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¹ Department of Pathology and Laboratory Medicine, ² Center for Human Nutrition, ³ Department of Human Genetics, and ⁴ Department of Neurology, David Geffen School of Medicine, ⁵ Molecular Shared Screening Resources, California NanoSystems Institute, ⁶ Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095

CORRESPONDENCE Richard A. Gatti: rgatti{at}mednet.ucla.edu OR Liutao Du: Ldu{at}mednet.ucla.edu

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.

Abbreviations used: A-T, ataxia-telangiectasia; ATM, A-T mutated; CSA, colony survival assay; FC, flow cytometry; FI, fluorescence intensity; HRP, horseradish peroxidase; HTS, high-throughput screening; IR, ionizing radiation; IRIF, irradiation-induced foci; LCL, lymphoblastoid cell line; mRNA, messenger RNA; NMD, nonsense-mediated mRNA decay; PTC, premature termination codon; PTT, protein transcription/translation; RTC, readthrough compound; SMC, structural maintenance of chromosome.

© 2009 Du et al.
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