Design and Synthesis of Highly Sensitive Fluorogenic Substrates for Glutathione S-Transferase and Application for Activity Imaging in Living Cells

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.

Discovery of pyrido[3,4-g]quinazoline derivatives as CMGC family protein kinase inhibitors: Design, synthesis, inhibitory potency and X-ray co–crystal structure

“…The isoquinoline moiety was formed under microwave irradiation of 8 in the presence of ammonia in methanol [25]. Finally, oxidation of 9 using MnO 2 led to 6-chloroisoquinoline-7-carbaldehyde 10 in 88% yield (Scheme 1) [26].…”

Functionalized Nano-adsorbent for Affinity Separation of Proteins

“…The separated proteins can be used for antigen and vaccine production, molecular immunology and structural, and biochemical and cell biological studies. Glutathione S -transferase (GST) represents a major group of detoxification isoenzymes which can be used in GST gene fusion system and medicine effect targeting field or as tumor markers [7, 8]. Various methods such as precipitation, chromatography, ultrafiltration, and dialysis are currently available for purifying various proteins, and in particular, affinity separation based on the natural biological affinity between biological macromolecules and complementary ligands is of extraordinary significance [9–14].…”

A new ensemble approach based chemosensor for the reversible detection of bio-thiols and its application in live cell imaging

“…While the elevated levels of intracellular biothiols are causal to or can exacerbate pathogenesis of Alzheimer's disease, cardiovascular disease, osteoporosis, and neurotoxicity [8][9][10][11][12][13][14]. Therefore, the development of simple and effective methods for the rapid and sensitive detection of biothiols in aqueous solution, and especially in living systems are of great importance for understanding their functions in cells and disease diagnosis [15].…”