Nonaminoglycoside compounds induce readthrough of nonsense mutations.
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 approximately 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.
Du
Liutao
L
Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
Damoiseaux
Robert
R
Nahas
Shareef
S
Gao
Kun
K
Hu
Hailiang
H
Pollard
Julianne M
JM
Goldstine
Jimena
J
Jung
Michael E
ME
Henning
Susanne M
SM
Bertoni
Carmen
C
Gatti
Richard A
RA
D000617:Aminoglycosides / Q000494:pharmacology*; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D018389:Codon, Nonsense; D004268:DNA-Binding Proteins / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D008810:Mice, Inbred C57BL; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D000098550:Structural Maintenance of Chromosome Protein 1
D000617:Aminoglycosides; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D018389:Codon, Nonsense; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D000098550:Structural Maintenance of Chromosome Protein 1; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases