NTPDase1), −2, −3 and −8 are the major ectonucleotidases responsible for nucleotide hydrolysis at the cell surface under physiological conditions, and NTPDase1 is predominantly located at the surface of vascular endothelial cells and leukocytes. Efficacious inhibitors of NTPDase1 are required to modulate responses induced by nucleotides in a number of pathological situations such as thrombosis, inflammation and cancer. Experimental Approach Here, we present the synthesis and enzymatic characterization of five 8‐BuS‐adenine nucleotide derivatives as potent and selective inhibitors of NTPDase1. Key Results The compounds 8‐BuS‐AMP, 8‐BuS‐ADP and 8‐BuS‐ATP inhibit recombinant human and mouse NTPDase1 by mixed type inhibition, predominantly competitive with Ki values <1 μM. In contrast to 8‐BuS‐ATP which could be hydrolyzed by other NTPDases, the other BuS derivatives were resistant to hydrolysis by either NTPDase1, −2, −3 or −8. 8‐BuS‐AMP and 8‐BuS‐ADP were the most potent and selective inhibitors of NTPDase1 expressed in human umbilical vein endothelial cells as well as in situ in human and mouse tissues. As expected, as a result of their inhibition of recombinant human NTPDase1, 8‐BuS‐AMP and 8‐BuS‐ADP impaired the ability of this enzyme to block platelet aggregation. Importantly, neither of these two inhibitors triggered platelet aggregation nor prevented ADP‐induced platelet aggregation, in support of their inactivity towards P2Y1 and P2Y12 receptors. Conclusions and Implications The 8‐BuS‐AMP and 8‐BuS‐ADP have therefore potential to serve as drugs for the treatment of pathologies regulated by NTPDase1.

Ticlopidine in Its Prodrug Form Is a Selective Inhibitor of Human NTPDase1

“…We previously observed that ticlopidine inhibited recombinant human NTPDase1 [46]. In this work we verified if this compound can be used as a specific inhibitor of the enzyme.…”

“…Sections of 6  μ m were obtained and fixed in 10% phosphate-buffered formalin mixed with cold acetone as before [46]. Localization of ectonucleotidase activities was determined using the Wachstein/Meisel lead phosphate method [47].…”

Big opportunities for small molecules in immuno-oncology

“…However, these tool compounds have many shortcomings 108 . ARL 67176 and 8-butylthio-ATP are nucleotide analogues with marginal selectivity and potency, and although they have been used for in vivo experiments, the pharmacokinetic behaviour of these compounds is unknown 109,110 . Polyoxotungstate (POM-1), which is a high-molecular-mass metal complex, raises similar concerns.…”

Fluorescence polarization immunoassays for monitoring nucleoside triphosphate diphosphohydrolase (NTPDase) activity

“…36,37 Briey, COS-7 cells (in 15 cm dishes) were transfected with an expression vector (pcDNA3) incorporating the cDNA that encodes each ectonucleotidase using Lipofectamine (Invitrogen) and harvested 40-72 h later. Cell membranes were prepared as described elsewhere.…”