Rankin P W, Jacobson E L, Benjamin R C, Moss J, Jacobson M K
Department of Biochemistry, Texas College of Osteopathic Medicine, University of North Texas, Fort Worth 76107.
J Biol Chem. 1989 Mar 15;264(8):4312-7.
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.
NAD⁺的ADP - 核糖基部分在三类酶催化的反应中被消耗：聚（ADP - 核糖）聚合酶、蛋白质单（ADP - 核糖基）转移酶和NAD⁺糖水解酶。在本研究中，我们评估了最初被鉴定为聚（ADP - 核糖）聚合酶抑制剂的化合物对这三类酶成员的选择性。在含有300微摩尔NAD⁺的测定中，体外测定了20多种化合物对聚（ADP - 核糖）聚合酶和单（ADP - 核糖基）转移酶A的50%抑制浓度（IC50）。在所测试的化合物中，苯甲酰胺是聚（ADP - 核糖）聚合酶最有效的抑制剂，IC50为3.3微摩尔。苯甲酰胺对单（ADP - 核糖基）转移酶A的IC50为4.1毫摩尔，另外四种细胞单（ADP - 核糖基）转移酶也观察到类似的值。苯甲酰胺对NAD⁺糖水解酶的IC50约为40毫摩尔。对于七种最佳抑制剂，研究了完整C3H1OT1/2细胞中聚（ADP - 核糖）聚合酶的抑制作用与培养基中抑制剂浓度的关系，并确定了50%抑制浓度（培养基IC50）。苯甲酰胺及其衍生物的培养基IC50值与体外IC50值非常相似。对于其他抑制剂，如烟酰胺、5 - 甲基烟酰胺和5 - 溴脱氧尿苷，培养基IC50值比体外IC50值高3 - 5倍。这些结果表明，培养基中微摩尔水平的苯甲酰胺应能选择性抑制完整细胞中的聚（ADP - 核糖）代谢。此外，比较定量抑制研究对于确定这些抑制剂的生物学效应是对聚（ADP - 核糖）还是单（ADP - 核糖）代谢的影响应该是有用的。
