Non-steroidal inhibitors of Drosophila melanogaster steroidogenic glutathione S-transferase Noppera-bo.

Insect growth regulators (IGRs) can be developed by elucidating the molecular mechanisms of insect-specific biological events. Because insect molting, and metamorphosis are controlled by ecdysteroids, their biosynthetic pathways can serve as targets for IGR development. The glutathione S-transferase Noppera-bo (Nobo), which is conserved in dipteran and lepidopteran species, plays an essential role in ecdysteroid biosynthesis. Our previous study using 17ß-estradiol as a molecular probe revealed that Asp113 of Drosophila melanogaster Nobo (DmNobo) is essential for its biological function. However, to develop IGRs with a greater Nobo inhibitory activity than 17ß-estradiol, further structural information is warranted. Here, we report five novel non-steroidal DmNobo inhibitors. Analysis of crystal structures of complexes revealed that DmNobo binds these inhibitors in an Asp113-independent manner. Among amino acid residues at the substrate-recognition site, conformation of conserved Phe39 was dynamically altered upon inhibitor binding. Therefore, these inhibitors can serve as seed compounds for IGR development.

4-Bromo-1,8-naphthalimide derivatives as fluorogenic substrates for live cell imaging of glutathione S-transferase (GST) activity.

Fluorogenic substrates are used to visualize the activity of cancer-associated enzymes and to interpret biological events. Certain types of glutathione S-transferase (GST), such as Pi class GST (referred to as GSTP1), are more highly expressed in a wide variety of human cancer tissues compared to their corresponding normal tissues. Pi class GST is thus a cancer cell molecular marker and potential target for overcoming resistance to chemotherapy. Here, we report that 4-bromo-1,8-naphthalimide (BrNaph) is a practical fluorogenic GST substrate. We have found that HE-BrNaph, an N-hydroxyethyl derivative, shows remarkable fluorescence enhancement upon GST-catalyzed SNAr replacement of the bromo group with a glutathionyl group. This substitution was highly selective and occurred only in the presence of GSH/GSTs; no non-enzymatic reaction was observed. We demonstrated that HE-BrNaph allows visualization of GST activity in living cells and enables to distinguish cancer cells from normal cells. Further, various N-substitutions in BrNaph retain susceptibility to enzymatic activity and isozyme selectivity, suggesting the applicability of BrNaph derivatives. Thus, BrNaph and its derivatives are GST substrates useful for fluorescence imaging and the intracellular detection of GSTP1 activity in living cells.

Fluorescein diacetate (FDA) and its analogue as substrates for Pi-class glutathione S-transferase (GSTP1) and their biological application.

Pi class glutathione S-transferase (GSTP1) is highly expressed in various cancerous cells and pre-neoplastic legions, where it is involved in apoptotic resistance or metabolism of several anti-tumour chemotherapeutics. Therefore, GSTP1 is a marker of malignant and pre-malignant cells and is a promising target for visualization and drug development. Here we demonstrate that fluorescein diacetate (FDA), a fluorescent probe used for vital staining, is a fluorescently activated by esterolytic activity of human GSTP1 (hGSTP1) selectively among various cytosolic GSTs. Fluorescence activation of FDA susceptible to GST inhibitors was observed in MCF7 cells exogenously overexpressing hGSTP1, but not in cells overexpressing hGSTA1 or hGSTM1. Inhibitor-sensitive fluorescence activation was also observed in several cancer cell lines endogenously expressing GSTP1, suggesting that GSTP1 is involved in FDA esterolysis in these cells. Among the FDA derivatives examined, FOMe-Ac, the acetyl ester of fluorescein O-methyl ether, was found to be a potential reporter for GSH-dependent GSTP1 activity as well as for carboxylesterase activity. Since GSTP1 is highly expressed in various types of cancer cells compared to their normal counterparts, improving the fluorogenic substrates to be more selective to the esterolysis activity of GSTP1 rather than carboxylesterases should lead to development of tools for detecting GSTP1-overexpressing cancer cells and investigating the biological functions of GSTP1.