title: Aminoglycoside-induced suppression of CYP2C19*3 premature stop codon.
abstract: BACKGROUND
  CYP2C19 is a polymorphic enzyme that plays a pivotal role in the metabolism of 10% of clinically used drugs worldwide. The CYP2C19*3 allele is characterized by a premature stop codon that leads to a truncated nonfunctional protein and consequently a poor metabolizer phenotype. Aminoglycoside antibiotics have been shown to induce readthrough of premature stop codons and partial restoration of protein function. We investigated the ability of the aminoglycosides gentamicin and G418 to induce readthrough of CYP2C19*3 premature stop codon in human cells.
  
  
  METHODS
  A CYP2C19*3 expression model in HeLa cells was used in all experiments. CYP2C19-EGFP expression was assessed by flow cytometry, immunoblotting, and fluorescence microscopy, whereas CYP2C19 enzymatic activity was quantified by hydroxylation of 3-cyano-7-ethoxycoumarin.
  
  
  RESULTS
  G418 and gentamicin promoted readthrough of the CYP2C19*3 premature stop codon in a concentration-dependent manner. Flow cytometry revealed a maximum 23% protein restoration with the highest aminoglycoside concentrations tested, namely 300 mg/ml G418 and 1000 mg/ml gentamicin. At these concentrations, G418 was more effective than gentamicin in restoring CYP2C19 expression in immunoblotting and fluorescence microscopy assays, as well as in restoring CYP2C19 enzymatic activity.
  
  
  CONCLUSIONS
  This is the first demonstration of readthrough of a stop codon in a pharmacogenetic target of clinical relevance, namely CYP2C19*3. The experimental models may be adapted to explore readthrough of stop codons in other genes of pharmacogenetic interest.
authors.affiliation: Instituto Nacional de Câncer, Divisão de Farmacologia, Instituto Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Mateus
authors.initials: M
authors.lastname: Fuchshuber-Moraes
authors.forename: Renato Sampaio
authors.initials: RS
authors.lastname: Carvalho
authors.forename: Christian
authors.initials: C
authors.lastname: Rimmbach
authors.forename: Dieter
authors.initials: D
authors.lastname: Rosskopf
authors.forename: Marcelo Alex
authors.initials: MA
authors.lastname: Carvalho
authors.forename: Guilherme
authors.initials: G
authors.lastname: Suarez-Kurtz
mesh_terms: D000617:Aminoglycosides / Q000494:pharmacology*; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D001681:Biological Assay; D018389:Codon, Nonsense / Q000235:genetics*; D065731:Cytochrome P-450 CYP2C19; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic
chemical_list: D000617:Aminoglycosides; D018389:Codon, Nonsense; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19