Quantitative studies of inhibitors of ADP-ribosylation in vitro and in vivo.
The ADP-ribosyl moiety of NAD+ is consumed in reactions catalyzed by three classes of enzymes: poly(ADP-ribose) polymerase, protein mono(ADP-ribosyl)transferases, and NAD+ glycohydrolases. In this study, we have evaluated the selectivity of compounds originally identified as inhibitors of poly(ADP-ribose) polymerase on members of the three classes of enzymes. The 50% inhibitory concentration (IC50) of more than 20 compounds was determined in vitro for both poly(ADP-ribose) polymerase and mono(ADP-ribosyl)transferase A in an assay containing 300 microM NAD+. Of the compounds tested, benzamide was the most potent inhibitor of poly(ADP-ribose) polymerase with an IC50 of 3.3 microM. The IC50 for benzamide for mono(ADP-ribosyl)transferase A was 4.1 mM, and similar values were observed for four additional cellular mono(ADP-ribosyl)transferases. The IC50 for NAD+ glycohydrolase for benzamide was approximately 40 mM. For seven of the best inhibitors, inhibition of poly(ADP-ribose) polymerase in intact C3H1OT1/2 cells was studied as a function of the inhibitor concentration of the culture medium, and the concentration for 50% inhibition (culture medium IC50) was determined. Culture medium IC50 values for benzamide and its derivatives were very similar to in vitro IC50 values. For other inhibitors, such as nicotinamide, 5-methyl-nicotinamide, and 5-bromodeoxyuridine, culture medium IC50 values were 3-5-fold higher than in vitro IC50 values. These results suggest that micromolar levels of the benzamides in the culture medium should allow selective inhibition of poly(ADP-ribose) metabolism in intact cells. Furthermore, comparative quantitative inhibition studies should prove useful for assigning the biological effects of these inhibitors as an effect on either poly(ADP-ribose) or mono(ADP-ribose) metabolism.
Rankin
P W
PW
Department of Biochemistry, Texas College of Osteopathic Medicine, University of North Texas, Fort Worth 76107.
Jacobson
E L
EL
Benjamin
R C
RC
Moss
J
J
Jacobson
M K
MK
D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002417:Cattle; D009243:NAD / Q000494:pharmacology; D009244:NAD+ Nucleosidase / Q000037:antagonists & inhibitors*; D009536:Niacinamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000067856:Poly(ADP-ribose) Polymerase Inhibitors
D001549:Benzamides; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D009536:Niacinamide; C037689:benzamide; C024785:5-methylnicotinamide; C025160:3-aminobenzamide; D036002:ADP Ribose Transferases; D009244:NAD+ Nucleosidase; D001973:Bromodeoxyuridine; C025159:3-methoxybenzamide