title: 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.
authors.affiliation: Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: Su H
authors.initials: SH
authors.lastname: Hung
authors.forename: K S
authors.initials: KS
authors.lastname: Madhusoodanan
authors.forename: Robert L
authors.initials: RL
authors.lastname: Boyd
authors.forename: James L
authors.initials: JL
authors.lastname: Baldwin
authors.forename: Roberta F
authors.initials: RF
authors.lastname: Colman
authors.forename: Robert W
authors.initials: RW
authors.lastname: Colman
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D004305:Dose-Response Relationship, Drug; D007313:Insecta; D008958:Models, Molecular; D010726:Phosphodiesterase Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: C451332:8-((4-bromo-2,3-dioxobutyl)thio)adenosine 3',5'-cyclic S-(methyl)monophosphorothioate; D010726:Phosphodiesterase Inhibitors; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517287:PDE3A protein, human