NMR spectra were recorded on a Bruker Avance 300. Chemical shifts are in parts per million (ppm). Mass spectra were recorded with a LCMS (Waters ZQ Micromass). HPLC analyses were performed using a C18 XBridge 3.5 μm particle size column (50 x 4.6 mm). HPLC gradient started from 98% H2O/0.1% formic acid, reaching 98% CH3CN/0.1% formic acid within 5 or 10 min at a flow rate of 2 mL/min. All commercial reagents and solvents were used without further purification. Purification yields were not optimized.
3-(N-Hydroxycarbamimidoyl)-benzoic acid methyl ester.
3.398 mmol of 3-Cyano-benzoic acid (Sigma Aldrich Fluka, 15,716-3, CAS 1877-72-1) were suspended in dichloromethane (10 mL) containing 5% of methanol. A 0.1 equivalent of 4-dimethylaminopyridine and 1.1 equivalent of dicyclohexyl carbodiimide was added at 0°C and stirred for 5 hours. Dicyclohexylurea was then filtered, and the solvent was removed by evaporation. After a second precipitation of dicyclohexylurea in diethylether, filtration and evaporation to dryness, 3-Cyano-benzoic acid methyl ester was obtained as a white powder. The crude residue was then dissolved in absolute ethanol (10 mL) containing a 1.4 equivalent of diisopropylethylamine. Hydroxylamine chlorhydrate (1.3 eq) was added to the mixture, which was refluxed for 4 hours and then ethanol was evaporated to dryness. The residue was dissolved in ethyl acetate and washed with water and brine. The organic layer was dried over magnesium sulfate and evaporated to dryness to yeld 510 mg of a white powder (yield
77%, Purity LC
1.07 min (5 min), MS (ESI+): m/z
The 3-(N-Hydroxycarbamimidoyl)-benzoic acid methyl ester (2.626 mmol) was suspended in toluene (10 mL) in presence of pyridine (1.1 eq), followed by the addition of 2-Fluoro-benzoyl chloride (Sigma Aldrich Fluka, 120847-25 G, CAS 393-52-2) and 3 hours of reflux. Toluene and pyridine were removed by evaporation. The crude residue was then dissolved in ethyl acetate and washed with aqueous acidic and basic solutions. The organic layer was dried over magnesium sulfate and evaporated to dryness to give 700 mg of a white powder (yield
89%, Purity LC
3.35 min (on 5 min), MS (ESI+): m/z
] oxadiazol-3-yl]-benzoic acid methyl ester (2.35 mmol) was suspended in a 1 M NaOH solution in methanol 1 M (10 mL). After 2 hours, the suspension was heated to 50°C and stirred overnight. After 16 hours, the reaction mixture was acidified to pH 4 with 1 M chlorhydric acid. The methanol was evaporated and the product was extracted from aqueous solution with ethyl acetate. The organic layer was then dried over MgSO4
. The product was crystallized in a mixture 60/40 ethanol/DCM and yielded 245 mg of a white crystal (yield
36%, purity LC
6.64 min (on 10 min), MS (ESI+): m/z
1 H NMR (CD2Cl2): δ 8.61 (t, J
1.4 Hz, 1 H), 8.30 (dt, J
7.8 Hz and J
1.2 Hz 1 H), 8.24 (td, J
7.6 Hz and J
1.7 Hz, 1 H), 8.15 (dt, J
7.8 Hz and J
1.4 Hz, 1 H), 7.9 (m, 1 H), 7.72 (t, J
7.7 Hz, 1 H), 7.55 (d, J
8.5 Hz, 1 H), 7.49 (q, J
7.6 Hz, 1 H).
The library of drugs contains more than 1200 pure active pharmaceutical compounds from international pharmacopeias as pure powders. Screening of the compounds was carried out from 96-well plates at 10 μM in DMSO.
Cell culture and chemical exposure
HeLa cells, expressing firefly luciferase mRNA and one of the fusion protein MS2-UPF, were grown in DMEM supplemented with 10% FBS, 2.5 mg/ml blasticidin and a mixture of 1 U/ml penicillin and 1 mg/ml of streptomycin at 37°C and 5% CO2
. Calu-3 and Calu-6 cells were grown in RPMI medium supplemented with 10% FBS and a mixture of 1 U/ml penicillin and 1 mg/ml of streptomycin at 37°C and 5% CO2
. DMD cells were grown in DMEM/199 medium (4:1) supplemented with 20% FBS, 10-7
M dexamethasone, 2.5 ng/ml HGF and a mixture of 1 U/ml penicillin and 1 mg/ml streptomycin at 37°C and 5% CO2
. Cell differentiation was initiated in DMEM supplemented with 10 μg/ml insulin and 100 μg/ml transferrin. 6CFSMEo- cells
] were grown in α-MEM supplemented with 10% FBS, 1 mM L-glutamine and a mixture of 1 U/ml of penicillin and 1 mg/ml of streptomycin at 37°C and 5% CO2
. Compounds were added 20 hours before harvesting the cells with the exception of the DMD cells which were exposed to the compounds for 48 hours starting with the addition of the differentiation medium.
Immortalization of DMD cells
hTERT and Cdk4 cDNA were cloned into distinct lentiviral vectors containing respectively the puromycin and neomycin selection markers. Transduction with lentiviral vectors were performed overnight in the presence of Polybrene (4 mg/ml; Sigma-Aldrich). Transduced cell cultures were submitted to selection in the presence of puromycin (0.2 μg/ml) and/or neomycin (0.3 mg/ml) for 8 days. The infected cells were then seeded at clonal density. Clones selected were isolated using glass cylinders and their proliferation and differentiation capacities were characterized in the culture conditions described above. The clone used in this study presented growth and differentiation capacity similar to that observed on the original primary culture.
HeLa cells stably expressing firefly luciferase mRNA with MS2 binding sites (bs) in the 3’UTR were obtained by transferring Fluc cDNA with MS2 bs from pCFluc (gift from Dr. Jens Lykke-Andersen) to the pLenti6/V5 using the PCR-based TOPO directional cloning system (Life Technologies) (sense primer: 5’CACCATGGAAGACGCCAAAAACAT3’ and antisense primer: 5’TGACACTATAGAATAGGGCC3’). The lentiviral particles produced with ViraPower Lentiviral Expression System (Life Technologies) were used to transduce the HeLa cells. Stable Fluc expressing clones were selected and isolated using a selective medium containing 2.5μg/ml Blasticidin.
The MS2-UPF expression vectors were constructed by transferring MS2-UPF cDNA from the pMS2-UPF proteins vector (gift from Dr. Jens Lykke-Andersen) into pLenti4/V5-DEST vector using Gateway Cloning according the manufacturer’s instructions. Each MS2-UPF cDNA was cloned using the following primers: sense: 5’GGGGACAAGTTTGTACAAAAAAGCAGGCTCACCATGGCTTCTAACTTTACTCAG3’; and antisense: 5’GGGGACCACTTTGTACAAGAAAGCTGGGTTA\ATTTAGGTGACACTATAGAA3’.
HeLa cells stably expressing firefly luciferase were transduced with the recombinant lentiviral constructs containing one of the four pLenti4/V5-MS2-UPF plasmids. Cells were seed into 96 well plates two days after infection and then exposed to the chemical compounds for 20 hours. Plates were loaded into a Tristar LB 941 microplate reader (Berthold) after the addition of Steadylite luciferase substrate (PerkinElmer) in order to measure Luciferase activity in each well for 1 second. Each plate was read 3 times.
RNA was purified using RNazol (MRC) according to the manufacturer’s instructions. A portion (1/5) of the RNA preparation was evaluated by reverse-transcription (RT) PCR (RT-PCR) using Superscript II (Life Technologies) for 2 hours at 42°C in presence of random hexamer for RT. The resultant cDNAs were PCR amplified in presence of dCTP( α33P) (Perkin Elmer) with primers indicated in Table
listing of primer sequences used in this study for PCR amplification
PCR products were quantified using Personal Molecular Imager and QuantityOne quantification software (Bio-Rad).
Glycosylation analysis of CFTR and CFTR immunoprecipitation
Cells were lysed in RIPA buffer (1% deoxycholic acid, 1% Triton x100, 0.1% SDS, 50 mM Tris pH7.4 and 150 mM NaCl) containing HALT protease inhibitor cocktail (Pierce). Then lysats were incubated with 10,000 units of PNGase (New England Biolabs) or 10,000 units of EndoH (New England Biolabs) for 4 hours at 37°C. Cells extracts were then used for the immunoprecipitation of CFTR. Briefly, non specific interactions with protein G agarose were removed by incubating protein extracts with 50 μl of protein G agarose beads (Pierce) for 30 minutes at 4°C. Then protein extracts were incubated with mAb 24–1 antibody for 2 hours at 4°C before to add 50 μl of protein G agarose beads and incubate for an additional 2 hours at 4°C. Beads were finally washed three times with RIPA buffer. Elution of CFTR was obtained by adding 50 μl of 2x sample buffer and vortexing before centrifuging samples and collecting the supernatant.
Western blot hybridization
Proteins were isolated in the following lysis buffer: 50 mM Tris pH7, 20 mM EDTA and 5% SDS. All protein extracts, with the exception of dystrophin, were analyzed by 10% SDS-PAGE. Dystrophin analysis was carried out using 7.5% SDS-PAGE. After migration, proteins were transferred onto a nitrocellulose membrane and then exposed to the primary antibody. The primary antibodies used were: anti-p53 (D01; Santa-Cruz), anti-dystrophin against an N-terminal epitope (4C7; Santa-Cruz), anti-dystrophin against a C-terminal epitope (exons 77–78) (Abcam), anti-CFTR against an N-terminal epitope (MM13-4; Millipore), anti-tubulin (Epitomics) and anti-phosphorylated eIF2α (Epitomics). The primary antibody incubation is followed by a secondary antibody incubation using anti-mouse or rabbit antibody (Jackson Immuno Research). Proteins finally detected using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce).
SPQ Halide-efflux Assay
Cells were seeded in 96-well plates and loaded overnight with 10 mM SPQ fluorophore (6-methoxy-N-(3-sulfopropyl)-quinolinium) (Life technologies). Cells were washed twice with I- buffer (135 mM NaI, 2.4 mM K2HPO4, 0.6 mM KH2PO4, 1 mM MgSO4, 1 mM CaSO4, 10 mM dextrose and 10 mM Hepes, pH 7.3) and then incubated in iodide buffer for 30 min. After establishing the basal fluorescence (2 min), the iodide buffer was replaced with NO3- buffer (135 mM NaNO3 instead of NaI) containing 20 μM forskolin and 200 μM IBMX. Fluorescence was then evaluated for a further 10 min. Fluorescence intensities were measured every 15 seconds using the Tristar LB 941 microplate reader (Berthold) equipped with a 340 nm excitation filter and a 450 nm emission filter.
Calu-6 cells were incubated with DMSO or 25 μM amlexanox for 20 hours before harvesting cells, or with cycloheximide (200 μg/ml) 4 hours before harvesting cells. Newly synthesized proteins were measured using Click-iT AHA for Nascent Protein Synthesis kit (Life Technologies). Briefly, 30 min. before harvesting cells, L-AHA (L-azidohomoalanine) is added to the cell culture medium in order to be incorporated into newly synthesized proteins. Then Cells were washed three times with PBS and lysis buffer was added to the cells. After 30 min. on ice, lysat was centrifuged and supernatant was collected. TAMRA molecule was then bound to the L-AHA modified amino acid using Clik-iT reaction buffer (Life technologies) by following the recommendations of the manufacturer. Finally, TAMRA was detected and quantified by western-blot using a DNR MF-ChemiBIS 3,2 (DNR) and Multi Gauge software (FUJIFILM).
Cells were harvested after trypsin treatment, centrifuged 3 min at 200 g and resuspended in PBS. Tali dead cell red (propidium iodide) reagent was then added to the cells. After a 5 min incubation in the dark, the cells were loaded into Tali image-based cytometer (Life Technologies).
DMD cells were grown on ibiTreat surface μ-dishes (Ibidi) and differentiated as described above. Cells were fixed 10 min in 95% ethanol at room temperature 48 hours after initiating differentiation. The fixed cells were washed twice in PBS and incubated with the C-terminal anti-dystrophin antibody for 1 hour at room temperature, washed twice with PBS and incubated with the secondary anti-rabbit antibody conjugated with Alexa 488 fluorophore (Life Technologies) for 1 hour at room temperature. The cells were then incubated with Hoechst stain for 1 min at room temperature before adding Vectashield (ABCYS) mounting medium and sealing a coverslip.