Probing Aplysia californica Adenosine 5'-Diphosphate Ribosyl Cyclase for Substrate Binding Requirements: Design of Potent Inhibitors

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• 作者：Marie E. Migaud ; Richard L. Pederick ; Victoria C. Bailey ; and Barry V. L. Potter
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：July 13, 1999
• 年：1999
• 卷：38
• 期：28
• 页码：9105 - 9114
• 全文大小：239K
• 年卷期：v.38,no.28(July 13, 1999)
• ISSN：1520-4995

文摘

Readily synthesized nicotinamide adenine dinucleotide (NAD⁺) analogues have been used toinvestigate 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 potentinhibitors of this enzyme. In all cases, inhibition of Aplysia californica cyclase by various substrateanalogues was found to be competitive while inhibition by nicotinamide exhibited mixed-behaviorcharacteristics. Nicotinamide hypoxanthine dinucleotide (NHD⁺), nicotinamide guanine dinucleotide(NGD⁺), C1'-m-benzamide adenine dinucleotide (Bp₂A), and C1'-m-benzamide nicotinamide dinucleotide(Bp₂N) were found to be nanomolar potency inhibitors with inhibition constants of 70, 143, 189, and 201nM, respectively. However, NHD⁺ and NGD⁺ are also known substrates and are slowly converted tocyclic products, thus preventing their further use as inhibitors. The symmetrical bis-nucleotides, bis-adenine dinucleotide (Ap₂A), bis-hypoxanthine dinucleotide (Hp₂H), and bis-nicotinamide dinucleotide(Np₂N), exhibited micromolar competitive inhibition, with Ap₂A displaying the greatest affinity for theenzyme. 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 thecyclase by adenosine 5'-O-diphosphate ribose (ADPR) and inosine 5'-O-diphosphate ribose (IDPR) wasobserved at millimolar concentration. The nicotinamide aromatic ring appears to be the optimal motifrequired for enzymatic recognition, while modifications of the 2'- and 3'-hydroxyls of the nicotinamideribose seem to hamper binding to the enzyme. Stabilizing enzyme/inhibitor interactions and the inabilityof 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 nowprovides the first potent nonhydrolyzable sea urchin ADP ribosyl cyclase and cADPR hydrolase inhibitorBp₂A, with inhibition observed at the micromolar and nanomolar level, respectively. The benzamidederivatives did not inhibit CD38 cyclase or hydrolase activity when NGD⁺ was used as substrate. Theseresults emphasize the difference between CD38 and other enzymes in which the cADPR cyclase activitypredominates.