A direct interaction between NQO1 and a chemotherapeutic dimeric naphthoquinone
© Pidugu et al. 2016
Received: 5 November 2015
Accepted: 19 January 2016
Published: 28 January 2016
Multimeric naphthoquinones are redox-active compounds that exhibit antineoplastic, antiprotozoal, and antiviral activities. Due to their multimodal effect on perturbation of cellular oxidative state, these compounds hold great potential as therapeutic agents against highly proliferative neoplastic cells. In our previous work, we developed a series of novel dimeric naphthoquinones and showed that they were selectively cytotoxic to human acute myeloid leukemia (AML), breast and prostate cancer cell lines. We subsequently identified the oxidoreductase NAD(P)H dehydrogenase, quinone 1 (NQO1) as the major target of dimeric naphthoquinones and proposed a mechanism of action that entailed induction of a futile redox cycling.
Here, for the first time, we describe a direct physical interaction between the bromohydroxy dimeric naphthoquinone E6a and NQO1. Moreover, our studies reveal an extensive binding interface between E6a and the isoalloxazine ring of the flavin adenine dinucleotide (FAD) cofactor of NQO1 in addition to interactions with protein side chains in the active site. We also present biochemical evidence that dimeric naphthoquinones affect the redox state of the FAD cofactor of NQO1. Comparison of the mode of binding of E6a with those of other chemotherapeutics reveals unique characteristics of the interaction that can be leveraged in future drug optimization efforts.
The first structure of a dimeric naphthoquinone-NQO1 complex was reported, which can be used for design and synthesis of more potent next generation dimeric naphthoquinones to target NQO1 with higher affinity and specificity.
KeywordsNQO1 Dimeric naphthoquinone Oxidative stress Anti-cancer agents
Multimeric naphthoquinones are reduction/oxidation (redox)-active compounds that possess a wide array of therapeutic activities. In particular, these compounds have exhibited well tolerated antibacterial, antifungal, antiviral, and antithrombotic activities . One of the most notable members of this class of compounds is conocurvone, a naturally-occurring trimeric naphthoquinone with a potent anti-HIV activity . Synthetic and natural naphthoquinones have demonstrated significant antineoplastic activity against hematologic and solid malignant cells [3–5]. In an effort to regiospecifically synthesize conocurvone, we previously developed a series of novel dimeric naphthoquinones and showed that they were selectively cytotoxic to human acute myeloid leukemia (AML), breast and prostate cancer cell lines and in particular those cell lines that rely on oxidative phosphorylation [6–8]. To better understand the mechanism of action of these agents, we performed a chemical genetic screen in yeast and identified the yeast oxidoreductase Nde1 as the major target of dimeric naphthoquinones [6, 9]. The human homologue of Nde1 is NAD(P)H quinone oxidoreductase 1 (E.C. 18.104.22.168, hereon referred to as NQO1, also known as DT-diaphorase and NAD(P)H dehydrogenase, quinone 1).
NQO1 is a quinone detoxifying flavoenzyme that catalyzes the two-electron reduction of quinones to hydroquinones. For dimeric naphthoquinones, the resulting hydroquinone is highly unstable and spontaneously gives electrons to oxygen and reverts to the oxidized form of quinone, producing two moles of superoxide per one mole of NAD(P)H . The ultimate outcome is a futile redox cycle in NQO1-overexpressing cells, such as many cancer cells, which can culminate in formation of substantial reactive oxygen species (ROS), oxidative damage to DNA and single- and double-strand DNA breaks. NQO1 exists as a homodimer with two tightly-associated flavin adenine dinucleotide (FAD) cofactors that reside at the deepest point of each active site of two monomers of 274 residues . The two active sites reside at opposite ends of the dimer and incorporate residues from each monomer. The normal biological function of NQO1 is to protect cells from the mutagenic, cytotoxic, and carcinogenic effects of natural and synthetic quinones . The obligate two electron reduction performed by NQO1 averts one-electron reduction of quinones by other flavoproteins such as cytochrome P450, which produces highly reactive radical semiquinone.
The role of NQO1 in cancer varies due to its role in redox biology. NQO1 exhibits tumor suppressor properties by modulating the stability of p53 [13, 14] and participating in suppression of the inflammatory response . Conversely, increased expression of NQO1 can confer a growth advantage in some cancers such as melanoma, pancreatic adenocarcinoma, non-small cell lung cancer, and prostate cancer [16, 17]. The association between the NQO1 C609T polymorphism and increased risk of AML and acute lymphoblastic leukemia (ALL) has also been reported [18, 19]. Exploitation of NQO1 as a target for cancer therapy typically entails two strategies. In some cases, inhibition of NQO1 can suppress cancer cell growth and potentiate chemotherapeutic cytotoxicity . In other cases, NQO1 can be used to activate particular quinone-based chemotherapeutics via its redox activity [21, 22]. For dimeric naphthoquinones, we have proposed that their unique chemical structures undergo NQO1-dependent redox cycling that produces an insurmountable amount of ROS that ultimately lead to mitochondrial dysfunction, DNA damage and cell death .
In the present study, we have determined the crystal structure of the novel dimeric naphthoquinone, 3-bromo-3′-hydroxy-2,2′-binaphthalenyl-1,4,1′,4′-tetraone (E6a , Additional file 1: Figure S1) bound to NQO1. This structure represents the first evidence of a direct interaction between a dimeric naphthoquinone and NQO1. Moreover, we present biochemical evidence that this interaction affects the redox state of the FAD cofactor. Our structure reveals an extensive binding interface between E6a and the isoalloxazine ring of the FAD cofactor of NQO1 in addition to interactions with protein side chains in the active site. This structure can be used as a starting point to design and synthesize more potent dimeric naphthoquinones tailored to target NQO1 with high affinity and specificity.
Results and discussion
Overall quality of crystal structure
Data collection and refinement statistics
Unit Cell (Å)
a = 56.93
a = 95.60
b = 107.16
b = 210.77
c = 99.76
c = 228.08
β = 100.68
Multiplicity (Last shell)
Completeness (Last shell)
E6a interactions with the NQO1 active site
Increase in FAD fluorescence upon E6a binding
Comparison of E6a binding to that of other quinone-based chemotherapeutic agents
Comparison of E6a binding to coumarin-based inhibitors of NQO1
The crystal structure of hNQO1 complexed with FAD and E6a presented here is the first evidence of direct interaction of the dimeric naphthoquinones with NQO1 at the active site. This is a valuable starting point for better understanding of the mode of binding of dimeric naphthoquinones to NQO1. Such data are required in order to establish structure activity relationships that support further structure-based optimization to improve the anti-neoplastic efficacy of this novel class of chemotherapeutics. High resolution crystal structures of hNQO1 with more dimeric naphthoquinones would help to understand the complete mechanism of activation of these agents by hNQO1.
Expression and purification
DNA for hNQO1 was codon optimized for expression in Escherichia coli and subcloned into a modified pET19b vector containing N-terminal 10XHis tag and PreScission protease cleavage site preceding the insert. The expression plasmid was transformed into BL21 (DE3) cells. Cells were grown at 37 °C to an OD600 of 0.6 and induced with 0.3 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 18 °C overnight. The cells were harvested at 4000 rpm for 20 min and resuspended in lysis buffer containing, 50 mM Tris pH 8.0, 500 mM NaCl and 2 mM beta-mercaptoethanol. The cells were lysed by sonication and soluble proteins were separated by centrifugation at 15000 rpm for 45 min. The clarified lysate was first purified using Ni-affinity chromatography. The histidine tag was removed from the purified hNQO1 by preScission protease cleavage, combined with a dialysis against 50 mM Tris pH 8.0, 150 mM NaCl and 1 mM DTT. Cleaved hNQO1 was further subjected to a final purification step using size exclusion chromatography.
Crystallization and structure determination
hNQO1 stored at a concentration of approximately 18–20 mg/ml in 50 mM Tris pH 8.0, 50 mM NaCl and 5 μM FAD was used for crystallization screening. Initial screening with JCSG+, Classics suite I and II from Qiagen resulted in initial hits in more than 20 conditions. Native data up to 2.0 Å resolution were collected using P21 crystals obtained in 20 % (w/v) PEG3350 and 0.2 M Ammonium Citrate. Another crystal form in the space group P212121 were obtained using 20 % PEG3350 and 0.2 M Potassium Sodium Tartarate. The complex between NQO1 and E6a was obtained by soaking the P212121 native crystals in mother liquor containing 1 mM E6a. X-ray diffraction data were collected in house and at beam line 5.0.3 of the Advanced Light source at Lawrence Berkeley National Laboratory for the holo and E6a-bound hNQO1 crystals respectively. The data were reduced using iMosflm  and Aimless  from the CCP4 program suite. Initial phases for both holo and E6a-bound hNQO1 were determined by molecular replacement using the program Phaser [40–43] using the coordinates of an hNQO1 monomer from a previously reported holo-structure (PDB accession code: 1D4A) . The initial molecular replacement solution for the hNQO1-E6a complex contained only 8 of the 14 monomers in the asymmetric unit. Using these 8 monomers as a fixed solution, the remaining monomers were placed iteratively using a combination of Phaser [40–43] and Molrep . The structure was refined using Refmac5 [45–49] from CCP4 program suite. Iterative cycles of model building using COOT [50–53] and refinement by refmac5 and TLS- and NCS- restrained  refinement using buster  yielded final structures with Rwork/Rfree of 18.0/21.6 for native and 18.3/22.0 for the E6a complex. Final Structures were deposited in PDB (Accession Codes: holo–hNQO1: 5EA2, hNQO1-E6a: 5EAI).
All fluorescence measurements were done using SpectraMax M5 plate reader at room temperature. An absorbance scan for 100 μM FAD in 50 mM Tris pH 8.0 and 50 mM NaCl resulted in a peak at 440 nm. The excitation wavelength was chosen to be 425 nm for FAD to avoid overlap of excitation and emission peaks. An emission scan with an excitation wavelength at 425 nm resulted in a peak at 525 nm. These wavelengths were confirmed by performing similar scans with FAD bound to hNQO1 in the same buffer and thus were used for all of the fluorescence experiments. Approximately 100 μl of each of the following samples in 50 mM Tris pH 8.0, 50 mM NaCl and 5 μM FAD were added in the wells of a costar Black/clear bottom 96 well plate. Fluorescence of hNQO1 alone in the above buffer was initially recorded. Next, NQO1 supplemented with 1 mM E6a in the above buffer was analyzed. A mixture of 5 μM FAD and 1 mM E6a mixture in the above buffer was used as a negative control. Approximately 100 μM hydrogen peroxide was added to the NQO1 sample and used as a positive control for the oxidized FAD. These data were analyzed using softMax Pro software.
Availability of supporting data
The atomic coordinates and structure factor amplitudes are available in the Protein Data Bank repository (PDB), Accession Codes 5ea2 (holo-NQO1) and 5eai (NQO1-E6a complex).
acute lymphoblastic leukemia
acute myeloid leukemia
flavin adenine dinucleotide
nicotinamide adenine dinucleotide
relative fluorescence units
reactive oxygen species
The X-ray data for holo-hNQO1 structure were collected at W.M. Keck/NIST X-ray Crystallography Core Facility at the Institute for Bioscience and Biotechnology Research and for hNQO1-E6a complex were collected at beam line 5.0.3 of the Advanced Light source at Lawrence Berkeley National Laboratory. We thank Dr. Daniel Nelson and Sara Linden for letting us use their SpectraMax M5 for the fluorescence experiments and useful discussions in analyzing the data. This work was supported by institutional funds from the Center for Biomolecular Therapeutics.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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