Inhibition of adenylyl cyclase by a family of newly synthesized adenine nucleoside 3'-polyphosphates

The synthesis of a number of adenine nucleoside 3'-polyphosphates has been devised via a phosphotriester approach that combines the method of alkoxide activation with the use of 2,2,2-tribromoethyl phosphoromorpholinochloridate as a phosphorylating agent, The family of compounds included 3'ADP, 3'ATP, 2'-deoxy-3'ADP, 2'-deoxy-3'ATP, 2',5'-dideoxy-3'ADP, and 2',5'-dideoxy-3'ATP. Potency as inhibitors of adenylyl cyclases followed the order: 3'-mono- < 3'-di- < 3'-triphosphate and adenosine (Ado) < 2'-d-Ado < 2',5'-dd-Ado derivatives, with 2',5'-dideoxy-3'ATP exhibiting an IC50 of similar to 40 nM. This order was maintained with purified and recombinant forms of the type I enzyme, The nucleoside 3'-phosphates caused noncompetitive inhibition of the type I adenylyl cyclase from bovine brain, consistent with inhibition via the P-site, Inhibition was not due to hydrolytic products because this was minimal and inhibition kinetics by inorganic polyphosphates were inconsistent with those caused by the nucleoside 3'-polyphosphates. Only 3'ATP underwent cation-catalyzed, nonenzymatic hydrolysis, with the primary product being 2':3'-cAMP. Because 3'-ADP and 3'-ATP are naturally occurring, this class of compounds may physiologically regulate adenylyl cyclases and possibly other enzymes, mediating responses that include a reduction in 3':5'-cAMP levels and consequent reductions in protein kinase A-activated pathways.