Pro-nucleotide Inhibitors of Adenylyl Cyclases in Intact Cells

Abstract

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 ␤-L-2,5-ddAdo, protected 5-phosphoryl derivatives of ␤-L-2,3-ddAdo, 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 ϳ2 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 ϳ30 nM) and suppressed opening of cAMP-dependent Cl ؊ channels in cardiac myocytes (IC 50 ϳ800 nM). 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.

International Union of Basic and Clinical Pharmacology. CI. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases

“…Membrane-permeable Membrane Adenylyl Cyclase Inhibitors. The issues with impermeability of the plasma membrane for nucleotides can be avoided by applying membrane-permeable nucleotide prodrugs (Laux et al, 2004), but this strategy has not yet found broad application in the field, leaving many of the most potent AC inhibitors with little cellular utility. As an alternative strategy, membrane-permeable and hence lipophilic nucleoside analogs can be used in intact cell studies.…”

Differential Inhibition of Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by Purine and Pyrimidine Nucleotides

“…Our data clearly show that the future design of competitive AC inhibitors should not be limited to adenine nucleotides (30) but should also include guanine, hypoxanthine, and uracil nucleotides. By analogy to P-site inhibitors (85), prodrugs of the competitive AC inhibitors described in this study may become valuable tools for the analysis of the function of specific AC isoforms in intact cells. It is also promising to develop P-site inhibitors with a polyphosphate chain at the 3Ј-O-ribosyl position but bases other than adenine (24,25,28,83).…”

Characterization of Mouse Heart Adenylyl Cyclase

“…By analogy to antiviral drugs, lipophilic nucleotide prodrugs with high bioavailability have to be designed. Conversion of the nucleotide prodrug to the active nucleoside 5Ј-triphosphate by phosphorylation is then accomplished by specific kinases (Laux et al, 2004). It is noteworthy that MANTNTPs and the corresponding MANT-NTP␥Ss possess comparable potencies on heart AC.…”