Leporizines A–C: Epithiodiketopiperazines Isolated from an

Article pubs.acs.org/jnp

Leporizines A−C: Epithiodiketopiperazines Isolated from an Aspergillus Species Ricardo Reategui,* Joshua Rhea, Janet Adolphson, Kathryn Waikins, Ryan Newell, John Rabenstein, Ulla Mocek, Michele Luche, and Grant Carr AMRI, Bothell Research Center, 22215 26th Avenue SE, Bothell, Washington 98021, United States S Supporting Information *

ABSTRACT: Three new compounds named leporizines A−C (1−3) have been isolated from an Aspergillus sp. strain. Their structures were elucidated by analysis of 1D and 2D NMR spectra. Leporizines A and B were isolated during dereplication of hits from a high-throughput screening campaign for correctors of the cystic fibrosis transmembrane conductance regulator (CFTR), and leporizine C was isolated while preparing additional material for characterization of leporizines A and B. CFTR activity observed for leporizines A and B was highly correlated with cell toxicity and was determined to be a nonspecific effect. Leporizine C was not cytotoxic to cells and did not elicit a response in the CFTR assays. To the best of our knowledge, leporizines A−C represent the first examples of this unusual epithiodiketopiperazine skeleton. ystic fibrosis (CF) is a recessive, genetic disease characterized by chronic lung illness, pancreatic dysfunction, and impaired electrolyte secretion and is linked to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR).1−3 CFTR is a cAMP-regulated chloride channel expressed in the epithelial membranes of the respiratory and digestive tissues and also influences other ion channels involved in electrolyte and fluid movements across the epithelia.1−4 In the United States, 90% of all CF patients carry at least one gene with a mutation at loci 508, where a phenylalanine is deleted from the amino acid sequence (F508del). This mutation generates a F508del-CFTR protein that is not folded properly and is subject to increased degradation by the proteasome. In addition, the F508del-CFTR protein that can translocate to the cell surface has diminished functional activity compared to normal CFTR.2 The loss of CFTR function causes decreased chloride and water excretion in epithelial-lined tissues, resulting in thick mucus, which obstructs the airways of the lungs and the pancreatic ducts. Compounds that facilitate movement of the F508del-CFTR protein to the cell surface are termed “correctors”, while compounds that restore function to the F508del-CFTR protein at the cell surface are termed “potentiators”. The presence of additional and more functional CFTR protein on the epithelial surface should restore function to these tissues and alleviate the pathology of the disease. In the search for new therapies for CF, a research collaboration was initiated between AMRI and the Cystic Fibrosis Foundation Therapeutics (CFFT) to screen AMRI’s Natural Products Libraries as a source of correctors and potentiators of F508del-CFTR function. More than 280 000 natural product samples (whole extracts and fractionated

C

© XXXX American Chemical Society and American Society of Pharmacognosy

samples) were screened in a FLIPR-based NaI Flux assay for corrector or potentiator activity. Samples that produced positive results were rescreened in 4-point dose responses in the presence and absence of a specific CFTR blocker in the FLIPR-based assay. Approximately 200 natural product sources were selected for refermentation (if microbial), extraction, and fractionation to produce enough material for dereplication. Isolated compounds from fractions that appeared to have activity in the NaI Flux assay were also tested in a conductance assay, which measures loss of voltage resistance over a monolayer of cells. While the NaI Flux assay measures the ability of I− ions to enter the cell, the conductance assay measures the total change in conductance from all ion movement, including ion movement due to loss of tight junction integrity. Nonspecific permeability due to compound toxicity is measurable by comparison of the baseline resistance to that of a nontreated control. The change in conductance can be measured after the addition of reagents (forskolin, potentiator, and CFTR blocker) to each well, allowing a more detailed observation of the effect of each reagent on the cell layer. Although the throughput for the conductance assay is considerably lower that than of the NaI Flux assay, the conductance assay measurements are more physiologically relevant to the CF disease state. As a result of this collaboration, 28 compounds with possible potentiator and/or corrector activities were isolated and fully identified, including one lead candidate. In the course of this collaboration one natural product extract from a fungal source that had been selected as a possible corrector was found to Received: December 21, 2012

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produce three new structurally related epithiodiketopiperazines named leporizines A−C (1−3). While leporizines A and B were responsible for the apparent activity, leporizine C was inactive. Details of the fermentation, isolation, structure elucidation, and biological activity of these compounds are discussed here.

Article

RESULTS AND DISCUSSION

An active ethyl acetate extract of an Aspergillus sp. strain (AMRI-13820) was fractionated into a 96-well plate by LCMS. Active fractions identified in the NaI flux screening assay were correlated with the UV, ELS, and MS data. This information guided the isolation of leporizines A−C from the extract by HPLC. Leporizine A (1) was obtained as a white solid. Its molecular formula was determined to be C25H25N3O8S3 (15 unsaturations) on the basis of HRESIMS. Analysis of 1H NMR, 13C NMR, and HSQC (Table 1) data of 1 indicated the presence of three methyl, one methylene, five methine (three of them oxygenated), five quaternary, eight olefinic, and three carbonyl carbons. Three spin systems could be revealed by analysis of COSY correlations corresponding to the C-8′−C-12, C-10′−C11′, and C-6−C-9 fragments. The presence of an orthosubstituted aromatic structure in 1 was indicated by the signals that appeared at δC 126.8 (C-5) and 153.5 (C-10) and those at δH/δC 7.18/125.9, 6.71/119.5, 7.18/132.4, and 6.18/110.9, which were assigned to C-6, C-7, C-8, and C-9, respectively (Table 1). HMBC correlations from H-6 to C-4 and C-10, from H-9 to C-5, and from H-11 to C-4 and C-10 suggested that this aromatic structure is part of a 2,3-substituted indole ring system. The connectivity of this ring to C-2 and C-3 was established by the additional HMBC correlations of H-11 to C2, C-3, C-4, and C-5, which determined the existence of partial structure I (Figure 1). The presence of an additional six-membered ring in 1 can be proposed on the basis of HMBC correlations from the methine

Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Spectroscopic Data for Leporizines A−C (1−3) in CD3OD leporizine A (1) position 1 2 3 4 5 6 7 8 9 10 11 12 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′

δC, type 164.8, 84.3, 80.8, 86.1, 126.8, 125.9, 119.5, 132.4, 110.9, 153.5, 80.6,

C C CH C C CH CH CH CH C CH

164.6, 78.7, 77.2, 79.1, 190.2, 120.9, 44.2, 93.1, 175.9, 22.2,

C C CH C C C C CH C CH2

62.7, 13.9, 20.3, 24.5,

CH CH3 CH3 CH3

δH (J in Hz)

4.56, s

7.18, 6.71, 7.18, 6.68,

m t (7.6) m d (7.9)

5.35, s

4.48, s

4.32, q (6.6) 4.23, 2.87, 4.71, 1.31, 1.07, 1.08,

d (19.6) dd (6.3, 19.6) d (6.3) d (6.6) s s

leporizine B (2) δC

leporizine C (3)

δH (J in Hz)

163.8 81.3 78.4 88.2 129.5 125.5 120.1 131.9 111.0 151.5 82.5 164.0 75.5 78.0 78.7 190.8 120.8 44.7 93.6 175.0 23.4 62.0 14.2 20.6 25.2

B

δC

4.75, s

7.29, 6.77, 7.14, 6.64,

br dt dt br

d (7.6) (0.6, 7.6) (1.3, 7.9) d (7.9)

5.48, s

4.79, s

4.32, q (6.6) 3.62, 2.86, 4.50, 1.29, 1.10, 1.17,

dd (3.8, 18.9) dd (6.3, 18.9) dd (3.8, 6.3) d (6.6) s s

164.3 74.0 78.4 86.4 130.3 124.3 119.3 131.3 110.5 152.1 81.7 14.6 165.4 72.9 76.1 78.8 189.7 120.7 44.2 92.6 173.3 25.5 61.0 14.8 20.4 25.4 15.0

δH (J in Hz)

4.60, s

7.27, 6.71, 7.12, 6.60,

br dt dt br

d (7.6) (1.0, 7.6) (1.3, 7.9) d (7.9)

5.57, s 1.93, s

4.65, s

4.45, q (6.6) 2.95, m 4.52, 1.31, 1.18, 1.28, 2.15,

dd (2.5, 6.0) d (6.6) s s s

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15 degrees of unsaturation. This connection can only be accomplished through the quaternary carbons C-2 and C-2′, each forming bonds to a nitrogen atom and to a sulfur atom. Each nitrogen atom had to be subsequently linked to their corresponding C-1 and C-1′ to form two amide carbonyls, and the sulfur atoms had to be linked together to form a bridge connecting C-2 and C-2′. The gross structure of 1 thus generated corresponded to an epithiodiketopiperazine. Leporizine B (2) was also isolated as a white solid. Its molecular formula had one sulfur less than 1 and was determined as C25H25N3O8S2 (15 unsaturations) on the basis of HRESIMS as well as 1H NMR and 13C NMR (Table 1) data. The UV, IR, and NMR spectra of 2 were very similar to those of 1. In addition, analysis of the COSY and HMBC correlations of 2 revealed identical spin systems and connectivities with those found in 1, strongly suggesting that the only difference between 1 and 2 is in the sulfur bridge. All data supported the structure of leporizine B (2) with a disulfur group connecting C-2 and C-2′. Leporizine C (3) was isolated as colorless wax. The molecular formula of 3 was determined as C27H31N3O8S2 (14 unsaturations) by HRESIMS, consistent with the 1H NMR, 13C NMR, and HSQC data (Table 1). The UV and IR spectra of 3 were similar to those of 1 and 2, but its 1H NMR and 13C NMR spectra revealed the presence of two additional methyl singlets. The same spin systems present in 1 and 2 were also present in 3, as the analysis of the COSY experiment revealed. Analysis of the HMBC correlations of 3 indicated identical connectivities

Figure 1. Selected HMBC correlations supporting the presence of fragments I and II in leporizine A (1).

proton H-11′ to C-4′ and C-5′ and from the methylene protons Ha-10′/Hb-10′ to C-4′, C-6′, C-9′, and C-11′. This ring should contain an α,β-unsaturated ketone, supported by the observed chemical shifts of C-5′, C-6′, and C-9′. A connection between C-7′ and C-6′ can be proposed by the presence of HMBC correlations from H-13′ and H-14′ to C-6′, C-7′, and C-8′. Additional HMBC correlations from H-8′ to C-9′ and the chemical shifts of H-8′ and C-8′ could be explained by a linkage between C-8′ and C-9′ via an oxygen atom. These considerations indicated the presence of a furan fused to the six-membered ring. Additional HMBC correlations from the methine hydrogen H-3′ to C-1′, C-2′, C-4′, C-5′, and C-11′ led us to propose partial structure II (Figure 1). The remaining structural elements were composed of two nitrogen atoms and three sulfur atoms that had to establish the connection of fragments I and II and generate a structure with

Figure 2. Leporizines in corrector conductance assay. Leporizines were tested in the conductance corrector assay using a 2-fold dilution series with the highest testing dose at 20 μM. (a) Responses by leporizines A and B in the conductance assay at 10 and 20 μM are not reversible by the addition of CFInh-172. Leporizine C response is similar to that of the DMSO control. (b) Leporizines A and B cause a severe loss of baseline transepithelial resistance at concentrations ≥ 10 μM. Baseline transepithelial resistance for leporizine C does not decrease at higher concentrations. Although treatment with 10 μM C17 causes a slight loss of baseline transepithelial resistance, the forskolin/PG-01-induced increase in conductance is reversible by the addition of CFInh-172. C

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49.5 for CD3OD or δ 2.49/39.5 for (CD3)2SO). HRESIMS data were acquired on a Waters Q-Tof Premier mass spectrometer by direct infusion of isolated samples diluted with CH3CN−H2O (1:1) + 0.1% HCOOH. The LCMS system consisted of an Agilent 1100 solvent pump, a degassing system, an LC flow-splitter, an ELS Sedere detector, and an Agilent photodiode array detector. Separation was achieved on a Phenomenex Gemini C18 (250 × 10 mm, 5 μm) HPLC column with a gradient of H2O (0.05% TFA)−CH3CN (0.05% TFA) at 5 mL/min, 95:5 to 100% CH3CN over 15 min. Biological Material. The Aspergillus sp. strain AMRI-13820 was isolated from a soil sample collected from sage rangeland south of Bridger, Montana. The sample was air-dried for 7 days, ground using a sterile mortar and pestle, and plated onto PDA (Difco) using a previously described replica-stamping technique.12 All plates were incubated in the dark at 25 °C for 5 days. Observed colonies were transferred to and maintained on yeast malt extract agar plates. The taxonomic name was assigned by phenotypic observations of various traits on Czapek’s agar and malt extract agar and comparison to published strain descriptions. The observed features, including globose olive-colored conidial heads becoming columnar with age, biseriate sterigmata, and dark colored, vertically elongated sclerotia with a white apical portion, closely matched the description of Aspergillus leporis by States and Christensen.13 Fermentation. The fermentation procedure utilized was a twostep process in which a suspension of culture macerate (mycelium and spores) was inoculated into 250 mL flasks containing 30 mL of a nutrient seed medium having the following composition per liter: 20 g D-glucose (Mallinckrodt 4912), 15 g Pharmamedia (Traders protein), 5 g yeast extract (Difco 0127-17), 4 g CaCO3 (Sigma C-6763), 3 g (NH4)2(SO4) (Mallinckrodt 3512), and 0.03 g ZnSO4·7H2O (Sigma Z-4750), adjusted to pH 6.5 prior to autoclaving. After inoculation, the flasks were incubated on a rotary shaker at 250 rpm (2 in. throw) and 25 °C for 2 days. Aliquots of 1 mL of the seed culture were then used to inoculate one hundred 250 mL flasks containing 30 mL of a production medium with the following composition per liter: 40 g alpha lactose (Sigma L-3625), 30 g Pharmamedia (Traders protein), 5 g D-glucose (Mallinckrodt 4912), 5 g Bacto peptone (Difco 0118-170), 3 g CaCO3 (Sigma C-6763), 0.5 g K2HPO4, Dibasic (Mallinckrodt 7092), 0.5 g MgSO4·7H2O (Sigma M-1880), and 0.3 g KCl (Sigma P4504), adjusted to pH 7.0 prior to autoclaving. Following inoculation, the production flasks were incubated on a rotary shaker at 250 rpm and 25 °C for 6 days. Extraction and Isolation. The fermentation mixture from each flask was pooled into a single vessel for extraction (∼3 L) with an equal volume of ethyl acetate. The ethyl acetate fraction was treated with sodium sulfate and then evaporated under reduced pressure. Isolation of compounds 1−3 was guided by LCMS data obtained during the dereplication process. Approximately 40 mg of the dried ethyl acetate extract was applied repeatedly in several portions onto a Phenomenex Gemini C18 column (150 × 10 mm, 5 μm) and eluted with a mobile phase consisting of solvents A (water and 0.05% trifluoroacetic acid) and B (acetonitrile and 0.05% trifluoroacetic acid), delivered in a gradient mode at 5 mL/min, starting with B from 5% to 100% in 30 min, and then B at 100% for 5 min. Semipurified compounds 1−3 were subjected again to HPLC purification using the same column and mobile phase in an isocratic mode with B at 30%. The new epithiodiketopiperazines 1 (9 mg), 2 (5 mg), and 3 (1 mg) were isolated with retention times of 23.8, 16.4, and 19.1 min, respectively. Sodium Iodide (NaI) Flux Assay for CFTR Corrector Activity. Fischer rat thyroid (FRT) epithelial cells co-expressing F508del-CFTR and yellow fluorescent protein (YFP-H148Q/I152L) were provided by Dr. A. S. Verkman, UCSF.2 YFP fluorescence is strongly quenched by iodide ions, but only weakly quenched by chloride ions.4 CFTR chloride channel opening is measured by monitoring the quenching of fluorescence by addition of NaI buffer after cAMP stimulation of the channel by forskolin treatment.2 Compounds used in this study (4cyclohexyloxy-2-{1-[4-(4-methoxybenzensulfonyl)piperazin-1-yl]ethyl}quinazoline (C3), N-[2-(5-chloro-2-methoxyphenylamino)-4′methyl[4,5′]bithiazolyl-2′-yl]benzamide (C4), N-(2-(5-chloro-2-me-

with those found in 1 and 2 in addition to the observed correlations of the methyl singlets to their respective C-2 and C-2′. These analyses indicated that 3 has C-2 and C-2′ linked to a sulfur atom, as in 1 and 2, and each sulfur atom has to be subsequently connected to a methyl group. All these considerations allowed proposing a gross structure of 3 that is similar to 1 and 2 but without a sulfur bridge, consistent with its molecular formula and unsaturation number. The relative stereochemistry of leporizines A−C (1−3) was largely based on NOESY correlations observed for 3. For example, the NOESY experiment in CD3OD revealed correlations of H-3 to H-11 and H3CS-2, of H3CS-2′ to H11, H-3′, and H3-14′, and of H3-14′ to H-8′ and H2-10′, which established the orientation of all these groups on the same face of the molecule. The orientations of OH-4, OH-4′, and H-11′ could not be assigned from NOESY experiments in CD3OD or DMSO-d6. However, biosynthetic considerations seem to suggest that OH-4 may have the same orientation as H-3 and H-11, since the stereocenters in the pyrroloindole moiety in 3 are identical to those of sporidesmin.5 Likewise, it is reasonable to propose that OH-4′ and H-11′ may be oriented opposite the aforementioned groups based on the similarity to the known compound phomalirazine.6 Several attempts to crystallize 1 and 2 did not produce material suitable for X-ray experiments, but the observation that the optical rotations of the three leporizines were of the same sign strongly suggested that the relative stereochemistry of 3 is the same as 2 and 1. Although the pyrroloindole system encountered in 1−3 could be found as part of epithiodiketopiperazines in several fungal metabolites (i.e., chaetomins,7,8 verticillins,9 sporidesmins,5,10 gliocladins11), the ring system depicted in fragment II is very rare and only found in phomalirazine (5).6 Combining this ring system with pyrroloindoles to form epithiodiketopiperazines is unprecedented among natural products. Leporizines A (1) and B (2) did not show activity significantly greater than the DMSO control in the conductance corrector assay at concentrations lower than or equal to 5 μM (Figure 2). Higher concentrations than 5 μM showed loss of reversibility and severe loss of starting transepithelial resistance, suggesting possible toxicity. Leporizine C (3) did not have activity or loss of starting transepithelial resistance (Figure 2). The results of the conductance assays indicated that leporizines A and B had predominantly non-CFTR-specific activities and showed indications of toxicity. Leporizines A and B were also found to be cytotoxic against HepG2 cells (data not shown). Biological experiments on these compounds were discontinued in order to pursue more promising leads from other natural product sources. Leporizines A and B have a sulfur bridge, and its presence or the conformation it provides may play a role in both their apparent activity and toxicity. Leporizine C has no such bridge and did not show any activity or toxicity in either of the assays. Additional studies to identify the mode of action of these compounds are necessary to fully explain these results.

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were acquired on a Bruker DRX 500 spectrometer equipped with a 5 mm inverse probe and operating at 500 MHz for 1H NMR and 125 MHz for 13C NMR. Deuterated solvents of 100% grade from Cambridge Isotopes and 5 mm NMR tubes from Wilmad and Shigemi were used. 1H/13C chemical shifts were referenced to the residual solvent peak (δ 3.30/ D

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thoxyphenylamino)-4′-methyl-4,5′-bithiazol-2′-yl)pivalamide (C17), 2-[(2-1H-indol-3-ylacetyl)methylamino]-N-(4-isopropylphenyl)-2phenylacetamide (PG-01), and 4-[4-oxo-2-thioxo-3-(3trifluoromethylphenyl)thiazolidin-5-ylidenemethyl]benzoic acid (CFInh-172)) were provided by the CFTR Compound Repository. A combination of two known correctors, C3 (12.5 μM) and C4 (25 μM), was found to give a higher signal window than each compound alone, and this combination was used as the positive control. F508delFRT cells were plated in a 384-well plate and incubated for 16−24 h at 37 °C under 5% CO2. Serially diluted compounds (100 μM highest final concentration) were added, and the plates were incubated at 37 °C for 16−20 h. Cell plates were washed, and 20 μM forskolin (for cAMP stimulation) and 50 μM genistein (as a potentiator) were added and incubated for 20 min at room temperature. A FLIPR TetraPlus (Molecular Devices) was used to read fluorescence at 2 reads/s for 12 s before and 20 s after addition of NaI buffer. A single phase exponential decay model was used, and the initial rate of iodide influx was calculated as the initial slope at the T0 of the NaI addition. Activity specific to CFTR can be determined by performing the assay in the presence of 10 μM CFInh-172, which blocks CFTR function.14 Compound-treated cells that have a fluorescence signal lower than the fluorescence of the control before addition of forskolin and genistein may have lost cell membrane integrity as a result of compound toxicity. Conductance Corrector Assay. The conductance assay was developed by Dr. Robert Bridges of Rosalind Franklin University of Medicine and Science. Custom equipment for this assay was kindly provided by Dr. Bridges for this collaboration. The F508del-CFTR FRT cells provided by Dr. Verkman were also used for this assay. FRT cells were grown on Transwell plates (Corning) for 7−10 days before use in the assay. Media was replaced every 2 days and the day before the assay. Serial dilutions of compounds (20 μM highest concentration) were added to both the apical and the basal sides of the plates, and the plates were incubated at 37 °C and 5% CO2 for 16−20 h. On the day of the assay the media was replaced and the Transwell plate was placed on a custom plate incubator to maintain the cell temperature at 37 °C. The baseline transepithelial resistance of each well was measured using an EVOMX voltohmmeter and STX-100 electrode (World Precision Instruments). Forskolin (10 μM) was added to the basal side, and after 20 min the transepithelial resistance was read. PG-013 (10 μM) (a potentiator) was added to the basal side, and after 20 min the transepithelial resistance was read. CFInh-172 (10 μM) was added to both the apical and the basal sides, and after 30 min the transepithelial resistance was read. Conductance was calculated as 1/R where R is the transepithelial resistance. The change in conductance was calculated for each addition, and the total response was calculated as the forskolin response plus potentiator response. Leporizine A (1): white solid; mp 123 °C (dec); [α]D −60 (c 14.8, CH3OH); UV (CH3CN) λmax 282 nm; IR (film on KBr plate) νmax 3409, 1679, 1611, 1427, 1383, 1206, 1135, 843, 801, 753, 723 cm−1; 1 H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data, see Table 1; ESIMS m/z 592 [M + H]+; HRESIMS m/z 592.0900 (calcd for C25H26N3O8S3, 592.0882) m/z 614.0721 (calcd for C25H25N3O8S3Na, 614.0702). Leporizine B (2): white solid; mp 120 °C (dec); [α]D −46 (c 5.0, CH3OH); UV (CH3CN) λmax 283 nm; IR (film on KBr plate) νmax 3396, 1679, 1611, 1427, 1378, 1205, 1138, 840, 797, 752, 722 cm−1; 1 H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data, see Table 1; ESIMS m/z 560 [M + H]+; HRESIMS m/z 560.1177 (calcd for C25H26N3O8S2, 560.1161); m/z 582.1000 (calcd for C25H25N3O8S2Na, 582.0981). Leporizine C (3): colorless wax; [α]D −12 (c 1.1, CH3OH); UV (CH3CN) λmax 287 nm; IR (film on KBr plate) νmax 3429, 1679, 1620, 1439, 1208, 1139, 844, 803, 725 cm−1; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data, see Table 1; ESIMS m/z 590 [M + H]+; HRESIMS m/z 590.1652 (calcd for C27H32N3O8S2, 590.1631); m/z 612.1472 (calcd for C27H31N3O8S2Na, 612.1450).

Article

ASSOCIATED CONTENT

S Supporting Information *

Pictures of strain AMRI-13820 grown under different conditions. 1D and 2D NMR spectra of leporizines A−C. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS AMRI would like to thank the Cystic Fibrosis Foundation Therapeutics, Bethesda, MD, and especially Dr. M. Ashlock, for support in this collaboration. We also thank Dr. R. Bridges of the Rosalind Franklin University of Medicine and Science, North Chicago, IL, for use of the conductance assay protocols and equipment and for scientific support of the project. We also thank Dr. A. Verkman of the University of California, San Francisco, CA, for the use of the F508del/YFP FRT cell line and the NaI flux assay protocols for high-throughput screening.

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REFERENCES

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