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 Ki = 47.6 and 4400 μM, 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 time-dependent, irreversible reaction with kmax = 0.0116 min−1 and KI = 10.1 μM. The order of effectiveness of protectants in decreasing the rate of inactivation (with Kd in μM) 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.