A high-throughput assay for modulators of NNT activity in permeabilized yeast cells

J Biomol Screen. 2011 Aug;16(7):734-43. doi: 10.1177/1087057111408088. Epub 2011 May 20.

Abstract

Nicotinamide nucleotide transhydrogenase (NNT) mutant mice show glucose intolerance with impaired insulin secretion during glucose tolerance tests. Uncoupling of the β cell mitochondrial metabolism due to such mutations makes NNT a novel target for therapeutics in the treatment of pathologies such as type 2 diabetes. The authors propose that increasing NNT activity would help reduce deleterious buildup of reactive oxygen species in the inner mitochondrial matrix. They have expressed human Nnt cDNA for the first time in Saccharomyces cerevisiae, and transhydrogenase activity in mitochondria isolated from these cells is six times greater than is seen in wild-type mitochondria. The same mitochondria have partially uncoupled respiration, and the cells have slower growth rates compared to cells that do not express NNT. The authors have used NNT's role as a redox-driven proton pump to develop a robust fluorimetric assay in permeabilized yeast. Screening in parallel a library of known pharmacologically active compounds (National Institute of Neurological Disorders and Stroke collection) against NNT ± cells, they demonstrate a robust and reproducible assay suitable for expansion into larger and more diverse compound sets. The identification of NNT activators may help in the elucidation of the role of NNT in mammalian cells and assessing its potential as a therapeutic target for insulin secretion disorders.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Enzyme Activation / drug effects
  • Gene Expression Regulation, Fungal
  • High-Throughput Screening Assays*
  • Humans
  • Mitochondria / genetics
  • Mitochondria / metabolism
  • NADP Transhydrogenases / genetics
  • NADP Transhydrogenases / metabolism*
  • Reproducibility of Results
  • Saccharomyces cerevisiae / drug effects*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae / metabolism*
  • Small Molecule Libraries / pharmacology

Substances

  • Small Molecule Libraries
  • NADP Transhydrogenases