Specificity of the ecto-ATPase inhibitor ARL 67156 on human and mouse ectonucleotidases.
OBJECTIVE
ARL 67156, 6-N,N-Diethyl-D-beta-gamma-dibromomethylene adenosine triphosphate, originally named FPL 67156, is the only commercially available inhibitor of ecto-ATPases. Since the first report on this molecule, various ectonucleotidases responsible for the hydrolysis of ATP at the cell surface have been cloned and characterized. In this work, we identified the ectonucleotidases inhibited by ARL 67156.


METHODS
The effect of ARL 67156 on recombinant NTPDase1, 2, 3 & 8 (mouse and human), NPP1, NPP3 and ecto-5'-nucleotidase (human) have been evaluated. The inhibition of the activity of NTPDases (using the following substrates: ATP, ADP, UTP), NPPs (pnp-TMP, Ap(3)A) and ecto-5'-nucleotidase (AMP) was measured by colorimetric or HPLC assays.


RESULTS
ARL 67156 was a weak competitive inhibitor of human NTPDase1, NTPDase3 and NPP1 with K(i) of 11+/-3, 18+/-4 and 12+/-3 microM, respectively. At concentrations used in the literature (50-100 microM), ARL 67156 partially but significantly inhibited the mouse and human forms of these enzymes. NTPDase2, NTPDase8, NPP3 and ecto-5'-nucleotidase activities were less affected. Importantly, ARL 67156 was not hydrolysed by either human NTPDase1, 2, 3, 8, NPP1 or NPP3.


CONCLUSIONS
In cell environments where NTPDase1, NTPDase3, NPP1 or mouse NTPDase8 are present, ARL 67156 would prolong the effect of endogenously released ATP on P2 receptors. However, it does not block any ectonucleotidases efficiently when high concentrations of substrates are present, such as in biochemical, pharmacological or P2X(7) assays. In addition, ARL 67156 is not an effective inhibitor of NTPDase2, human NTPDase8, NPP3 and ecto-5'-nucleotidase.
Lévesque
S A
SA
Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Université Laval, Québec, QC, Canada.
Lavoie
E G
EG
Lecka
J
J
Bigonnesse
F
F
Sévigny
J
J
D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001081:Apyrase / Q000037:antagonists & inhibitors / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D009708:Nucleotidases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013379:Substrate Specificity; D013938:Thymidine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
C092431:6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP; D015703:Antigens, CD; D015226:Dinucleoside Phosphates; C427944:ENPP3 protein, human; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C032579:thymidine 5'-4-nitrophenyl phosphate; D013938:Thymidine Monophosphate; D000249:Adenosine Monophosphate; C011440:diadenosine triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009708:Nucleotidases; D015720:5'-Nucleotidase; D010727:Phosphoric Diester Hydrolases; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D000251:Adenosine Triphosphatases; D011755:Pyrophosphatases; C027201:ectoATPase; C439652:nucleoside-triphosphate diphosphohydrolase 3; C518948:nucleoside triphosphate diphosphohydrolase 8, human; C489209:nucleoside triphosphate diphosphohydrolase 8, mouse; D001081:Apyrase; C067869:CD39 antigen; D014544:Uridine Triphosphate