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Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
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Gene induction for the treatment of methylmalonic aciduria

Ruimei Hu1, Nicole E Buck, Mahmoud S Khaniani

  • 1Cell and Gene Therapy Group, Murdoch Childrens Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Victoria, Australia.

The Journal of Gene Medicine
|February 10, 2009
PubMed
Abstract

Insights

Researchers developed a reporter assay to find drugs for methylmalonic aciduria. This assay identified cisplatin, zidovudine, and adefovir as compounds that increase methylmalonyl-coenzyme A mutase (MCM) expression, offering potential treatments.

Area of Science:

  • Biochemistry
  • Genetics
  • Pharmacology

Background:

  • Methylmalonic aciduria is an inherited metabolic disorder affecting propionate metabolism.
  • Mutations in methylmalonyl-coenzyme A mutase (MCM) cause a common form of this disorder, reducing enzyme activity.
  • Pharmacological upregulation of residual MCM activity is a potential therapeutic strategy.

Purpose of the Study:

  • To construct and characterize a cellular genomic reporter assay for MCM expression.
  • To utilize the assay to identify compounds that modulate MCM expression for methylmalonic aciduria treatment.

Main Methods:

  • A genomic reporter construct was created using homologous recombination to insert an EGFP cassette into the MCM (MUT) gene in a BAC clone.
  • Stable HeLa cell lines were generated from the construct.
  • MCM mRNA levels and EGFP expression were quantified using RT-PCR and flow cytometry, respectively.

Main Results:

  • The reporter assay was used to screen various compounds.
  • Cisplatin, zidovudine, and adefovir were identified as compounds that significantly increased MCM mRNA and EGFP expression.
  • These findings support the potential efficacy of these compounds in treating methylmalonic aciduria.

Conclusions:

  • The developed assay is effective for identifying compounds that modulate MCM expression.
  • This assay has potential for high-throughput screening of chemical libraries.
  • Novel compounds specifically targeting MCM expression can be discovered using this screening approach.

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