Design and synthesis of highly sensitive fluorogenic substrates for glutathione S-transferase and application for activity imaging in living cells

J Am Chem Soc. 2008 Nov 5;130(44):14533-43. doi: 10.1021/ja802423n. Epub 2008 Oct 9.

Abstract

Here we report the development of fluorogenic substrates for glutathione S-transferase (GST), a multigene-family enzyme mainly involved in detoxification of endogenous and exogenous compounds, including drug metabolism. GST is often overexpressed in a variety of malignancies and is involved in the development of resistance to various anticancer drugs. Despite the medical significance of this enzyme, no practical fluorogenic substrates for fluorescence imaging of GST activity or for high-throughput screening of GST inhibitors are yet available. So, we set out to develop new fluorogenic substrates for GST. In preliminary studies, we found that 3,4-dinitrobenzanilide (NNBA) is a specific substrate for GST and established the mechanisms of its glutathionylation and denitration. Using these results as a basis for off/on control of fluorescence, we designed and synthesized new fluorogenic substrates, DNAFs, and a cell membrane-permeable variant, DNAT-Me. These fluorogenic substrates provide a dramatic fluorescence increase upon GST-catalyzed glutathionylation and have excellent kinetic parameters for the present purpose. We were able to detect nuclear localization of GSH/GST activity in HuCCT1 cell lines with the use of DNAT-Me. These results indicate that the newly developed fluorogenic substrates should be useful not only for high-throughput GST-inhibitor screening but also for studies on the mechanisms of drug resistance in cancer cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anilides / chemistry
  • Anilides / metabolism
  • Catalysis
  • Cell Line, Tumor
  • Drug Design
  • Fluorescent Dyes / analysis
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / metabolism
  • Glutathione / chemistry
  • Glutathione / metabolism
  • Glutathione S-Transferase pi / chemistry
  • Glutathione S-Transferase pi / metabolism
  • Glutathione Transferase / analysis
  • Glutathione Transferase / chemistry*
  • Glutathione Transferase / metabolism
  • HeLa Cells
  • Humans
  • Kinetics
  • Microscopy, Fluorescence / methods
  • Models, Molecular
  • Spectrometry, Fluorescence / methods
  • Substrate Specificity

Substances

  • Anilides
  • Fluorescent Dyes
  • GSTP1 protein, human
  • Glutathione S-Transferase pi
  • Glutathione Transferase
  • Glutathione