Synthesis and Characterization of 8-Nitroguanosine 3',5'-Cyclic Monophosphorothioate Rp-Isomer as a Potent Inhibitor of Protein Kinase G1α

Biol Pharm Bull. 2017 Mar 1;40(3):365-374. doi: 10.1248/bpb.b16-00880. Epub 2016 Dec 16.


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.

MeSH terms

  • Acetylcholine
  • Animals
  • Aorta
  • Cyclic GMP / analogs & derivatives*
  • Cyclic GMP / chemical synthesis
  • Cyclic GMP / metabolism
  • Cyclic GMP / pharmacology
  • Cyclic GMP-Dependent Protein Kinase Type I / antagonists & inhibitors*
  • Cyclic GMP-Dependent Protein Kinase Type I / metabolism
  • Guanosine / analogs & derivatives*
  • Guanosine / metabolism
  • Guanosine / pharmacology
  • Isomerism
  • Male
  • Mice, Inbred C57BL
  • Nitro Compounds / metabolism
  • Nitro Compounds / pharmacology*
  • Protein Processing, Post-Translational
  • Signal Transduction
  • Thionucleotides / chemical synthesis
  • Thionucleotides / metabolism
  • Thionucleotides / pharmacology*
  • Vasodilation / drug effects*


  • 8-nitroguanosine
  • 8-nitroguanosine 3',5'-cyclic monophosphate
  • Nitro Compounds
  • Thionucleotides
  • Guanosine
  • 8-bromoguanosino-3',5'-cyclic monophosphorothioate
  • 8-bromocyclic GMP
  • guanosine-3',5'-cyclic phosphorothioate
  • Cyclic GMP-Dependent Protein Kinase Type I
  • Cyclic GMP
  • Acetylcholine