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

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.

Figure 1.

Chemical structure of tested aminopyrimidines.

Figure 2.

Inhibition of PKA, cGKI, and cGKII by compounds AP-C5 and AP-C6. A, phosphorylation of peptide substrate by PKA, cGKI, and cGKII was assessed at compound concentrations ranging between 1.0·10⁻⁹ and 3.0·10⁻⁴ mol/liter. The concentration dependence of enzyme inhibition was analyzed, yielding the pIC₅₀ values shown in the inset. The number of technical replicates (n) is indicated in parentheses. Combined with the K_m(ATP) values determined in a separate set of experiments, these were used to estimate the pIC₅₀ values at cellular ATP levels (44). B, rate of cGKII-dependent ³²P_i-peptide production (V) as a function of the ATP concentration in the absence (control) or presence of AP-C5 (0.1 μmol/liter) or AP-C6 (0.5 μmol/liter). Data were derived from a single experiment performed in triplicate. *, no complete inhibition was attained in the concentration range tested. Error bars represent S.D.

Figure 3.

Schematic of the docking of AP-C5 at the ATP-binding domain of cGKII. This model was based on the X-ray structure of the highly homologous catalytic subunit of PKA. The numbered amino acids designate residues involved in ligand docking. Crucial structural elements of the active site are highlighted as follows: G-loop, green; Hyd1 (AXK β3-strand) residues, orange; αC-helix, magenta; hinge, blue; HRD motif, red; DFG motif, turquoise. The inset shows an alignment of the ATP-binding pockets of cGKI, cGKII, and PKA. *, residues that are within a 4.5-Å radius of the ligand. A boxed asterisk indicates a residue that is not conserved in cGKI and/or PKA.

Figure 4.

cGKII-, but not PKA-, dependent VASP phosphorylation in intestinal organoids is blocked by AP-C5. VASP phosphorylation at Ser-239 was detected by immunoblotting in mouse intestinal organoids. A, effect of AP-C5 on 8-pCPT-cGMP– and VIP-dependent VASP phosphorylation (p-VASP). Note that phosphorylation of Ser-157, a PKA-preferred residue, leads to a shift in the electrophoretic mobility of VASP (upper band of the doublet). Numerals to the left of the blot refer to the molecular mass (kDa) of protein standards shown in the left outer lane. B, aggregate data depicting the fluorescence intensity of the VASP signal relative to the villin signal of the same sample. Each data point represents one technical replicate. **, p < 0.01. Error bars represent S.D.

Figure 5.

Concentration-dependent inhibition of cGKII-dependent anion secretion in mouse ileum by AP-C5 and AP-C6. A, representative Ussing chamber experiment illustrating the procedure for testing inhibition of cGKII-dependent, CFTR-mediated anion secretion. Anion secretion was stimulated by addition of 8-pCPT-cGMP. After the ensuing Isc response had reached a plateau, compound or vehicle (DMSO) was added to the luminal bath. Inhibition of ileal anion secretion was calculated as the reduction in the Isc response 20 min after addition of compound relative to the initial full Isc response assessed just before addition of the compound. B, inhibition of 8-pCPT-cGMP–dependent anion secretion as a function of compound concentration. Data of six (AP-C5) or five (AP-C6) biological replicates were analyzed by nonlinear regression, yielding the IC₅₀ values (mean with 95% confidence interval in square brackets) shown in the inset. Error bars represent S.D.

Figure 6.

Inhibition of cGKII by AP-C5 or AP-C6 reduces STa-dependent anion secretion in mouse ileum. A, representative experiment illustrating the procedure for testing inhibition of STa-dependent anion secretion. Note that in the sustained presence of compound the tissue remained responsive to forskolin. B, both compounds (20 μmol/liter) reduced STa-dependent anion secretion relative to a solvent (0.1% DMSO) control. Each data point represents one biological replicate. ***, p < 0.001 versus DMSO. Error bars represent S.D.

Figure 7.

AP-C5 and AP-C6 potentiate cAMP signaling by PDE inhibition. A, in ileum, AP-C5 and AP-C6 did not reduce the Isc response elicited by forskolin (10 μmol/liter). Each data point represents one biological replicate. B, forskolin-dependent anion secretion in the presence of AP-C5 (50 μmol/liter) or vehicle (DMSO). AP-C5 lowered the forskolin concentration required to attain half-maximal stimulation of the Isc (p < 0.0001; inset). Data of 10 biological replicates per group were analyzed. C, compounds AP-C5 and AP-C6 potentiate forskolin-induced cAMP production in T84 cells. T84 cells were incubated with cGKII inhibitor compound (50 μmol/liter), DMSO (0.1%), or isobutylmethylxanthine (IBMX) (0.5 mmol/liter) in the absence or presence of forskolin (0.5 μmol/liter). Each data point represents one technical replicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus forskolin/DMSO. Error bars represent S.D.