A Nonhydrolyzable Reactive cAMP Analogue, (S_p)-8-[(4-Bromo-2,3-dioxobutyl)thio]adenosine 3‘,5‘-Cyclic S-(Methyl)monophosphorothioate, Irreversibly Inactivates Human Platelet cGMP-Inhibited cAMP Phosphodiesterase at Micromolar Concentrations

Abstract

We previously showed that 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate inactivates cAMP phosphodiesterase (PDE3A); however, millimolar concentrations were needed to inactivate PDE3A because of ongoing hydrolysis. We have now synthesized a nonhydrolyzable reactive cAMP analogue, (S(p))-8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic S-(methyl)monophosphorothioate (S(p)-8-BDB-TcAMPSMe). S(p)-8-BDB-TcAMPSMe inactivates PDE3A in a time-dependent, irreversible manner, exhibiting saturation kinetics with a k(max) of (19.5 +/- 0.3) x 10(-3) min(-1) and a K(I) of 3.5 +/- 0.3 muM. To ascertain whether S(p)-8-BDB-TcAMPSMe reacts in the active site, nonhydrolyzable analogues of the substrate cAMP, or the competitive inhibitor cGMP, were included to protect against the inactivation of PDE3A. The order of effectiveness of protectants in decreasing the rate of inactivation (with K(d) values in micromolar) is as follows: S(p)-cAMPS (18) > R(p)-cGMPS (560) and S(p)-cGMPS (1260) > 5'-AMP (17 660), R(p)-cAMPS (30 110), and 5'-GMP (42 170). We docked S(p)-8-BDB-TcAMPSMe into PDE3A, based on the structural model of PDE3A-cAMP and the kinetic data from site-directed mutants. The S(p)-8-BDB-TcAMPSMe fits into the active site in the model. These results suggest that inactivation of PDE3A by the affinity reagent is a consequence of reaction at the overlap between cAMP and cGMP binding regions in the active site. S(p)-8-BDB-TcAMPSMe has proven to be an effective active site-directed irreversible cAMP affinity label for platelet PDE3A and can be used to identify amino acids in the active site of PDE3A as well as in other cAMP phosphodiesterases.

A new nonhydrolyzable reactive cGMP analogue, (Rp)-guanosine-3′,5′-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A

“…Sp-cGMPS and Rp-cGMPS are both competitive inhibitors of PDE3A [13]. The K d for both Sp-cGMPS and Rp-cGMPS are similar, indicating that there is no sterospecificity when PDE3A binds to either isomer of cGMPS.…”

“…However, the utility of 8-BDB-TcAMP was limited since it only inactivates PDEs at millimolar concentrations because of continuous hydrolysis to the 5′-AMP derivative by the enzymes under investigation. We previously reported synthesis of the new nonhydrolyzable reactive cAMP derivative (Sp)-adenosine-3′,5′-cyclic-S-(4-bromo-2,3-dioxobutyl) phosphorothioate (Sp-cAMPS-BDB), which contains both reactive bromoketo and dioxo groups [13]. The bromoketo group can form covalent bonds with the nucleophilic side chains of many amino acids including cysteine, aspartate, glutamate, histidine, tyrosine and lysine, while the dioxo provides the ability to react with arginine residues [29].…”

“…Sp-cGMPS and Rp-cGMPS act as competitive inhibitors of PDE3A when tested using the substrate cAMP [13]. The K i values for Sp-cGMPS and Rp-cGMPS are 305 ± 54 and 210 ± 33 μM (Table 1), respectively, indicating that the enzyme binds the Rp isomer better than it binds the Sp-cGMPS.…”

“…Enzyme activity was measured by comparing the amount of cAMP hydrolyzed in PDE3A containing samples with that of controls without enzyme. These data were then used to calculate the enzyme specific activity in nmoles of cAMP hydrolyzed per mg of protein per minute [13].…”

“…Aliquots of each final reaction mixture were removed at timed intervals, diluted in a buffer containing 47.5 mM HEPES (pH 7.04), 20 mM MgCl 2 and 4 mM MES, and then assayed in triplicate for residual PDE3A activity. NAD + was used as control for the protection experiments since it is neither the substrate nor the inhibitor of PDE3A [10,13].…”