WS14.04Small molecules acting as eRF degraders differently rescue G542X and W1282X-CFTR function

Objectives

Nonsense mutations produce severely truncated forms of the CFTR chloride channel that are insensitive to presently available CFTR modulators. Pharmacological read-through (RT) of the ribosome stalled at the PTC site allow continuation of the protein synthesis. RT is limited by the nonsense mediated mRNA decay (NMD), that degrades mutant mRNA. We reported that G542X-CFTR is the most sensitive mutation to RT and the W1282X is the most responding to NMD inhibition. eRF degraders have been reported as effective on PTCs. We evaluated CC-90009 and SRI41315, an eRF3a and eRF1 degrader, on G542X-CFTR and W1282X-CFTR.

Methods

16HBE14o- cells with the G542X-CFTR mutation were treated for 24 h with different drug combinations containing CC-90009 (0.1 µM) or SRI-41315 (15 µM) with/without VX-809 (1 µM), and ELX-02 (200 µM), as a RT agent, or SMG1i (1 µM), as a NMD inhibitor. We evaluated the rescue of CFTR function using Isc recordings. CFTR rescue was also evaluated with the halide-sensitive yellow protein (HS-YFP) assay on 16HBE14o- cells expressing W1282X-CFTR.

Results

In 16HBE14o- cells we found little effect of CC-90009 in rescuing W1282X-CFTR. CC-90009 was effective on G542X-CFTR in combination with a RT agent. We found a significant effect of SRI-41315 on W1282X-CFTR in HS-YFP assay when combined with SMG1i. No significant effect was observed by Isc experiments when we treated G542X-CFTR cells with SRI-41315, either with RT agents or NMD inhibitor.

Conclusions

The eRF3a degrader CC-90009 has a role in potentiating RT, since it is effective on G542X-CFTR in combination with ELX-02, while the eRF1 degrader SRI-41315 is particularly active on W1282X-CFTR together with SMG1i. These results suggest that eRF degraders have different involvement in RT and NMD mechanisms.

This work was supported by: the Cystic Fibrosis Foundation (GALIET22I0), the Italian Cystic Fibrosis Foundation (FFC#9/2022 and GMRF#1/2024), and the Italian Ministry of Health (GR-2018-12367126).