Pyridopyrimidine derivatives as inhibitors of cyclic nucleotide synthesis: Application for treatment of diarrhea

Abstract

Acute secretory diarrhea induced by infection with enterotoxigenic strains of Escherichia coli involves binding of stable toxin (STa) to its receptor on the intestinal brush border, guanylyl cyclase type C (GC-C). Intracellular cGMP is elevated, inducing increase in chloride efflux and subsequent accumulation of fluid in the intestinal lumen. We have screened a library of compounds and identified a pyridopyrimidine derivatives {5-(3-bromophenyl)-1,3-dimethyl-5,11-dihydro-1H-indeno[2',1':5,6]pyrido[2,3-d]pyrimidine-2,4,6-trione; BPIPP} as an inhibitor of GC-C that can suppress STa-stimulated cGMP accumulation by decreasing GC-C activation in intact T84 human colorectal carcinoma cells. BPIPP inhibited stimulation of guanylyl cyclases, including types A and B and soluble isoform in various cells. BPIPP suppressed stimulation of adenylyl cyclase and significantly decreased the activities of adenylyl cyclase toxin of Bordetella pertussis and edema toxin of Bacillus anthracis. The effects of BPIPP on cyclic nucleotide synthesis were observed only in intact cells. The mechanism of BPIPP-dependent inhibition appears to be complex and indirect, possibly associated with phospholipase C and tyrosine-specific phosphorylation. BPIPP inhibited chloride-ion transport stimulated by activation of guanylyl or adenylyl cyclases and suppressed STa-induced fluid accumulation in an in vivo rabbit intestinal loop model. Thus, BPIPP may be a promising lead compound for treatment of diarrhea and other diseases.

Fig. 1.

Effect of BPIPP on cGMP accumulation in cultured cells. (A) T84 cells were pretreated with 50 μM BPIPP or vehicle (0.1% DMSO) with or without 1 mM IBMX for 10 min and then treated with 0.1 μM STa (ST0.1) or 1 μM STa (ST1) for 10 min; intracellular (i) and extracellular (e) cGMP were assayed in the extract or in the treatment medium, respectively. n = 3–6. (B) Membranes were isolated from T84 cells and GC-C activity was assayed without stimulation (basal) or with 0.1 μM STa (STa 0.1), 1 μM STa (STa 1), or 3 mM MnCl₂ (Mn) in the presence of 50 μM BPIPP or vehicle. Similar data were obtained in membranes isolated in buffer containing protein phosphatase inhibitors from cells pretreated for 10 min with BPIPP. n = 3–4. (C) T84 cells were pretreated with 50 μM BPIPP or vehicle for 10 min and then with indicated concentrations of STa for 10 min, and cGMP accumulation was measured. n = 3. (D) T84 cells were pretreated with indicated concentrations of BPIPP or vehicle (Con) for 10 min and then with 0.5 μM guanylin or 100 nM STa for 10 min, and cGMP accumulation was measured. n = 6. (E) BE-2 cells were pretreated with indicated concentrations of BPIPP or vehicle (Con) for 10 min and then treated with 1 μM atrial natriuretic peptide (ANP) or 0.5 μM C-type natriuretic peptide (CNP) for 10 min, and intracellular cGMP was measured. n = 4. (F) RFL-6 cells were pretreated with 50 μM BPIPP or vehicle for 10 min and then with 10 μM nitric oxide donor benzotrifuroxan (NO donor), 1 μM ANP, or 1 μM CNP for 4 min. *, P < 0.01; n = 3.

Fig. 2.

Effect of BPIPP on cAMP accumulation in cultured cells. (A) RFL-6 cells were pretreated with 50 μM BPIPP or vehicle for 10 min and then with 50 μM forskolin (For) or 100 μM isoproterenol (Iso); cells were pretreated with 1 μg/ml cholera toxin for 30 min (CT30) or 60 min (CT60) and then, after a 10-min incubation with IBMX and BPIPP, cAMP accumulation was measured. *, P < 0.01; n = 4. (B) T84 cells were pretreated in serum-free growth medium in the presence of 1 μg/ml adenylyl cyclase toxin from Bordetella pertussis (BAC), a combination of 2 μg/ml protective antigen and 0.1 μg/ml edema factor from Bacillus anthracis (edema toxin; ET), or 1 μg/ml Vibrio cholera toxin (CT) for 1 h (pretreatment phase; pretreat); cells were washed and further incubated in DBPS containing 1 mM IBMX for 10 min to measure cAMP accumulation (incubation phase; incubate); cells were treated with vehicle or with 50 μM BPIPP during the corresponding phase as indicated. Control (100% values) for BAC, ET, and CT were 9.98 ± 0.72, 0.14 ± 0.01, and 7.85 ± 0.82 nmol of cAMP per mg of protein. *, P < 0.05; **, P < 0.01; n = 3. (C) T84 cells were pretreated with 50 μM BPIPP or vehicle and 1 mM IBMX for 10 min and then treated with indicated concentrations of forskolin for 10 min. n = 4.

Fig. 3.

Antisecretory activity of BPIPP in cell culture and in vivo. (A) T84 cells were pretreated with 50 μM BPIPP or vehicle in solution 1 containing NaCl, and chloride transport was measured in the presence of indicated concentrations of STa in solution 2 containing NaNO₃ by using 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ) fluorescence as a Cl⁻ sensor. n = 8. (B) Effect of 50 μM BPIPP on Cl⁻ transport in T84 cells stimulated by various agents in solution 1 containing NaCl; 100 μM isoproterenol (Iso), 10 μg/ml cholera toxin (CT; CT-ptr, BPIPP was present during pretreatment similar to Fig. 2B; CT-inc, BPIPP was present only during incubation and assay phase; CT-both, BPIPP was present during pretreatment and incubation-assay phase), 25 μM forskolin (For), 1 mM or 0.1 mM 8-bromo-cAMP (BcA-1 or BcA-0.1), or 10 μM ionomycin (Iono). Fluorescence was measured by using SPQ or N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) as indicator. Absolute F_t − F_c fluorescence values for vehicle control were 138 ± 6 for isoproterenol, 74 ± 5 for cholera toxin, 1,031 ± 36 for forskolin, 52 ± 2 and 780 ± 84 for 0.1 and 1 mM 8-bromo-cAMP in the presence of SPQ, and 635 ± 75 and 1,945 ± 102 for 0.1 and 1 mM 8-bromo-cAMP and 720 ± 81 for ionomycin in the presence of MQAE. *, P < 0.05 BPIPP vs. vehicle; n = 8. (C) Effect of compounds on secretion in rabbit intestinal segments (ileal loops) injected with 1 ml of PBS containing indicated concentrations of STa [none (Control), 0.1, 0.2, or 1.0 μM] with vehicle, 10 or 50 μM BPIPP, and 50 μM compound IVa. Incubations were continued for 5 h, and then volume/length ratios of the segments were determined. *, P < 0.05; n = 4–8.

Fig. 4.

Stimulation of activity of a tyrosine-specific protein kinase in T84 cells by BPIPP. (A) Cells were treated with 50 μM BPIPP or vehicle for 20 min and protein kinase activity was measured in the extract by using various tyrosine-containing protein kinase substrates. E4Y, (Glu)₄-Tyr; EAY, Glu-Ala-Tyr; EY, Glu-Tyr. n = 3. (B) Cells were pretreated in a similar manner and then homogenized with a glass–glass homogenizer in a hypotonic buffer and fractionated to separate crude membrane fraction from the cytosol. Protein kinase activity was measured by using EAY as the substrate. *, P < 0.05; n = 6.