A new nonhydrolyzable reactive cAMP analog, (Sp)-adenosine-3′,5′-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate irreversibly inactivates human platelet cGMP-inhibited cAMP phosphodiesterase

Levels of cAMP that control critical platelet functions are regulated by cGMP-inhibited cAMP phosphodiesterase (PDE3A). We previously showed that millimolar concentrations of the hydrolyzable 8-[(4-bromo-2,3-dioxobutyl)thioadenosine 3'.5'-cyclic monophosphate (8-BDB-TcAMP) inactivate PDE3A. We have now synthesized a nonhydrolyzable affinity label to probe the active site of PDE3A. The nonhydrolyzable adenosine 3'.5'-cyclic monophosphorothioates. Sp-cAMPS and Rp-cAMPS. function as competitive inhibitors of PDE3A with K-l = 47.6 and 4400 muM. respectively. We therefore coupled Sp-cAMPS with 1,4-dibromobutanedione to yield (Sp)-adenosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, [Sp-cAMPS-(BDB)]. Sp-cAMPS-(BDB) inactivates PDE3A in a tithe-dependent, irreversible reaction with k(max) = 0.0116 min(-1) and K-l = 10.1 muM. The order of effectiveness of protectants in decreasing the rate of inactivation (with K-d in muM) is: Sp-cAMPS (24) > Rp-cGMPS (1360). Sp-cGMPS (1460) > GMP (4250), AMP (10600), Rp-cAMPS (22170). These results suggest that the inactivation of PDE3A by Sp-cAMPS-(BDB) is a consequence of reaction at the overlap of the cAMP and cGMP binding regions in the active site. (C) 2002 Elsevier Science (USA).