The quinone reductase enzyme NAD(P)H: quinone oxidoreductase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes the two-electron reduction of quinones. This Perspective briefly reviews the structure and mechanism, physiological role, and upregulation and induction of the enzyme, but focuses on the synthesis of new heterocyclic quinones and their metabolism by recombinant human NQO1. Thus a range of indolequinones, some of which are novel analogues of mitomycin C, benzimidazolequinones, benzothiazolequinones and quinolinequinones have been prepared and evaluated, leading to detailed knowledge of the structural requirements for efficient metabolism by the enzyme. Potent mechanism-based inhibitors (suicide substrates) of NQO1 have also been developed. These indolequinones irreversibly alkylate the protein, preventing its function both in standard enzyme assays and also in cells. Some of these quinones are also potent inhibitors of growth of human pancreatic cancer cells, suggesting a potential role for such compounds as therapeutic agents.
NAD(P)H:quinone oxidoreductase 1 (NQO1) is currently an emerging target in pancreatic cancer. In this report, we describe a series of indolequinones, based on 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), and evaluate NQO1 inhibition and growth inhibitory activity in the human pancreatic MIA PaCa-2 tumor cell line. The indolequinones with 4-nitrophenoxy, 4-pyridinyloxy, and acetoxy substituents at the (indol-3-yl)methyl position were NADH-dependent inhibitors of recombinant human NQO1, indicative of mechanism-based inhibition. However, those with hydroxy and phenoxy substituents were poor inhibitors of NQO1 enzyme activity, due to attenuated elimination of the leaving group. The ability of this series of indolequinones to inhibit recombinant human NQO1 correlated with NQO1 inhibition in MIA PaCa-2 cells. The examination of indolequinone interactions in complex with NQO1 from computational-based molecular docking simulations supported the observed biochemical data with respect to NQO1 inhibition. The design of both NQO1-inhibitory and noninhibitory indolequinone analogues allowed us to test the hypothesis that NQO1 inhibition was required for growth inhibitory activity in MIA PaCa-2 cells. ES936 and its 6-methoxy analogue were potent inhibitors of NQO1 activity and cell proliferation; however, the 4-pyridinyloxy and acetoxy compounds were also potent inhibitors of NQO1 activity but relatively poor inhibitors of cell proliferation. In addition, the phenoxy compounds, which were not inhibitors of NQO1 enzymatic activity, demonstrated potent growth inhibition. These data demonstrate that NQO1 inhibitory activity can be dissociated from growth inhibitory activity and suggest additional or alternative targets to NQO1 that are responsible for the growth inhibitory activity of this series of indolequinones in human pancreatic cancer.
The indolequinone ES936 {5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione} was previously developed in our lab as an antitumor agent against pancreatic cancer. The objective of this study was to identify indolequinones with improved potency against pancreatic cancer and to define their mechanisms of action. Pancreatic cancer cell lines PANC-1, MIA PaCa-2, and BxPC-3 were used in in vitro assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) and clonogenic assays]; indolequinones displayed potent cytotoxicity against all three cell lines, and two specific classes of indolequinone were particularly potent agents. These indolequinones induced caspase-dependent apoptosis but no redox cycling or oxidative stress in MIA PaCa-2 and BxPC-3 cells. Selected indolequinones were also screened against the NCI-60 cell line panel and were found to be particularly effective against colon, renal, and melanoma cancer cells. A potential target of these indolequinones was identified as thioredoxin reductase. Indolequinones were found to be potent inhibitors of thioredoxin reductase activity both in pancreatic cancer cells and in cellfree systems. The mechanism of action of the indolequinones was shown to involve metabolic reduction, loss of a leaving group to generate a reactive electrophile resulting in alkylation of the selenocysteine residue in the active site of thioredoxin reductase. In vivo efficacy of the indolequinones was also tested in the MIA PaCa-2 pancreatic tumor xenograft in nude mice, and lead indolequinones demonstrated high efficacy and low toxicity. Inhibition of thioredoxin reductase represents a potential novel target in pancreatic cancer and may provide a biomarker of effect of lead indolequinones in this type of cancer.Pancreatic cancer is the fourth leading cause of cancer death in the United States (Jemal et al., 2008), with a 5-year survival rate of Ͻ5%. Current treatment options of radiation therapy, chemotherapy, and surgery have been ineffective at improving the survival rate (Ghaneh et al., 2007). Development of novel targeted therapeutic approaches is desperately needed.We have reported previously the development of an indolequinone, 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]-indole-4,7-dione (ES936, 1), that exhibited potent growth inhibition effects against human pancreatic cancer cell lines (Dehn et al., 2006). The antitumor activity of ES936 was originally attributed to its role as a mechanism-based inhibitor of human NQO1 [NAD(P)H:quinone oxidoreductase 1 (DT-diaphorase; EC 1.6.99.2)] (Winski et al., 2001). NQO1 inhibition by dicumarol, a nonspecific inhibitor, has been shown to be cytotoxic in human pancreatic cancer cells (Cullen et al., 2003;Lewis et al., 2004). However, when a series of indolequinone compounds
A role for the flavoprotein NRH:quinone oxidoreductase 2 (NQO2, QR2) in human diseases such as malaria, leukemia and neurodegeneration has been proposed. In order to explore the potential of NQO2 as a therapeutic target, we have developed potent and selective mechanism-based inhibitors centered on the indolequinone pharmacophore. The compounds show remarkable selectivity for NQO2 over the closely related flavoprotein NQO1 with small structural changes defining selectivity. Biochemical studies confirmed mechanism-based inhibition while X-ray crystallography and mass spectrometry revealed the nature of the inhibitor interaction with the protein. These indolequinones represent the first mechanism-based inhibitors of NQO2, and their novel mode of action involving alkylation of the flavin cofactor, provides significant advantages over existing competitive inhibitors in terms of potency and irreversibility, and will open new opportunities to define the role of NQO2 in disease.
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