(660) Triple potentiator-mediated activation of CFTR mutants with gating defects is independent of channel phosphorylation and NBD dimerization

Background: Recently, we and other described the potentiator activity of elexacaftor (VX-445), which indicated a distinct VX-445 mechanism of action that is additive to ivacaftor (VX-770), suggesting that the VX-770+VX-445 combination therapy provides benefit to patients with gating mutations [1, 2]. In this study we investigated whether addition of previously identified co-potentiators [3] to the VX-770+VX-445 combination would further enhance mutant channel activity and interrogated which steps of channel gating are required for the potentiator action.

Methods: Combinatorial profiling and cluster analysis of R352Q-, S549N-, S549R, W1282X- and N1303K-CFTR showed additivity of VX-445 to both class I potentiators [4] (VX-770, ABBV-974) and class II potentiators [4] (apigenin, bDMC). Triple potentiator-mediated activation was confirmed by short-circuit current measurement in gene-edited 16HBE14o- and primary human nasal epithelia (HNE). The dependence of the triple potentiator activation on channel phosphorylation, nucleotide binding domain (NBD) dimerization or tetrahelix bundle formation were investigated in CFTR variants lacking 15 serine-phosphorylation sites (15SA), the NBD2 domain (1218X) or containing the mutations E267R and K1060E in the tetrahelix bundle, respectively.

Results: Introduction of a third potentiator significantly increased the G551D-, N1303K- and W1282X-mediated current in gene-edited 16HBE14o- and primary HNE. In 15SA-CFTR in the presence of PKA-inhibitor and in 1218X in the absence of PKA-activation, triple potentiator-mediated activation increased the channel-density normalized CFTR function close to the WT-level. Conversely, mutations in the terahelix bundle attenuated the potentiator efficacy.

Conclusions: The results suggest that triple-potentiator mediated CFTR activation is independent of channel phosphorylation and NBD1-2 dimerization, however structural re-arrangement of the intra-cellular loops 2 and 4 leading to tetrahelix bundle formation are required to support potentiator-mediated channel opening. Moreover, introduction of a third potentiator with distinct mechanism-of-action in addition to VX-770+VX-445 further increases channel function, which may particularly be beneficial in mutations with mild or correctable folding defect but impaired NBD dimerization such as W1282X and N1303K.

Acknowledgements: We thank the CFFT for the gene-edited 16HBE14o- cells and Dr. Jack Riordan (UNC-Chapel Hill) for the CFTR-15SA plasmid. This work was supported by the Canadian Institutes of Health Research (PJT-153095 and PJT-173342 to G.V., E.M. and G.L.L.), the Cystic Fibrosis Foundation Therapeutics to G.L.L., as well as Cystic Fibrosis Canada to G.L.L.

References: [1] Veit, G., Vaccarin, C. & Lukacs, G. L. Elexacaftor co-potentiates the activity of F508del and gating mutants of CFTR. J Cyst Fibros 20, 895-898, doi:10.1016/j.jcf.2021.03.011 (2021).
[2] Shaughnessy, C. A., Zeitlin, P. L. & Bratcher, P. E. Elexacaftor is a CFTR potentiator and acts synergistically with ivacaftor during acute and chronic treatment. Sci Rep 11, 19810, doi:10.1038/s41598-021-99184-1 (2021).
[3] Veit, G. et al. Mutation-specific dual potentiators maximize rescue of CFTR gating mutants. J Cyst Fibros 19, 236-244, doi:10.1016/j.jcf.2019.10.011 (2020).
[4] Phuan, P. W. et al. Nanomolar-potency 'co-potentiator' therapy for cystic fibrosis caused by a defined subset of minimal function CFTR mutants. Sci Rep 9, 17640, doi:10.1038/s41598-019-54158-2 (2019).