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CELLULAR AND MOLECULAR

-Aminoazaheterocyclic-Methylglyoxal Adducts Do Not Inhibit Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Activity

Departments of Medicine and Physiology, University of California, San Francisco, California (N.D.S., W.N., A.S.V.); and Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, Genova, Italy (O.Z.-M., L.J.V.G.)

Received September 28, 2007; accepted February 12, 2008.

	Abstract

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Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have potential applications in the therapy of secretory diarrheas and polycystic kidney disease. In a recent study, several highly polar -aminoazaheterocyclic-methylglyoxal adducts were reported to reversibly inhibit CFTR chloride channel activity with IC₅₀ values in the low picomolar range (J Pharmacol Exp Ther 322:1023–1035, 2007), more than 10,000-fold better than that of thiazolidinone and glycine hydrazide CFTR inhibitors previously identified by high-throughput screening. In this study, we resynthesized and evaluated the -aminoazaheterocyclic-methylglyoxal adducts reported to have high CFTR inhibition potency (compounds 5, 7, and 8). We verified that the reported synthesis procedures produced the target compounds in high yield. However, we found that these compounds did not inhibit CFTR chloride channel function in multiple cell lines at up to 100 µM concentration, using three independent assays of CFTR function including short-circuit current analysis, whole-cell patch-clamp experiments, and yellow fluorescence protein-fluorescence quenching. As positive controls, approximately 100% of CFTR inhibition was found by thiazolidinone and glycine hydrazide CFTR inhibitors. Our data provide direct evidence against CFTR inhibition by -aminoazaheterocyclic-methylglyoxal adducts.

Routaboul et al. (2007) recently reported that -aminoazaheterocyclic-methylglyoxal adducts inhibit CFTR chloride channel function completely and reversibly, with low picomolar IC₅₀ values, more than 10,000-fold better than the most potent known CFTR inhibitors. The picomolar-potency compounds were identified by screening a collection of seven -aminoazaheterocyclic-methylglyoxal adducts. These adducts had been reported previously in a study that described an efficient, one-step synthesis approach involving the reaction of methylglyoxal with -aminoazaheterocycles (Routaboul et al., 2002). The exceptionally high potency and reversibility of the -aminoazaheterocyclic-methylglyoxals was surprising for several reasons. First, prior screening of 200,000 compounds for CFTR inhibition produced only one compound with an IC₅₀ less than 1 µM, with an overall low verified hit rate of 1/50,000 (Ma et al., 2002b; Muanprasat et al., 2004). Discovery of picomolar-potency CFTR inhibitors by screening seven compounds of a single chemical class is surprising, particularly because the compounds were tested for CFTR inhibition without a rational basis. Second, most channel inhibitors with picomolar potency are animal toxins rather than small, synthetic molecules (Favreau et al., 2001; Grunnet et al., 2001; Benton et al., 2003; Castle et al., 2003). Last, contrary to the data of Routaboul et al. (2007), very slow (more than hours) reversibility of picomolar-affinity inhibitors is expected even in the diffusion limit (Bkaily et al., 1985).

For these reasons, and because of the potential major therapeutic implications of picomolar-potency CFTR inhibitors, we reevaluated the findings of Routaboul et al. (2007). We verified the high-yield synthesis of -aminoazaheterocyclic-methylglyoxal adducts, as originally reported by Routaboul et al. (2002); however, using multiple assays, cell lines, and concentrations, and with appropriate positive controls, we were unable to demonstrate CFTR inhibition by the -aminoazaheterocyclic-methylglyoxals.

	Materials and Methods

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Synthesis Procedures. ¹H NMR spectra were obtained in D₂O, pH 7, using a 400 MHz Varian spectrometer. Mass spectrometry was done on a Waters LC/MS System (Alliance HT 2790+ZQ, HPLC; model 2690; Waters, Milford, MA). Flash chromatography was performed using EM silica gel (230–400 mesh), and thin-layer chromatography (TLC) was done on Merck Silica Gel 60 F254 plates.

Synthesis of -Aminoazaheterocycle-Methylglyoxal Adducts. Compounds 2, 3, 4, 5, 6, and 8 were synthesized in high yield by using the methods of Routaboul et al. (2002, 2007).

On average, a mixture of the aminoazaheterocycle and a dilute solution of methylglyoxal were stirred under argon at 50°C for 1 to 2 days (until reaction was completed by LC/MS). The residue after evaporation was purified by a combination of precipitation, recrystallization, normal/reverse-phase column chromatography, and preparative TLC (Routaboul et al., 2002, 2007). Compound 7 was not synthesized due to commercially unavailable starting materials. Original NMR and MS data are included in the supplemental data.

2-Amino-Pyridine Adducts 2ab. Yield, 76%; ¹H NMR: 8.15–7.88 (1H, m, Ar-H), 7.76–7.48 (1H, m, Ar-H), 6.82–6.80 (1H, d, Ar-H), 6.78–6.715 (1H, t, Ar-H), 4.30/3.93 (1H, s, CH), 1.50 (3H, s, CH), 1.39 (3H, s, CH); MS (ES⁺)(m/z): 239 [M + 1]⁺.

2-Amino-3-Hydroxypyridine Adducts 3ab. Yield: 76%; m.p.: 141–142°C (decomposition); ¹H NMR: 7.31–7.21 (1H, m, CH), 6.64–6.52 (2H, m, CH), 4.30/3.89 (1H, s, m, CH), 1.52–1.42 (3H, bs, CH₃), 1.38 (3H, s, CH₃); MS (ES⁺)(m/z): 255 [M + 1]⁺.

Adenine Adducts 4ab. Yield, 66%; ¹H NMR: 8.29 (1H, s, CH); 8.01 (1H, s, CH); 4. 69 (1H, s, CH); 1.86/1.47 (3H, s, CH₃); MS (ES⁺)(m/z): 280 [M + 1]⁺.

Deoxy-Adeninosine Adducts 5ab. Yield: 67%; ¹H NMR:; 8.23 (s, 1H, Ar-CH), 8.16 (d, 1H, Ar-CH), 6.30 (t, 1H, N-CH), 4.79 (d, 1H, adduct CH), 4.50–4.44 (m, 1H, CH), 4.01 (q, 1H, CH), 3.68–3.58 (2 dd or q, 2H, DO-CH₂), 2.70–2.63 (m, 1H, CH₂), 2.42–2.36 (m, 1H, CH₂), 1.93 (s, 3H, CH₃), 1.46 (s, 3H, CH₃); MS (ES⁺)(m/z): 396 [M + 1]⁺.

Adenosine Adducts 6ab. Yield, 59%; ¹H NMR: 8.32 (s, 1H, Ar-H), 8.18 (s, 1H, Ar-H), 5.89 (d, 1H, N-CH), 5.10/4.74 (s, 1H, adduct CH), 4.34 (t, 1H, CH), 4.09 (q, 1H, CH), 4.01 (q, 1H, CH₂), 3.76–3.62 (2 dd or q, 2H), 3.45/3.36 (q, 2H), 2.15 (s, 3H, CH₃), 1.86 (s, 3H, CH₃); MS (ES⁺)(m/z): 412 [M + 1]⁺.

1-Aminoisoquinoline Adducts 8ab. Yield, 71%; m.p. 175–177°C (decomposition); ¹H NMR: 8.31–8.21 (1H, m, Ar-H), 7.86–7.47 (4H, m, Ar-H) 7.14–6.99 (1H, m, Ar-H), 4.37 and 4.05 (1H, s, CH), 1.64 (3H, s, CH₃), 1.57 (3H, s, CH₃); MS (ES⁺)(mz): 289 [M + 1]⁺.

Fluorescence Cell-Based Assay of CFTR Inhibition. Fisher rat thyroid (FRT) cells stably cotransfected with human wild-type (WT) CFTR and yellow fluorescence protein (YFP)-H148Q, as described previously (Ma et al., 2002a), were plated in black-walled, 96-well plates with transparent plastic bottoms (Costar; Corning Life Sciences, Acton, MA), cultured overnight to confluence, washed three times with phosphate-buffered saline (PBS), and incubated with test compounds in a final volume of 60 µl. YFP-H148Q fluorescence was measured using a fluorescence plate reader (FLUOstar Optima; BMG Labtech, Offenburg, Germany) equipped with custom excitation and emission filters (500 and 544 nm, respectively; Chroma Technology Corp., Brattleboro, VT). Fluorescence intensity in each well was measured for a total of 14 s. In each well, 100 µlof PBS/I^– (PBS with 100 mM Cl^– replaced by I^–) was injected by a syringe pump at 2 s after the start of data collection. The initial rate of fluorescence decay caused by I^– influx was measured to determine CFTR activity (Ma et al., 2002a).

Short-Circuit Current Measurements. FRT cells (stably expressing human WT CFTR) were cultured on Snapwell filters with 1-cm² surface area (Costar; Corning Life Sciences) to resistance >1000 ·cm², as described previously (Ma et al., 2002a). Filters were mounted in an EasyMount Chamber System (Physiologic Instruments, San Diego, CA). For apical Cl^– current measurements, the basolateral hemichamber contained the following: 130 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 1 mM CaCl₂, 0.5 mM MgCl₂, 10mM Na-HEPES, and 10 mM glucose, pH 7.3. The basolateral membrane was permeabilized with amphotericin B (250 µg/ml) for 30 min. In the apical solution, 65 mM NaCl was replaced by sodium gluconate, and CaCl₂ was increased to 2 mM. Solutions were bubbled with 95% O₂/5% CO₂ and maintained at 37°C. Current was recorded using a DVC-1000 voltage clamp (World Precision Instruments, Inc., Sarasota, FL) using Ag/AgCl electrodes and 1 M KCl agar bridges. Some measurements were done using T84 colonic epithelial cells and primary cultures of human bronchial epithelial cells, which natively express human WT CFTR), as described previously (Galietta et al., 2000). Short-circuit current was also measured in freshly isolated mouse ileum using symmetric bicarbonate-containing solutions, as described previously (Thiagarajah et al., 2004).

Whole-Cell Patch Clamp. Patch-clamp experiments were performed at room temperature on FRT cells stably expressing human WT CFTR, as described previously (Taddei et al., 2004). Some experiments were done using Chinese hamster ovary (CHO) cells after transient transfection with human WT CFTR. In brief, the pipette solution contained the following: 120 mM CsCl, 10 mM TEA-Cl, 0.5 mM EGTA, 1 mM MgCl₂, 40 mM mannitol, 10 mM Cs-HEPES, pH 7.3, 1 mM MgATP, and 0.4 µg/ml catalytic subunit of protein kinase A. The bath solution contained the following: 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, 10 mM mannitol, and 10 mM Na-HEPES, pH 7.4. The cell membrane was clamped at specified voltages using an EPC-7 patch-clamp amplifier (List Medical Instruments, Darmstadt, Germany). Voltage stimulation consisted of alternate pulses of –100 and +100 mV from a holding potential of 0 mV. Where needed, a full range of voltage pulses was applied in 20-mV steps, with pulse duration of 800 ms. Data were filtered at 500 Hz and digitized at 1000 Hz using an Instrutech ITC-16 AD/DA interface and the PULSE (HEKA, Lambrecht, Germany) software. Inhibitors were applied by extracellular perfusion.

Transient Transfection of CHO Cells. CHO cells, plated in 6-well plates (500,000 cells/well), were transfected with 4 µg of plasmid DNA and 10 µl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) per well according to the manufacturer's instructions. Cells were cotransfected with two plasmids carrying the coding sequence for CFTR and the yellow fluorescent protein in a 3:1 M ratio. After 24 h, cells were detached by trypsinization and sorted with a fluorescence-activated cell sorter. Fluorescent cells were isolated and plated in 35-mm Petri dishes for whole-cell patch-clamp recordings.

	Results

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Synthesis and Characterization of -Aminoazaheterocyclic-Methylglyoxal Adducts. Figure 1A shows the chemical structures of the two CFTR inhibitors, CFTR_inh-172 and MalH-1, identified previously by high-throughput screening. Figure 1B shows the -aminoazaheterocyclic-methylglyoxal adducts reported by Routaboul et al. (2007) to strongly inhibit CFTR with IC₅₀ values in the low picomolar to nanomolar range. All adducts were a mixture of at least two diastereomers, in approximately 50:50 proportion, and were highly soluble in water, except for 8ab, which had low solubility. By preparative TLC and column chromatography, we were successful in separating diastereomers 3ab but not 5ab. Routaboul et al. (2007) tested all adducts as diastereomeric mixtures, with the exception of the adduct 5a. Because we found that mixture 5ab was inactive at concentrations to 10⁶-fold greater than the reported IC₅₀ by Routaboul et al. (2007), we did not work further in separating 5a and 5b.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Structures and synthesis of -aminoazaheterocyclic-methylglyoxal adducts. A, structures of thiazolidinone CFTR inhibitor CFTRinh-172 and malonic acid hydrazide CFTR inhibitor MalH-1. B, structures of -aminoazaheterocyclic-methylglyoxals (from Routaboul et al., 2007) reported to inhibit CFTR. C, reaction scheme for synthesis of -aminoazaheterocyclic-methylglyoxals adducts. Aqueous mixture of starting materials stirred at 50°C until disappearance of -aminoheterocycle.

CFTR Inhibition Measurements. Measurements of CFTR inhibition were done first using FRT cells expressing human wild-type CFTR, an established cell line that was used for primary high-throughput screening to identify inhibitors and activators of wild-type CFTR (Ma et al., 2002a,b; Muanprasat et al., 2004), and for secondary electrophysiological assays (Taddei et al., 2004). This cell line expresses CFTR strongly and contains no other cAMP-regulated anion current. Figure 2A shows representative short-circuit current measurements, in which apical membrane current provides a direct measure of CFTR chloride conductance. Apical membrane current rapidly increased after addition of the cell-permeable cAMP agonist forskolin. However, compounds 3ab, 4ab, 5ab, and 8ab did not demonstrably reduce apical membrane current, which was rapidly reduced to approximately zero after addition of the CFTR inhibitor MalH-1 at the end of each measurement. A similar lack of CFTR inhibition was found for compounds 2ab and 6ab (data not shown). In control experiments, apical membrane current was reduced in a concentration-dependent manner by the CFTR inhibitors, CFTR_inh-172 (Fig. 2B) and MalH-1 (data not shown), with approximately 100% inhibition seen at higher concentrations (Ma et al., 2002b; Sonawane et al., 2006).

View larger version (29K): [in this window] [in a new window]   Fig. 2. CFTR inhibition studies in FRT cells expressing human WT CFTR. A, short-circuit current analysis, showing apical membrane current in permeabilized FRT cells in the presence of a transepithelial chloride gradient. The curves show responses to cAMP agonist forskolin (20 µM) followed by indicated concentrations of test compounds. Each experiment is representative of three to five measurements on separate cell cultures. B, a control study using the CFTR inhibitor CFTRinh-172. C, concentration-inhibition data from YFP fluorescence quenching plate-reader assay.

Short-circuit current measurements were also done on two cell types that natively express human wild-type CFTR, T84 colonic epithelial cells and primary cultures of human bronchial epithelial cells, as well as in mouse intestine. Figure 3 shows short-circuit current data for active chloride secretion, in which measurements were done in nonpermeabilized cells in the absence of a chloride gradient. As described above, the increased current corresponds to apical membrane CFTR chloride conductance. Forskolin produced a prompt increase in chloride current as a result of CFTR activation in T84 cells. Each of the -aminoazaheterocyclic-methylglyoxals tested did not reduce chloride current (Fig. 3A). In each case, the CFTR inhibitor CFTR_inh-172 reduced current to near zero at the end of the experiment. A similar lack of inhibition was found in human bronchial epithelial cells for compound 8ab (Fig. 3B). In addition, the -aminoazaheterocyclic-methylglyoxals at high concentrations did not inhibit short-circuit current in mouse ileal epithelium (Fig. 3C).

View larger version (20K): [in this window] [in a new window]   Fig. 3. Short-circuit analysis of epithelial cells that natively express wild-type CFTR: T84 colonic cells, primary cultures of human bronchial cells, and mouse intestine. A, short-circuit current in nonpermeabilized T84 cells in the absence of a chloride gradient. The curves show responses to forskolin (20 µM) followed by indicated concentrations of test compounds. B, short-circuit current recording from human bronchial epithelial cells. C, compound effects on short-circuit current in mouse ileum. CFTR chloride current stimulated by forskolin (20 µM) and isobutylmethylxanthine (100 µM). Compounds tested at 10 µM (S.E., n = 3). The data show that the percentage of inhibition by 5ab and 8ab is not significantly different from zero.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Whole-cell patch-clamp study of CFTR inhibition. A, time course of whole-cell membrane currents elicited at +100 (open symbols) and –100 mV (filled symbols) in a CFTR-expressing FRT cell. Where indicated, cells were stimulated with 100 µM CPT cAMP and challenged with 5ab (100 µM) and CFTRinh-172 (10 µM). Where indicated by lowercase letters, the stimulation was interrupted to apply scaled voltage steps. B, current-voltage relationships generated from the experiment in A (baseline current subtracted). C, superimposed membrane currents induced at different membrane potentials (from –100 to +100 mV) in 20-mV steps (from the same experiment). D, membrane currents from CHO cells expressing CFTR. The first three panels show superimposed membrane currents induced at different membrane potentials with 100 µM CPT cAMP alone or in combination with 5ab (100 µM) or CFTRinh-172 (10 µM). The fourth panel shows the corresponding current-voltage relationship (one set of experiments typical of three).

Thus, the short-circuit current, fluorescence, and patch-clamp analyses provide direct evidence against CFTR inhibition by -aminoazaheterocyclic-methylglyoxal adducts.

	Discussion

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The goal of our study was to evaluate the CFTR inhibition potency of the -aminoazaheterocyclic-methylglyoxal adducts reported by Routaboul et al. (2007). This investigation was motivated by the potential usefulness of potent small-molecule CFTR inhibitors in the therapy of secretory diarrheas and polycystic kidney disease. Based on the much greater reported potency of the -aminoazaheterocyclic-methylglyoxals than known CFTR inhibitors, verification of their potency and mechanism of action is important in selecting compound classes for clinical development. Although our experiments reproduced the synthesis procedures and analytical data of several -aminoazaheterocyclic-methylglyoxals, we were unable to demonstrate CFTR inhibition using multiple cell lines and several established assay methods, with experiments done in two independent laboratories in San Francisco, CA, and Genoa, Italy. In each case, positive controls with known CFTR inhibitors validated the sensitivity of the assays. As mentioned in the Introduction, for a number of reasons, it was quite surprising a priori that CFTR inhibitors with low picomolar potency could be discovered after screening of a very small set of compounds of a single chemical class. These compounds had been synthesized previously as examples of a novel, one-step stereoselective reaction of methylglyoxal with -aminoazaheterocycle (Routaboul et al., 2002).

We cannot account for the CFTR inhibition potency data reported by Routaboul et al. (2007). The simple, one-step synthesis, done exactly as reported by Routaboul et al. (2002), yielded the compounds in high yield. Analytical data for the compounds reported by Routaboul et al. (2002) and those synthesized here were in agreement. Mass spectra showed the correct molecular weights, which indicated the adduct formation from two methylglyoxal molecules with one -aminoazaheterocycle.

With regard to the CFTR inhibition studies reported by Routaboul et al. (2007), there seem to be technical concerns and inconsistencies. Our whole-cell patch-clamp recordings show that the lack of inhibition in short-circuit current and fluorescence experiments could not be due to an unusual voltage dependence of -aminoazaheterocyclic-methylglyoxals. We found no significant inhibition at all of the applied voltages in the –100 to +100 mV range, the same range used by Routaboul et al. (2007). We also tested concentrations up to 10⁶-fold higher than the reported IC₅₀, making it unlikely that a lack of inhibition was due to insufficient concentration or membrane permeability. Our whole-cell patch-clamp results in FRT and CHO cells do not agree with those of Routaboul et al. (2007) in which 10 pM, a concentration that is 10 times lower than the IC₅₀ reported by the same authors, fully blocked CFTR currents. The 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence measurements (see Fig. 7 in Routaboul et al., 2007) appear to be internally inconsistent as well, because inhibition was determined from the initial upslope of the curves in the first few seconds just after nitrate addition in the presence of the inhibitor. Inhibition at this early time is inconsistent with their data in Fig. 4C, showing 1-min kinetics for onset of inhibition. We also note that such a rapid onset of inhibition is inconsistent with the high polarity and solubility of the -aminoazaheterocyclic-methylglyoxals, because their permeation across a cell plasma membrane to bind to an inhibition site in the cell interior should be very slow. An interior binding site is mandated by the data in Fig. 8 of Routaboul et al. (2007), showing remarkably weaker CFTR inhibition after mutation (G551D) at a residue located in a cytosolic domain of CFTR. Finally, the intestinal short-circuit current measurements in Fig. 10 of Routaboul et al. (2007) appear to be flawed in that inhibitors were added before rather than after forskolin simulation of CFTR. Whereas an appropriate forskolin response was seen in their Fig. 10A, the near-zero baseline current in the intestinal strips studied in Fig. 10, B and C, before inhibitor addition, suggests nonviable tissue. The reduced forskolin response in these panels is thus a consequence of poor tissue viability rather than CFTR inhibition. We found no inhibition of CFTR-dependent short-circuit current in mouse intestine by the -aminoazaheterocyclic-methylglyoxals.

In conclusion, the -aminoazaheterocyclic-methylglyoxal adducts reported by Routaboul et al. (2007) lack CFTR inhibition activity and are thus not candidates for further development.

	Footnotes

 
This study was supported by Grants DK72517, HL73854, EB00415, EY13574, DK35124, and DK43840 from the National Institutes of Health and Drug Discovery and Research Development Program grants from the Cystic Fibrosis Foundation.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.132357.

ABBREVIATIONS: CFTR, cystic fibrosis transmembrane conductance regulator; LC/MS, liquid chromatography/mass spectrometry; NMR, nuclear magnetic resonance; TLC, thin-layer chromatography; 3ab, 2-amino-3-hydroxypyridine-methylglyoxal adducts; 4ab, adenine-methylglyoxal adducts; 5ab, 2-deoxyadenosine-methylglyoxal adducts; 6ab, adenosine-methylglyoxal adducts; 8ab, 1-aminoisoquinoline-methylglyoxal adducts; FRT, Fisher rat thyroid; WT, wild type; YFP, yellow fluorescence protein; PBS, phosphate-buffered saline; CHO, Chinese hamster ovary; CFTR_inh-172, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone; MalH-1, 2-naphthalenylamino-bis[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]propanedioic acid dihydrazide.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Alan S. Verkman, Departments of Medicine and Physiology, 1246 Health Sciences East Tower, University of California, San Francisco, CA 94143-0521. E-mail: Alan.Verkman{at}ucsf.edu

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