Involvement of NADPH: cytochrome P450 reductase in the activation of indoloquinone EO9 to free radical and DNA damaging species5
Introduction
The bioreductive alkylating agent mitomycin C has been used clinically for the treatment of several tumour types. Despite significant antitumour activity, its widespread use is limited as a result of its extensive and unpredictable dose-limiting myelosuppression [1]. An indoloquinone analogue of mitomycin C, EO9 (Fig. 1; [2]), has undergone preclinical and clinical development under the auspices of the EORTC. EO9 was found to have a different spectrum of preclinical antitumour activity to mitomycin C, showed preference for solid tumours over leukaemias, and most importantly was non-myelopsuppressive 3, 4. As a result of these features and information regarding its mechanism of enzymatic activation (see below), EO9 entered Phase I and II clinical trial 5, 6, 7. Although responses were seen in Phase I trials [6], activity was not confirmed in Phase II [7]. Nevertheless, clinical development of EO9 continues, potentially to define a clinical role for this agent in a setting where its short plasma half-life is not so critical, e.g. intravescicular therapy for bladder cancer. In addition, studies continue with the aim of identifying bioreductive agents that will be clinically more effective than EO9 [8].
A characteristic of bioreductive alkylating agents such as EO9 is that they are designed to require reduction for activation to a cytotoxic species. Several enzymes are known to be involved in the reduction of quinone compounds [8]. These include those catalysing one-electron reduction, e.g. NADPH: cytochrome P450 reductase and cytochrome b5 reductase as well as DT-diaphorase, which carries out two-electron reduction. As part of an ‘enzyme-directed approach’ to bioreductive drug development 8, 9, 10, we were interested in elucidating the molecular enzymology and mechanism of action of EO9. We had previously focused on the involvement of the two-electron reducing flavoenzyme DT-diaphorase and provided data to support a role for this enzyme in the activation of EO9 to a cytotoxic species 11, 12, 13, 14, 15.
Initially, extracts of the high DT-diaphorase expressing rat Walker tumour and human HT29 colon carcinoma cells were shown to catalyse reduction of EO9 12, 15. The rate of reduction decreased in the presence of the potent DT-diaphorase inhibitor dicoumarol. Dicoumarol is, however, also known to affect other enzyme systems. Proof of the ability of DT-diaphorase to reduce EO9 was provided by use of purified rat, mouse, and human recombinant DT-diaphorase 11, 16. Activation of EO9 catalysed by DT-diaphorase resulted in generation of a DNA-damaging metabolite(s) able to induce strand breaks and interstrand cross-links in DNA [13]. Studies correlating cellular sensitivity with enzyme expression provided further evidence for the importance of DT-diaphorase in activation of EO9 under aerobic conditions 17, 18, 19, 20, 21 but not in hypoxia [22]. Interestingly, cells expressing low levels of DT-diaphorase were greatly sensitised to EO9 under hypoxic compared with aerobic conditions 19, 23. Also, inclusion of dicoumarol in cytotoxicity assays did not provide complete protection against the cytotoxic effects. These data suggested that other enzymes may also be important in reductive activation of EO9, especially under hypoxia.
Malipaard et al.[24] have shown that xanthine oxidase is able to reduce EO9 to generate a DNA-damaging species. NADPH: cytochrome P450 reductase is known to reduce other bioreductive quinone compounds such as AZQ and mitomycin C 25, 26, 27. Furthermore, recent experiments by Saunders and co-workers [28], involving transfection and overexpression of the NADPH: cytochrome P450 reductase gene, have strongly implicated a role for this enzyme in the cellular reduction of EO9. Here, we provide additional evidence in support of a role for this enzyme in activation of EO9 to free radical and DNA-damaging species. The experiments were carried out under predominantly aerobic conditions and are therefore more directly relevant to cytotoxic effects that EO9 may generate in well-oxygenated normal cells (contributing to toxicity) and in the aerobic population of tumour cells (contributing to therapeutic response). Understanding the enzymology of EO9 metabolism could be useful in the future clinical development of the drug, as well in the development of analogues of EO9 and related indoloquinone bioreductive agents, which continues to be an area of significant activity 8, 28.
Section snippets
Materials
EO9 was a generous gift from the EORTC New Drugs Development Office, Amsterdam. Rat NADPH: cytochrome P450 reductase was kindly supplied by Professor C. R. Wolf, Dundee, Scotland and plasmid pBR322 DNA was obtained from Boehringer Manheim UK Ltd., Lewis, East Sussex, UK.
Electron spin resonance spectroscopy
Electron spin resonance spectroscopy was carried out using a Varian E109 Century X-band (9.3 GHz) spectrometer. ESR parameters were in general: scan range ± 50 G, time constant 1 sec, modulation amplitude 1.25 G, receiver gain
Reduction of EO9 and free radical formation catalysed by NADPH: cytochrome P450 reductase
Reproducible ESR spectra were obtained following aerobic incubation of EO9 with NADPH and NADPH: cytochrome P450 reductase. Fig. 2a shows a typical 7-line hyperfine spectrum superimposed over a broad single spectrum (see Fig. 2a). This free radical, attributable to the EO9 drug radical, persisted for 30 min and was dependent on the presence of NADPH, EO9, and enzyme (results not shown). ESR spectrometry in combination with DMPO, a spin trap for short-lived radicals [29], was used to investigate
Discussion
We and others (see Introduction) have provided considerable evidence to suggest that the obligate two-electron reducing enzyme DT-diaphorase is able to catalyse reduction of EO9 and play a major role in the mechanism of the drug cytotoxicity under aerobic conditions. However, as also outlined in the Introduction, the results of these studies indicated that other enzymes are also likely to be involved, particularly under hypoxic conditions [22]. In the present studies, we show that purified rat
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Tumor hypoxia: From basic knowledge to therapeutic implications
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2019, Clinical and Translational Radiation OncologyCitation Excerpt :There is a need for predictive biomarkers to guide clinical development of HAPs, including identification of the oxidoreductase enzymes necessary to catalyze their activation via electron donation. The human flavoproteome, comprising 79 unique flavoenzymes [74], likely plays a major role in the bioreductive transformation of HAPs, which is in agreement with the known involvement of individual oxidoreductases [75–89]. Approaches aimed at identifying these key catalytic proteins, their relative contributions and tissue distributions will ultimately guide the clinical application of HAPs.
Aziridinyl-substituted benzo-1,4-quinones: A preliminary investigation on the theoretical and experimental studies of their structure and spectroscopic properties
2017, Spectrochimica Acta - Part A: Molecular and Biomolecular SpectroscopyCitation Excerpt :Bioreductive agents could be activated by both two-electron reductive flavoprotein enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase (EC 1.6.99.2)) [8] and a single-electron reductive enzyme such as NADPH cytochrome P450 reductase (P450R) (EC:1.6.2.4) [9]. Currently, it is known that a heightened concentration of reductive enzyme DT-diaphorase (NQO1) is present in a hypoxic cancer cell [10]. The activation of quinonic anticancer agents by enzyme NQO1 has also been extensively studied [11].
Synthesis and cytotoxicity of pyranonaphthoquinone natural product analogues under bioreductive conditions
2013, Bioorganic and Medicinal Chemistry
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Abbreviations: EORTC, European Organisation for the Research and Treatment of Cancer; DMPO, 5,5-dimethyl-1-pyrroline-1-oxide; and AN and AH, hyperfine-splitting constants for nitrogen and hydrogen, respectively.
- 1
Bristol-Myers Squibb Pharmaceuticals Limited. 141-149 Staines Road, Hounslow, Middlesex, TW3 3JA, UK.
- 2
Wood Mackenzie, Kintore House, 74-77 Queen Street, Edinburgh EH2 4NS, UK.
- 4
The School of Pharmacy, University of London, Brunswick Square, London, WC1N 1AX, UK.
- 5
Metabolic, CV Risk Factors and Oncology Clinical Development, Glaxo Wellcome, Greenford Road, Middlesex, UB6 OHE, UK.
- 3
Enact Pharma, Building 115, Porton Down Science Park, Salisbury SP4 OJQ, UK.