Figure legends:
Figure 1 – RDR01752 rescues F508del-CFTR processing and PM expression. (A) CFBE cells stably expressing F508del-CFTR were incubated for 24 h with DMSO (negative control), VX-809 (3.7 µM), VX-661 (5 µM) or an increasing concentration of RDR01752. (B) CFTR processing (C/B+C) was quantified and normalized to tubulin levels (loading control). N = 4. Vs. DMSO: *P <0.05, ***P <0.001. Vs. VX-809:#P <0.05,##P <0.01. (C) CFBE stably expressing mCherry-Flag-F508del-CFTR were incubated for 48 h with DMSO (negative control), VX-809 (3.7 µM) or an increasing concentration of RDR01752. (D) Immunostaining was performed and fluorescence images of extracellularly exposed Flag-tags were quantified to determine CFTR PM expression. Data are normalized to the negative control (DMSO). N = 4. Vs. VX-809: #P <0.05,##P <0.01.
Figure 2 – RDR01752 rescues F508del-CFTR function. (A)Representative cell fluorescence recording acquired with a microplate reader. CFBE cells stably co-expressing F508del-CFTR and the HS-YFP were incubated for 24h with DMSO (vehicle), VX-809 (3.7 µM) or increasing concentrations of RDR01752. Cells were then acutely (30 min) stimulated with Fsk (20 µM) and Gen (50 µM). (B) CFTR activity was quantified based on the rate of YFP quenching and normalized to the negative control (DMSO, dashed line). N = 4. Vs. VX-809:#P <0.05. (C-F) Monolayers of CFBE cells stably expressing F508del-CFTR were incubated for 24 h with (C) DMSO (negative control), (D) VX-809 (3.7 µM), or (E) RDR01752 (10 µM). Original Ussing chamber (open-circuit) recordings depicting transepithelial voltage measurements (Vte). There is an absence of response in cells treated with DMSO, while negative deflections are observed in cells treated with VX-809 or RDR01752 following the application of Fsk+IBMX and genistein, which are reverted by application of Inh172.(F) Data are expressed as Isc calculated from voltage deflections obtained for the responses to Fsk+IBMX+Gen. N = 3.Vs. DMSO: *P <0.05, **P <0.01.Vs. VX-809: #P <0.05.
Figure 3 – Intestinal organoids (F508del/F508del) respond positively to RDR01752. (A) Bright-field images of organoids incubated for 24 h with DMSO (negative control), VX-809 (3.7 µM), VX-661 (5 µM) or RDR01752 (10 µM) and acutely stimulated with forskolin (Fsk, 0.128 µM) with VX-770 (3 µM) or genistein (Gen, 50 µM). (B) Data of FIS of organoids are expressed as the absolute area under the curve (AUC; baseline = 100%, t = 60 min, 0.128 µM Fsk). Absence of bars indicates there was no swelling (NS). N = 3. Vs. DMSO: *P <0.05, **P <0.01, ***P <0.001.
Figure 4 – RDR01752 increases the rescue of F508del-CFTR PM expression in low temperature and in cells expressing in cis the genetic revertants G550E and 4RK, but not in R1070W. CFBE stably expressing mCherry-Flag-CFTR (WT, F508del, DD/AA variants or carrying G550E, R1070W, 4RK in cis with F508del) were incubated for 24 h with DMSO (negative control), RDR01752 (10 µM), VX-809 (3.7 µM) or VX-661 (5 µM) and then maintained for additional 24h at (A)37°C or (B) in low temperature (27°C). Data are represented as heatmaps of the quantification from fluorescence images of extracellularly exposed Flag-tags to determine CFTR PM expression. The values for each cell line are normalized to DMSO at 37°C. N = 6.
Figure 5 – RDR01752 is not additive to VX-809 or VX-661 in rescuing F508del-CFTR traffic. (A) CFBE stably expressing mCherry-Flag-F508del-CFTR were incubated for 48 h with the following compounds individually or in combination: DMSO (negative control), RDR01752 (10 µM), VX-809 (3.7 µM), VX-661 (5 µM) and VX-770 (3 µM).(B) Immunostaining was performed and fluorescence images of extracellularly exposed Flag-tags were quantified to determine CFTR PM expression. Data are normalized to the negative control (DMSO). N = 4.Vs. single corrector (white bars): *P <0.05, ***P < 0.001. n.s.: no significant.
Figure 6 – Assessment of RDR01752 effects on rare CFTR mutants by FLIPR membrane potential (FMP) assay. FRT cells stably expressing CFTR variants (wt, G85E, R334W, T338I, R347P, F508del, V520F, S549F, G551D, M1101K or N1303K) were incubated for 24 h with DMSO (negative control), RDR01752 (10 µM) or VX-809 (1 µM). FMP assay was performed to monitor membrane depolarization induced by stimulation with forskolin (10 µM) plus genistein (50 µM) as a measurement of CFTR function. N = 3.Vs. DMSO (black bars): *P <0.05.
Figure 7 – Effect of RDR01752 and VX-809 individually or in combination on functional rescue of CFTR carrying F508del, G85E, N1303K or R334W. (A) Monolayers of FRT cells stably expressing CFTR variants (F508del, G85E, N1303K or R334W) were incubated for 24 h with DMSO (negative control), VX-809 (1 µM) and RDR01752 (10 µM) alone or combined. Representative recordings of Isc measurements of Ussing chamber for each CFTR mutant. CFTR currents were stimulated using forskolin (FSK; 10 µM) and genistein (GST; 50 µM) and inhibited by CFTRinh -172 (172; 10 µM). ATP (100µM) was added at the end of each experiment as a positive control for viability. (B)Data are represented as mean increase in Isc induced by FSK+GST. N = 3-6. Vs. DMSO: *P < 0.05, **P < 0.01.