Design and synthesis of versatile GSTP1-specific fluorogenic substrates for the highly sensitive detection of GSTP1 activity in living cells.

Pi-class glutathione S-transferase (GSTP1) is a detoxification enzyme that is highly expressed in various types of cancer cells and is a promising target for cancer imaging and therapy. Ps-TAc, an acetylated derivative of the GSTP1-specific fluorogenic substrate Ps-TG, is attracting attention as an effective GSTP1 fluorescent probe, and has been successfully used to visualize intracellular GSTP1 activity. Ps-TAc is a prodrug type fluorescent probe in which the phenolic hydroxyl group of Ps-TG is acetylated and thus is susceptible to nonspecific hydrolysis, potentially compromising its ability to detect GSTP1 activity. Here, we describe the development of a highly selective fluorogenic GSTP1 substrate that is membrane permeable and does not involve esterification and show its application to live-cell imaging and FACS analysis. We designed and synthesized several compounds with benzylsulfone substituents instead of the mesyl group of Ps-TG and tested their fluorescence activation by GSTP1 catalysis in vitro and in cellulo. Of the test compounds, Ps-TG3 was the most suitable for the visualization of intracellular GSTP1 activity because the signal from living cells increased significantly when MK-571, an inhibitor of multidrug resistance proteins (MRPs), was simultaneously loaded. The results obtained by co-loading Ps-TG3 and MK571 into GSTP1-nonexpressing cells suggest that Ps-TG3 can be a substrate for MRPs. The usefulness of Ps-TG3 was demonstrated by fluorescence imaging of several cancer cell cultures and FACS analysis of lymphoma cells. The results presented here suggest that Ps-TG3, in combination with MK571, is useful for visualizing and detecting intracellular GSTP1 activity in cancer cells that highly express GSTP1.