Use of NanoLuc-Based Reporters that Monitor Nonsense Suppression and Inhibition of Nonsense-Mediated mRNA Decay to Identify Potent New Agents That Partially Reverse the Effects of Nonsense Mutations

ABSTRACT: Premature termination codons (PTCs) lead to loss of protein function by 1) ending mRNA translation before a full-length protein is made, and 2) by triggering nonsense-mediated mRNA decay (NMD), a pathway that targets a PTC-containing mRNA for degradation. Nonsense suppression therapy aims to restore protein function by suppressing termination at PTCs (called readthrough, or RT). However, the efficiency of current readthrough agents is generally quite low. We reasoned that by stimulating both readthrough and increasing mRNA levels, greater protein function can be restored than with readthrough alone. In this study, we developed a series of novel NanoLuc reporters designed to identify and differentiate compounds that induce readthrough, enhance mRNA abundance (by NMD inhibition or other mechanisms), or target both mechanisms simultaneously (referred to as dual RT/NMD reporters). We show that treatments that promote readthrough and NMD inhibition led to synergistic increases in NanoLuc activity of the RT/NMD reporter over either condition alone. We then used a dual RT/NMD reporter to carry out a high throughput screen of a library of 771,345 compounds. We identified 180 compounds with the ability to increase NanoLuc activity in our dual RT/NMD reporter assay. To confirm that such synergy can also amplify protein function in a disease model, we treated cells expressing a CFTR-Y122X mini-intron construct with one hit from this screen, SRI-37240. We found that this compound produced a much larger increase in CFTR activity compared to either the RT compound G418 +/- the NMD inhibitor amlexanox, and the activity obtained could be further enhanced with CFTR modulators. These results confirm the utility of these new reporters to identify many potent new molecules that counteract the effects of nonsense mutations.

ORGANISM(S): Homo sapiens

PROVIDER: GSE144140 | GEO | 2020/12/01

REPOSITORIES: GEO