NAD(P)H:Quinone Oxidoreductase 1 (NQO1) - American Chemical

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NAD(P)H:Quinone Oxidoreductase 1 (NQO1) as a Therapeutic and Diagnostic Target in Cancer Kuojun Zhang,† Dong Chen,† Kun Ma,‡ Xiaoxing Wu,† Haiping Hao,*,† and Sheng Jiang*,† †

J. Med. Chem. 2018.61:6983-7003. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 08/23/18. For personal use only.

State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 210009, China ‡ Center for Drug Evaluation, China Food and Drug Administration, Beijing 100038, China ABSTRACT: NAD(P)H:quinone oxidoreductase 1 (NQO1) is a two-electron reductase responsible for detoxification of quinones and also bioactivation of certain quinones. It is abnormally overexpressed in many tumors and intimately linked with multiple carcinogenic processes. NQO1 is considered to be a cancer-specific target for therapy but currently available NQO1 inhibitors have not yet led to chemotherapeutic success. Utilization of NOQ1’s ability to bioactivate chemotherapeutic quinones, however, has emerged as a promising selective anticancer therapy. On the basis of the different levels of NQO1 between cancer and normal cells, the catalytic property of NQO1 has recently been exploited to develop effective probes for cancer detection. This article summarizes the most significant advances concerning the discovery and development of NQO1 inhibitors, NQO1-directed chemotherapeutic quinones, and NQO1-activated optical probes, along with the prospects and potential obstacles in this research area.

1. INTRODUCTION Cancer remains one of the leading causes of death worldwide. According to the American Cancer Society, 1.6 million new cancer cases and 600920 cancer deaths were estimated to have occurred in the United States in 2017.1 Despite enormous efforts in past decades, conquering this devastating disease still poses huge challenges. One major hurdle is the difficulty of securing early diagnosis, thereby perhaps missing the best time for treatment. Nonselective chemotherapy has a central role in cancer therapy regimens and produces undesirable toxic effects and additional challenges, such as acquired chemoresistance and high rates of recurrence, which further diminish the odds of therapeutic success. Thus, early detection of cancer biomarkers and development of innovative therapeutics are urgently needed for effective cancer control and management. Targeting enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1, EC 1.6.99.2) may improve these aspects of cancer treatment. NQO1 was isolated in the late 1950s, characterized from the soluble fraction of rat liver homogenates by Ernster and Navazio,2 and named DT-diaphorase (DTD). In subsequent years, the functional importance of NQO1 has been an active area of research. It is well-known that NQO1 is a flavoenzyme capable of catalyzing obligatory two-electron reductions of a wide range of quinones to the hydroquinones in an NAD(P)Hdependent manner. Depending on the chemical reactivities of the hydroquinones formed, this reaction is either a detoxification or bioactivation process.3 Generation of stable hydroquinones by NQO1 is believed to be a detoxification mechanism because this process can avoid one-electron © 2018 American Chemical Society

reductions that can produce toxic semiquinone radicals and reactive oxygen species (ROS) and direct reactions with intracellular sulfhydryl groups.4,5 Rather than detoxifying, NQO1-directed reduction in some cases can bioactivate certain antitumor quinones into potent cytotoxic compounds through the formation of an unstable hydroquinone that can alkylate DNA or rapidly produce amounts of ROS via redox cycling.6 Moreover, NQO1 can reduce the drug-derived quinone-like species such as quinone-imine metabolites that are formed by cytochromes P450 (CYP), thereby playing an important role in relieving the adverse drug reactions (ADRs) and mediating a protective mechanism for detoxification.7,8 In addition to its enzymatic activity, NQO1 has been found to be involved in other biological processes such as antioxidant activity and the recently recognized stabilization of key regulatory proteins under stress.9 Interest in NQO1 has been largely sparked by the findings that it is expressed in many human solid tumors at levels of 5− 200-fold above that in normal tissue, such as breast,10−12 lung,13−15 prostate,16 stomach,17 colon,18 pancreatic19,20 and head and neck cancer,21 and cholangiocarcinoma22,23 among others.24−26 Further, its elevated activity is closely associated with tumor progression, aggressiveness, resistance to chemotherapy, and poor patient prognosis. Some effort has been made to explore small-molecule NQO1 inhibitors as an anticancer strategy but little success has been achieved. Received: January 23, 2018 Published: April 30, 2018 6983

DOI: 10.1021/acs.jmedchem.8b00124 J. Med. Chem. 2018, 61, 6983−7003

Journal of Medicinal Chemistry

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Figure 1. Structure of human NQO1 in complex with FAD (apoenzyme) (PDB 1D4A). (A) Overall structure of NQO1 apoenzyme. One monomer is shown in green and the other in orange. Carbon atoms of FAD are shown in purple. (B) One monomer of NQO1 apoenzyme. The catalytic domain is shown in purple and the C-terminal domain in blue. Carbon atoms of FAD are shown in green.

lower levels being detected in the Golgi complex, mitochondria, endoplasmic reticulum, cellular membrane, nucleus, mitotic spindle, and extracellular components.32,38,39 The X-ray crystallographic structures of rat, mouse, and human NQO1 alone, and of complexes in which these enzymes are bound to cofactors, inhibitors, and substrates, have been determined.40−44 Analysis of these crystal structures revealed high homology between samples from rat, mouse, or humans.41,42 The structures of the apo recombinant human NQO1 (hNQO1) containing only the FAD and the complex with the substrate duroquinone (2,3,5,6-tetramethyl-1,2-benzoquinone (1) are shown in Figures 1 and 2, respectively.

Considering the different levels of NQO1 in tumors compared to that in normal tissues, the compounds containing a quinone pharmacophore that are bioactivated by NQO1 should show remarkable tumor-selectivity, leaving normal cells intact. This constitutes an explicit example of “enzyme-directed bioreductive drug development” and has led to the development of NQO1-directed antitumor agents. To date, numerous NQO1 substrates have been reported and a few are in clinical development.6 For example, indolequinone apaziquone is now being evaluated in phase III trials as an intravesical therapeutic agent in the treatment of nonmuscle invasive bladder cancer (NMIBC).27 Such reports have aroused further interest in the development of more efficient NQO1 substrates as bioreductive drugs. The catalytic properties of NQO1 together with the fact that NQO1 is overexpressed early in the carcinogenic process has prompted the design of various NQO1-activated optical probes for cancer cell imaging. Such probes are invaluable for accurate and early cancer diagnosis and optimization of surgical and personalized chemotherapeutic treatments.28−31 Thus, NQO1 is an attractive target for selective anticancer therapy and accurate cancer detection. In this report, we review advances with NQO1 as an anticancer target. Potential obstacles and future directions in this research area are also presented.

2. AN OVERVIEW OF NQO1 NQO1 is considered to be a ubiquitous soluble enzyme present in nearly all animal species. With the development of detection technology, its distribution across tissues and localization in cells has been assessed in several animal species, including humans. NQO1 is distributed at various levels in almost all normal human tissues. In general, it is expressed at high levels in adipocytes, vascular endothelium, and epithelial cells and at lower levels in human hepatocytes and myocardia.32 Because NQO1 expression is both inducible and genetically determined,8,33,34 significant intra- and interindividual variability has been found in NQO1 levels. For example, despite low levels in normal hepatic tissues, NQO1 can be highly inducible in preneoplastic lesions and liver cancers.35,36 Several lines of evidence also suggest that some factors such as obesity and ethnicity may result in different hepatic NQO1 levels in different individuals.8,33,34,37 In terms of cellular localization, more than 90% of NQO1 is found to be in cytosol with much

Figure 2. Co-crystal structure of NQO1 apoenzyme with 1 bound to the active site (PDB 1DXO). (A) Binding surface of 1 to the active site. (B) Detailed binding mode of 1. (C) Chemical structure of 1. Carbon atoms of FAD are shown in purple and 1 in yellow. Hydrogen bonds are represented by green dashed lines.

hNQO1 is a ∼60 kDa protein consisting of two highly identical subunits intertwined antiparallel to one another, each consisting of 274 amino acids with one noncovalently bound FAD. Each subunit has two separate domains, in which residues 1−220 constitute a major catalytic domain and residues 221−273 are a small C-terminal domain. The catalytic domain consists a central parallel β-sheet surrounded by helices, while the Cterminal domain is composed of an antiparallel hairpin motif, an α-helix and several loops (Figure 1B). Each catalytic domain 6984



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Figure 3. NQO1’s biological role in cancer. NQO1 can provide normal cells with multiple layers of protection to resist carcinogenesis. On the other hand, NQO1 is overexpressed in cancer cells, where it promotes cell proliferation, malignant transformation, and drug resistance.

NQO1. NQO2 has a 54% and 49% identity with NQO1 in cDNA and protein sequence, respectively. NQO1 and NQO2 have significant structural similarity, particularly in the conserved features of the active sites observed in X-ray crystallographic studies.5,50 NQO2 is also able to reduce a quinone to hydroquinone, but it cannot use NAD(P)H as reducing cofactor unless the pH is substantially lower. Instead, NQO2 utilizes dihydronicotinamide riboside (NRH) to develop its enzymatic activity. Furthermore, in contrast to NQO1, which is overexpressed in solid tumors, NQO2 is highly expressed in red blood cells and in leukemias.51 In addition, typical NQO1 inhibitors such as dicoumarol (DIC, 2) and ES936 (6) are poor inhibitors of NQO2, while potent NQO2 inhibitors such as resveratrol and quercetin only weakly inhibit NQO1.5,8,50 Although NQO1 and NQO2 share overlapping substrate specificity, remarkable differences have been found in terms of affinities toward various substrates.52 Due to its biological significance, there is growing interest in NQO2, and a great deal of information about this quinone reductase is accumulating. Because this subject is outside of the scope of this review, a brief overview of NQO2 will suffice here.

contains three binding sites: (i) the FAD binding site, (ii) a hydrophilic adenine-ribose portion of a NAD(P)H binding site, and (iii) a hydride donor/acceptor binding site, which is a catalytic pocket that binds either the nicotinamide portion of NAD(P)H, the hydride donor, or a hydride acceptor substrate.5,44 The NQO1-catalyzed reaction proceeds via a ping-pong, enzyme-substituted mechanism in which NAD(P)H and the substrate alternately occupy the same binding site. It involves a double hydride transfer in which one hydride is transferred from NAD(P)H to N5 of the FAD and is followed by the release of NAD(P)+ from the catalytic site; the other hydride is transferred across a 4 Å distance45 from the N5 of FADH2 to the quinone substrate, and this is followed by the release of the hydroquinone formed from the catalytic binding pocket.41,42 The two independent but almost identical active sites formed by residues from each subunit are positioned at opposite ends of the dimer interface. The FAD moiety in the catalytic domain of one of the monomers forms one wall of the catalytic pocket.42,46 The major molecular interactions of duroquinone (1) with the enzyme are shown in Figure 2. Duroquinone binds to the NQO1 active site in a position similar to that adopted by the nicotinamide ring of NADP+ by interacting with FAD and several hydrophobic and hydrophilic residues. It aligns with the isoalloxazine ring of FAD, the distance between the two rings being 3.4 Å, and with five aromatic residues (Trp-105′, Phe106′, Phe-178, Tyr-126, and Tyr-128) and Gly-149′ and enjoys H-bond interactions with the hydroxyl groups of Tyr128 and Tyr-126.42 Together with the data from the cocrystal structures with other quinone substrates, it can be concluded that the active site of NQO1 is a flexible and changeable hydrophobic pocket with three potential hydrogen-bonding residues (i.e., Tyr-126, Tyr-128, and His-161) that can accommodate consecutive binding of NAD(P)H and ligands of different sizes and shapes, adopting a variety of binding modes and interactions.42,44 Such high plasticity may provide significant advantages in the design of NQO1-directed antitumor agents by tolerating different sizes and diversified chemical properties. However, caution needs to be exercised because quinone substrates can bind to the active site in more than one orientation, and analogue compounds with different substituents may bind to the enzyme in different orientations. NQO1 crystal structure-based molecular modeling studies have been applied in structure-based design efforts for the optimization of several groups of inhibitors and substrates.45,47−49 An isoenzyme of NQO1 is NRH:quinone oxidoreductase 2 (NQO2) that, at its C-terminus, is 43 residues shorter than

3. ROLE OF NQO1 IN CANCER A great deal of evidence has been published suggesting a “Janus” effect of NQO1 in cancer biology, where it behaves as either a tumor suppressor or a tumor promotor (Figure 3). NQO1 is constitutively expressed at relatively low levels in various normal tissues under physiological conditions. Under oxidative or electrophilic stress, it can be transcriptionally induced in concert with a battery of defensive genes under the control of NF-E2 p45-related factor 2 (Nrf2)/Kelch-like ECHassociated protein 1(Keap1)/− antioxidant response element (ARE) signaling pathway, providing cells with multiple layers of protection against carcinogenesis. These include detoxification of quinones, instant scavenging of superoxides, maintenance of lipid-soluble antioxidants in their reduced forms, and serving as a gatekeeper of the 20S proteasome which can stabilize key regulatory proteins.53 Cancer chemoprevention by dietary phytochemicals that can induce the expression of NQO1 has been considered as a practical and economic approach to cancer control and management. However, NQO1 has been found to be abnormally up-regulated in numerous human solid tumors and high levels of NQO1 correlate with poorer patient prognosis. Moreover, NQO1 appears to be overexpressed very early in carcinogenesis, even in precancerous lesions, and it is a potentially useful clinical cancer diagnostic biomarker.54 Cancer cells are known to have an apparent increase in their ROS 6985



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Figure 4. Chemical structures of the most studied NQO1 inhibitors.

also correlated with the level of NQO1, and these results were further validated in NQO1-null mice. In addition, it has been demonstrated that NQO1 can act as a chaperone, preventing the proteasome-mediated degradation of hypoxia inducible factor α (HIF-1α) and indicating that its tumor-promoting effects are in part associated with HIF-1α.60 Indeed, RNAimediated knockdown of NQO1 has been found to suppress HIF-1α signaling and tumor growth of human colorectal and breast cancer cells. Madajewski and co-workers have determined that NQO1 is critical for tumor growth at both primary and secondary nonsmall cell lung cancer sites because depletion of NQO1 expression in vitro causes an increase in ROS formation, inhibits anchorage-independent growth, increases anoikis sensitization, and decreases cell invasion.55 This result was confirmed in a lung tumor xenograft model. It is important to note that NQO1 overexpression is linked to cancer stem cell phenotypes, as depletion of NQO1 significantly diminishes the proportion of aldehyde dehydrogenase (ALDH)high cancer cells within the tumor population. In cholangiocarcinoma (CCA), a rare type of liver cancer, inhibition of NQO1 with DIC (2), a potent NQO1 inhibitor, was reported by Prawan et al.61 to markedly potentiate the cytotoxicity of gemcitabine to CCA. Further, RNAi knockdown and NQO1 knock-in cell lines were used to validate the role of NQO1 in chemotherapy resistance.62 The results shown that knockdown of NQO1 sensitizes CCA to an array of commonly used chemotherapeutic agents, including 5-fluorouracil (5-FU), doxorubicin, and gemcitabine, while overexpressed NQO1 on the other hand, protects CCA cells from these chemotherapeutic agents, indicating the possibility for combination therapy using wide-ranging traditional cytotoxic agents.62 Inhibition of NQO1 by RNAi significantly inhibits the colony formation capacity of CCA and arrested cells at the G1 phase, causing inhibition of cell proliferation by up-regulation of p21 and down-regulation of cyclin D1.63 Significantly, knockdown of NQO1 also suppresses the metastasis of CCA by upregulation of tissue inhibitors of metalloproteinases 1 (TIMP1) and down-regulation of matrix metalloproteinase 9 (MMP9).63

production relative to normal cells. In this context, high levels of NQO1 in cancers are postulated to assist cancer cells in dealing with elevated oxidative stress, as they do in normal cells.55 This hypothesis has been confirmed, to some extent, from studies of Nrf2, the transcription factor controlling NQO1 expression. In many cancers, including cancers of the lung, liver, gall bladder, pancreas, and ovarian, Nrf2 is constantly overactivated by gain-of-function mutations of itself or lossof-function mutations of Keap1.56,57 Hyperactivation of Nrf2 helps transformed or malignant cells to evade extreme oxidative stress through the induction of cytoprotective genes, e.g., the overexpression of NQO1.56 The mechanism by which NQO1 is overexpressed through Nrf2 may vary among different types of solid tumors. For example, mutations of Keap1 in gall bladder cancer lead to the decreased binding with Nrf2 and drive the expression of NQO1,56,57 while in pancreatic cancer, K-ras mutations promote the dimerization of c-Jun with Nrf2 independently of Keap1 to drive NQO1 induction.58 Besides, Nrf2-induced overexpression of NQO1 may play a role in the disappointing outcomes of chemotherapy, where it may be an adaptive response to oxidative stress and cytotoxicity and may confer protective effects on cancer cells.56,57 In this regard, inhibition of NQO1 would prevent cancer cell growth and malignant transformation as such inhibition could result in toxic oxidative stress levels. The crosstalk between NQO1 and signaling networks integrating their activity ultimately determines the fate of cells. Li et al.24 demonstrated that NQO1 was up-regulated with respect to melanocytes in most melanoma cell lines. This overexpression significantly induces cell cycle progression by up-regulation of cyclins A2, B1, and D1. Furthermore, it was found that NQO1 binds to and stabilizes trans-activator BCL3, which in turn up-regulates NF-κB p50, promoting the proliferation of melanoma cells. A study by Ahn et al.59 reported that NQO1 was required for the tumor necrosis factor (TNF) and other inflammatory stimuli-induced activation of IKK, NF-κB, JNK, p38 MAPK, p44/p42 MAPK, and Akt. Moreover, TNF-induced expressions of NF-κB-targeted gene implicated in cell proliferation, antiapoptosis, and invasion were 6986



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Thus, NQO1 plays an important role in cancer cell survival, proliferation, invasion, metastasis, and angiogenesis, as well as chemosensitivity, via interaction with various oncogenic signaling pathways. There are two common single nucleotide polymorphisms (SNPs) in NQO1 with defined phenotypes and population frequencies. These are NQO1*2 (ID, rs180056; NQO1 609C>T; NQO1 P187S; allelic frequency, 0.22−0.47) and NQO1*3 (ID, rs1131341; NQO1 465C>T; NQO1 R139W; allelic frequency, 0.00−0.05).64 NQO1*2 is known to have a profound effect on cellular NQO1 activity by highly decreasing its stability, as the mutant form of protein is rapidly ubiquitinated and degraded by the proteasome.65 Individuals homozygous for the NQO1*2 polymorphism have negligible NQO1 enzymatic activity, whereas individuals genotyped as heterozygous have decreased levels of NQO1 activity. While many recently performed meta-analyses have suggested a correlation between NQO1*2 polymorphism and increased cancer risk, other studies question its significance.66 Individuals with this polymorphism who have cancer would obviously not benefit from a therapy that targets overexpression of NQO1. NQO1*3 polymorphism is rarely found in homozygosis, but such polymorphism in heterozygosis has been shown to be correlated with increased risk of pediatric acute lymphoblastic leukemia.67 The phenotypical implications of NQO1*3 vary according to the substrate, showing native activity using dichlorophenol indophenols as substrates but reduced activity toward mitomycin C (MMC, 14).

Figure 5. Co-crystal structure of NQO1 apoenzyme with 2 bound to the active site (PDB 2F1O). (A) Binding surface of 2 to the active site. (B) Detailed binding mode of 2. Carbon atoms of FAD are shown in purple and 2 in yellow. Hydrogen bonds are represented by green dashed lines.

of pancreatic cancer cells. However, substantial evidence reveals that DIC’s growth inhibitory effects on pancreatic cancer cells were due to other properties of DIC, such as mitochondrial uncoupling, rather than inhibition of NQO1.68,69 The lack of selectivity of 2 has prompted searches for structurally novel NQO1 inhibitors. Virtual screening of the National Cancer Institute (NCI) database identified 3 as a potent competitive NQO1 inhibitor. This compound displays potency comparable to that of 2 and, more importantly, has “off-target” effects remarkably reduced from those of 2.68 Subsequently, a wide investigation of inhibitors structurally related to 2 led to the discovery of several NQO1 inhibitors that are endowed with equal or greater potency and improved selectivity toward NQO1. These include the symmetric analogues such as 4 and asymmetric analogues such as 5.69 Unfortunately, it was finally concluded that the growth inhibitory effects of newly synthesized coumarin-based NQO1 inhibitors, similar to 2, were independent of the extent of NQO1 inhibition. This fact did not favor NQO1 as a druggable target. Nevertheless, the improved selectivity profiles of these compounds allow them to serve as pharmacological probes with which to study other biological functions of NQO1, particularly the ability to regulate oncoprotein stability. 4.2. Irreversible Inhibitors. As described above, a number of well-established “‘off-target”’ effects limit the use of compound 2 in exploration of NQO1 function. The nature of competitive inhibition and ping-pong catalytic mechanism of NQO1 associated with other DIC derivatives also impedes their use in the evaluation of the activating or detoxifying properties of NQO1. To overcome these hurdles, an alternative approach was adopted to develop mechanism-based (suicide) inhibitors exemplified by indolequinone ES936 (6), whose Ki = 0.45 ± 0.7 μM and kinact = 0.78 ± 0.12 min−1.70 The partition ratio (the number of inhibitor molecules released from the active site in relation to the number which remain to inactivate the

4. NQO1 INHIBITORS Preliminary validation of NQO1 as a therapeutic target has been achieved mainly through RNAi knockdown experiments. RNAi-based studies, however, do not necessarily recapitulate the effects observed with small molecule inhibitors. This has led to interest in the development of NQO1 inhibitors, but studies with currently available NQO1 inhibitors show no correlation between NQO1 inhibition and anticancer activity. Further study is still required to determine if the inhibition of NQO1 by small molecules can be leveraged for therapeutic applications. Some inhibitors exhibit excellent anticancer activity, especially in combination with other chemotherapeutics, and in addition, efficient and selective NQO1 inhibitors can serve as tools with which to study the biology of NQO1 beyond RNAi knockdown experiments. Figure 4 summarizes the most studied NQO1 inhibitors, which are generally classified into two types based on the mechanism of action, namely competitive (reversible) inhibitors and mechanism-based (irreversible) inhibitors. 4.1. Typical Competitive Inhibitors. A range of structurally diverse NQO1 competitive inhibitors have been reported. These compete with NAD(P)H for binding to NQO1, thus preventing the hydride transfer to FAD. Among known inhibitors, DIC (2), an anticoagulant isolated from sweet clover (Melilotusalba), is the most potent competitive inhibitor with Ki = 1−10 nM.43 The cocrystal structure reported by Asher et al.43 showed that 2 binds to the active site through a series of hydrophobic and hydrogen bonds with residues of enzyme and the cofactor FAD. One coumarin ring of 2 lies parallel to the isoalloxazine ring of the FAD and forms two hydrogen bonds, between O-5 of DIC and Tyr 128 and between O-17 and His 161′ (Figure 5). DIC has been frequently used as a pharmacological tool with which to study the cellular function of NQO1, and considerable research has focused on the ability of 2 to inhibit the malignant phenotype 6987



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(SAR) explored, no correlation was observed between the two variants in identical cancer cell lines. In fact, some analogues that are potent inhibitors of NQO1 are relatively ineffective inhibitors of cell proliferation, whereas those compounds that are poor inhibitors of NQO1 exhibit robust growth inhibitory effects.71,73 This suggests that other targets apart from NQO1, such as thioredoxin reductase need to be considered in order to understand the potent efficiency of this series of indolequinones in human pancreatic cancer cells.74 In addition to the indolequinones, 7-aminoquinoline-5,8dione antibiotics (7, 8) have been identified as NQO1 inhibitors. As with ES936 (6), 7 and 8 may irreversibly inhibit NQO1 activity due to the presence of effective leaving groups at the (quinoline-3-yl)methyl position that can be eliminated upon reduction of the quinone moiety by NQO1 followed by the formation of a iminium intermediate.75 4.3. New Scaffolds of Competitive Inhibitors. In the case of DIC (2) and ES936 (6), a better understanding of NQO1’s biological functions requires some new scaffolds for NQO1 inhibitors. Using compounds 2 and 6 for reference, Bian et al.76 recently identified compounds 9−11 with different chemical scaffolds as NQO1 inhibitors with IC50 values of 0.92, 2.04, and 1.47 μM, respectively, through a shape-based similarity search and a second cascade docking filtering. By comparing the docked pose of 9 within the NQO1 active site to that of 2, it was found that the general molecular orientation and the spatial location of 2 and 9 are very similar, and the modifications of fragments A and C in 9 possibly increased its affinity toward NQO1.49 Accordingly, SAR studies of 9 led to the discovery of several potent NQO1 inhibitors. In particular, 12 and 13 show more potent activity than 9 with inhibition rates of 89.2%, 99.6%, and 81.2% at 10 μM, respectively. Due to the superior aqueous solubility than that of 13, compound 12 was selected for the design of an affinity-based probe (see section 6.2, below).

enzyme) is a more significant parameter describing the mechanism-based inhibitor. The partition ratio for ES936 is 1.40 ± 0.03, suggesting that ES936 is a potent inhibitor of NQO1.70 Compound 6 was discovered serendipitously during the process of characterization of NQO1 substrates in the indolequinone series. In the mechanism of action, this compound irreversibly alkylates either Tyr126 or Tyr128 within the NQO1 active site after it undergoes reduction by NQO1 followed by loss of the 4-nitrophenoxy group, generating a reactive iminium intermediate (Figure 6).71 The

Figure 6. Proposed mechanism for irreversible inhibition of NQO1 by ES936.

plane of ES936 is parallel to the isoallozaxine ring of the FAD. Together, the 1- and 2-methyl and the bulky group at the indole 3-position support its deep insertion into the catalytic site. Enzyme−ES936 interactions are mostly hydrophobic contacts, with only one hydrogen bond formed between the O-7 of ES396 and Tyr 127′ (Figure 7).70 Compound 6 shows potent NQO1 inhibition and a growth inhibitory effect in the low nanomolar range in the human pancreatic cancer cell line MIA PaCa-2, implying a positive correlation between inhibition of NQO1 activity and anticancer activity.72 However, when a series of ES936-based indolequinones was synthesized and their structure−activity relationships

5. BIOREDUCTIVE SUBSTRATES FOR NQO1 Hydroquinones generated from the NQO1-mediated twoelectron reduction of quinones are much more stable and readily conjugate with endogenous ligands, such as GSH and glucuronic acid, thereby facilitating their elimination. However, hydroquinones are not necessarily an innocuous species and in some cases can induce cancer cell death by any of three mechanisms: (i) chemical rearrangement leading to alkylation of DNA, (ii) oxidization back to parent quinones with production of significant amount of reactive oxygen species (ROS) which can directly cause DNA damage, and (iii) inhibition of heat shock protein (Hsp90) (Figure 8).77 In principle, NQO1-bioactivated anticancer agents can be classified into two types: (i) typical quinone substrates and (ii) quinone-containing prodrugs, where the quinone serves as the moiety for the targeted delivery of cytotoxic agents to prescribed sites. 5.1. Typical Quinone Substrates. Numerous naturally occurring and synthetic quinone substrates have been reported and reviewed previously elsewhere.6,46 They are in different classes based on their structural skeletons and the most relevant compounds of each class are enumerated, followed by a summary of the mode of action, substrate specificity, and reported clinical data. On the basis of mechanism of action, they can be classified as DNA alkylators (Figure 9), protein inhibitors (Figure 10), or redox cyclers (Figure 11).

Figure 7. Cocrystal structure of NQO1 apoenzyme with 6 bound to the active site (PDB 1KBQ). (A) Binding surface of 6 to the active site. (B) Detailed binding mode of 6. Carbon atoms of FAD are shown in purple and 2 in yellow. Hydrogen bonds are represented by green dashed lines. 6988



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Figure 8. Detoxification and activation pathways of quinones upon reduction by NQO1. ROS = reactive oxygen species; Hsp90 = heat shock protein 90.

Figure 9. Chemical structures of NQO1-bioreductive agents that are converted into DNA alkylators upon reduction by NQO1.

5.1.1. Indolequinones. MMC (14), isolated from Streptomyces caespitosus and considered to be an archetypical bioreductive alkylating agent, was produced commercially in Japan in 1958 but not approved for clinical use in the USA until 1974.78 Its cytotoxic mechanism is initiated by enzymatic reduction of the quinone moiety, followed by generation of electrophilic centers at C1 and C10 that can alkylate DNA causing the damage.79 Although it is clear that NQO1 is capable of activating 14, the tumor response to 14 cannot be simply related to NQO1 levels, and many studies have failed to identify an association between the two variables.80 MMC has been shown to undergo activation by other enzymes, including NADPH:cytochrome P450 reductase (P-450R), xanthine oxidase (XO), NADPH:cytochrome b5 reductase (b-5R) and NAD(P)H:quinone oxidoreductase 2 (NQO2). This, together with the fact that the reduction of 14 by NQO1 is pHdependent, suggests that 14 is a poor substrate for NQO1.81 Despite a wide antitumor scope with effectiveness in many solid tumors, including gastric, pancreatic, breast, bladder, cervical, and bladder, the clinical application of 14 has been limited by its rapid clearance and cumulative and unpredictable toxic effects. Systemic use of 14 has decreased in the past decades, with the exception of its treatment of anal squamous cell carcinoma in combination with radiotherapy and 5-FU.82

Because 14 is generally not associated with multidrug resistance, extensive efforts in recent years have been focused on reducing its toxicity. In particular, a lipid-based prodrug of 14 (MLP) was prepared and formulated in long-circulating pegylated liposomes (PL-MLP, Promitil). Notably, this formulation demonstrates increased efficacy, improved pharmacokinetic profiles, and reduced toxicity in various tumor types when compared to the parent compound (14) and has recently entered into phase I study for advanced solid tumor patients (NCT01705002).83 Apaziquone (15), originally known as EO9, was developed in the mid-1980s in a project sponsored by the Dutch Cancer Society (KWF Kankerbestrijding) at the University of Amsterdam.84 It is a simpler synthetic analogue of 14 with an aziridine directly attached to the indolequinone. Although 15 is structurally related to 14, it exhibits better antitumor activity than 14 and no significant bone marrow toxicity was detected in animal toxicology studies.85 The mechanism of 15 has been extensively studied and is believed to involve three main pathways: (i) generation of ROS via redox cycling to cause DNA strand breaks following the reduction of quinone moiety of apaziquone by one-electron reductases, (ii) alkylation of DNA by electrophilic centers at the (indol-3-yl)methyl and the (indol-2-yl)methyl upon the 6989



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by NQO1 both in vitro and in vivo.97 Preclinical studies demonstrated significant efficacy of RH1 in breast, colorectal, ovarian, and nonsmall cell lung cancer, Ewing’s sarcoma, and osteosarcoma xenograft models.98,99 Compound 20, formulated in 20% cyclodextrin, was selected by Cancer Research UK (CRUK) and NCI (NCT00558727) for a dose-escalation phase I clinical study in patients with advanced solid tumors, and it was found to be well tolerated with a maximum tolerated dose (MTD) of 1430 μg/m2/day.100 However, the paired tumor biopsy data show that DNA cross-linking can occur in the presence of relatively low levels of NQO1 activity and, conversely, that higher tumor NQO1 activity can be associated with relatively lower tumor DNA cross-linking.100 It is plausible that RH1 activation in vivo occurs at nontumor sites or that RH1 can be activated by non-NQO1 mechanisms such as p450R101 and NQO2.102 5.1.3. Benzoquinones Ansamycins. Heat shock protein 90 (Hsp90) is a molecular chaperone responsible for the ATPdependent folding, maturation, stability, and function of a number of client proteins implicated in cancer progression. As a consequence, Hsp90 is an attractive drug target for cancer therapy because its inhibition has the potential for simultaneous interference of multiple oncogenic signaling proteins (Figure 10).103 Benzoquinone ansamycins including the quinone-

reduction of quinone moiety by one-electron or two-electron reductases, and (iii) alkylation of DNA by opening of the aziridine ring after protonation.27 Under aerobic conditions, NQO1-rich tumors are sensitive to 15 and a good correlation exists between NQO1 level and tumor sensitivity. However, under hypoxic conditions, one-electron reductases such as P450R play a prominent role and tumors with low NQO1 level are sensitive to 15. Compound 15 is therefore a unique bioreductive agent because it is able to target either the aerobic fraction of NQO1-rich tumors or the hypoxic fraction of NQO1-deficient tumors. Significantly, the capacity of 15 to target hypoxic cells suggests its potential utility in combination with radiotherapy.86 This compound has a chequered history progressing from the clinic to the laboratory and back again.27 Due to its outstanding antitumor profile and ability to avoid myelosuppression, 15 soon proceeded into clinical evaluation, but it was found that intravenous administration of 15 for advanced breast, gastric, pancreatic, colorectal, and nonsmall lung cancers in phase II studies failed to generate the desired response.87,88 This was ascribed to poor delivery to tumors caused by short half-life in circulation and poor penetration through avascular tissue. While these properties represent a serious problem in the treatment of systemic disease, there are, paradoxically, significant advantages in local therapies required for nonmuscle invasive bladder cancer (NMIBC). EOquin, a new formulation of 15 specially developed for intravesical administration, has entered clinical studies. Significant activity against NMIBC and no systemic side effects are observed when 15 is administered intravesically to marker lesions in multiple phase I/II studies.89−91 Spectrum Pharmaceuticals Inc. has recently completed two phase III clinical trials (NCT00461591 and NCT00598806) with 15.92,93 These two trials did not reach statistically significant goals in the two-year recurrence rate between treatment and placebo arms when analyzed individually. When the results of the two trials were combined, however,92,93 statistical significance was obtained in both the two-year recurrence rate and time to recurrence. Posthoc analysis of the pooled data exhibits significant efficacy of 15, especially when the administration of 15 is completed 30 min or more after surgery.92 The FDA’s Oncologic Drugs Advisory Committee questioned the validity of this statistical analysis and requested a further phase III trial to test the hypotheses arising from the completed studies.27 5.1.2. Diaziridinylbenzoquinones. Aziridine-substituted benzoquinones were among the earliest quinone-containing alkylating agents evaluated as potential anticancer drugs. The early representative compounds containing aziridine include triaziquone (16), carbazilquinone (17), and diaziquone (AZQ, 18) that are NQO1 substrates and have been clinically evaluated. However, their clinical use was precluded for various reasons, including severe toxic side effects, undesirable pharmacokinetic properties, and lack of efficacy superior to that of existing chemotherapeutic agents.94 Studies of a large number of AZQ analogues led to the identification of MeDZQ (19), which has greater cytotoxicity to NQO1-rich tumors and a faster reduction rate by NQO1 than AZQ (18).95 However, 19 has not been clinically evaluated due to its low solubility. A more soluble diaziridinylbenzoquinone, RH1 (20), is rapidly taken up by cancer cells and induces preferential DNA cross-linking, cell cycle disturbance, and apoptosis in an NQO1-dependent manner.96 A study conducted using established isogenic cell line pairs demonstrated that RH1 can be effectively bioactivated

Figure 10. Chemical structures of NQO1-bioreductive agents that are converted into Hsp90 inhibitors more potent than the parent compounds upon reduction by NQO1.

containing polyketide antibiotics geldanamycin (GA, 21), 17(allylamino)-17-demethoxygeldanamycin (17-AAG, 22), and 17-(dimethyl-aminoethylamino)-17-demethoxygeldanamycin (17-DMAG, 23), represent the first-generation of Hsp90 inhibitors. This type of Hsp90 inhibitor competitively binds to the N-terminal ATP binding pocket of Hsp90, abolishing the ATPase activity, leading to the destabilization of oncogenic clients and the inhibition of tumor growth. As reported, the hydroquinone forms of benzoquinone ansamycins, formed by the metabolism of NQO1, are relatively stable and are more potent Hsp90 inhibitors than the parent compounds.104−107 Molecular modeling studies suggest that a number of hydrogen bond interactions are formed between the hydroquinone and the key residues within the ATP binding pocket of Hsp90, resulting in enhanced binding affinity for Hsp90 compared to the parent quinones.104,105 The benzoquinone ansamycins can also be reduced by one-electron reductases and can interact with glutathione (GSH). These reactions are correlated with toxicity, especially liver toxicity, because the liver contains high levels of one-electron reductases.108,109 6990



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Figure 11. Chemical structures of NQO1-bioreductive agents that are converted into redox cyclers upon reduction by NQO1.

Compound 21 was first isolated from the mycelia and broth of Streptomyces geldanus in 1970 and is a prototypical benzoquinone ansamycin. This Hsp90 inhibitor acted as an important chemical probe with which to validate the druggability of Hsp90, with potent and broad anticancer properties. Despite its promise in preclinical studies, its use in clinical settings was terminated because of unacceptable hepatotoxicity.110 17-AAG (tanespimycin, KOS953, 22) shows good activity against a wide range of solid and hematologic malignancies in preclinical models and is the first Hsp90 inhibitor to enter clinical evaluation. It displays a decreased hepatotoxicity that is mediated by one-electron reductases and lower reactivity than 21 toward GSH at the C-19 position of the ansa ring.109 Compound 22 has completed multiple phase I/II clinical studies for various malignancies as a monotherapy and a combination therapy. The phase III clinical studies for patients with relapsed and refractory multiple myeloma (NCT00546780 and NCT00514371) have been reviewed elsewhere.111 However, 22 suffers from P-glycoprotein mediated multidrug resistance and formulation problems stemming from poor solubility and low bioavailability.111 In an attempt to simplify formulation, further efforts were undertaken by the NCI and led to the identification of 17DMAG (alvespimycin, KOS-1022, 23). Compound 23 has superior preclinical activities and displays reduced metabolic liability on NQO1 and improved formulation and pharmacokinetic properties.112 Significantly, this molecule is orally available, facilitating administration and probably increasing patient compliance during treatment. Thus, 23 has been

investigated in multiple phase I clinical trials for many malignancies, as well as in phase II studies for patients with advanced leukemia.111 However, researchers at Kosan Biosciences discontinued the development of 23 due to its toxicity profile. Infinity Pharmaceuticals, Inc., has published two more soluble GM analogues, IPI-504 (retaspimycin hydrochloride, 24) and IPI-493 (25). Compound 24 is a highly soluble hydroquinone hydrochloride derivative of 17-AAG (22), and 22 and 24 can rapidly interconvert under redox conditions.107 There is evidence that NQO1 does not determine the activity of 24.113 A phase III study of 24 for the treatment of nonsmall cell lung cancer and other solid tumors is currently in progress.114 Compound 25, a metabolite of 17-AAG (22), is less sensitive and dependent on NQO1. Phase I clinical trials of 25 in patients for solid tumors were initiated in 2009,115 but no further developments have been reported. 5.1.4. Quinolinequinones. Streptonigrin (STN, 26) is a naturally occurring quinolinequinone antibiotic that was first isolated from the broth of Streptomyces flocculus by Rao and Cullen in 1959.116 This is a moderate NQO1 substrate, causing DNA and chromosome damage via a redox cycling process.47 Structurally, the 5,8-quinolinedione moiety in 26 is essential for its cytotoxicity and can be reduced by NQO1117 and/or oneelectron reductases118 to a hydroquinone or semiquinone intermediate, respectively. Either form spontaneously reverts back to the parent compound in the presence of molecular oxygen accompanied by generation of superoxide anions (O2•−), which can be converted to hydrogen peroxide (H2O2). Hydrogen peroxide can be reduced to hydroxyl radical 6991



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phosphorylation (OXPHOS),136,137,141 which is independent of p53, caspase, cell cycle status, oncogenic driver, and pro- or antiapoptotic proteins, implying great potential for circumvention of drug resistance.130,131,136,137,141,142 Few drugs can harness such cell death responses in a tumor-specific manner at clinically achievable doses, while cancer cells with >100 units of NQO1 catalytic activity are extremely sensitive to 31 with normal tissues intact.143 31 takes advantage of the elevated NQO1:CAT ratios to elicit selective killing toward many solid tumors, especially refractory pancreatic, prostate, breast, and nonsmall cell lung cancer.15,16,130,132,137,141 Importantly, in drug-resistant triplenegative breast cancers, Cao et al.130 found that 31-mediated bystander effects could accomplish higher efficacy via killing both NQO1-rich and adjacent heterogeneous NQO1-deficient tumors, while normal tissues are protected by their low NQO1:CAT ratio. On the other hand, 31 can also induce NQO1-independent cytotoxicity in NQO1-null or NQO1-rich cancer cells treated with DIC (2) at higher concentrations and with prolonged exposure. This may be related to the elevated oxidative stress arising from one-electron reduction by other enzymes, such as P-450R and b-5R,140 or from nonenzymatic catalyzed two-electron reduction.144 Despite its therapeutic promise, the (pre)clinical evaluation of 31 has been largely hampered by its poor solubility (0.038 mg/mL), short half-life in vivo (blood t1/2 = 24 min), and narrow therapeutic window resulting from methemoglobinemia.145−147 In an effort to overcome these undesirable properties, active research has been directed to the prodrug strategy combined with specific types of delivery systems.148 In the design of β-lap prodrugs, a Schiff’s base leaving group was engineered, leading to a series of nontoxic and inactive mono(arylimino) derivatives of β-lap such as 34, which undergoes hydrolysis to release active β-lap under acidic conditions.149 The hydroquinone analogue of 31 complexed with hydroxypropyl β-cyclodextrin (HPβ-CD), i.e., the clinical forms ARQ501 and ARQ761, have entered into multiple phase I/II clinical trials as monotherapies for patients with various cancer types and also in combination with other cytotoxic drugs.150−153 While increased aqueous solubility and tumor response were observed, these studies were limited due to hemolytic anemia and rapid clearance caused by the HPβ-CD carrier and the dose-limiting methemoglobinemia and nonspecific distribution. Further efforts have employed nanotherapeutic delivery vehicles, such as poly(ethylene glycol)block-poly(D, L -lactide) (PEG−PLA) polymer micelles,145,154,155 gold nanoparticles,156 polymer millirods,157 and superparamagnetic iron oxide nanoparticles.158 On the basis of the unique mechanism associated with 31, strategies were carried out combining radiotherapy15,16 or other anticancer drugs that act on cancer-specific NADPH-biogenesis pathways but lack tumor selectivity and adequate efficiency when serving as a single agent.138,159 These include various PARP inhibitors,137 base excision repair (BER) inhibitor methoxyamine, 135 nicotinamidephospho-ribosyltransferase (NAMPT) inhibitor FK866,136,160 and mitochondrial glutaminase 1 (GLS1) inhibitor CB839.161 These agents share a distinct but highly complementary mechanism of action with 31, affording a synergistic effect which reduces required doses and treatment times for both agents, enhances their efficacy, and expands the therapeutic window. Tan IIA (32) is a natural o-naphthoquinone occurring abundantly in the rhizome of the Chinese herb Salvia

(·OH) via a metal-catalyzed Fenton reaction. The hydroxyl radical is ultimately responsible for the DNA degradation. Compound 26 was studied in clinical trials in the 1960s and 1970s but was discontinued due to severe and prolonged myelosuppression.119 Lavendamycin (27), originally isolated in 1982 from Streptomyces lavendulae by Balitz et al.120 is structurally and biosynthetically similar to 26 (Figure 11). While 27 is not suitable for clinical development due to its low aqueous solubility and high degree of toxicity, its analogues with less toxicity and improved solubility should have potential as anticancer agents. Some of these, e.g., compounds 28−30, can be metabolized quickly by recombinant human NQO1 in vitro and show more toxicity toward the BE-NQ human colon adenocarcinoma cell lines that are transfected with NQO1 than to the BE-WT (NQO1-deficient) cell lines.45,48,121 5.1.5. Naphthoquinones. Structurally, the NQO1 substrates discussed above are all p-quinones. The known o-quinone substrates for NQO1 are limited to three natural naphthoquinones, β-lapachone (β-lap, 31), tanshinone IIA (Tan IIA, 32), and dunnione (33), and their synthetic analogues. β-Lapachone (31), a tetrahydropyran-fused o-naphthoquinone first isolated from the bark of the Lapacho tree (genus Tabebuia) native to South America, has received considerable attention due to its wide variety of pharmacological properties, which include anti-infective, anti-inflammatory, and particularly outstanding anticancer effects.122 The cytotoxicity of 31 was originally hypothesized to impede DNA damage repair due to inhibition of topoisomerases.123 It is now known that it is a specific NQO1 substrate, and many valuable models have been utilized to research its cytotoxic mechanism. In isogenic cell line pairs, isogenically matched NQO1-deficient cancer cells were resistant to 31, while NQO1-containing cells were sensitive to 31.122,124,125 Heat and radiation upregulate the NQO1 level, enhancing the anticancer activity of 31 in both cultured and xenografted tumors.126−128 Furthermore, lethality induced by 31 could be significantly inhibited by NQO1 inhibitor 2 or be limited by knockdown of NQO1 via siRNA. Together, the cytotoxicity of 31 is predominately determined by the metabolic activation of NQO1. 31 is reduced by NQO1 to the corresponding hydroquinone and rapidly autoxidized back to the parent quinone through the semiquinone intermediate, allowing for a futile redox cycle which consumes ∼60 mol equivalents of NAD(P)H and produces ∼120 mol equivalents of O2−•, which are rapidly transformed into H2O2 by superoxide dismutase (SOD).124,129 Most solid tumors have a higher NQO1 expression than surrounding normal tissues, particularly >85% of nonsmall cell lung cancer, have NQO1 at levels of 10−50-fold above normal tissues, ∼85% pancreatic cancer at levels of 10−100-fold, ∼60% prostate and breast cancer at levels of 5−100-fold, but with a low level of catalase (CAT), the reverse of normal tissues.15,16,130−132 As a consequence, the resulting H2O2 is long-lived and rapidly diffuses into the nucleus, leading to oxidative base and DNA single-strand breaks (SSB), intracellular Ca2+ increase,133,134 poly(ADP-ribose)polymerase-1 (PARP-1) hyperactivation, NAD+/ATP depletion, and ultimately cell death as a form of programmed necrosis, known as NAD+-Keresis.15,130,135−138 It is important to note that cell death mediated by 31 is driven by nuclear translocation of apoptosis-inducing factor (AIF),139 and activation of μcalpain,15,124,140 as well as inhibition of glyceraldehyde 3phosphate dehydrogenase (GAPDH)/glycolysis and oxidative 6992



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Figure 12. NQO1-activated prodrugs. The trigger group that can be activated by NQO1 is shown in red, the linker in blue, and the parent drug in green.

miltiorrhiza Bunge (Danshen). It exhibits significant cytotoxicity against a wide range of human cancers in a broad concentration range from submicromolar to high micromolar. Recently, it has been found to be a specific NQO1 substrate with a metabolic activation mechanism similar to that of 31 that initiates a p53independent but caspase-dependent mitochondrial apoptotic pathway relying on NQO1-mediated ROS production.162 You et al.163 first hybridized 32 and 31, generating the scaffold of 3-methylnaphtho[1,2-b]furan-4,5-dione. By analysis of the binding mode with NQO1, a series of L-shaped oquinone analogues derived from this hybrid scaffold have been prepared and could occupy the side binding pocket by interacting with residues Tyr128, Phe232, Phe236, and His194. Compound (35) was found to be an efficient substrate for NQO1 and to exert cytotoxicity through NQO1-driven ROS production with tumor selectivity and safety better than that of 31. Unfortunately, 35 is even less soluble than 31 (0.021 and 0.038 mg/mL, respectively). To identify more drug-like molecules, another new scaffold of naphtho[2,1-d]oxazole-4,5dione with improved solubility was produced by incorporating a variety of water-soluble side chains.164 The most soluble and promising compound in this series, 36, displays significant activity in NQO1-expressing cancer cells and in tumor xenograft mouse models.164 As mentioned above, the NQO1 specific activity of 31 is limited to a relatively narrow concentration range and exposure time, possibly because it is metabolized by one-electron reductases, especially P-450R.140 The same group yielded diversified β-lap-inspired scaffolds in an attempt to obtain novel quinone compounds and reduce the nonspecific ROS production by P-450R.165 A specific substrate (37) was identified, affording a much higher selectivity toward NQO1 versus P-450 than that of 31 with selectivity ratios of 6.37 and 1.36, respectively, indicating a superior safety profile. Docking analysis has suggested that aside from the π-stacking of the isoalloxazine moiety of FAD, other factors contribute to its excellent performance. These include hydrogen bonding interactions between two carbonyl groups and Tyr126 and

Tyr128 residues that were similar to hydrogen bonds in 31, and additional C−H···π interactions formed by the 7,8-dimethyl groups of 37 with Trp105 and Phe178 residues.165 An L-shape type of nitrogen-containing chain was attached to the C2 position of 37, resulting in the more effective and selective NQO1 substrate 38.166 The naturally occurring tetrahydrofuran fused o-naphthoquinone(±)-dunnione (33) has been recently identified by the same group as an efficient NQO1 substrate, and the mechanism of action of the cytotoxicity was found to involve NQO1-dependent ROS production.167 5.1.6. Diazaanthraquinones. Deoxynyboquinone (DNQ, 39) was originally synthesized in 1961 during a study of the natural antibiotic nybomycin and more recently was found to be a natural product present in a deep-sea Actinomycete Pseudonocardia sp. SCSIO 01299 in 2011.168 Compound 39 was found to possess potent antitumor activity in vitro and in vivo, which is dependent on quantities of toxic ROS produced by NQO1-catalyzed redox cycling.47,77 While DNQ 39 killed cancer cells by inducing a PARP-1 hyperactivation-mediated programmed necrosis pathway identical to that produced by 31, 39 is about 20−100-fold more potent than 31, with a dramatically expanded therapeutic window, longer in vivo half-life, and enhanced metabolic stability in intact hepatocytes in vitro, albeit with more severe dose-limiting methemaglobinemia than 31.169 According to the documented cocrystal structure, a good relationship exists between the NQO1 processing activity and the distance between the N5 of FAD and carbon α to the quinone carbonyl in the substrate, with compounds that have a shorter predicted “hydride donor−acceptor distance” in computational docking proving to be better NQO1 substrates.38 Guided by this rule, a diverse set of DNQ derivatives with a range of NQO1 processing activities were reported by Parkinson et al.47,77 Results show that the anticancer activity of these DNQ derivatives was strongly correlated with NQO1 processing ability. The compounds that behave as excellent NQO1 substrates are also potent cancer cell death inducers. In 6993



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Figure 13. NQO1-activated fluorescent probes. The trigger group is present in red, the linker in blue, and the dye in green.

reliable early cancer diagnosis, the collection of temporal and spatial information about tumor microenvironment, exploration of cellular machinery of the disease, and optimization of surgical and personalized chemotherapeutic treatments. Despite its great significance, it remains a challenging task. First, it is necessary to identify a cancer specific trait, a biomarker that is stable and dependable and not shared with normal cells. Second, development of rapid and highly sensitive reporter systems or probes is required to specifically monitor the trait. Enzyme NQO1 is such a biomarker, given the fact that it is upregulated in many solid tumors relative to the normal counterparts and its activity is strongly affected by the life cycle of cancer cells. To date, a number of NQO1 activity-based and affinity-based optical probes have been successfully developed for detection and cellular imaging of NQO1. 6.1. Activity-Based Probes. Enzyme-activated, or “turnon” probes can generate rapid, highly sensitive, and selective signals associated with cancer cells, and this type of probe has seen growing interest. In the case of NQO1, the explored probes are commonly composed of a quinone-based enzyme substrate as a trigger group and a dye as a reporter, two moieties directly bonded or bridged by a linker. The intense fluorescent reporter signature of these probes is unveiled upon the specific activation of the quinone moiety by NQO1, as described below (Figure 13). 6.1.1. Fluorescent Probes. The trimethyl-locked quinone propionic acid (Q3PA) has been extensively used as the trigger group because of its rapid and highly selective reduction by NQO1 and the subsequent facile intramolecular lactonization which releases the reporter group rapidly.38 Rhodamines have been a favored choice in fluorescent probe-based applications. McCarley et al.175 reported an activatable probe (45) consisting of a Q3PA trigger group and an N-morpholino-capped rhodamine, named MorphR110. The intense fluorescence of 45 is not revealed until the Q3PA is reduced by hNQO1, followed by the lactonize-and-release sequence. While 45 is very sensitive to the presence of purified hNQO1 in solution under physiological conditions, it fails to image NQO1-positive cancer cells. This is possibly due to the remarkable decrease in the fluorescence and absorbance of the reporter (MorphR110) upon its reaction with NADH. The same group discovered a seminaphthorhodamine compound (MJSNR) with excellent

the presence of DIC (2), the cell death induced by compounds that are better NQO1 substrates diminishes to a greater extent. In particular, the pharmacokinetic and toxicological data of isobutyldeoxy-nyboquinone (IB-DNQ, 40) in the domestic felid species are promising and support further development of IB-DNQ as an anticancer agent targeting NQO1-expressing cancers.170 Significantly, DNQs serving as model compounds clearly show that there is a strong correlation between NQO1 processing ability and anticancer activity. 5.2.2. Quinone-Containing Prodrugs. Indolequinone-based prodrugs have received considerable attention because they can avoid the toxic effects of the parent drugs and indolequinone moieties can efficiently detach parent drugs from the (indol-3yl)methyl position upon reduction by NQO1. The indolequinone structure itself can be converted to an electrophilic iminium intermediate, which can also induce cytotoxicity to cancer cells, thereby triggering a synergistic effect with the parent drug. Representative indolequinone-based prodrugs are shown in Figure 12. Camptothecin (CPT) attached at the C3 position of the indolequinone nucleus through an N,N′-dimethyl-1-aminoethylcarbamate linker affords the water-soluble prodrug 41.171 This CPT prodrug is an efficient substrate for NQO1 and exhibits much lower toxic effects and higher hypoxia selectivity than the parent drug. Molecule 42 with oridonin attached at the indolequinone nucleus via an ester linkage exhibits potent anticancer activity in NQO1-rich tumor cells and in a liver cancer xenograft mouse model.172 This induced NQO1dependent apoptosis proceeds through a ROS-triggered mitochondrial apoptotic pathway. An indolequinone-diazeniumdiolate (43) was reported to release NO upon metabolism by NQO1 and shows significant antiproliferative activity in NQO1-expressing cancer cells.173 Huang et al.174 reported a highly cancer-targeting prodrug (44) containing moieties of a therapeutic drug SN38 (7-ethyl-10- hydroxycamptothecin), an indolequinone structure, and an αvβ3 integrin-targeting cyclic peptide RGDyK. Compound 44 was found to target αvβ3 integrins, releasing the drug SN38 in the presence of NQO1.

6. NQO1 AND CANCER DETECTION Achieving highly selective and sensitive detection or visualization of cancer-associated events and targets is invaluable for 6994



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Figure 14. NQO1-activated theranostic prodrugs. The trigger group is present in red, the linker in blue, and the dye in green.

Q3PA and the linker self-cleavage with unprecedented, catalytically efficient formation of a fluorescent reporter. Significantly, although it requires a special camera,30,31 this probe provides high-fidelity detection and visualization of endogenous intracellular NQO1 activity in 2D or 3D cancer cell cultures and in a preclinical in vivo model of peritoneally disseminated ovarian cancer. Fei et al.178 synthesized another profluorophore (51) by conjugating Q3PA with a red fluorescent dye OH-BODIPY via a self-degrading linker, p-aminobenzyl alcohol. A polymeric micelle assembly was employed to encapsulate this profluorophore (51), leading to a nanoprobe with enhanced solubility, photostability, and biocompatibility. More importantly, this nanoprobe has been successfully utilized for discriminative imaging of NQO1-positive cells via favorable ratiometric measurement of NQO1 activity. 6.1.2. Theranostic Prodrug. Theranostic prodrugs, as the name suggests, possess the dual-function of diagnosis and therapy. This type of drug has emerged as an invaluable technology in biomedicine. An NQO1-activable theranostic prodrug (52) was constructed by Liu et al.179 The trigger group and fluorescence quencher Q3PA is conjugated via a selfcleavable linker p-aminobenzyl alcohol to an anticancer drug CPT that also serves as a fluorescent dye. Free CPT was released, and its fluorescence was recovered by dequenching from Q3PA. In this way, this prodrug can not only detect NQO1 activity and provide the feedback in real-time on quinone reduction and drug release but also selectively kill cancer cells with overexpressed NQO1. In an effort to further enhance the cancer-targeting property, Shin et al.180 disclosed a tumor-targeting enzyme-triggered theranostic prodrug (53) that is composed of three moieties, a therapeutic drug SN38, Q3PA, and a cancer-targeting unit, biotin. Upon preferential uptake by cancer cells with the aid of biotin, 53 is cleaved by NQO1 accompanied by the release of SN38, inducing cancer cell apoptosis (Figure 14). Simultaneously, the drug release and apoptosis of cancer cells expressing both biotin receptors and high levels of NQO1 was imaged and monitored via the native fluorescence of the free SN38. This type of enzyme-triggered targeted prodrug therapy is a promising approach for future cancer treatment. 6.2. Affinity-Based Probes. An affinity-based smallmolecule probe 54 was developed by linking the NQO1 inhibitor (12) as the recognition group and fluorescein isothiocyanate (FITC) with an aliphatic amine. This probe demonstrates good inhibitory activity against NQO1 and has been successfully exploited to label the enzyme in lung adenocarcinoma A549 cells that have robust expression of NQO1 at micromolar concentrations (Figure 15).49

photophysical properties and stability in the presence of NADH.176 They constructed the turn-on probe (46) in which MJSNR is attached to Q3PA. Although the positive-to-negative ratios (PNR) upon exposure to cancer cells is not high, 46 possesses some desirable characteristics, including rapid uptake and turn-on and high selectivity with respect to potential chemical or enzymatic interference.176 Such pioneering work has inspired extensive efforts for the development of a variety of NQO1-responsive, turn-on fluorescence probes. McCarley et al.28,177 developed two quenched probes (47, 48) based on naphthalimide, whose fluorescence is efficiently masked by photoinduced electron transfer (PeT) quenching arising from the covalently attached Q3PA, a quencher subunit. Upon activation of Q3PA and subsequent detachment, the reporter naphthalimide can be dequenched and exhibits highly intense and Stokes-shifted emission due to the push−pull internal charge-transfer (ICT) mechanism of the naphthalimide scaffold. The difference in chemical structural between the two probes lies only in the linker between Q3PA and fluorescent dye. The probe (48) contains a shorter electron-donating linker in contrast to the electron-drawing carbamate-based linking unit in probe 47. For probe 48, this produces a greater increase in brightness for reporter versus a probe that achieves a higher PNR value upon exposure to cancer cells and a more efficient NQO1 catalytic efficiency, which in turn results in a faster release of free reporter.177 More importantly, these two probes, especially probe 48, which generates a green energy-range reporter upon activation, can readily penetrate the membrane of human cancer cells and allow discrimination between NQO1-positive and NQO1-negative cancer cell lines by the naked eye without influence on cell viability in the longer-term cellular studies. This highlights the feasibility of the application to living-cell imaging.28,177 A self-eliminating linker N-methyl-p-aminobenzyl alcohol (NMPABA) was used by the McCarley group to generate a tripartite quenched fluorophore Q3PA-naphthalimide (49).29 Interestingly, reduced Q3PA is rapidly detached from the linker NMPABA, which subsequently undergoes degradation to release naphthalimide. This reporter possesses highly intense, red-shifted fluorescence emission, and NQO1positive cancer cells can be imaged and identified with a PNR value of 500:1. Near-infrared (NIR) probes are ideal tools for in vivo imaging as this type of probe offers deep tissue penetration and low background fluorescence. McCarley et al.30 reported an NIR triggerable probe (50) in which Q3PA was attached to tricarbocyanine (TCy) by a self-degrading linker, 2-mercaptoethanol, through a carbamate to the amine of TCy. The fluorescence of 50 is greatly increased after the activation of 6995



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therapeutic window. The combination of 31 with other drugs that target cancer-specific NADPH-biogenesis pathways have distinct but complementary mechanisms with 31 shows immense promise to enhance efficacy and expand the therapeutic window. For example, 31 (depletion of NAD+) and NAMPT inhibitor FK866 (inhibition of NAD+ production) together can induce a more remarkable depletion of NAD+, exerting a synergistic effect to induce cancer cell death. Encouragingly, DNQ (39) is an excellent NQO1 substrate and its anticancer activity is strictly dependent on the bioactivation of NQO1, indicating promise as a personalized anticancer therapy. 39 and its analogues have been used as model compounds to elucidate a key question regarding the relationship between the ability of substrates to be processed by NQO1 and their anticancer activity. This provides a design criterion for new NQO1-activated bioreductive agents. DNQ analogues like 40 are also excellent substrates for NQO1 with high efficacy, cancer selectivity, and desirable pharmacokinetic properties in in vivo models. These profiles make them attractive for translational development. The early detection of cancer is critical to successful treatment. Enzyme-activated probes have emerged as a promising tool for accurate early cancer detection. Currently, there have been a number of NQO1-activated fluorescence probes published, and some of them can effectively distinguish cancer cells from healthy cells. For example, the NIR probe (50) has achieved in vitro and in vivo imaging of NQO1 and is capable of sensitively detecting peritoneal ovarian cancer metastases in an animal model. Of great significance, theranostic prodrugs are highly desirable for both precise diagnosis and cancer treatment. NQO1-based theranostic prodrugs provide not only real-time monitoring of the reduction of quinone moiety and parent drug release but also elicit selective cytotoxicity toward cancer cells, two birds with one stone. We anticipate that more effective, sensitive, and targeted optical probes and theranostic prodrugs will be developed to improve cancer detection and treatment.

Figure 15. An affinity-based probe. The trigger group is present in red, the linker in blue and the dye in green.

7. CONCLUSION AND OUTLOOK A major goal in current cancer therapy is to improve selectivity for cancer cells over that of normal cells. One approach to achieve this is targeting a specific trait peculiar to cancer cells. Due to its well-documented overexpression in many human cancers together with its bioactivation of certain quinone substrates, NQO1 is considered as a desirable target for cancer therapy. To date, NQO1 inhibitors and chemotherapeutic quinones with different chemotypes are available for further exploration of NQO1 as a therapeutic target for cancer therapy. However, some critical issues remain to be resolved. While RNAi-mediated silencing of NQO1 has provided strong evidence to support the promotional effect of NQO1 in cancer development, studies with the currently available NQO1 inhibitors have been unsuccessful and translating this approach into clinical trials is currently problematic. It is unknown whether this is just due to the ineffectiveness of the NQO1 inhibitors utilized. To date, only a limited number of NQO1 inhibitors have been reported. Most of them suffer some limitations such as selectivity issues and the high concentrations required for inhibition. Many important aspects of NQO1 function in cancer are not completely understood. For example, why NQO1 is overexpressed in tumors and whether NQO1 is the main driver of the cancer are still questions. Therefore, there is an urgent need for the development of highly potent and selective NQO1 inhibitors in order to further probe the NQO1 biology and validate the druggability of NQO1, proceeding beyond RNAi knockdown studies. Personalized medicine means more efficient and effective disease diagnosis and treatment because this strategy enables the preselection of cancer patients based on molecular biomarkers and utilization of drugs whose therapeutic efficiency can be predicted. NQO1 is an ideal target in this regard, given its overexpression in many solid tumors coupled with existing diagnostic tests for NQO1 polymorphism and overexpression. However, the development of NQO1 substrates has encountered some challenges. A few of the bioreductive agents developed to date have been specific for a single enzyme, NQO1. Hence, the separate NQO1 level may not be a suitable indicator to predict clinical response to the therapy of these bioreductive agents. Again, most of the reported NQO1 substrates, such as MMC (14), apaziquone (15), AZQ (18), and RH1 (20), are DNA alkylators. Their clinical efficacy may be limited due to resistance originating in the DNA repair machinery. The head-to-head assays conducted in vitro and cultured cell systems convincingly demonstrate that only two classes of compounds, β-lap (31) and DNQ (39), induce cancer cell death through NQO1-mediated activation.47,77 While β-lap (31), a redox cycler, elicits tumor-selective programmed necrosis, it has modest potency in vitro and has other drawbacks such as poor solubility and a narrow

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AUTHOR INFORMATION

Corresponding Authors

*For S.J.: phone, 86-18688888237; E-mail, jiangsh9@gmail. com. *For J.H.: E-mail, [email protected]. ORCID

Haiping Hao: 0000-0002-5142-1134 Sheng Jiang: 0000-0002-4550-5024 Notes

The authors declare no competing financial interest. Biographies Kuojun Zhang received her Master’s degree at China Pharmaceutical University. Currently, she is pursuing her Ph.D. under the supervision of Professor Sheng Jiang. Her research topics are focused on the discovery of effective inhibitors of NQO1. Dong Chen received his Bachelor’s degree from China Pharmaceutical University and earned his Ph.D. in Medicinal Chemistry from Kunming Institute of Botany, Chinese Academy of Sciences. He is currently a member of the Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, and spends much of his time on synthesis of natural products. Kun Ma received his Bachelor’s degree from China Pharmaceutical University and earned his Master’s degree from Shenyang 6996



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push−pull internal charge transfer; NIR, near-infrared; FITC, fluorescein isothiocyanate

Pharmaceutical University. He is currently a member of the Centre for Drug Evaluation, China Food and Drug Administration.

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Xiaoxing Wu received his Ph.D. from Brandeis University in 2009 and continued his postdoctoral research at the Massachusetts Institute of Technology. After two years of working in academia and four years in top pharmaceutical companies, he joined in faculty at China Pharmaceutical University as a Principal Investigator in 2017. His current research interests are in the discovery of small-molecule drugs for treatment of metabolic diseases or immuno-oncology.

(1) Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer statistics, 2017. CaCancer J. Clin. 2017, 67, 7−30. (2) Eranster, L.; Navazio, F. Soluble diaphorase in animal tissues. Acta Chem. Scand. 1958, 12, 595−602. (3) Ross, D.; Kepa, J. K.; Winski, S. L.; Beall, H. D.; Anwar, A.; Siegel, D. NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms. Chem.-Biol. Interact. 2000, 129, 77−97. (4) Talalay, P.; Dinkova-Kostova, A. T. Role of nicotinamide quinone oxidoreductase 1 (NQO1) in protection against toxicity of electrophiles and reactive oxygen intermediates. Methods Enzymol. 2004, 382, 355−364. (5) Bianchet, M. A.; Erdemli, S. B.; Amzel, L. M. Structure, function, and mechanism of cytosolic quinone reductases. Vitam. Horm. 2008, 78, 63−84. (6) Siegel, D.; Yan, C.; Ross, D. NAD(P)H:quinone oxidoreductase 1 (NQO1) in the sensitivity and resistance to antitumor quinones. Biochem. Pharmacol. 2012, 83, 1033−1040. (7) Vredenburg, G.; Elias, N. S.; Venkataraman, H.; Hendriks, D. F.; Vermeulen, N. P.; Commandeur, J. N.; Vos, J. C. Human NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated inactivation of reactive quinoneimine metabolites of diclofenac and mefenamic acid. Chem. Res. Toxicol. 2014, 27, 576−586. (8) den Braver-Sewradj, S. P.; den Braver, M. W.; Toorneman, R. M.; van Leeuwen, S.; Zhang, Y.; Dekker, S. J.; Vermeulen, N. P. E.; Commandeur, J. N. M.; Vos, J. C. Reduction and scavenging of chemically reactive drug metabolites by NAD(P)H:quinone oxidoreductase 1 and NRH:quinone oxidoreductase 2 and variability in hepatic concentrations. Chem. Res. Toxicol. 2018, 31, 116−126. (9) Dinkova-Kostova, A. T.; Talalay, P. NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme and exceptionally versatile cytoprotector. Arch. Biochem. Biophys. 2010, 501, 116−123. (10) Yang, Y.; Zhang, Y.; Wu, Q.; Cui, X.; Lin, Z.; Liu, S.; Chen, L. Clinical implications of high NQO1 expression in breast cancers. J. Exp. Clin. Cancer Res. 2014, 33, 14−14. (11) Marin, A.; Lopez de Cerain, A.; Hamilton, E.; Lewis, A. D.; Martinez-Penuela, J. M.; Idoate, M. A.; Bello, J. DT-diaphorase and cytochrome B5 reductase in human lung and breast tumours. Br. J. Cancer 1997, 76, 923−929. (12) Bentle, M. S.; Reinicke, K. E.; Dong, Y.; Bey, E. A.; Boothman, D. A. Nonhomologous end joining is essential for cellular resistance to the novel antitumor agent, beta-lapachone. Cancer Res. 2007, 67, 6936−6945. (13) Cui, X.; Jin, T.; Wang, X.; Jin, G.; Li, Z.; Lin, L. NAD(P)H:quinone oxidoreductase-1 overexpression predicts poor prognosis in small cell lung cancer. Oncol. Rep. 2014, 32, 2589−2595. (14) Li, Z.; Zhang, Y.; Jin, T.; Men, J.; Lin, Z.; Qi, P.; Piao, Y.; Yan, G. NQO1 protein expression predicts poor prognosis of non-small cell lung cancers. BMC Cancer 2015, 15, 207. (15) Bey, E. A.; Bentle, M. S.; Reinicke, K. E.; Dong, Y.; Yang, C. R.; Girard, L.; Minna, J. D.; Bornmann, W. G.; Gao, J.; Boothman, D. A. An NQO1- and PARP-1-mediated cell death pathway induced in nonsmall-cell lung cancer cells by beta-lapachone. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 11832−11837. (16) Dong, Y.; Bey, E. A.; Li, L. S.; Kabbani, W.; Yan, J.; Xie, X. J.; Hsieh, J. T.; Gao, J.; Boothman, D. A. Prostate cancer radiosensitization through poly(ADP-Ribose) polymerase-1 hyperactivation. Cancer Res. 2010, 70, 8088−8096. (17) Lin, L.; Qin, Y.; Jin, T.; Liu, S.; Zhang, S.; Shen, X.; Lin, Z. Significance of NQO1 overexpression for prognostic evaluation of gastric adenocarcinoma. Exp. Mol. Pathol. 2014, 96, 200−205. (18) Ji, L.; Wei, Y.; Jiang, T.; Wang, S. Correlation of Nrf2, NQO1, MRP1, cmyc and p53 in colorectal cancer and their relationships to

Haiping Hao received his Ph.D. from China Pharmaceutical University in 2006 and was a visiting scholar at the National Cancer Institute (USA) during 2012−2013. Professor Hao is the Dean of College of Pharmacy and the Associate Director of the State Key Laboratory of Natural Medicines at China Pharmaceutical University. His major scientific interests focus on the elucidation of metabolic regulation in the pathogenesis of chronic diseases such as tumor and atherosclerosis and the translational link to target discovery and drug development. Sheng Jiang received his Ph.D. degree from Shanghai Institute of Organic Chemistry (China) in 2003. After four years of postdoctoral training, he started his independent career in 2008 at Guangzhou Biomedicine and Health, CAS. In 2017, Professor Jiang moved his research group to the China Pharmaceutical University. His scientific interest is mainly focused on development of novel small drug-like molecules and peptidomimetic derivatives acting on targets of epigenetics and immuno-oncology.

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REFERENCES

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation (21472191, 81773559), the National Major Scientific and Technological Program for Drug Discovery Grant (2018ZX09301045002), the International Cooperation Special Grant (2016A050502036) from the Science and Technology Development Project of Guangdong Province, the International Cooperation Grant (201704030099) of Guangzhou, and the Science and Technology Planning Project of Guangdong Province (2013A022100019) and Chinese Pharmaceutical AssociationYiling Biopharmaceutical Innovation Foundation.

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ABBREVIATIONS USED NQO1, NAD(P)H:quinone oxidoreductase 1; DTD, DTdiaphorase; Nrf2, NF-E2 p45-related factor 2; Keap1, Kelchlike ECH-associated protein 1; ARE, antioxidant response element; TNF, tumor necrosis factor; HIF-1α, hypoxia inducible factor α; ALDH, aldehyde dehydrogenase; CCA, cholangiocarcinoma; DIC, dicumarol; 5-FU, 5-fluorouracil; TIMP1, tissue inhibitors of metalloproteinases 1; MMP9, matrix metalloproteinase 9; SNPs, single nucleotide polymorphisms; SAR, structure−activity relationships; MMC, mitomycin C; ROS, reactive oxygen species; Hsp90, heat shock protein; P-450R, NADPH:cytochrome P450 reductase; XO, xanthine oxidase; b-5R, NADPH:cytochrome b5 reductase; NMIBC, nonmuscle invasive bladder cancer; AZQ, diaziquione; STN, streptonigrin; β-lap, β-lapachone; Tan IIA, tanshinone IIA; PARP-1, poly(ADP-ribose)polymerase-1; HPβ-CD, hydroxylpropyl β-cyclodextrin; PEG−PLA, poly(ethylene glycol)-block-poly(D,L-lactide); BER, base excision repair; NAMPT, nicotinamidephosphoribosyltransferase; GLS1, glutaminase 1; DNQ, deoxynyboquinone; CPT, camptothecin; PeT, photoinduced electron transfer; ICT, 6997



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