Small molecule anionophores promote transmembrane anion permeation matching CFTR activity

Anion selective ionophores, anionophores, are small molecules capable of facilitating the transmembrane transport of anions. Inspired in the structure of natural product prodigiosin, four novel anionophores 1a-d, including a 1,2,3-triazole group, were prepared. These compounds proved highly efficient anion exchangers in model phospholipid liposomes. The changes in the hydrogen bond cleft modified the anion transport selectivity exhibited by these compounds compared to prodigiosin and suppressed the characteristic high toxicity of the natural product. Their activity as anionophores in living cells was studied and chloride efflux and iodine influx from living cells mediated by these derivatives was demonstrated. These compounds were shown to permeabilize cellular membranes to halides with efficiencies close to the natural anion channel CFTR at doses that do not compromise cellular viability. Remarkably, optimal transport efficiency was measured in the presence of pH gradients mimicking those found in the airway epithelia of Cystic Fibrosis patients. These results support the viability of developing small molecule anionophores as anion channel protein surrogates with potential applications in the treatment of conditions such as Cystic Fibrosis derived from the malfunction of natural anion transport mechanisms.

The spectroscopic and analytical data for the new aldehydes and their precursors are reported below:

S10
Computational methods. Calculations of the molecular electronic structure were done using the Gaussian 09 program 4 . The geometries of all species were fully optimized at the B3LYP/6-31+G** level. The environmental effects were taken into account by the Polarizable Continuum Mode (PCM) using the CPCM model 5 . The nature of all optimized structures was determined using harmonic frequency analysis as true minima with no imaginary frequencies.
Supplementary  For the valinomycin experiments, 20 mg/ml of Asolectin from soybean (Sigma-Aldrich 11145, mixture of phospholipids) were dissolved in chloroform, and lipid films were obtained by evaporation of the solvent under a gentle nitrogen flux; in order to remove all chloroform, films were further dried overnight in vacuum. The phospholipids were hydrated in chloride buffer (in mM: NaCl 450, 20 mM HEPES; pH 7.0), and vigorously vortex mixed and, to ensure equilibration, sonicated in 5 cycles of 1.5 min each, with 1 min rest, in ice. Liposomes were centrifuged at 2000 g for 5-10 minutes to remove any titanium particle released by the sonicator tip and larger aggregates. LUV were then obtained by extrusion through polycarbonate filters mounted in extruder. Samples were subjected to 19 passes through a single 100 nm mesh filter. External solution was exchanged twice in a Sephadex G25 column previously equilibrated with the external chloride-free solution in mM: NaNO 3 450, 20 mM HEPES; pH 7.0).

Chloride efflux measurements in LUV.
Efflux of chloride from LUV was measured with a chloride ion-sensitive electrode (Vernier, Beaverton, Oregon, USA) in a constantly stirred 3.5 to 5.0 ml LUV suspension. Data was acquired using a LabQuest mini interface (Vernier). Ionophores were dissolved in DMSO to a concentration of 10 mM. After an initial equilibration, chloride efflux was induced by a small volume (<1%) of ionophore. Control experiments where similar amounts of DMSO (without anionophores) were added demonstrated that these concentrations of DMSO do not induce any chloride efflux (see below). The measurement was concluded with the addition of the detergent Triton-X or polyoxyethylene 10 tridecyl ether (C13E10) to break off the bilayers and measure the maximum chloride content in the LUV's. where Cl(300) / Cl max i s the ratio between the external chloride concentration after 300 s and the maximum chloride concentration upon application of detergent; A is the concentration of the anion carrier; EC 50 is the concentration to obtain the half of the maximum effect; n is the Hill's coefficient.

Determination of the dose-response (EC 50
Figures S10-S13 show the transport data driven by the application of 1a-d and prodigiosine on LUV containing NaCl (489 mM NaCl and 5 mM phosphate buffer, pH 7.2), and immersed in NaNO 3 (489 mM NaNO 3 and 5 mM phosphate buffer, pH 7.2). At the end of the experiment the vesicles were lysed with detergent to release all chloride ions and the resulting value was considered to represent the 100% and was used as such. Each trace represents the average of at least three different experiments, carried out with at least three different batches of vesicles. Figures S15-S19 show the transport data driven by the application of 1a-d and prodigiosine on LUV containing 451 mM NaCl and 20 mM phosphate buffer, pH 7.2, were immersed in150 mM Na 2 SO 4 , 40 mM HCO 3 and 20 mM phosphate buffer, pH 7.2. At the end of the experiment the vesicles were lysed with detergent to release all chloride ions and the resulting value was considered to represent the 100% and was used as such. Each trace represents the average of at least three different experiments, carried out with at least three different batches of vesicles. 2) The nonencapsulated lucigenin was removed by size exclusion chromatography, using a Sephadex G-50 column, eluted with a lucigenin-free nitrate solution. The fluorescence emission at 503 nm after excitation at 372 nm was recorded using a Hitachi F-7000 fluorescence spectrophotometer, at 25 ºc. After recording the fluorescence baseline for 60 s, an aliquot of the anion carrier in methanol was added. The fluorescence emission was then recorded during five minutes.

Supplementary
Safranin O assays. LUV were prepared as described above. Phospholipids were rehydrated with a chloride solution, and the non-encapsulated solution was replaced by sulphate by size exclusion chromatography. At the beginning of the assay Safranin O was added for a final concentration of 0.2 µM.The fluorescence at 580 nm after exciting at 520 nm was recorded for two minutes, and an aliquote of theanion carrier, in DMSO solution, was added; fluorescence changes were monitored over time for 10 minutes. The fluorescence intensity relationship I(t) / I(120) was plotted as a function of time.

Human embryonic kidney (HEK) cell lines were grown in standard conditions, in
Ham's F10 medium supplemented with 2 mM L-glutamine and 10% foetal bovine serum. All culture reagents were purchased from Sigma Aldrich. Cells were grown at 37 °C under a 5% CO 2 atmosphere.
Cell viability assays. Cell viability was determined by the MTT assay. Briefly, cells (1×10 5 cells/mL) were seeded in 96-well plates and allowed to grow for 24 h. Acridine Orange assay. A549 cells (10 5 cells/mL) were seeded onto glass coverslips in a 12-well plate and 24 h later they were treated with 10 µM of the studied compounds. DMSO (1% v/v) was added to control cells. After 1 h, the cells were washed twice with PBS and incubated in 5 µg/mL acridine orange solution (Sigma-Aldrich) for 30 min at room temperature. Finally, they were washed with PBS-10% FBS twice and fluorescence was examined with a NIKON eclipse E800 microscope (SCT filter 440/480 nm, Nikon Instruments, Melville, NY, USA).
Supplementary Figure 28. Acridine orange assay in A549 after treatment with 1a -1d. To explore the effect of lowering the extracellular pH, the NaI solution was buffered at pH 6.9 with HEPES, or at pH 6.6 and 6.2 using MES. The activity of anionophores was determined using a fluorescence plate reader equipped with 500 nm excitation and 535 emission filters, as previously described 8 . The assay is based in the fact that the fluorescence of the YFP is greater quenched by iodide than by chloride7. Functional assays were done at 37°C.

Measurement of iodide influx in cells. FTR-cells expressing
If not otherwise stated, 30 minutes before the assay, the cells were washed twice with a PBS containing 137 mM NaCl. The cells were incubated in 60 µl PBS at 37°C with anionophores or with DMSO as control. Once the assay started, the fluorescence was recorded every 0.2 seconds for as long as 14-40 s for each well. At 2 seconds after the start of fluorescence recording were injected 165 µl of a PBS containing 137 mM NaI instead of NaCl, so that the final concentration of NaI in the well is 100 mM. If the anionophore tested is active and allows the influx of I -, this anion binds to the YFP and quenches the fluorescence. After background subtraction and normalization for the maximal value before NaI addition, the signal decay was fitted with a double exponential function and the maximum rate of fluorescence decay (QR) was derived. This parameter is a direct indication of the activity of the tested compound. Ionophores were dissolved in DMSO to a concentration of 10 mM. After an initial equilibration, chloride efflux was induced by a small volume (<1%) of ionophore. The measurement was concluded with the addition of the sodium dodecyl sulphate (SDS) to break off the membranes and measure the total chloride content in the cells.