title: 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.
abstract: The amino acids involved in substrate (cAMP) binding to human platelet cGMP-inhibited cAMP phosphodiesterase (PDE3A) are identified. Less is known about the inhibitor (cGMP) binding site. We have now synthesized a nonhydrolyzable reactive cGMP analog, Rp-guanosine-3',5'-cyclic-S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate (Rp-cGMPS-BDB). Rp-cGMPS-BDB irreversibly inactivates PDE3A (K(I)=43.4+/-7.2muM and k(cart)=0.007+/-0.0006 min(-1)). The effectiveness of protectants in decreasing the rate of inactivation by Rp-cGMPS-BDB is: Rp-cGMPS (K(d)=72 microM)>Sp-cGMPS (124), Sp-cAMPS (182)>GMP (1517), Rp-cAMPS (3762), AMP (4370 microM). NAD(+), neither a substrate nor an inhibitor of PDE3A, does not protect. Nonhydrolyzable cGMP analogs exhibit greater affinity than the cAMP analogs. These results indicate that Rp-cGMPS-BDB targets favorably the cGMP binding site consistent with a docking model of PDE3A-Rp-cGMPS-BDB active site. We conclude that Rp-cGMPS-BDB is an effective active site-directed affinity label for PDE3A with potential for other cGMP-dependent enzymes.
authors.affiliation: The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 North Broad Street, OMS 418, Philadelphia, PA 19140, USA.
authors.forename: Su H
authors.initials: SH
authors.lastname: Hung
authors.forename: Andy H
authors.initials: AH
authors.lastname: Liu
authors.forename: Robin A
authors.initials: RA
authors.lastname: Pixley
authors.forename: Penelope
authors.initials: P
authors.lastname: Francis
authors.forename: LaTeeka D
authors.initials: LD
authors.lastname: Williams
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Matsko
authors.forename: Karine D
authors.initials: KD
authors.lastname: Barnes
authors.forename: Sharmila
authors.initials: S
authors.lastname: Sivendran
authors.forename: Roberta F
authors.initials: RF
authors.lastname: Colman
authors.forename: Robert W
authors.initials: RW
authors.lastname: Colman
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis / Q000737:chemistry*; D001665:Binding Sites; D001792:Blood Platelets; D006152:Cyclic GMP / Q000031:analogs & derivatives*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D058987:Phosphodiesterase 3 Inhibitors; D011485:Protein Binding
chemical_list: D000345:Affinity Labels; D004791:Enzyme Inhibitors; D058987:Phosphodiesterase 3 Inhibitors; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517287:PDE3A protein, human; D006152:Cyclic GMP