In silico identification and biochemical characterization of novel inhibitors of NQO1

From in silico docking and COMPARE analysis, novel inhibitors of human NAD(P)H quinone oxidoreductase (NQO1) have been identified from the NCI compound database, the most potent of which has an observed IC₅₀ of 0.7 μM. The inhibitors exhibit a diverse range of scaffolds. The ability of docking calculations to predict experimentally determined binding affinities for NQO1 is discussed, considering the influence of target flexibility and scoring function.     

Evidence for NQO1 and NQO2 catalyzed reduction of ortho- and para-quinone methides

Abstract

NAD(P)H:quinone oxidoreductase (NQO1) and NRH:quinone oxidoreductase 2 (NQO2) catalyze the two-electron reduction of quinones and thereby prevent generation of toxic radicals. Quinone methides (QMs) covalently react with cellular macromolecules to form DNA adducts and/or protein conjugates resulting in toxicity and carcinogenesis. Based on similar structural features of quinones and QMs, it is logical to assume that NQO1 and/or NQO2 could also catalyze the two-electron reduction of QMs. However, hitherto the reduction of QMs, as both endogenous and/or exogenous biological substrates, by either NQO1/NQO2 has never been demonstrated. Here we show for the first time that both NQO1 and NQO2 can catalyze the reduction of electrophilic ortho-/para-QMs. The involvement of the enzyme in the reduction of p-cresol quinone methide (PCQM) and o-cresol quinone methide (OCQM) was demonstrated by reappearance of NQO1/NQO2-FAD peak at 450 nm after addition of the QMs to the assay mixture. Further reduction of methides by NQO1/NQO2 was confirmed by analyzing the assay mixture by tandem mass spectrometry. Preliminary kinetic studies show that NQO2 is faster in reducing QMs than its homolog NQO1, and moreover, ortho-QMs are reduced faster than para-QMs. Enzyme-substrate docking studies showed results consistent with enzyme catalysis. Thus, NQO1/NQO2 can play a significant role in deactivation of QMs.     

In silico identification and biochemical evaluation of novel inhibitors of NRH:quinone oxidoreductase 2 (NQO2)

The NCI chemical database has been screened using in silico docking to identify novel inhibitors of NRH:quinone oxidoreductase 2 (NQO2). Compounds identified from the screen exhibit a diverse range of scaffolds and inhibitory potencies are generally in the micromolar range. Some of the compounds also have the ability to inhibit NQO1. The modes of binding of the different compounds to the two enzymes are illustrated and discussed.     

Reduction of nitroaromatic compounds by NAD(P)H:quinone oxidoreductase (NQO1): the role of electron-accepting potency and structural parameters in the substrate specificity

We aimed to elucidate the role of electronic and structural parameters of nitroaromatic compounds in their two-electron reduction by NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). The multiparameter regression analysis shows that the reactivity of nitroaromatic compounds (n=38) increases with an increase in their single-electron reduction potential and the torsion angle between nitrogroup(s) and the aromatic ring. The binding efficiency of nitroaromatics in the active center of NQO1 exerted a less evident role in their reactivity. The reduction of nitroaromatics is characterized by more positive entropies of activation than the reduction of quinones. This points to a less efficient electronic coupling of nitroaromatics with the reduced isoalloxazine ring of FAD, and may explain their lower reactivity as compared to quinones. Another important but poorly understood factor enhancing the reactivity of nitroaromatics is their ability to bind at the dicumarol/quinone binding site in the active center of NQO1.     

Development of indolequinone mechanism-based inhibitors of NAD(P)H:quinone oxidoreductase 1 (NQO1): NQO1 inhibition and growth inhibitory activity in human pancreatic MIA PaCa-2 cancer cells

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.     

NAD(P)H:Quinone Oxidoreductase 1 (NQO1) Localizes to the Mitotic Spindle in Human Cells

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an FAD containing quinone reductase that catalyzes the 2-electron reduction of a broad range of quinones. The 2-electron reduction of quinones to hydroquinones by NQO1 is believed to be a detoxification process since this reaction bypasses the formation of the highly reactive semiquinone. NQO1 is expressed at high levels in normal epithelium, endothelium and adipocytes as well as in many human solid tumors. In addition to its function as a quinone reductase NQO1 has been shown to reduce superoxide and regulate the 20 S proteasomal degradation of proteins including p53. Biochemical studies have indicated that NQO1 is primarily located in the cytosol, however, lower levels of NQO1 have also been found in the nucleus. In these studies we demonstrate using immunocytochemistry and confocal imaging that NQO1 was found associated with mitotic spindles in cells undergoing division. The association of NQO1 with the mitotic spindles was observed in many different human cell lines including nontransformed cells (astrocytes, HUVEC) immortalized cell lines (HBMEC, 16HBE) and cancer (pancreatic adenocarcinoma, BXPC3). Confocal analysis of double-labeling experiments demonstrated co-localization of NQO1with alpha-tubulin in mitotic spindles. In studies with BxPc-3 human pancreatic cancer cells the association of NQO1 with mitotic spindles appeared to be unchanged in the presence of NQO1 inhibitors ES936 or dicoumarol suggesting that NQO1 can associate with the mitotic spindle and still retain catalytic activity. Analysis of archival human squamous lung carcinoma tissue immunostained for NQO1 demonstrated positive staining for NQO1 in the spindles of mitotic cells. The purpose of this study is to demonstrate for the first time the association of the quinone reductase NQO1 with the mitotic spindle in human cells.     

Implications of NQO1 in cancer therapy

NAD(P)H:quinone oxidoreductase (NQO1), an obligatory two-electron reductase, is a ubiquitous cytosolic enzyme that catalyzes the reduction of quinone substrates. The NQO1- mediated two-electron reduction of quinones can be either chemoprotection/detoxification or a chemotherapeutic response, depending on the target quinones. When toxic quinones are reduced by NQO1, they are conjugated with glutathione or glucuronic acid and excreted from the cells. Based on this protective effect of NQO1, the use of dietary compounds to induce the expression of NQO1 has emerged as a promising strategy for cancer prevention. On the other hand, NQO1-mediated two-electron reduction converts certain quinone compounds (such as mitomycin C, E09, RH1 and β-lapachone) to cytotoxic agents, leading to cell death. It has been known that NQO1 is expressed at high levels in numerous human cancers, including breast, colon, cervix, lung, and pancreas, as compared with normal tissues. This implies that tumors can be preferentially damaged relative to normal tissue by cytotoxic quinone drugs. Importantly, NQO1 has been shown to stabilize many proteins, including p53 and p33ING1b, by inhibiting their proteasomal degradation. This review will summarize the biological roles of NQO1 in cancer, with emphasis on recent findings and the potential of NQO1 as a therapeutic target for the cancer therapy. [BMB Reports 2015; 48(11): 609-617]     

Evidence for NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated quinone-dependent redox cycling via plasma membrane electron transport: A sensitive cellular assay for NQO1

2,3-Dimethoxy 1,4-naphthoquinone (DMNQ), which redox cycles via two-electron reduction, mediates reduction of the cell-impermeative tetrazolium dye WST-1 in kidney epithelial cells (MDCK), which express high levels of NQO1, but not in HL60 or CHO cells, which are NQO1 deficient. DMNQ-dependent WST-1 reduction by MDCK cells was strongly inhibited by low concentrations of the NQO1 inhibitor dicoumarol and was also inhibited by diphenyleneiodonium, capsaicin, and superoxide dismutase (SOD), but not by the uncoupler FCCP or the complex IV inhibitor cyanide. This suggests that DMNQ-dependent WST-1 reduction by MDCK cells is catalyzed by NQO1 via redox cycling and plasma membrane electron transport (PMET). Interestingly, we observed an association between DMNQ/WST-1 reduction and extracellular H₂O₂ production as determined by Amplex red. Exposure of MDCK cells to DMNQ for 48 h caused cellular toxicity that was extensively reversed by co-incubation with dicoumarol or exogenous SOD, catalase, or N-acetylcysteine. No effects were observed in NQO1-deficient CHO and HL60 cells. In conclusion, we have developed a simple real-time cellular assay for NQO1 and show that PMET plays a significant role in DMNQ redox cycling via NQO1, leading to cellular toxicity in cells with high NQO1 levels.     

NRH:quinone oxidoreductase2 (NQO2)

The quinone oxidoreductases [NAD(P)H:quinone oxidoreductase1 (NQO1) and NRH:quinone oxidoreductase2 (NQO2)] are flavoproteins. NQO1 is known to catalyse metabolic detoxification of quinones and protect cells from redox cycling, oxidative stress and neoplasia. NQO2 is a 231 amino acid protein (25956 mw) that is 43 amino acids shorter than NQO1 at its carboxy-terminus. The human NQO2 cDNA and protein are 54 and 49% similar to the human liver cytosolic NQO1 cDNA and protein. Recent studies have revealed that NQO2 differs from NQO1 in its cofactor requirement. NQO2 uses dihydronicotinamide riboside (NRH) rather than NAD(P)H as an electron donor. Another difference between NQO1 and NQO2 is that NQO2 is resistant to typical inhibitors of NQO1, such as dicoumarol, Cibacron blue and phenindone. Flavones, including quercetin and benzo(a)pyrene, are known inhibitors of NQO2. Even though overlapping substrate specificities have been observed for NQO1 and NQO2, significant differences exist in relative affinities for the various substrates. Analysis of the crystal structure of NQO2 revealed that NQO2 contains a specific metal binding site, which is not present in NQO1. The human NQO2 gene has been precisely localized to chromosome 6p25. The human NQO2 gene locus is highly polymorphic. The NQO2 gene is ubiquitously expressed and induced in response to TCDD. Nucleotide sequence analysis of the NQO2 gene promoter revealed the presence of several cis-elements, including SP1 binding sites, CCAAT box, xenobiotic response element (XRE) and an antioxidant response element (ARE). The complement of these elements regulates tissue specific expression and induction of the NQO2 gene in response to xenobiotics and antioxidants. The in vivo role of NQO2 and its role in quinone detoxification remains unknown.     

Triazoloacridin-6-ones as novel inhibitors of the quinone oxidoreductases NQO1 and NQO2

A range of triazoloacridin-6-ones functionalized at C5 and C8 have been synthesized and evaluated for ability to inhibit NQO1 and NQO2. The compounds were computationally docked into the active site of NQO1 and NQO2, and calculated binding affinities were compared with IC₅₀ values for enzyme inhibition. Excellent correlation coefficients were demonstrated suggesting a predictive QSAR model for this series of structurally similar analogues. From this we have identified some of these triazoloacridin-6-ones to be the most potent NQO2 inhibitors so far reported.     

Structure-function studies of DT-diaphorase (NQO1) and NRH: quinone oxidoreductase (NQO2)

DT-diaphorase, also referred to as NQO1 or NAD(P)H: quinone acceptor oxidoreductase, is a flavoprotein that catalyzes the two-electron reduction of quinones and quinonoid compounds to hydroquinones, using either NADH or NADPH as the electron donor. NRH (dihydronicotinamide riboside): quinone oxidoreductase, also referred to as NQO2, has a high nucleotide sequence identity to DT-diaphorase and is considered to be an isozyme of DT-diaphorase. These enzymes transfer two electrons to a quinone, resulting in the formation of a hydroquinone product without the accumulation of a dissociated semiquinone. Steady and rapid-reaction kinetic experiments have been performed to determine the reaction mechanism of DT-diaphorase. Furthermore, chimeric and site-directed mutagenesis experiments have been performed to determine the molecular basis of the catalytic differences between the two isozymes and to identify the critical amino acid residues that interact with various inhibitors of the enzymes. In addition, functional studies of a natural occurring mutant Pro-187 to Ser (P187S) have been carried out. Results obtained from these investigations are summarized and discussed.     

NQO1 and NQO2 regulation of humoral immunity and autoimmunity

NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2) are cytosolic enzymes that catalyze metabolic reduction of quinones and derivatives. NQO1-null and NQO2-null mice were generated that showed decreased lymphocytes in peripheral blood, myeloid hyperplasia, and increased sensitivity to skin carcinogenesis. In this report, we investigated the in vivo role of NQO1 and NQO2 in immune response and autoimmunity. Both NQO1-null and NQO2-null mice showed decreased B-cells in blood, lower germinal center response, altered B cell homing, and impaired primary and secondary immune responses. NQO1-null and NQO2-null mice also showed susceptibility to autoimmune disease as revealed by decreased apoptosis in thymocytes and pre-disposition to collagen-induced arthritis. Further experiments showed accumulation of NADH and NRH, cofactors for NQO1 and NQO2, indicating altered intracellular redox status. The studies also demonstrated decreased expression and lack of activation of immune-related factor NF-kappaB. Microarray analysis showed altered chemokines and chemokine receptors. These results suggest that the loss of NQO1 and NQO2 leads to altered intracellular redox status, decreased expression and activation of NF-kappaB, and altered chemokines. The results led to the conclusion that NQO1 and NQO2 are endogenous factors in the regulation of immune response and autoimmunity.     

In vivo role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the regulation of intracellular redox state and accumulation of abdominal adipose tissue

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein that utilizes NAD(P)H as an electron donor, catalyzing the two-electron reduction and detoxification of quinones and their derivatives. NQO1-/- mice deficient in NQO1 activity and protein were generated in our laboratory (Rajendirane, V., Joseph, P., Lee, Y. H., Kimura, S., Klein-Szanto, A. J. P., Gonzalez, F. J., and Jaiswal, A. K. (1998) J. Biol. Chem. 273, 7382-7389). Mice lacking a functional NQO1 gene (NQO1-/-) were born normal and reproduced adeptly as the wild-type NQO1+/+ mice. In the present report, we show that NQO1-/- mice exhibit significantly lower levels of abdominal adipose tissue as compared with the wild-type mice. The NQO1-/- mice showed lower blood levels of glucose, no change in insulin, and higher levels of triglycerides, beta-hydroxy butyrate, pyruvate, lactate, and glucagon as compared with wild-type mice. Insulin tolerance test demonstrated that the NQO1-/- mice are insulin resistant. The NQO1-/- mice livers also showed significantly higher levels of triglycerides, lactate, pyruvate, and glucose. The liver glycogen reserve was found decreased in NQO1-/- mice as compared with wild-type mice. The livers and kidneys from NQO1-/- mice also showed significantly lower levels of pyridine nucleotides but an increase in the reduced/oxidized NAD(P)H:NAD(P) ratio. These results suggested that loss of NQO1 activity alters the intracellular redox status by increasing the concentration of NAD(P)H. This leads to a reduction in pyridine nucleotide synthesis and reduced glucose and fatty acid metabolism. The alterations in metabolism due to redox changes result in a significant reduction in the amount of abdominal adipose tissue.     

Evidence from ESI-MS for NQO1-catalyzed reduction of estrogen ortho-quinones

Estrogen ortho-quinones have been implicated as ultimate carcinogenic metabolites of estrogens. The present conclusion that estrogen ortho-quinones are not substrates for NAD(P)H:quinone oxidoreductase (NQO1) stems from earlier reports. In this investigation, we were successful in circumventing the problem of nonenzymatic reduction of estrogen quinone by NAD(P)H, which led to the above conclusion, and for the first time we show that NQO1 catalyzes the reduction of estrogen quinones. Mass spectrometric binding studies involving estradiol-3,4-quinone or menadione with NQO1 clearly support the formation of an enzyme-substrate physical complex. However, the NQO1 mass spectrum did not alter after addition of cholesterol, the control. Two different strategies were employed to ascertain the NQO1 activity in estrogen quinone reduction. First, the ping-pong mechanism of NQO1 catalysis was utilized to overcome the problem of nonenzymatic reduction of the substrate by NADH. Second, tetrahydrofolic acid, which has a lower reducing potential, was used as an alternate cofactor. Both of these methods confirmed the reduction of estradiol-3,4-quinone by NQO1, when the assay mixtures were analyzed by UV or liquid chromatography-mass spectrometry. Furthermore, reduction of 9,10-phenanthrene quinone or menadione was observed using the reported assay conditions. Thus, clear evidence for the catalytic reduction of estrogen ortho-quinones by NQO1 has been obtained; its mechanism and implications are discussed.     

Evidence for NQO2-mediated reduction of the carcinogenic estrogen ortho-quinones

The physiological function of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase) is to detoxify potentially reactive quinones by direct transfer of two electrons. A similar detoxification role has not been established for its homologue NRH:quinone oxidoreductase 2 (NQO2). Estrogen quinones, including estradiol(E(2))-3,4-Q, generated by estrogen metabolism, are thought to be responsible for estrogen-initiated carcinogenesis. In this investigation, we have shown for the first time that NQO2 catalyzes the reduction of electrophilic estrogen quinones and thereby may act as a detoxification enzyme. ESI and MALDI mass spectrometric binding studies involving E(2)-3,4-Q with NQO2 clearly support the formation of an enzyme-substrate physical complex. The problem of spontaneous reduction of substrate by cofactor, benzyldihydronicotinamide riboside (BNAH), was successfully overcome by taking advantage of the ping-pong mechanism of NQO2 catalysis. The involvement of the enzyme in the reduction of E(2)-3,4-Q was further supported by addition of the inhibitor quercetin to the assay mixture. NQO2 is a newly discovered binding site (MT3) of melatonin. However, addition of melatonin to the assay mixture did not affect the catalytic activity of NQO2. Preliminary kinetic studies show that NQO2 is faster in reducing estrogen quinones than its homologue NQO1. Both UV and liquid chromatography-tandem mass spectrometry assays unequivocally corroborate the reduction of estrogen ortho-quinones by NQO2, indicating that it could be a novel target for prevention of breast cancer initiation.     

Role of NAD(P)H:quinone oxidoreductase (NQO1) in apoptosis induction by aziridinylbenzoquinones RH1 and MeDZQ

We aimed to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1) in apoptosis induction by antitumour quinones RH1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-1,4-benzoquinone) and MeDZQ (2,5-dimethyl-3,6-diaziridinyl-1,4-benzoquinone). Digitonin-permeabilized FLK cells catalyzed NADPH-dependent single- and two-electron reduction of RH1 and MeDZQ. At equitoxic concentrations, RH1 and MeDZQ induced apoptosis more efficiently than the nonalkylating duroquinone or H(2)O(2). The antioxidant N,N'-diphenyl-p-phenylene diamine, desferrioxamine, and the inhibitor of NQO1 dicumarol, protected against apoptosis induction by all compounds investigated, but to a different extent. The results of multiparameter regression analysis indicate that RH1 and MeDZQ most likely induce apoptosis via NQO1-linked formation of alkylating species but not via NQO1-linked redox cycling.     

Immunodetection of NAD(P)H:quinone oxidoreductase 1 (NQO1) in human tissues

Despite the extensive interest in NADPH:quinone oxidoreductase (NQO1, DT-diaphorase), there is little immunohistochemical information regarding its distribution in either normal human tissues or in human tumors. Using immunohistochemistry (IHC), we have examined cell-specific expression of NQO1 in many normal tissues and tumors as a step toward defining the distribution of NQO1 in humans. NQO1 was detected by IHC in respiratory, breast duct, thyroid follicle, and colonic epithelium, as well as in the corneal and lens epithelium of the eye. NQO1 was also detected by IHC in vascular endothelium in all tissues examined. NQO1 could also readily be detected in the endothelial lining of the aorta but was not detected using immunoblot analysis in the myocardium. Adipocytes stained positive for NQO1, and the enzyme was also detected by both IHC and immunoblot analysis in parasympathetic ganglia in the small intestine and in the optic nerve and nerve fibers. NQO1 was not highly expressed in five different human liver samples using immunoblot analysis, whereas studies using IHC demonstrated only trace NQO1 staining in isolated bile duct epithelium. NQO1 expresion was also examined by IHC in a variety of solid tumors. Marked NQO1 staining was detected in solid tumors from thyroid, adrenal, breast, ovarian, colon, and cornea and in non-small cell lung cancers. The NQO1 content of many solid tumors supports the use of NQO1-directed anticancer agents for therapeutic purposes, but the distribution of NQO1 in normal tissues suggests that potential adverse effects of such agents need to be carefully monitored in preclinical studies.