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

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Author(s)

Abstract

<p>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>i.e.</i>, the process called protein <i>S</i>-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 <i>S</i>-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 <i>S</i>-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.</p>

Journal

  • Biological and Pharmaceutical Bulletin

    Biological and Pharmaceutical Bulletin 40(3), 365-374, 2017

    The Pharmaceutical Society of Japan

Codes

  • NII Article ID (NAID)
    130005398514
  • NII NACSIS-CAT ID (NCID)
    AA10885497
  • Text Lang
    ENG
  • ISSN
    0918-6158
  • NDL Article ID
    028006066
  • NDL Call No.
    Z53-V41
  • Data Source
    NDL  J-STAGE 
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