Isozyme-Specific Fluorescent Inhibitor of Glutathione S-Transferase Omega 1

Abstract

Recently, the glutathione S-transferase omega 1 (GSTO1) is suspected to be involved in certain cancers and neurodegenerative diseases. However, profound investigation on the pathological roles of GSTO1 has been hampered by the lack of specific methods to determine or modulate its activity in biological systems containing other isoforms with similar catalytic function. Here, we report a fluorescent compound that is able to inhibit and monitor the activity of GSTO1. We screened 43 fluorescent chemicals and found a compound (6) that binds specifically to the active site of GSTO1. We observed that compound 6 inhibits GSTO1 by covalent modification but spares other isoforms in HEK293 cells and demonstrated that compound 6 could report the activity of GSTO1 in NIH/3T3 or HEK293 cells by measuring the fluorescence intensity of the labeled amount of GSTO1 in SDS-PAGE. Compound 6 is a useful tool to study GSTO1, applicable as a specific inhibitor and an activity reporter.

The Binding of Fluorophores to Proteins Depends on the Cellular Environment

“…From these results, we envisioned that sufficient structural modifications of fluorophore scaffold could lead us to develop probes that label specific proteins from whole proteomes. [8] As an extension of our recent finding of a fluorescein derivative labeling glutathione s-transferase, [9] here we report a rosamine derivative that labels tubulin in vitro and a mitochondrial protein in live cells.…”

A high-performance liquid chromatography-based assay of glutathione transferase omega 1 supported by glutathione or non-physiological reductants

“…Using the HPLC-based procedure, we studied the effect of specific GSTO1 inhibitors, namely KT53 [11] and CMFDA [12], as well as the non-specific GSTO1 inhibitor zinc [13] on 4-NPG reduction by rat liver cytosol. Importantly, each of these inhibitors inactivates GSTO1 by reacting with the active site cysteine.…”

“…Therefore, we set out to develop a high-performance liquid chromatography (HPLC)-based procedure, anticipating that it will be sensitive enough to quantify the conversion of 4-NPG to 4-NAP in the presence of GSH as a measure of GSTO1 activity. In this endeavor, our main objectives were (i) to identify and quantitate the product(s) of 4-NPG reduction; (ii) to determine the conditions under which product formation, in the presence of GSH, 2-ME, or the disulfide reducing agent tris(2-carboxyethyl)phosphine (TCEP), is linear with respect to time and cytosolic protein concentration; and (iii) to ascertain the inhibitory effect on product formation of chemicals that inactivate GSTO1, such as the specific GSTO1 inhibitor KT53 [11] and 5-chloromethylfluorescein diacetate (CMFDA) [12], as well as zinc ion [13]. Development of the spectrophotometric GSTO1 assay into a more sensitive HPLC-based procedure is reported below, whereas studies on the involvement of GSTO1 in reduction of dimethylarsinic acid are intended to be communicated separately.…”

Small-molecule probes elucidate global enzyme activity in a proteomic context

“…However, CDy2 exhibited extraordinary selectivity for a single protein, ALDH2, which is one isoform of aldehyde dehydrogenase in a complex proteome. Studying ABPs containing a benzyl halide (59) and α,β-unsaturated ketone (61) revealed ABPs that labels a single protein in whole proteome.…”