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

• Published in: Biological & pharmaceutical bulletin 2017/03/01, Vol.40(3), pp.365-374
• Main Authors: Ahmed, Khandaker Ahtesham, Zhang, Tianli, Ono, Katsuhiko, Tsutsuki, Hiroyasu, Ida, Tomoaki, Akashi, Soichiro, Miyata, Keishi, Oike, Yuichi, Akaike, Takaaki, Sawa, Tomohiro
• Format: Article
• Language: English
• Subjects: 8-nitro-cGMP; Acetylcholine; Adducts; Animals; Aorta; Cyclic GMP; 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; Cysteine; Enzyme inhibitors; Enzymes; Glutathione; Guanosine - analogs & derivatives; Guanosine - metabolism; Guanosine - pharmacology; guanosine 3′,5′-cyclic monophosphate (cGMP)-dependent protein kinase; guanosine 3′,5′-cyclic monophosphorothioate Rp-isomer; Inhibitors; Isomerism; Kinases; Male; Mice, Inbred C57BL; Muscle contraction; Nitric oxide; Nitro Compounds - metabolism; Nitro Compounds - pharmacology; Protein kinase; protein kinase G (PKG); Protein Processing, Post-Translational; Protein S; protein S-guanylation; Proteins; Reactive oxygen species; Residues; Signal Transduction; Smooth muscle; Sodium; Sodium nitrite; Synaptic plasticity; Thionucleotides - chemical synthesis; Thionucleotides - metabolism; Thionucleotides - pharmacology; vascular relaxation; Vasodilation - drug effects; Western blotting
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.b16-00880

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.