Pro-nucleotide Inhibitors of Adenylyl Cyclases in Intact Cells

• Published in: The Journal of biological chemistry 2004-04, Vol.279 (14), p.13317-13332
• Main Authors: Laux, Wolfgang H G, Pande, Praveen, Shoshani, Ilana, Gao, Junyuan, Boudou-Vivet, Valérie, Gosselin, Gilles, Johnson, Roger A
• Format: Article
• Language: English
• Subjects: Adenosine Monophosphate - analogs & derivatives; Adenosine Monophosphate - chemistry; Adenosine Monophosphate - pharmacology; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases - metabolism; Adipocytes - cytology; Animals; Cell Line; Cyclic AMP - metabolism; Guinea Pigs; Heart - drug effects; Humans; Macrophages - cytology; Macrophages - enzymology; Male; Mice; Myocardium - metabolism; Prodrugs - chemistry; Prodrugs - pharmacology; Stem Cells - cytology; Stem Cells - enzymology; Tritium
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M309535200

9-Substituted adenine derivatives with protected phosphoryl groups were synthesized and tested as inhibitors of adenylyl cyclase in isolated enzyme and intact cell systems. Protected 3′-phosphoryl derivatives of 2′,5′-dideoxyadenosine (2′,5′-dd-Ado) and β- l -2′,5′-dd-Ado, protected 5′-phosphoryl derivatives of β- l -2′,3′-dd-Ado, and protected phosphoryl derivatives of two 9-(2-phosphonomethoxy-acyl)-adenines were synthesized. Protection was afforded by two cyclosaligenyl- or three S- acyl-2-thioethyl-substituents. These pro-nucleotides were tested for their capacity to block forskolin-induced increases in [ 3 H]cAMP in OB1771 and F442A preadipocytes and human macrophages prelabeled with [ 3 H]adenine. A striking selectivity for 2′,5′-dd-Ado-3′-phosphoryl derivatives was observed. Cyclosaligenyl-derivatives (IC 50 ∼2 μ m ) were much less potent than S -acyl-2-thioethyl-derivatives. Best studied of these was 2′,5′-dd-Ado-3′ -O -bis( S -pivaloyl-2-thioethyl)-phosphate, which blocked [ 3 H]cAMP formation in preadipocytes (IC 50 ∼30 n m ) and suppressed opening of cAMP-dependent Cl – channels in cardiac myocytes (IC 50 ∼800 n m ). None of the pro-nucleotides inhibited adenylyl cyclase per se , whether isolated from rat brain or OB1771 cells. These compounds exhibit the hallmarks of prodrugs. Data suggest they are taken up, are deprotected, and are converted to a potent inhibitory form to inhibit adenylyl cyclase, but only by intact cells. The availability and characteristics of these prodrugs should make them useful for blocking cAMP-mediated pathways in intact cell systems, in biochemical, pharmacological, and potentially therapeutic contexts.