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  2. Selective inhibition of intestinal guanosine 3',5'-cyclic monophosphate signaling by small-molecule protein kinase inhibitors

Selective inhibition of intestinal guanosine 3',5'-cyclic monophosphate signaling by small-molecule protein kinase inhibitors

  • J Biol Chem. 2018 May 25;293(21):8173-8181. doi: 10.1074/jbc.RA118.002835.
Marcel J C Bijvelds 1 Gary Tresadern 2 Ann Hellemans 3 Karine Smans 2 Natascha D A Nieuwenhuijze 4 Kelly F Meijsen 4 Jean-Pierre Bongartz 2 Luc Ver Donck 2 Hugo R de Jonge 4 Jan A J Schuurkes 3 Joris H De Maeyer 3
Affiliations

Affiliations

  • 1 Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, P. O. Box 2040, 3000CA Rotterdam, The Netherlands. Electronic address: m.bijvelds@erasmusmc.nl.
  • 2 Janssen Research and Development, a Division of Janssen Pharmaceutica NV, Turnhoutseweg 30, B-2340 Beerse, Belgium.
  • 3 Shire-Movetis NV, Veedijk 58, B-2300 Turnhout, Belgium.
  • 4 Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, P. O. Box 2040, 3000CA Rotterdam, The Netherlands.
Abstract

The guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase II (cGKII) serine/threonine kinase relays signaling through guanylyl cyclase C (GCC) to control intestinal fluid homeostasis. Here, we report the discovery of small-molecule inhibitors of cGKII. These inhibitors were imidazole-aminopyrimidines, which blocked recombinant human cGKII at submicromolar concentrations but exhibited comparatively little activity toward the phylogenetically related protein kinases cGKI and cAMP-dependent protein kinase (PKA). Whereas aminopyrimidyl motifs are common in protein kinase inhibitors, molecular modeling of these imidazole-aminopyrimidines in the ATP-binding pocket of cGKII indicated an unconventional binding mode that directs their amine substituent into a narrow pocket delineated by hydrophobic residues of the hinge and the αC-helix. Crucially, this set of residues included the Leu-530 gatekeeper, which is not conserved in cGKI and PKA. In intestinal organoids, these compounds blocked cGKII-dependent phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). In mouse small intestinal tissue, cGKII inhibition significantly attenuated the anion secretory response provoked by the GCC-activating Bacterial heat-stable toxin (STa), a frequent cause of infectious secretory diarrhea. In contrast, both PKA-dependent VASP phosphorylation and intestinal anion secretion were unaffected by treatment with these compounds, whereas experiments with T84 cells indicated that they weakly inhibit the activity of cAMP-hydrolyzing phosphodiesterases. As these protein kinase inhibitors are the first to display selective inhibition of cGKII, they may expedite research on cGMP signaling and may aid future development of therapeutics for managing diarrheal disease and other pathogenic syndromes that involve cGKII.

Keywords

CFTR; ETEC; PKG; cGMP; cyclic GMP; cystic fibrosis transmembrane conductance regulator; drug discovery; enterotoxigenic E. coli; intestinal epithelium; protein kinase G; secretory diarrhea; signal transduction; vasodilator-stimulated phosphoprotein.

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