Functional rescue of other CFTR mutants by RDR01752
To evaluate the ability of RDR01752 to rescue other CFTR mutants, we used an FMP assay in FRT cells to measure the depolarization that occurs when CFTR PM channels are activated. The nine missense mutants studied are located across the different CFTR domains, namely in: TMD1 (G85E, R334W, T338I and R347P); NBD1 (V520F, S549F and G551D); TMD2 (M1101K); and NBD2 (N1303K). Cells expressing these CF-causing mutations were incubated with either RDR01752 or VX-809 for 24h (Fig. 6). An increase in the Fsk+Gen response was observed in F508del-expressing FRT cells treated with either RDR01752 or VX-809, indicating that CFTR function was rescued by RDR01752, consistent with both the ΔIsceqdata and the rate of HS-YFP quenching observed in CFBE cells. Both RDR010752 and VX-809 also rescue CFTR function in R334W-, V520F- and M1101K-expressing cells. However, in this assay only RDR01752 (and not VX-809) demonstrated an effect on G85E- and T338I-expressing cells, while R347P-, S549F- and N1303K-expressing cells only responded to VX-809 treatment, albeit the latter at very low levels (Fig. 6). No significant differences were observed in CFTR function between G551D- and WT-expressing cells after treatment with either RDR01752 or VX-809 compared to control.
To confirm these findings, we investigated the effects of RDR01752 and VX-809, alone or combined, in polarized FRT cells expressing the mutants that showed rescue, namely F508del-, G85E-, N1303K- or R334W-CFTR by Ussing chamber measurements (Fig. 7). An increase of CFTR-dependent Cl secretion was observed in F508del-expressing cells incubated with either VX-809 or RDR01752, albeit much lower for the latter. Incubation with both compounds combined resulted in a significantly greater rescue of F508del-CFTR function in comparison to VX-809 alone. Furthermore, although the rescue of R334W-CFTR by RDR01752 was not higher than that by VX-809 (as in the FMP assay), co-administering the two compounds did increase CFTR-dependent Cl secretion. No significant effects were found in G85E- and N1303K-expressing cells in CFTR function after incubation with either RDR01752 or VX-809, alone or combined.