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N4-monobutyryl-cCMP activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo

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Abstract

There is increasing evidence for a role of cytidine 3′,5′-cyclic monophosphate (cCMP) as second messenger. In a recent study, we showed that cCMP activates both purified guanosine 3′,5′-cyclic monophosphate (cGMP)-dependent protein kinase Iα (PKG Iα) and adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) isoenzymes with the regulatory subunits RIα and RIIα. Moreover, the membrane-permeant cCMP analog dibutyryl (DB)-cCMP induces effective vasodilation and inhibition of platelet aggregation via PKG Iα, but not via PKA. These data prompted us to conduct a systematic analysis of the effects of cyclic nucleotide (cNMP) analogs on purified PKG Iα and PKA RIα and RIIα We also studied the effect of DB-cCMP on PKA-dependent phosphorylation of the transcription factor cAMP response-binding protein (CREB) in S49 wild-type lymphoma cells and S49 kin− cells, devoid of the catalytic subunit of PKA. The major cellular metabolite of the prodrug DB-cCMP, N4-monobutyryl (4-MB)-cCMP, was a partial and low-potency activator of purified PKG Iα and a full and moderate-potency activator of PKA RIα and RIIα. Sp-cCMPS and Sp-cAMPS activated PKA RIα and RIIα with much higher potency and efficacy than PKG Iα. Molecular modeling suggested that the cytidine ring interacts with PKG Iα mainly via hydrophobic interactions, while the butyryl group projects away from the kinase. In contrast to DB-cAMP, DB-cCMP did not induce PKA-dependent phosphorylation in intact cells. Taken together, our data show that N4-monobutyryl-cCMP (4-MB-cCMP) activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo. cNMP phosphorothioates constitute a starting point for the development of PKA activators with high selectivity relative to PKG.

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Abbreviations

cAMP:

Adenosine 3′,5′-cyclic monophosphate

cGMP:

Guanosine 3′,5′-cyclic monophosphate

cCMP:

Cytidine 3′,5′-cyclic monophosphate

CREB:

cAMP response-binding protein

cNMP:

Nucleoside 3′,5′-cyclic monophosphate

PKA:

cAMP-dependent protein kinase

PKG Iα:

cGMP-dependent protein kinase Iα

AM:

Acetoxymethyl ester

MB:

Monobutyryl

DB:

Dibutyryl

PDE:

Phosphodiesterase

MRP:

Multidrug resistance protein

NBD:

Nucleotide-binding domain

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Acknowledgments

This work was supported by grants of the Deutsche Forschungsgemeinschaft to R.S. We also thank the reviewers for their constructive critique.

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Authors and Affiliations

  1. Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany

    Sabine Wolter, Marina Golombek & Roland Seifert

  2. Department of Pharmaceutical and Medicinal Chemistry II, Institute of Pharmacy, University of Regensburg, D-93040, Regensburg, Germany

    Stefan Dove

  3. Biolog Life Science Institute, Flughafendamm 9a, D-28199, Bremen, Germany

    Frank Schwede

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  1. Sabine Wolter
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  2. Stefan Dove
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  3. Marina Golombek
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  4. Frank Schwede
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  5. Roland Seifert
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Correspondence to Roland Seifert.

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Wolter, S., Dove, S., Golombek, M. et al. N4-monobutyryl-cCMP activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo. Naunyn-Schmiedeberg's Arch Pharmacol 387, 1163–1175 (2014). https://doi.org/10.1007/s00210-014-1042-9

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