title: Synthesis and Characterization of 8-Nitroguanosine 3',5'-Cyclic Monophosphorothioate Rp-Isomer as a Potent Inhibitor of Protein Kinase G1α.
abstract: Guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinases (PKG) are kinases regulating diverse physiological functions including vascular smooth muscle relaxation, neuronal synaptic plasticity, and platelet activities. Certain PKG inhibitors, such as Rp-diastereomers of derivatives of guanosine 3',5'-cyclic monophosphorothioate (Rp-cGMPS), have been designed and used to study PKG-regulated cell signaling. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is an endogenous cGMP derivative formed as a result of excess production of reactive oxygen species and nitric oxide. 8-Nitro-cGMP causes persistent activation of PKG1α through covalent attachment of cGMP moieties to cysteine residues of the enzyme (i.e., the process called protein S-guanylation). In this study, we synthesized a nitrated analogue of Rp-cGMPS, 8-nitroguanosine 3',5'-cyclic monophosphorothioate Rp-isomer (Rp-8-nitro-cGMPS), and investigated its effects on PKG1α activity. We synthesized Rp-8-nitro-cGMPS by reacting Rp-8-bromoguanosine 3',5'-cyclic monophosphorothioate (Rp-8-bromo-cGMPS) with sodium nitrite. Rp-8-Nitro-cGMPS reacted with the thiol compounds cysteine and glutathione to form Rp-8-thioalkoxy-cGMPS adducts to a similar extent as did 8-nitro-cGMP. As an important finding, a protein S-guanylation-like modification was clearly observed, by using Western blotting, in the reaction between recombinant PKG1α and Rp-8-nitro-cGMPS. Rp-8-Nitro-cGMPS inhibited PKG1α activity with an inhibitory constant of 22 µM in a competitive manner. An organ bath assay with mouse aorta demonstrated that Rp-8-nitro-cGMPS inhibited vascular relaxation induced by acetylcholine or 8-bromo-cGMP more than Rp-8-bromo-cGMPS did. These findings suggest that Rp-8-nitro-cGMPS inhibits PKG through induction of an S-guanylation-like modification by attaching the Rp-cGMPS moiety to the enzyme. Additional study is warranted to explore the potential application of Rp-8-nitro-cGMPS to biochemical and therapeutic research involving PKG1α activation.
authors.affiliation: Department of Pathology, University of Alabama at Birmingham.
authors.forename: Khandaker Ahtesham
authors.initials: KA
authors.lastname: Ahmed
authors.forename: Tianli
authors.initials: T
authors.lastname: Zhang
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Ono
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Tsutsuki
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Ida
authors.forename: Soichiro
authors.initials: S
authors.lastname: Akashi
authors.forename: Keishi
authors.initials: K
authors.lastname: Miyata
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Oike
authors.forename: Takaaki
authors.initials: T
authors.lastname: Akaike
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Sawa
mesh_terms: D000109:Acetylcholine; D000818:Animals; D001011:Aorta; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D007536:Isomerism; D008297:Male; D008810:Mice, Inbred C57BL; D009574:Nitro Compounds / Q000378:metabolism / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction; D013873:Thionucleotides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D014664:Vasodilation / Q000187:drug effects*
chemical_list: C481144:8-nitroguanosine; C525106:8-nitroguanosine 3',5'-cyclic monophosphate; D009574:Nitro Compounds; D013873:Thionucleotides; D006151:Guanosine; C083763:8-bromoguanosino-3',5'-cyclic monophosphorothioate; C016276:8-bromocyclic GMP; C017352:guanosine-3',5'-cyclic phosphorothioate; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D006152:Cyclic GMP; D000109:Acetylcholine