Activation of Bioreductive Antitumour Agents by DT-Diaphorase

Bailey, Susan Maria (1995) Activation of Bioreductive Antitumour Agents by DT-Diaphorase. PhD thesis, University of Glasgow.

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Bioreductive anticancer agents possess two potential targets for selectivity, hyperexpressed activating enzymes and hypoxia. The subject of this thesis is part of an 'enzyme directed approach' to rational drug development in which compounds are tailored to suit the catalytic preferences of key bioreductive enzymes. Studies have focused on the two-electron reducing flavoenzyme DT-diaphorase due to its increased expression and activity in tumour versus normal tissue and its ability to catalyse metabolism of substrates with structural similarity to principal bioreductive agents. The overall aim of the thesis was to determine the involvement of DT-diaphorase in the activation of the novel indoloquinone EO9, aziridinylbenzoquinone AZQ and selected cyclopropamitosenes. Structure-activity relationships were examined using analogues of these compounds. Initial experiments to characterise metabolism of EO9 showed it to be efficiently reduced by DT-diaphorase present in sonicates of human HT29 and rat Walker cells as well as by a highly purified form of the rat enzyme. Chemosensitivity assays also demonstrated EO9 to be a highly potent cytotoxin for these cell lines, which express high levels of DT-diaphorase. Study of EO9 analogues showed that structural modification altered both ability to undergo metabolism catalysed by DT-diaphorase and cytotoxicity. A clear correlation was observed between cytotoxicity and metabolism with analogues which were better substrates for DT-diaphorase being more potent cytotoxins than the more poorly reduced derivatives. This supported the importance of DT-diaphorase in activation of the indoloquinones to their cytotoxic species. Inhibitor studies further substantiated this hypothesis but indicated involvement of additional enzymes. The latter possibility was supported by results of electron spin resonance spectroscopy experiments where NADPH: cytochrome P450 reductase was shown to metabolise EO9. The mechanism of reaction has to some extent been elucidated. Both DT-diaphorase and NADPH: cytochrome P450 reductase metabolised EO9 to highly oxygen sensitive metabolites which, in the presence of air, were auto-oxidised to generate highly reactive and potentially damaging oxygen and drug based radicals. In intact cells EO9 induced DNA damage in the form of strand breaks and interstrand cross-links at pharmacologically relevant concentrations. This damage was more extensive in the high DT-diaphorase expressing cell line HT29 than in the BE cell line which does not express a functional form of the enzyme. Using a cell free system the importance of DT-diaphorase activation for generation of both strand breaking and interstrand cross-linking species was determined. Monoadducts could be formed with DNA in the absence of activation although the sequence selectivity exhibited by the compound was found to be altered following reduction. Neither hypoxia or pH altered the initial rate of EO9 reduction catalysed by DT- diaphorase. Hypoxia did however alter the extent of DNA damage and other investigators have shown that both pH and hypoxia can influence cytotoxicity of this compound. Metabolism studies suggested that these parameters will influence the stability of metabolites formed. AZQ and some cyclopropamitosenes were also reduced by purified rat Walker DT- diaphorase but less efficiently than EO9. In a similar manner to EO9 some correlation between metabolism and cytotoxicity was observed with the aziridinylbenzoquinones although the differences were within a far narrower range than for the indoloquinones. To conclude, data presented in this thesis has confirmed a role for DT-diaphorase in activation of EO9, AZQ and cyclopropamitosenes. In addition NADPH: cytochrome P450 reductase has also been shown to metabolise EO9. The mechanism of reaction and cytotoxicity has been partially elucidated and structure-activity relationship studies have highlighted features of the drug molecules which are important for both metabolism and cytotoxicity. Such information may be important in the design of improved analogues and for optimisation of clinical protocols employing these novel, bioreductive agents.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Paul Workman
Keywords: Oncology, Pharmacology
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-76313
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 15:47
Last Modified: 19 Nov 2019 15:47

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