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Efficient NQO1 Substrates are Potent and Selective Anticancer Agents Elizabeth I. Parkinson, Joseph S. Bair, Megan Cismesia, and Paul J. Hergenrother* Department of Chemistry, Roger Adams Laboratory, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States S Supporting Information *
ABSTRACT: A major goal of personalized medicine in oncology is the identification of drugs with predictable efficacy based on a specific trait of the cancer cell, as has been demonstrated with gleevec (presence of Bcr-Abl protein), herceptin (Her2 overexpression), and iressa (presence of a specific EGFR mutation). This is a challenging task, as it requires identifying a cellular component that is altered in cancer, but not normal cells, and discovering a compound that specifically interacts with it. The enzyme NQO1 is a potential target for personalized medicine, as it is overexpressed in many solid tumors. In normal cells NQO1 is inducibly expressed, and its major role is to detoxify quinones via bioreduction; however, certain quinones become more toxic after reduction by NQO1, and these compounds have potential as selective anticancer agents. Several quinones of this type have been reported, including mitomycin C, RH1, EO9, streptonigrin, β-lapachone, and deoxynyboquinone (DNQ). However, no unified picture has emerged from these studies, and the key question regarding the relationship between NQO1 processing and anticancer activity remains unanswered. Here, we directly compare these quinones as substrates for NQO1 in vitro, and for their ability to kill cancer cells in culture in an NQO1-dependent manner. We show that DNQ is a superior NQO1 substrate, and we use computationally guided design to create DNQ analogues that have a spectrum of activities with NQO1. Assessment of these compounds definitively establishes a strong relationship between in vitro NQO1 processing and induction of cancer cell death and suggests these compounds are outstanding candidates for selective anticancer therapy.
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little-to-no NQO1 in normal tissues,9−13 compounds bioreduced by NQO1 to unstable hydroquinones could be potent and selective anticancer agents.2 Several examples of NQO1activated compounds are reported in the literature, including the alkylators mitomycin C (MMC),8,16,17 RH1,18−20 and EO917,21,22 and the redox cycling compounds streptonigrin (STN)20,23 and β-lapachone12,24 (β-lap, Figure 1B). However, none of these compounds have been able to demonstrate the full potential of an NQO1-activated anticancer agent either due to activation by other reductases (MMC,2,25,26 RH1,27,28 STN29), detoxification by NQO1 under select conditions (EO9,30,31 MMC31,32), short in vivo half-lives (RH1,33 EO9,34 β-lap35), or severe toxicity (STN36). We recently identified deoxynyboquinone (DNQ, Figure 1B) as a potent anticancer compound and developed an efficient, modular synthesis of this small molecule that allowed for further biological characterization.37 These studies show that cells treated with DNQ rapidly generate ROS,38 and this ROS
QO1 (NAD(P)H:quinone oxidoreductase-1) is a dimeric flavoprotein that catalyzes the 2-electron reduction of a variety of quinones, as well as quinoneimines, azoaromatic, and nitroaromatic compounds.1−3 NQO1 catalyzes this reduction via a ping-pong mechanism in which either NADH or NADPH can be utilized as the electron source.4 Typically, this reduction detoxifies quinones, resulting in the formation of a relatively stable hydroquinone that is subsequently conjugated (to glutathione, sulfate, or glucose) and eliminated.5,6 This process prevents 1-electron reductases (e.g., cytochrome P450 reductase and cytochrome b5 reductase) from converting the quinone to a highly reactive semiquinone that undergoes reduction−oxidation (redox) cycling, which would indiscriminately produce high levels of toxic reactive oxygen species (ROS) (Figure 1A).1,7 While NQO1 is usually a detoxifying enzyme, its reduction can turn certain quinones into potent cell death-inducing compounds. This occurs when the bioreduction of a quinone by NQO1 results in the formation of an unstable hydroquinone capable of either alkylating DNA or undergoing redox cycling, generating ROS (Figure 1A).2,8 Due to the dramatic elevation of NQO1 in many cancers (including lung,9−12 colon,9,11 pancreatic,13−15 and breast cancer9,10) and the expression of © XXXX American Chemical Society
Received: April 12, 2013 Accepted: August 12, 2013
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dx.doi.org/10.1021/cb4005832 | ACS Chem. Biol. XXXX, XXX, XXX−XXX
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Figure 1. (A) Reduction pathways and subsequent redox cycling of quinones with 1-electron reductases (such as cytochrome P450 reductase and cytochrome b5 reductase) and 2-electron reductases (such as NQO1). (B) Cytotoxic quinones reported to be bioactivated by NQO1.
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is the main cause of cancer cell death.37 The mechanism by which DNQ induces ROS formation has been determined to be via NQO1-mediated reduction followed by spontaneous reoxidation to the quinone (Figure 1A).38 Additionally, DNQ was shown to have potent activity in vivo against an aggressive orthotopic lung carcinoma model in athymic mice.38 Critically, the relationship between NQO1 processing and quinone anticancer activity is not well understood. A study that investigates the anticancer capacity of a single structural class of quinones in relation to their ability to be processed by NQO1 would greatly increase this understanding. Such a study would allow for the development of optimized compounds and would aid in tracking any on-target and off-target toxicities in vivo. The addition of DNQ to the spectrum of NQO1 substrates now affords the opportunity to fully interrogate this relationship between NQO1, quinones, and cancer cell death. Herein is described the head-to-head assessment of MMC, RH1, STN, βlap, and DNQ. Based on the superiority of DNQ in these experiments, computational guidance was used to design multiple DNQ derivatives that were predicted to vary in their ability to be processed by NQO1. These compounds were then synthesized and assessed for their ability to be substrates for NQO1 in vitro, and for their NQO1-dependent anticancer activity in cell culture. These experiments strongly suggest that highly efficient NQO1 substrates make outstanding anticancer agents.
RESULTS AND DISCUSSION Quinones as NQO1 Substrates In Vitro. The ability of NQO1 to process various quinones (DNQ, β-lap, STN, RH1, and MMC) in vitro was assessed. In this assay, the quinone is coincubated with both NQO1 as well as an excess of NADH, and oxidation of NADH is followed by the decrease in absorbance at 340 nm. DNQ, β-lap, and STN were all substrates for NQO1, and each utilized greater than 1 equiv. of NADH over the course of the assay, demonstrating the ability of these quinones to redox cycle (Supporting Information (SI) Figure 1). From these data, Michaelis−Menten curves were generated, and apparent catalytic efficiencies were calculated (apparent because they also reflect the kinetics of redox cycling for each compound). As shown in Figure 2, DNQ is a highly efficient substrate and redox cycler, with an apparent catalytic efficiency that approaches the diffusion controlled limit (kcat/KM = 6.2 × 107 M−1s−1). DNQ is processed over 9 times faster than the next best compound, β-lap (kcat/KM = 0.67 × 107 M−1·s−1), and 24 times faster than STN (kcat/KM = 0.26 × 107 M−1·s−1). RH1 and MMC are extremely poor substrates for NQO1 in this assay with observed activity less than 100 μmol/min/μmol. Quinones versus Cancer Cells in Culture. DNQ, β-lap, STN, RH1, and MMC were also investigated for their potency against cancer cells that overexpress NQO1. The lung adenocarcinoma cell line A549 has robust expression of NQO1,38 and we measured the NQO1 activity in the cell B
dx.doi.org/10.1021/cb4005832 | ACS Chem. Biol. XXXX, XXX, XXX−XXX
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tested against A549 cells in the presence of the NQO1 inhibitors dicoumarol (DIC, 25 μM, Figure 3A) and ES936 (100 nM, Figure 3B). DIC is a competitive inhibitor of NQO1 (KI = 1−10 nM) that interacts with the NAD(P)H binding site.39 ES936 is a potent mechanism based inhibitor which alkylates key tyrosine residues within the NQO1 active site.40 While neither compound has perfect specificity for NQO1,40−42 both DIC and ES936 are widely used in studies of NQO1-mediated cell death,12,24,38,43,44 as incubation of cells with these inhibitors is effective in blocking the enzymatic activity of NQO1.15,44−46 As shown in Figure 3A and B, coincubation with DIC and ES936 dramatically protects A549 cells from DNQ-mediated cell death, shifting the IC50 53-fold and >170-fold, respectively (the fold is the ratio of the IC50 of cotreatment with quinone and inhibitor to the IC50 of treatment with only quinone, and a higher ratio indicates greater protection and greater NQO1 selectivity). DIC and ES936 also protect cells from β-lap-induced cell death, shifting the IC50 10-fold and 6-fold (Figure 3A and B). DIC has littleto-no effect on STN, RH1, or MMC-induced cell death, suggesting that only DNQ and β-lap kill in this assay by an NQO1-dependent mechanism. Analogous results were observed with an NQO1-overexpressing breast cancer cell line, MCF-7 (NQO1 activity = 1900 nmol/min/μg protein), as shown in SI Figure 2. A small but statistically significant
Figure 2. Michaelis−Menten curves for DNQ, β-lap, and STN with NQO1. Virtually no activity (
