Journal Article

Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms

Goldman, Yale E., Ng, Martin Y., Ghelfi, Mikel D., Li, Hong, Cooperman, Barry S.

2021

Overview

During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers. Small molecule nonsense suppressors, known as TRIDs (translational read-through–inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously [1], we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell’s protein synthesis machinery. Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near-cognate tRNA ± TRIDs.

• Publisher: National Academy of Sciences
• Subject: Aminoglycosides - metabolism / Aminoglycosides - pharmacology / Animals / Artemia - genetics / Biochemistry / Biological Sciences / Codon, Nonsense - drug effects / Codon, Nonsense - metabolism / Codon, Terminator - drug effects / Codon, Terminator - metabolism / Cystic Fibrosis - genetics / Muscular Dystrophy, Duchenne - genetics / Oxadiazoles - metabolism / Oxadiazoles - pharmacology / Peptide Chain Elongation, Translational - drug effects / Protein Biosynthesis - drug effects / Protein Synthesis Inhibitors / Ribosomes - drug effects / RNA, Transfer - drug effects / RNA, Transfer - genetics / RNA, Transfer - metabolism / Saccharomyces - genetics