(645) A Novel eRF1 Degrader Induces Translational Readthrough of CFTR Nonsense Mutations to Therapeutically Relevant Levels in Combination with Aminoglycosides

Background: Premature termination codons (PTCs) in CFTR result in nonfunctional protein and affect ~11% of people with CF (PwCF). There are no approved therapies for CF caused by nonsense mutations. Nonsense suppression therapy utilizes small molecules to suppress translation termination at in-frame PTCs to restore partial levels of full-length, functional CFTR protein (i.e. readthrough). Following a high throughput screen to identify agents that suppress PTCs, we reported a small molecule chemical series that induces readthrough and synergizes with aminoglycosides by inducing proteasomal degradation of the translation termination factor eRF1 (Sharma, Du, et al., Nature Communications 2021; 12(1):4358). This increases the frequency of near-cognate amino acid insertion at the PTC, rescuing partial CFTR function in multiple cell lines and primary cells. Unfortunately, this series has off target effects that prevents further clinical development. Since then, we identified a distinct molecular series that also depletes eRF1 to suppress PTCs.

Methods: A high throughput screen was conducted using NanoLuc-based reporter cell lines to identify compounds that promote readthrough of PTCs. Validated readthrough hits were tested for the ability to suppress CF-associated PTCs and rescue CFTR expression and function in a variety of cell types, including 16HBEs and intestinal organoids by forskolin induced swelling and transepithelial chloride transport. The most promising hits were further examined for the mechanism by which they promote readthrough.

Results: SRI-41765, the lead compound from the series, induces readthrough of multiple PTCs in 16HBE14o- (16HBE) cells expressing Nanoluc readthrough reporters. Mechanistic evaluation demonstrated reduction in eRF1 levels by Western blot. SRI-41765 (15 µM) restored ~4.2% (SRI-41765+G418) and ~6.3% (SRI-41765+ELX-02) of wild type CFTR function in 16HBE cells expressing the G542X mutation when combined with submaximal (EC10) concentrations of aminoglycosides G418 or ELX-02; Maximally effective concentrations of ELX-02 (250 µg/ml) increased correction to 15% of WT when combined with SRI-41765. Rescue of CFTR activity was associated with increased CFTR mRNA levels, suggesting reduced nonsense mediated mRNA decay. In primary cell organoids derived from donors with only nonsense mutations for PTCs (G542X/R1162X, G542X/R553X, G542X/1717-1G- >A, G542X/W1089X), SRI-41765 combined with ELX-02 (250 µg/ml) increased forskolin (0.8 uM stimulus) induced swelling on par with bioactivity observed for F508del CFTR modulators (20%-40% of WT organoids, depending on genotype). We also observed improved CFTR activity in organoids from the same donors grown as 2D epithelial monolayers and assessed by transepithelial equivalent current. In each case, CFTR activity could be further enhanced by addition of CFTR modulators. Western blotting confirmed partially restored CFTR expression under these conditions. SRI-41765 has a favorable pharmacological profile (solubility and microsomal stability), no obvious cellular toxicity, and stable pharmacokinetics in mice.

Conclusions: SRI-41765 represents novel compound that induces translational readthrough by degrading eRF1 to restore substantial CFTR function in primary organoids with PTC alleles, particularly in combination with aminoglycosides. Given its favorable pharmacological properties, further development of SRI-41765 as a therapeutic agent for PwCF who carry PTCs is warranted.

Acknowledgements: NIH (P30DK072482) and Cystic Fibrosis Foundation

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