Migaud M E, Pederick R L, Bailey V C, Potter B V
Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, U.K.
Biochemistry. 1999 Jul 13;38(28):9105-14. doi: 10.1021/bi9903392.
Readily synthesized nicotinamide adenine dinucleotide (NAD(+)) analogues have been used to investigate aspects of the cyclization of NAD(+) to cyclic adenosine 5'-O-diphosphate ribose (cADPR) catalyzed by the enzyme adenosine 5'-O-diphosphate (ADP) ribosyl cyclase and to produce the first potent inhibitors of this enzyme. In all cases, inhibition of Aplysia californica cyclase by various substrate analogues was found to be competitive while inhibition by nicotinamide exhibited mixed-behavior characteristics. Nicotinamide hypoxanthine dinucleotide (NHD(+)), nicotinamide guanine dinucleotide (NGD(+)), C1'-m-benzamide adenine dinucleotide (Bp(2)A), and C1'-m-benzamide nicotinamide dinucleotide (Bp(2)N) were found to be nanomolar potency inhibitors with inhibition constants of 70, 143, 189, and 201 nM, respectively. However, NHD(+) and NGD(+) are also known substrates and are slowly converted to cyclic products, thus preventing their further use as inhibitors. The symmetrical bis-nucleotides, bis-adenine dinucleotide (Ap(2)A), bis-hypoxanthine dinucleotide (Hp(2)H), and bis-nicotinamide dinucleotide (Np(2)N), exhibited micromolar competitive inhibition, with Ap(2)A displaying the greatest affinity for the enzyme. 2',3'-Di-O-acetyl nicotinamide adenine dinucleotide (AcONAD(+)) was not a substrate for the A. californica cyclase but also displayed some inhibition at a micromolar level. Finally, inhibition of the cyclase by adenosine 5'-O-diphosphate ribose (ADPR) and inosine 5'-O-diphosphate ribose (IDPR) was observed at millimolar concentration. The nicotinamide aromatic ring appears to be the optimal motif required for enzymatic recognition, while modifications of the 2'- and 3'-hydroxyls of the nicotinamide ribose seem to hamper binding to the enzyme. Stabilizing enzyme/inhibitor interactions and the inability of the enzyme to release unprocessed material are both considered to explain nanomolar inhibition. Recognition of inhibitors by other ADP ribosyl cyclases has also been investigated, and this study now provides the first potent nonhydrolyzable sea urchin ADP ribosyl cyclase and cADPR hydrolase inhibitor Bp(2)A, with inhibition observed at the micromolar and nanomolar level, respectively. The benzamide derivatives did not inhibit CD38 cyclase or hydrolase activity when NGD(+) was used as substrate. These results emphasize the difference between CD38 and other enzymes in which the cADPR cyclase activity predominates.
易于合成的烟酰胺腺嘌呤二核苷酸（NAD(+)）类似物已被用于研究由腺苷5'-O-二磷酸（ADP）核糖基环化酶催化的NAD(+)环化为环状腺苷5'-O-二磷酸核糖（cADPR）的各个方面，并产生了该酶的首批强效抑制剂。在所有情况下，发现各种底物类似物对加州海兔环化酶的抑制作用具有竞争性，而烟酰胺的抑制作用表现出混合行为特征。烟酰胺次黄嘌呤二核苷酸（NHD(+)）、烟酰胺鸟嘌呤二核苷酸（NGD(+)）、C1'-间苯甲酰胺腺嘌呤二核苷酸（Bp(2)A）和C1'-间苯甲酰胺烟酰胺二核苷酸（Bp(2)N）被发现是纳摩尔效力的抑制剂，抑制常数分别为70、143、189和201 nM。然而，NHD(+)和NGD(+)也是已知的底物，会缓慢转化为环状产物，因此无法进一步用作抑制剂。对称的双核苷酸，双腺嘌呤二核苷酸（Ap(2)A）、双次黄嘌呤二核苷酸（Hp(2)H）和双烟酰胺二核苷酸（Np(2)N），表现出微摩尔级的竞争性抑制，其中Ap(2)A对该酶表现出最大的亲和力。2',3'-二-O-乙酰基烟酰胺腺嘌呤二核苷酸（AcONAD(+)）不是加州海兔环化酶的底物，但在微摩尔水平也表现出一定的抑制作用。最后，在毫摩尔浓度下观察到腺苷5'-O-二磷酸核糖（ADPR）和肌苷5'-O-二磷酸核糖（IDPR）对环化酶的抑制作用。烟酰胺芳香环似乎是酶识别所需的确最佳基序，而烟酰胺核糖2'-和3'-羟基的修饰似乎会阻碍与酶的结合。稳定的酶/抑制剂相互作用以及酶无法释放未加工的物质都被认为可以解释纳摩尔级的抑制作用。还研究了其他ADP核糖基环化酶对抑制剂的识别，并且本研究现在提供了第一种强效的不可水解的海胆ADP核糖基环化酶和cADPR水解酶抑制剂Bp(2)A，分别在微摩尔和纳摩尔水平观察到抑制作用。当使用NGD(+)作为底物时，苯甲酰胺衍生物不抑制CD38环化酶或水解酶活性。这些结果强调了CD38与其他以cADPR环化酶活性为主的酶之间的差异。
