*3.2. E*ff*ect of VX-809 on F508del-CFTR Variants Lacking RE and RI*

As for rescue of CFTR variants lacking RE and RI by CFTR modulators, VX-809 restored the processing of both ∆RES-∆RIL- and ∆RIL-F508del-CFTR variants equally well and to wt-CFTR levels (from 71-78% to 92-96%. These data suggest a strong synergistic effect between VX-809 and ∆RI<sup>L</sup> to rescue ∆RES-F508del-CFTR processing and thus some possible interference of the regulatory insertion with VX-809 binding to F508del-CFTR (see Figure 6). Indeed, although previous studies [34] suggested putative binding of VX-809 to NBD1:MSD2 (ICL4) interface (see Figure 6) or to TMD1 [35], they also suggested possibility of further F508del-CFTR correction at distinct conformational sites, so data shown here suggest that VX-809 may also bind to the regulatory insertion.

Interestingly, the processing defect of ∆RIS-wt-CFTR (but not of ∆RIS-F508del-CFTR) was rescuable by VX-809 to 90%, indicating that the amino acid stretches of the RI<sup>L</sup> that remain present in RI<sup>S</sup> do not affect the rescue of ∆RIS-wt-CFTR but preclude rescue of F508del-CFTR by VX-809.

We also tested the impact of removing helix 9 (H9) which precedes the RE (635Gln-Gly646), just after H8 (630Phe-Leu634), both helices proposed to interact with the NBD1:NBD2 heterodimer interface by folding onto the NBD1 β-subdomain [21,27] as well as to bind ICL4 near Phe508. When H9 is present, ∆REL-wt-CFTR processing appears to be favored by VX-809, suggesting some synergy of this small molecule with H9 helix to correct the conformational defect(s) caused by ∆RE<sup>L</sup> on wt-CFTR. These data are in contrast to ∆H9-F508del-CFTR which exhibits 0% processing with or without ∆RE<sup>L</sup> and no rescue by VX-809. We can assume that H9 also contacts RE, at least in some states, such as when the RE adopts different conformations as previously suggested [19].

Most strikingly, and in contrast to its effect on processing, was the effect of ∆RI<sup>L</sup> on the VX-770 stimulated currents which were decreased by almost half vs those stimulated by Gen/Fsk (92% to 58%). These data seem to indicate that the absence of the regulatory insertion could impair (and its presence favor) binding of VX-770 to CFTR (see Figure 6). Surprisingly, the removal of RE<sup>S</sup> from ∆RIL-F508del-CFTR could correct this defect and restore the maximal function by VX-770 (see Figure 6). Indeed, under VX-770, ∆RES-∆RIL-F508del-CFTR exhibited significantly higher activity (87%) than ∆RIL-F508del-CFTR (58%). In contrast, removal of RE<sup>S</sup> from wt-CFTR significantly reduced its functionin comparison with that of wt-CFTR (down to 70%) but had no effect on processing. Our study does not address the mechanism coupling the RI to F508del-CFTR pharmacological rescue, namely, why is the RI essential for rescue by VX-770 or why is it inhibitory for rescue by VX-809. Nevertheless, insight may be obtained from experimental structural data. In a recent cryo-EM structure of full-length human CFTR [29] VX-770 was found to bind in a transmembrane location coinciding with a hinge involved in gating. The authors suggested that populating this binding pocket may stabilize the intermolecular rotation opening the CFTR channel upon ATP binding at the NBD1-NBD2 interface. Given that the RI is located near the NBD interface and ATP binding site [29] we may speculate that RI deletion hampers NBD dimerization and, therefore, rescue by VX-770. Regarding VX-809, we may speculate that the RI interferes with drug binding.
