Spectroscopic and rapid kinetic experiments were performed to detail the interaction of human glutathione S-transferases GSTA1-1, GSTM2-2, and GSTP1-1 with 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX). This compound is a representative molecule of a new class of 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives (non-GSH peptidomimetic compounds) that have been designed both to give strong GST inhibition and to accumulate in tumor cells avoiding the extrusion mechanisms mediated by the multidrug resistance protein pumps. We have recently shown that submicromolar amounts of NBDHEX trigger apoptosis in several human tumor cell lines through the dissociation of the JNK⅐GSTP1-1 complex (Turella, P., Cerella, C., Filomeni, G., Bullo, A., De Maria, F., Ghibelli, L., Ciriolo, M. R., Cianfriglia, M., Mattei, M., Federici, G., Ricci, G., and Caccuri, A. M. (2005) Cancer Res. 65, 3751-3761). Results reported in the present study indicated that NBDHEX behaves like a suicide inhibitor for GSTs. It bound to the H-site and was conjugated with GSH forming a complex at the C-4 of the benzoxadiazole ring. This complex was tightly stabilized in the active site of GSTP1-1 and GSTM2-2, whereas in GSTA1-1 the release of the 6-mercapto-1-hexanol from the complex was the favored event. Docking studies demonstrated the likely localization of the complex in the GST active sites and provide a structural explanation for its strong stabilization.

Selected 7-nitro-2,1,3-benzoxadiazole derivatives have been recently found very efficient inhibitors of glutathione Stransferase (GST) P1-1, 5 an enzyme which displays antiapoptotic activity and is also involved in the cellular resistance to anticancer drugs. These new inhibitors are not tripeptide glutathione-peptidomimetic molecules and display lipophylic properties suitable for crossing the plasma membrane. In the present work, we show the strong cytotoxic activity of these compounds in the following four different cell lines: K562

It is now well established that exposure of cells and tissues to nitric oxide leads to the formation of a dinitrosyl-iron complex bound to intracellular proteins, but little is known about how the complex is formed, the identity of the proteins, and the physiological role of this process. By using EPR spectroscopy and enzyme activity measurements to study the mechanism in hepatocytes, we here identify the complex as a dinitrosyl-diglutathionyl-iron complex (DNDGIC) bound to Alpha class glutathione S-transferases (

The interaction of dinitrosyl-diglutathionyl-iron complex (DNDGIC), a natural carrier of nitric oxide, with representative members of the human glutathione transferase (GST) superfamily, i.e. GSTA1-1, GSTM2-2, GSTP1-1, and GSTT2-2, has been investigated by means of pre-steady and steady state kinetics, fluorometry, electron paramagnetic resonance, and radiometric experiments. This complex binds with extraordinary affinity to the active site of all these dimeric enzymes; GSTA1-1 shows the strongest interaction (K D Х 10 ؊10 M), whereas GSTM2-2 and GSTP1-1 display similar and slightly lower affinities (K D Х 10 ؊9 M). Binding of the complex to GSTA1-1 triggers structural intersubunit communication, which lowers the affinity for DNDGIC in the vacant subunit and also causes a drastic loss of enzyme activity. Negative cooperativity is also found in GSTM2-2 and GSTP1-1, but it does not affect the catalytic competence of the second subunit. Stopped-flow and fluorescence data fit well to a common minimal binding mechanism, which includes an initial interaction with GSH and a slower bimolecular interaction of DNDGIC with one high and one low affinity binding site. Interestingly, the Theta class GSTT2-2, close to the ancestral precursor of GSTs, shows very slow binding kinetics and hundred times lowered affinity (K D Х 10 ؊7 M), whereas the bacterial GSTB1-1 is not inhibited by DNDGIC. Molecular modeling and EPR data reveal structural details that may explain the observed kinetic data. The optimized interaction with this NO carrier, developed in the more recently evolved GSTs, may be related to the acquired capacity to utilize NO as a signal messenger.

Electron paramagnetic resonance and kinetics experiments have been made to determine the formation, stability, and fate of the natural nitric oxide carrier, dinitrosyl-diglutathionyl-iron complex (DNDGIC), in heterogeneous systems approaching in vivo conditions. Both in human placenta and rat liver homogenates DNDGIC is formed spontaneously from GSH, S-nitrosoglutathione, and trace amounts of ferrous ions. DNDGIC is unstable in homogenates depleted of glutathione Stransferase (GST); an initial phase of rapid decomposition is followed by a slower decay, which is inversely proportional to the concentration. In the crude human placenta homogenate, GSTP1-1, which represents 90% of the cytosolic GST isoenzymes, is the preferential target for DNDGIC. It binds the complex almost stoichiometrically and stabilizes it for several hours (t1 ⁄2 ‫؍‬ 8 h). In the presence of an excess of DNDGIC, negative cooperativity in GSTP1-1 opposes the complete loss of the usual detoxicating activity of this enzyme. In the rat liver homogenate, multiple endogenous GSTs (mainly Alpha and Mu class isoenzymes) bind the complex quantitatively and stabilize it (t1 ⁄2 ‫؍‬ 4.5 h); negative cooperativity is also seen for these GSTs. Thus, the entire pool of cytosolic GSTs, with the exception of the Theta GST, represents a target for stoichiometric amounts of DNDGIC and may act as storage proteins for nitric oxide. These results confirm the existence of a cross-link between NO metabolism and the GST superfamily.A crucial step in biochemical research is the demonstration that a specific phenomenon observed in vitro may actually occur in vivo. In the accompanying paper (1) we have characterized the peculiar interaction in vitro of an endogenous NO carrier, the dinitrosyl-diglutathionyl-iron complex (DNDGIC), 1 with representative members of the glutathione S-transferase (GST) superfamily. In this paper we extend these studies in order to verify the occurrence and the modality of this interaction in complex biological systems like crude tissue homogenates. The aim of the present paper is also to determine whether DNDGIC could be formed in multicomponent biological systems starting from GSNO, GSH, and trace amounts of ferrous ions. Although a homogenate is not representative of the environment found in the living cell, the use of homogenates allows direct measurements of GST activity, which at present cannot be determined in intact cells. A useful model for this investigation is the human placenta tissue which mainly expresses GSTP1-1, and the rat liver which represents a more complex system containing many different GST isoenzymes (2). Our results demonstrate that DNDGIC can be readily formed in homogenates and that GSTP1-1 is the main target of DNDGIC in the human placenta, whereas the entire pool of GSTs (mainly Alpha and Mu class GSTs) plays a similar role in rat liver. DNDGIC is greatly stabilized when bound to GSTs, and these enzymes may act as half-storage proteins for NO taking advantage of a common negative cooperativity induced by DNDGIC bi...