Nonaminoglycoside compounds induce readthrough of nonsense mutations

• DOI: 10.1084/jem.20081940
• Publication Date: 2009-09-22T01:48:21Z
• Authors: Liutao Du; Robert Damoiseaux; Shareef Nahas; Kun Gao; Hailiang Hu; Julianne M. Pollard; Jimena Goldstine; Michael E. Jung; Susanne M. Henning; Carmen Bertoni; Richard A. Gatti
• Keywords: 0301 basic medicine; 570; Biomedical and clinical sciences; Cell Survival; Chromosomal Proteins, Non-Histone; Immunology; Muscle Fibers, Skeletal; Cell Cycle Proteins; Enzyme-Linked Immunosorbent Assay; Ataxia Telangiectasia Mutated Proteins; Neurodegenerative; Protein Serine-Threonine Kinases; Inbred C57BL; Muscle Fibers; Medical and Health Sciences; Article; 576; Cell Line; Ataxia Telangiectasia; Mice; 03 medical and health sciences; Rare Diseases; Genetics; 2.1 Biological and endogenous factors; Animals; Phosphorylation; Codon; Pediatric; Biomedical and Clinical Sciences; Tumor Suppressor Proteins; Neurosciences; Health sciences; Non-Histone; Skeletal; 3. Good health; Chromosomal Proteins; DNA-Binding Proteins; Mice, Inbred C57BL; Aminoglycosides; Nonsense; Codon, Nonsense; Biotechnology

Large numbers of genetic disorders are caused by nonsense mutations for which compound-induced readthrough of premature termination codons (PTCs) might be exploited as a potential treatment strategy. We have successfully developed a sensitive and quantitative high-throughput screening (HTS) assay, protein transcription/translation (PTT)–enzyme-linked immunosorbent assay (ELISA), for identifying novel PTC-readthrough compounds using ataxia-telangiectasia (A-T) as a genetic disease model. This HTS PTT-ELISA assay is based on a coupled PTT that uses plasmid templates containing prototypic A-T mutated (ATM) mutations for HTS. The assay is luciferase independent. We screened ∼34,000 compounds and identified 12 low-molecular-mass nonaminoglycosides with potential PTC-readthrough activity. From these, two leading compounds consistently induced functional ATM protein in ATM-deficient cells containing disease-causing nonsense mutations, as demonstrated by direct measurement of ATM protein, restored ATM kinase activity, and colony survival assays for cellular radiosensitivity. The two compounds also demonstrated readthrough activity in mdx mouse myotube cells carrying a nonsense mutation and induced significant amounts of dystrophin protein.