Fluorescein diacetate (Di-O-acetylfluorescein) is a cell permeable esterase-substrate for hGSTP1-1.

Fluorescein diacetate (FDA) serves as a fluorogenic substrate in assessing the efficacy of inhibitors against the enzyme GSTP1-1 in vitro. Notably, Ethacrynic acid (EA) and NBDHEX are effective in inhibiting the enzyme-activated fluorescence of FDA, demonstrating concentration-dependent inhibition with IC50 values of 3.3±0.5 μM and 0.61±0.04 μM, respectively. FDA, an acetylated variant of fluorescein, is activated fluorescently through the esterolytic action of the human Pi-class glutathione S-transferase (hGSTP1), marking it specific among various cytosolic GSTs. This fluorogenic activity allows for the usage of FDA in vital staining. Particularly, when MCF7 cells overexpress hGSTP1, an observable fluorescence activation due to GST inhibitors is noted, an effect not seen in cells overexpressing hGSTA1 or hGSTM1. The enzyme-dependent fluorescence activation is further corroborated by incubating FDA with recombinant hGSTP1-1 and GSH, showing significant fluorescence only in the presence of both, indicating the necessity of hGSTP1-1 activity for such activation. This specificity is underscored by the established linear relation between fluorescence increase rate and hGSTP1-1 concentration, determining the enzyme's specific activity for FDA to be 79±15 nmol/min/mg protein.