7-Nitro-4-(phenylthio)benzofurazan is a potent generator of superoxide and hydrogen peroxide

Abstract

Here, we report on 7-nitro-4-(phenylthio) benzofurazan (NBF-SPh), the most potent derivative among a set of patented anticancer 7-nitrobenzofurazans (NBFs), which have been suggested to function by perturbing protein–protein interactions. We demonstrate that NBF-SPh participates in toxic redox-cycling, rapidly generating reactive oxygen species (ROS) in the presence of molecular oxygen, and this is the first report to detail ROS production for any of the anticancer NBFs. Oxygraph studies showed that NBF-SPh consumes molecular oxygen at a substantial rate, rivaling even plumbagin, menadione, and juglone. Biochemical and enzymatic assays identified superoxide and hydrogen peroxide as products of its redox-cycling activity, and the rapid rate of ROS production appears to be sufficient to account for some of the toxicity of NBF-SPh (LC50 = 12.1 µM), possibly explaining why tumor cells exhibit a sharp threshold for tolerating the compound. In cell cultures, lipid peroxidation was enhanced after treatment with NBF-SPh, as measured by 2-thiobarbituric acid-reactive substances, indicating a significant accumulation of ROS. Thioglycerol rescued cell death and increased survival by 15-fold to 20-fold, but pyruvate and uric acid were ineffective protectants. We also observed that the redox-cycling activity of NBF-SPh became exhausted after an average of approximately 19 cycles per NBF-SPh molecule. Electrochemical and computational analyses suggest that partial reduction of NBF-SPh enhances electrophilicity, which appears to encourage scavenging activity and contribute to electrophilic toxicity.

Synthesis and structure–activity relationship of new cytotoxic agents targeting human glutathione-S-transferases

“…A recent study analyzed the biological properties of an analog of compound 2, with the nitro group replaced by sulfonic acid. The lack of cytotoxic effects of this compound on EMT6 murine tumor cells supported the idea that the cytotoxicity of NBD compounds is a consequence of the generation of reactive oxygen species during the reduction of the nitro group [30]. Nevertheless, in our hands the C4-amino derivative 40, an analog of 1 with the C4-sulfur atom replaced by an amino group, retains the nitro group at the NBD portion but was ineffective in U-2OS cells (LC 50 > 100 mM) as well as against GSTP1-1 (IC 50 !…”

Proteomic and functional analyses reveal pleiotropic action of the anti-tumoral compound NBDHEX in Giardia duodenalis

“…More important as generator redox stress is that 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) derivatives can undergo, in the presence of oxygen and before complete nitroreduction of NBD-moiety, several redox-cycles thus efficiently consuming O 2 and dramatically generating ROS (Patridge et al., 2012). Simultaneously, the generated intermediates possess electrophilic scavenging activity and can react with cellular components.…”

Reactive Chemicals and Electrophilic Stress in Cancer

“…Structural analysis of the protein bound NBD-hexanol derivative revealed its covalent interaction with glutathione in the GSTP1, an isoenzyme of the GST superfamily [30]. Thereafter, compound 7-nitro-4(phenylthio)benzofurazan (NBF-SPh) has been found to be a potent generator of superoxide and hydrogen peroxide when compared to other ROS producing chemicals [42]. NBF-SPh promotes the consecutive production of O2 − and H2O2 through the reversible electrochemical redox cycling of molecular oxygen in cells (Figure 3).…”