NQO1 Mediates Lenvatinib Resistance by Regulating ROS-induced Apoptosis in Hepatocellular Carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated death worldwide. As a first-line drug for advanced HCC treatment, lenvatinib faces a significant hurdle due to the development of both intrinsic and acquired resistance among patients, and the underlying mechanism remains largely unknown. The present study aims to identify the pivotal gene responsible for lenvatinib resistance in HCC, explore the potential molecular mechanism, and propose combinatorial therapeutic targets for HCC management. METHODS: Cell viability and colony formation assays were conducted to evaluate the sensitivity of cells to lenvatinib and dicoumarol. RNA-Seq was used to determine the differences in transcriptome between parental cells and lenvatinib-resistant (LR) cells. The upregulated genes were analyzed by GO and KEGG analyses. Then, qPCR and Western blotting were employed to determine the relative gene expression levels. Afterwards, the intracellular reactive oxygen species (ROS) and apoptosis were detected by flow cytometry. RESULTS: PLC-LR and Hep3B-LR were established. There was a total of 116 significantly upregulated genes common to both LR cell lines. The GO and KEGG analyses indicated that these genes were involved in oxidoreductase and dehydrogenase activities, and reactive oxygen species pathways. Notably, NAD(P)H:quinone oxidoreductase 1 (NQO1) was highly expressed in LR cells, and was involved in the lenvatinib resistance. The high expression of NQO1 decreased the production of ROS induced by lenvatinib, and subsequently suppressed the apoptosis. The combination of lenvatinib and NQO1 inhibitor, dicoumarol, reversed the resistance of LR cells. CONCLUSION: The high NQO1 expression in HCC cells impedes the lenvatinib-induced apoptosis by regulating the ROS levels, thereby promoting lenvatinib resistance in HCC cells.

Hypoxia-responsive nanocarriers for chemotherapy sensitization via dual-mode inhibition of hypoxia-inducible factor-1 alpha.

The overexpression of hypoxia-inducible factor-1 alpha (HIF-1α) in solid tumor compromises the potency of chemotherapy under hypoxia. The high level of HIF-1α arises from the stabilization effect of reduced nicotinamideadeninedinucleotide(phosphate) NAD(P)H: quinone oxidoreductase 1 (NQO1). It was postulated that the inhibition of NQO1 could degrade HIF-1α and sensitize hypoxic cancer cells to antineoplastic agents. In the current work, we report hypoxia-responsive polymer micelles, i.e. methoxyl poly(ethylene glycol)-co-poly(aspartate-nitroimidazole) orchestrate with a NQO1 inhibitor (dicoumarol) to sensitize the ovarian cancer cell line (SKOV3) to a model anticancer agent (sorafenib) at low oxygen conditions. Both cargos were physically encapsulated in the nanoscale micelles. The placebo micelles transiently induced the depletion of reduced nicotinamideadeninedinucleotidephosphate (NADPH) as well as glutathione and thioredoxin under hypoxia, which further inactivated NQO1 because NADPH was the cofactor of NQO1. As a consequence, the expression of HIF-1α was repressed due to the dual action of dicoumarol and polymer. The degradation of HIF-1α significantly increased the vulnerability of SKOV3 cells to sorafenib-induced apoptosis, as indicated by the enhancement of cytotoxicity, and increase of caspase 3 and cytochrome C. The current work opens new avenues of addressing hypoxia-induced drug resistance in chemotherapy.

Aerobic Cytotoxicity of Aromatic N-Oxides: The Role of NAD(P)H:Quinone Oxidoreductase (NQO1).

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities, which are typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the role of NAD(P)H:quinone oxidoreductase (NQO1) in ArN→O aerobic cytotoxicity. We synthesized 9 representatives of ArN→O with uncharacterized redox properties and examined their single-electron reduction by rat NADPH:cytochrome P-450 reductase (P-450R) and Plasmodium falciparum ferredoxin:NADP+ oxidoreductase (PfFNR), and by rat NQO1. NQO1 catalyzed both redox cycling and the formation of stable reduction products of ArN→O. The reactivity of ArN→O in NQO1-catalyzed reactions did not correlate with the geometric average of their activity towards P-450R- and PfFNR, which was taken for the parameter of their redox cycling efficacy. The cytotoxicity of compounds in murine hepatoma MH22a cells was decreased by antioxidants and the inhibitor of NQO1, dicoumarol. The multiparameter regression analysis of the data of this and a previous study (DOI: 10.3390/ijms20184602) shows that the cytotoxicity of ArN→O (n = 18) in MH22a and human colon carcinoma HCT-116 cells increases with the geometric average of their reactivity towards P-450R and PfFNR, and with their reactivity towards NQO1. These data demonstrate that NQO1 is a potentially important target of action of heteroaromatic N-oxides.

A pharmacological review of dicoumarol: An old natural anticoagulant agent.

Dicoumarol is an oral anticoagulant agent prescribed in clinical for decades. It is a natural hydroxycoumarin discovered from the spoilage of Melilotus officinalis (L.) Pall and is originally discovered as a rodenticide. Due to its structural similarity to that of vitamin K, it significantly inhibits vitamin K epoxide reductase and acts as a vitamin K antagonist. Dicoumarol is mainly used as an anticoagulant to prevent thrombogenesis and to cure vascular thrombosis. Other biological activities besides anticoagulants such as anticancer, antimicrobial, antiviral, etc., have also been documented. The side effects of dicoumarol raise safety concerns for clinical application. In this review, the physicochemical property, the pharmacological activities, the side effects, and the pharmacokinetics of dicoumarol were summarized, aiming to provide a whole picture of the "old" anticoagulant.

ß-Lapachone Induces Acute Oxidative Stress in Rat Primary Astrocyte Cultures that is Terminated by the NQO1-Inhibitor Dicoumarol.

ß-lapachone (ß-lap) is reduced in tumor cells by the enzyme NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1) to a labile hydroquinone which spontaneously reoxidises to ß-lap, thereby generating reactive oxygen species (ROS) and oxidative stress. To test for the consequences of an acute exposure of brain cells to ß-lap, cultured primary rat astrocytes were incubated with ß-lap for up to 4 h. The presence of ß-lap in concentrations of up to 10 µM had no detectable adverse consequences, while higher concentrations of ß-lap compromised the cell viability and the metabolism of astrocytes in a concentration- and time-dependent manner with half-maximal effects observed for around 15 µM ß-lap after a 4 h incubation. Exposure of astrocytes to ß-lap caused already within 5 min a severe increase in the cellular production of ROS as well as a rapid oxidation of glutathione (GSH) to glutathione disulfide (GSSG). The transient cellular accumulation of GSSG was followed by GSSG export. The ß-lap-induced ROS production and GSSG accumulation were completely prevented in the presence of the NQO1 inhibitor dicoumarol. In addition, application of dicoumarol to ß-lap-exposed astrocytes caused rapid regeneration of the normal high cellular GSH to GSSG ratio. These results demonstrate that application of ß-lap to cultured astrocytes causes acute oxidative stress that depends on the activity of NQO1. The sequential application of ß-lap and dicoumarol to rapidly induce and terminate oxidative stress, respectively, is a suitable experimental paradigm to study consequences of a defined period of acute oxidative stress in NQO1-expressing cells.

Inhibition of pyruvate dehydrogenase kinase­1 by dicoumarol enhances the sensitivity of hepatocellular carcinoma cells to oxaliplatin via metabolic reprogramming.

The Warburg effect is a unique metabolic feature of the majority of tumor cells and is closely related to chemotherapeutic resistance. Pyruvate dehydrogenase kinase 1 (PDK1) is considered a 'switch' that controls the fate of pyruvate in glucose metabolism. However, to date, to the best of our knowledge, there are only a few studies to available which had studied the reduction of chemotherapeutic resistance via the metabolic reprogramming of tumor cells with PDK1 as a target. In the present study, it was found dicoumarol (DIC) reduced the phosphorylation of pyruvate dehydrogenase (PDH) by inhibiting the activity of PDK1, which converted the metabolism of human hepatocellular carcinoma (HCC) cells to oxidative phosphorylation, leading to an increase in mitochondrial reactive oxygen species ROS (mtROS) and a decrease in mitochondrial membrane potential (MMP), thereby increasing the apoptosis induced by oxaliplatin (OXA). Furthermore, the present study elucidated that the targeting of PDK1 may be a potential strategy for targeting metabolism in the chemotherapy of HCC. In addition, DIC as an 'old drug' exhibits novel efficacy, bringing new hope for antitumor therapy.

Spatio-Temporally Reporting Dose-Dependent Chemotherapy via Uniting Dual-Modal MRI/NIR Imaging.

Unpredictable in vivo therapeutic feedback of hydroxyl radical (. OH) efficiency is the major bottleneck of chemodynamic therapy. Herein, we describe novel Fenton-based nanotheranostics NQ-Cy@Fe&GOD for spatio-temporally reporting intratumor . OH-mediated treatment, which innovatively unites dual-channel near-infrared (NIR) fluorescence and magnetic resonance imaging (MRI) signals. Specifically, MRI signal traces the dose distribution of Fenton-based iron oxide nanoparticles (IONPs) with high-spatial resolution, meanwhile timely fluorescence signal quantifies . OH-mediated therapeutic response with high spatio-temporal resolution. NQ-Cy@Fe&GOD can successfully monitor the intracellular release of IONPs and . OH-induced NQO1 enzyme in living cells and tumor-bearing mice, which makes a breakthrough in conquering the inherent unpredictable obstacles on spatio-temporally reporting chemodynamic therapy, so as to manipulate dose-dependent therapeutic process.

Dicoumarol suppresses HMGA2-mediated oncogenic capacities and inhibits cell proliferation by inducing apoptosis in colon cancer.

Colon cancer is one of the leading causes of cancer-related deaths and its five-year survival rate remains low in locally advanced or metastatic stages of colon cancer. Overexpression of high mobility group protein AT-hook2 (HMGA2) is associated with cancer progression, metastasis, and poor prognosis in many malignancies. Oxidative stress regulates cellular mechanisms and provides an environment that favors the cancer cells to survive and progress, yet, at the same time, oxidative stress can also be utilized as a cancer-damaging strategy. The molecular regulatory roles of HMGA2 in oxidative stress and their involvement in cancer progression are largely unknown. In this study, we investigated the involvement of HMGA2 in regulation of oxidative stress responses by luciferase reporter assays. Moreover, we utilized dicoumarol (DIC), a derivative of coumarin which has been suggested to be involved in oxidation regulation with anticancer effects, and demonstrated that DIC could induce apoptosis and inhibit cell migration of HMGA2 overexpressing colon cancer cells. Further investigation also evidenced that DIC can enhance the cancer inhibition effect of 5-FU in colony formation assays. Taken together, our data revealed novel insights into the molecular mechanisms underlying HMGA2 and highlighted the possibility of targeting the cellular antioxidant system for treating patients and preventing from cancer progression in HMGA2 overexpressing colon cancer cells.

Cancer-associated variants of human NQO1: impacts on inhibitor binding and cooperativity.

Human NAD(P)H quinone oxidoreductase (DT-diaphorase, NQO1) exhibits negative cooperativity towards its potent inhibitor, dicoumarol. Here, we addressed the hypothesis that the effects of the two cancer-associated polymorphisms (p.R139W and p.P187S) may be partly mediated by their effects on inhibitor binding and negative cooperativity. Dicoumarol stabilized both variants and bound with much higher affinity for p.R139W than p.P187S. Both variants exhibited negative cooperativity towards dicoumarol; in both cases, the Hill coefficient (h) was approximately 0.5 and similar to that observed with the wild-type protein. NQO1 was also inhibited by resveratrol and by nicotinamide. Inhibition of NQO1 by resveratrol was approximately 10,000-fold less strong than that observed with the structurally similar enzyme, NRH quinine oxidoreductase 2 (NQO2). The enzyme exhibited non-cooperative behaviour towards nicotinamide, whereas resveratrol induced modest negative cooperativity (h = 0.85). Nicotinamide stabilized wild-type NQO1 and p.R139W towards thermal denaturation but had no detectable effect on p.P187S. Resveratrol destabilized the wild-type enzyme and both cancer-associated variants. Our data suggest that neither polymorphism exerts its effect by changing the enzyme's ability to exhibit negative cooperativity towards inhibitors. However, it does demonstrate that resveratrol can inhibit NQO1 in addition to this compound's well-documented effects on NQO2. The implications of these findings for molecular pathology are discussed.

Negative Cooperativity in NAD(P)H Quinone Oxidoreductase 1 (NQO1).

NAD(P)H quinone oxidoreductase-1 (NQO1) is a homodimeric protein that acts as a detoxifying enzyme or as a chaperone protein. Dicourmarol interacts with NQO1 at the NAD(P)H binding site and can both inhibit enzyme activity and modulate the interaction of NQO1 with other proteins. We show that the binding of dicoumarol and related compounds to NQO1 generates negative cooperativity between the monomers. This does not occur in the presence of the reducing cofactor, NAD(P)H, alone. Alteration of Gly150 (but not Gly149 or Gly174) abolished the dicoumarol-induced negative cooperativity. Analysis of the dynamics of NQO1 with the Gaussian network model indicates a high degree of collective motion by monomers and domains within NQO1. Ligand binding is predicted to alter NQO1 dynamics both proximal to the ligand binding site and remotely, close to the second binding site. Thus, drug-induced modulation of protein motion might contribute to the biological effects of putative inhibitors of NQO1.

Screening for inhibitor of episomal DNA identified dicumarol as a hepatitis B virus inhibitor.

Currently, there is no available therapy to eradicate hepatitis B virus (HBV) in chronically infected individuals. This is due to the difficulty in eliminating viral covalently closed circular (ccc) DNA, which is central to the gene expression and replication of HBV. We developed an assay system for nuclear circular DNA using an integration-deficient lentiviral vector. This vector produced non-integrated circular DNA in nuclei of infected cells. We engineered this vector to encode firefly luciferase to monitor the lentiviral episome DNA. We screened 3,840 chemicals by this assay for luciferase-reducing activity and identified dicumarol, which is known to have anticoagulation activity. We confirmed that dicumarol reduced lentiviral episome DNA. Furthermore, dicumarol inhibited HBV replication in cell culture using NTCP-expressing HepG2 and primary human hepatocytes. Dicumarol reduced intracellular HBV RNA, DNA, supernatant HBV antigens and DNA. We also found that dicumarol reduced the cccDNA level in HBV infected cells, but did not affect HBV adsorption/entry. This is a novel assay system for screening inhibitors targeting nuclear cccDNA and is useful for finding new antiviral substances for HBV.

Dicoumarol Inhibits Multidrug Resistance Protein 1-Mediated Export Processes in Cultured Primary Rat Astrocytes.

Dicoumarol is frequently used as inhibitor of the detoxifying enzyme NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1). In order to test whether dicoumarol may also affect the cellular glutathione (GSH) metabolism, we have exposed cultured primary astrocytes to dicoumarol and investigated potential effects of this compound on the cell viability as well as on the cellular and extracellular contents of GSH and its metabolites. Incubation of astrocytes with dicoumarol in concentrations of up to 100 µM did not acutely compromise cell viability nor was any GSH consumption or GSH oxidation to glutathione disulfide (GSSG) observed. However, unexpectedly dicoumarol inhibited the cellular multidrug resistance protein (Mrp) 1-dependent export of GSH in a time- and concentration-dependent manner with half-maximal effects observed at low micromolar concentrations of dicoumarol. Inhibition of GSH export by dicoumarol was not additive to that observed for the known Mrp1 inhibitor MK571. In addition, dicoumarol inhibited also the Mrp1-mediated export of GSSG during menadione-induced oxidative stress and the export of the GSH-bimane-conjugate (GS-B) that had been generated in the cells after exposure to monochlorobimane. Half-maximal inhibition of the export of Mrp1 substrates was observed at dicoumarol concentrations of around 4 µM (GSH and GSSG) and 30 µM (GS-B). These data demonstrate that dicoumarol strongly affects the GSH metabolism of viable cultured astrocytes by inhibiting Mrp1-mediated export processes and identifies for the first time Mrp1 as additional cellular target of dicoumarol.

NAD(P)H quinone oxidoreductase (NQO1): an enzyme which needs just enough mobility, in just the right places.

NAD(P)H quinone oxidoreductase 1 (NQO1) catalyses the two electron reduction of quinones and a wide range of other organic compounds. Its physiological role is believed to be partly the reduction of free radical load in cells and the detoxification of xenobiotics. It also has non-enzymatic functions stabilising a number of cellular regulators including p53. Functionally, NQO1 is a homodimer with two active sites formed from residues from both polypeptide chains. Catalysis proceeds via a substituted enzyme mechanism involving a tightly bound FAD cofactor. Dicoumarol and some structurally related compounds act as competitive inhibitors of NQO1. There is some evidence for negative cooperativity in quinine oxidoreductases which is most likely to be mediated at least in part by alterations to the mobility of the protein. Human NQO1 is implicated in cancer. It is often over-expressed in cancer cells and as such is considered as a possible drug target. Interestingly, a common polymorphic form of human NQO1, p.P187S, is associated with an increased risk of several forms of cancer. This variant has much lower activity than the wild-type, primarily due to its substantially reduced affinity for FAD which results from lower stability. This lower stability results from inappropriate mobility of key parts of the protein. Thus, NQO1 relies on correct mobility for normal function, but inappropriate mobility results in dysfunction and may cause disease.

Intracellular reduction of coenzyme Q homologues with a short isoprenoid side chain induces apoptosis of HeLa cells.

Coenzyme Q (CoQ) is an essential factor of the mitochondrial respiratory chain. CoQ homologues with different lengths of the isoprenoid side chain are widely distributed in nature, but little is known about the relationship between the isoprenoid side chain length and biological function; therefore, we examined the effects of CoQ homologues on HeLa cells. When CoQ homologues with a shorter isoprenoid side chain than CoQ4 were added to HeLa cells, they induced cell death, and the order of cytotoxic intensity was as follows: CoQ0 ≫ CoQ3 ≈ CoQ1 > CoQ2 ≫ CoQ4. Furthermore, we found that CoQ1, CoQ2 and CoQ3 could induce caspase-mediated apoptosis, and the order of intensity was as follows: CoQ3 > CoQ2 ≥ CoQ1. We could not identify the participation of reactive oxygen species in the apoptosis induction, but observed that an NAD(P)H dehydrogenase (quinone) 1 (NQO1) inhibitor, dicumarol, could inhibit not only the intracellular reduction of the homologues but also apoptosis. However, because dicumarol did not affect well-known apoptosis inducers, such as anti-Fas IgG, tumor necrosis factor (TNF)-α, TNF-related apoptosis-inducing ligand, UV-B and H2O2 of HeLa cells at all, we concluded that NQO1-related intracellular reduction of CoQ, or its reduced product, ubiquinol, may participate in the apoptosis induction of HeLa cells.

Dicumarol inhibits PDK1 and targets multiple malignant behaviors of ovarian cancer cells.

Pyruvate dehydrogenase kinase 1 (PDK1) is overexpressed in ovarian cancer and thus is a promising anticancer therapeutic target. Our previous work suggests that coumarin compounds are potential inhibitors of PDKs. In this study, we used the ovarian cancer cell line SKOV3 as the model system and examined whether dicumarol (DIC), a coumarin compound, could inhibit ovarian cancer through targeting PDK1. We showed that DIC potently inhibited the kinase activity of PDK1, shifted the glucose metabolism from aerobic glycolysis to oxidative phosphorylation, generated a higher level of reactive oxygen species (ROS), attenuated the mitochondrial membrane potential (MMP), induced apoptosis, and reduced cell viability in vitro. The same phenotypes induced by DIC also were translated in vivo, leading to significant suppression of xenograft growth. This study not only identifies a novel inhibitor for PDK1, but it also reveals novel anticancer mechanisms of DIC and provides a promising anticancer therapy that targets the Warburg effect.

Mitochondrial "power" drives tamoxifen resistance: NQO1 and GCLC are new therapeutic targets in breast cancer.

Here, we identified two new molecular targets, which are functionally sufficient to metabolically confer the tamoxifen-resistance phenotype in human breast cancer cells. Briefly, ~20 proteins were first selected as potential candidates, based on unbiased proteomics analysis, using tamoxifen-resistant cell lines. Then, the cDNAs of the most promising candidates were systematically transduced into MCF-7 cells. Remarkably, NQO1 and GCLC were both functionally sufficient to autonomously confer a tamoxifen-resistant metabolic phenotype, characterized by i) increased mitochondrial biogenesis, ii) increased ATP production and iii) reduced glutathione levels. Thus, we speculate that pharmacological inhibition of NQO1 and GCLC may be new therapeutic strategies for overcoming tamoxifen-resistance in breast cancer patients. In direct support of this notion, we demonstrate that treatment with a known NQO1 inhibitor (dicoumarol) is indeed sufficient to revert the tamoxifen-resistance phenotype. As such, these findings could have important translational significance for the prevention of tumor recurrence in ER(+) breast cancers, which is due to an endocrine resistance phenotype. Importantly, we also show here that NQO1 has significant prognostic value as a biomarker for the prediction of tumor recurrence. More specifically, higher levels of NQO1 mRNA strongly predict patient relapse in high-risk ER(+) breast cancer patients receiving endocrine therapy (mostly tamoxifen; H.R. > 2.15; p = 0.007).

Identification of novel ROS inducer by merging the fragments of piperlongumine and dicoumarol.

A series of novel ROS inducers were designed by merging the fragments of piperlongumine and dicoumarol. Most of these derivatives showed potent in vitro activity against three cancer cell lines and good selectivity towards normal lung cells. The most potent and selective compound 3e was proven to exhibit obvious ROS elevation and excellent in vivo antitumor activity with suppressed tumor growth by 48.46% at the dose of 5mg/kg. Supported by these investigation, these findings encourage further investigation around this interesting antitumor chemotype.

Dicoumarol: A Drug which Hits at Least Two Very Different Targets in Vitamin K Metabolism.

Dicoumarol, a symmetrical biscoumarin can be considered as the "parent" of the widely used anticoagulant drug, warfarin. The discovery of dicoumarol's bioactive properties resulted from an investigation into a mysterious cattle disease in the 1940s. It was then developed as a pharmaceutical, but was superseded in the 1950s by warfarin. Both dicoumarol and warfarin antagonise the blood clotting process through inhibition of vitamin K epoxide reductase (VKOR). This blocks the recycling of vitamin K and prevents the γ-carboxylation of glutamate residues in clotting factors. VKOR is an integral membrane protein and our understanding of the molecular mechanism of action of dicoumarol and warfarin is hampered by the lack of a three dimensional structure. There is consequent controversy about the membrane topology of VKOR, the location of the binding site for coumarin inhibitors and the mechanism of inhibition by these compounds. Dicoumarol (and warfarin) also inhibit a second enzyme, NAD(P)H quinone oxidoreductase 1 (NQO1). This soluble, cytoplasmic enzyme may also play a minor role in the recycling of vitamin K. However, its main cellular roles as an enzyme appear to be detoxification and the prevention of the build-up of reactive oxygen species. NQO1 is well characterised biochemically and structurally. Consequently, structure-based drug design has identified NQO1 inhibitors which have potential for the development of anti-cancer drugs. Many of these compounds are structurally related to dicoumarol and some have reduced "off target" effects. Therefore, it is possible that dicoumarol will become the "parent" of a second group of drugs.

Distinct responses of compartmentalized glutathione redox potentials to pharmacologic quinones targeting NQO1.

Deoxynyboquinone (DNQ), a potent novel quinone-based antineoplastic agent, selectively kills solid cancers with overexpressed cytosolic NAD(P)H:quinone oxidoreductase-1 (NQO1) via excessive ROS production. A genetically encoded redox-sensitive probe was used to monitor intraorganellar glutathione redox potentials (EGSH) as a direct indicator of cellular oxidative stress following chemotherapeutic administration. Beta-lapachone (ß-lap) and DNQ-induced spatiotemporal redox responses were monitored in human lung A549 and pancreatic MIA-PaCa-2 adenocarcinoma cells incubated with or without dicumarol and ES936, potent NQO1 inhibitors. Immediate oxidation of EGSH in both the cytosol and mitochondrial matrix was observed in response to DNQ and ß-lap. The DNQ-induced cytosolic oxidation was fully prevented with NQO1 inhibition, whereas mitochondrial oxidation in A549 was NQO1-independent in contrast to MIA-PaCa-2 cells. However, at pharmacologic concentrations of ß-lap both quinone-based substrates directly oxidized the redox probe, a possible sign of off-target reactivity with cellular thiols. Together, these data provide new evidence that DNQ's direct and discerning NQO1 substrate specificity underlies its pharmacologic potency, while ß-lap elicits off-target responses at its effective doses.

Novel high throughput pooled shRNA screening identifies NQO1 as a potential drug target for host directed therapy for tuberculosis.

UNLABELLED: Chemical regulation of macrophage function is one key strategy for developing host-directed adjuvant therapies for tuberculosis (TB). A critical step to develop these therapies is the identification and characterization of specific macrophage molecules and pathways with a high potential to serve as drug targets. Using a barcoded lentivirus-based pooled short-hairpin RNA (shRNA) library combined with next generation sequencing, we identified 205 silenced host genes highly enriched in mycobacteria-resistant macrophages. Twenty-one of these "hits" belonged to the oxidoreductase functional category. NAD(P)H: quinone oxidoreductase 1 (NQO1) was the top oxidoreductase "hit". NQO1 expression was increased after mycobacterial infection, and NQO1 knockdown increased macrophage differentiation, NF-κB activation, and the secretion of pro-inflammatory cytokines TNF-α and IL-1ß in response to infection. This suggests that mycobacteria hijacks NQO1 to down-regulate pro-inflammatory and anti-bacterial functions. The competitive inhibitor of NQO1 dicoumarol synergized with rifampin to promote intracellular killing of mycobacteria. Thus, NQO1 is a new host target in mycobacterial infection that could potentially be exploited to increase antibiotic efficacy in vivo. Our findings also suggest that pooled shRNA libraries could be valuable tools for genome-wide screening in the search for novel druggable host targets for adjunctive TB therapies.