Pharmacological inhibitors of the nonsense-mediated RNA decay identified by high-throughput screening.

Abstract

A relevant percentage of genetic diseases (~10%) are caused by premature termination codon (PTC) mutations, including cystic fibrosis (CF). CF patients carrying a nonsense mutation in the CFTR gene are insensitive to CFTR modulators and cannot benefit from the pharmacological therapy with Kaftrio®. The rescue of CFTR with PTCs faces two limiting steps: 1) the nonsense-mediated RNA decay (NMD) mechanism that, by degrading mRNAs carrying PTCs, strongly reduces CFTR transcript levels; 2) PTCs cause the arrest of protein synthesis, with the production of a truncated and nonfunctional CFTR protein. The combination of NMD inhibitors with readthrough agents, which overcome the translation arrest, can maximize rescue of CFTR with PTCs. 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. In this work, we screened a chemical library with a CFTR functional assay to find novel compounds able to modulate NMD mechanism. At the end of the screening, we identified and validated by secondary assays three possible candidates acting as NMD inhibitors/modulators that could increase CFTR rescue in patients with PTCs, namely CC-115, samotolisib and elimusertib. The study also provides new insights into the changes occurring to cells transcriptome after the inhibition of the physiological NMD pathway, improving our knowledge of possible side effects of NMD inhibitors. This could help in the development of new compounds with more efficacy and less toxicity, that could be useful for the treatment of different genetic disorders caused by PTCs.