WS14.03Novel pharmacological inhibitors of the NMD mechanism to rescue CFTR function in cystic fibrosis patients with nonsense mutations

Objectives

The pharmacological treatment for cystic fibrosis (CF) patients with premature termination codons (PTCs) requires a combinatorial approach that includes inhibitors of the nonsense-mediated RNA decay (NMD) mechanism as well as compounds that promote the ribosomal readthrough. NMD inhibitors can also be effective in the absence of a readthrough agent for PTCs localized at the carboxy-terminus of the CFTR sequence such as W1282X. The aim of our project was to identify novel molecules able to modulate the NMD mechanism.

Methods

We screened a chemical library of more than 9,000 compounds using the 16HBE14o^– cell line expressing W1282X-CFTR and the halide-sensitive yellow fluorescent protein (HS-YFP). Cells were incubated for 24 with test compounds plus CFTR correctors. The functional assay allowed to identify compounds enhancing CFTR function. The most effective molecules were characterized by secondary biochemical and functional assays to assess their mechanism of action.

Results

75 primary hits were identified by the screening. Molecules with largest effect on CFTR rescue were further tested in the presence/absence of CFTRinh-172 to confirm a direct activity on CFTR channel and not on other anion channels/transporters. We focused our attention on three different compounds: NMDi-01, NMDi-02, NMDi-03. They induced more than 10-fold increase in CFTR-mRNA transcripts and promoted the appearance of a signal for CFTR protein. NMDi-01 and NMDi-02, together with CFTR correctors, generated a marked increase in CFTR channel activity in short-circuit current recordings experiments. NMDi-03 per se elicited a significant increase in CFTR function that was further amplified when it was combined with correctors.

Conclusions

We identified three possible candidates acting as NMD inhibitors/modulators that could increase CFTR rescue in patients with PTCs.

This project is supported by CFF (GALIET22I0) and the ECFS (post-doctoral research fellowship to Arianna Venturini)