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.

Hypoxic erythrocytes mediate cardioprotection through activation of soluble guanylate cyclase and release of cyclic GMP

“…In these experiments we used 2 distinct inhibitors of PKG. In a first set of experiments, the inhibitor of cGMP-binding to PKG, Rp-8-bromoguanosine-3′,5′-cyclic monophosphorothioate (Rp-8-Br-cGMPS) ( 17 , 18 ), or the PKG inhibitor KT5823 were added to the supernatant from RBCs following exposure to hypoxia. The cardioprotective effect of the supernatant from hypoxic RBCs was abolished by both KT5823 ( Figure 6, A and B ) and Rp-8-Br-cGMPS ( Figure 6, C and D ), suggesting cGMP-dependent activation of PKG by the mediator released from RBCs.…”

Bond Formation at C8 in the Nucleoside and Nucleotide Purine Scaffold: An Informative Selection

“…Aryl and heteroaryl functions can be substituted into the C8-position in purines by organometallic-promoted cross-coupling reactions [14]. The Pd/Cu-mediated direct arylation of 2 ′ -deoxyadenosine (39) with aryl iodides in Scheme 11 affords C8-arylated 2 ′ -deoxyadenosine (40) derivatives. The combination of cesium carbonate with a secondary amine such as piperidine generates in situ a reagent complex [(CH 2 ) 5 NH] 2 Pd(OAc) 2 that promotes the trans-coupling.…”

“…A nitro group has been inserted into the C8-position in guanosine by a nucleophilic displacement reaction from the bromide 125 (Scheme 36) [40]. The (Rp)-C8-bromo-cGMPS (125) substrate is incubated with sodium nitrite in DMSO.…”

Nitrated Nucleotides: New Players in Signaling Pathways of Reactive Nitrogen and Oxygen Species in Plants

“…Cell exposure to the exogenous electrophile methylmercury, which triggers NO and ROS signaling, elevated intracellular 8-nitro-cGMP, depleted reactive persulfides and 8-SH-cGMP, increased S-guanylation and activation of H-Ras leading to damaged cerebellar neurons (Ihara et al, 2017). 8-nitro-cGMP was found to S-guanylate thiol groups of cGMP-dependent protein kinases (PKG), the primary sensor proteins of intracellular cGMP known to control an array of cellular reactions (Ahmed et al, 2017). S-guanylation of PKG occurs specifically at two susceptible residues Cys42 and Cys195 among 11 cysteine residues of PKG.…”