A High-Throughput Assay for In Vitro Determination of Release Factor-Dependent Peptide Release from a Pretermination Complex by Fluorescence Anisotropy-Application to Nonsense Suppressor Screening and Mechanistic Studies

• Published in: Biomolecules (Basel, Switzerland) Vol. 13; no. 2; p. 242
• Main Authors: Ghelfi, Mikel D, Bhat, Saleem Y, Li, Hong, Cooperman, Barry S
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 27-01-2023; MDPI
• Subjects: Anisotropy; ataluren; Catalysis; Codon, Nonsense; Codon, Terminator; Codons; Disease; Duchenne's muscular dystrophy; FDA approval; Fluorescence; High-throughput screening (Biochemical assaying); high-throughput screening (HTS); Humans; Labeling; Methods; Molecular biology; Mutation; Peptides; Peptides - metabolism; Physiological aspects; premature termination codon; Protein Biosynthesis; Proteins; readthrough; Suppression, Genetic; termination; Testing; Toxicity; Transfer RNA; translation readthrough-inducing drug (TRID); United States; termination; premature termination codon; high-throughput screening (HTS); readthrough; translation readthrough-inducing drug (TRID); ataluren
• ISSN: 2218-273X; 2218-273X
• DOI: 10.3390/biom13020242

Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases. We recently elucidated ataluren's mechanism of action as a competitive inhibitor of release factor complex (RFC) catalysis of premature termination and identified ataluren's binding sites on the ribosome responsible for such an inhibition. These results suggest the possibility of discovering new TRIDs, which would retain ataluren's low toxicity while displaying greater potency and generality in stimulating readthrough via the inhibition of termination. Here we present a detailed description of a new in vitro plate reader assay that we are using both to screen small compound libraries for the inhibition of RFC-dependent peptide release and to better understand the influence of termination codon identity and sequence context on RFC activity.