1887

Abstract

A nitroreductase with distinct properties that can activate the prodrug 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) was isolated from . The encoding gene was identified as a homologue of the of , and was obtained as a PCR product by reverse genetics, cloned and the entire nucleotide sequence determined. The gene was found to reside between homologues of the and genes; however, the and genes of were not separated by a fourth gene, . The gene was overexpressed, the recombinant protein purified and its properties were compared with those of two CB 1954-activating enzymes, B nitroreductase (NTR) and Walker DT-diaphorase (DTD). In common with these enzymes menadione was an electron acceptor ( 3 μM) and activity with this substrate was inhibited by the presence of dicoumarol ( 10 μM). In contrast, YwrO showed a marked preference for NADPH as a cofactor ( 40 μM) and therefore could not be classified as a DTD (EC 1.6.99.2). The flavin FMN was an acceptor with high affinity. YwrO was shown to be a flavoprotein with a monomeric molecular mass of 215 kDa by calculation and SDS-PAGE. The cytotoxic 4-hydroxylamine derivative was the single CB 1954 reduction product, but YwrO was inactive with the bischloroethyl analogue of CB 1954, SN 23862. In both of these properties YwrO more closely resembles DTD than NTR. Its for CB 1954 was lower than that of NTR (617 μM compared to 862 μM). Enhanced cytotoxicity of CB 1954 was demonstrated on incubation of V79 cells with prodrug, NADPH and YwrO. The work has led to the identification of a previously unknown nitroreductase, YwrO, with distinct properties which will aid the rational selection of appropriate genes for applications in directed enzyme prodrug therapy (DEPT).

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-1-297
2002-01-01
2019-10-17
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/1/1480297a.html?itemId=/content/journal/micro/10.1099/00221287-148-1-297&mimeType=html&fmt=ahah

References

  1. Anlezark, G. M., Melton, R. G., Sherwood, R. F., Coles, B., Friedlos, F. & Knox, R. J. ( 1992; ). The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) – I. Purification and properties of a nitroreductase enzyme from Escherichia coli – a potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT). Biochem Pharmacol 44, 2289-2295.[CrossRef]
    [Google Scholar]
  2. Anlezark, G. M., Melton, R. G., Sherwood, R. F., Wilson, W. R., Denny, W. A., Palmer, B. D., Knox, R. J., Friedlos, F. & Williams, A. ( 1995; ). Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase. Biochem Pharmacol 50, 609-618.[CrossRef]
    [Google Scholar]
  3. Anlezark, G. M., Cook, R., Melton, R. G., Freemantle, P., Pedley, R. B., Boden, J. & Knox, R. J. ( 1996; ). Biodistribution of A5B7 F(ab′)2:E. coli B nitroreductase conjugate in nude mice with human xenografts. Br J Cancer 73, 40.
    [Google Scholar]
  4. Bagshawe, K. D. ( 1987; ). Antibody directed enzymes revive anti-cancer prodrugs concept. Br J Cancer 56, 531-532.[CrossRef]
    [Google Scholar]
  5. Bagshawe, K. D., Springer, C. J., Searle, F., Antoniw, P., Sharma, S. K., Melton, R. G. & Sherwood, R. F. ( 1988; ). A cytotoxic agent can be generated selectively at cancer sites. Br J Cancer 58, 700-703.[CrossRef]
    [Google Scholar]
  6. Bailey, S. M., Knox, R. J., Hobbs, S. M., Jenkins, T. C., Mauger, A. B., Melton, R. G., Burke, P. J., Connors, T. A. & Hart, I. R. ( 1996; ). Investigation of alternative prodrugs for use with E. coli nitroreductase in ‘suicide gene’ approaches to cancer therapy. Gene Ther 3, 1143-1150.
    [Google Scholar]
  7. Boland, M. P., Knox, R. J. & Roberts, J. J. ( 1991; ). The differences in kinetics of rat and human DT diaphorase result in a differential sensitivity of derived cell lines to CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). Biochem Pharmacol 41, 867-875.[CrossRef]
    [Google Scholar]
  8. Bridgewater, J. A., Knox, R. J., Pitts, J. D., Collins, M. K. & Springer, C. J. ( 1997; ). The bystander effect of the nitroreductase/CB1954 enzyme/prodrug system is due to a cell-permeable metabolite. Hum Gene Ther 8, 709-717.[CrossRef]
    [Google Scholar]
  9. Bryant, D. W., McCalla, D. R., Leeksma, M. & Laneuville, P. ( 1981; ). Type I nitroreductases of Escherichia coli. Can J Microbiol 27, 81-86.[CrossRef]
    [Google Scholar]
  10. Chen, S., Knox, R., Wu, K., Deng, P. S., Zhou, D., Bianchet, M. A. & Amzel, L. M. ( 1997; ). Molecular basis of the catalytic differences among DT-diaphorase of human, rat and mouse. J Biol Chem 272, 1437-1439.[CrossRef]
    [Google Scholar]
  11. Clark, A. J., Iwobi, M., Cui, W., Crompton, M., Harold, G., Hobbs, S., Kamalati, T., Knox, R., Neil, C., Yull, F. & Gusterson, B. ( 1997; ). Selective cell ablation in transgenic mice expression E. coli nitroreductase. Gene Ther 4, 101-110.[CrossRef]
    [Google Scholar]
  12. Connors, T. A. ( 1995; ). The choice of prodrugs for gene directed enzyme prodrug therapy of cancer. Gene Ther 2, 702-709.
    [Google Scholar]
  13. Cornish-Bowden, A. (1979). Fundamentals of Enzyme Kinetics, pp. 73–96. Boston: Butterworths.
  14. Dachs, G. U., Dougherty, G. J., Stratford, I. J. & Chaplin, D. J. ( 1997; ). Targeting gene therapy to cancer: a review. Oncol Res 9, 313-325.
    [Google Scholar]
  15. Denny, W. A. & Wilson, W. R. ( 1998; ). The design of selectively-activated anti-cancer prodrugs for use in antibody-directed and gene-directed enzyme-prodrug therapies. J Pharm Pharmacol 50, 387-394.[CrossRef]
    [Google Scholar]
  16. Drabek, D., Guy, J., Craig, R. & Grosveld, F. ( 1997; ). The expression of bacterial nitroreductase in transgenic mice results in specific cell killing by the prodrug CB1954. Gene Ther 4, 93-100.[CrossRef]
    [Google Scholar]
  17. Hoffman, R. ( 1993; ). To do tissue culture in two or three dimensions? That is the question. Stem Cells (Miamisburg) 11, 105-111.[CrossRef]
    [Google Scholar]
  18. Kinouchi, T. & Ohnishi, Y. ( 1983; ). Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis. Appl Environ Microbiol 46, 596-604.
    [Google Scholar]
  19. Knox, R. J., Boland, M. P., Friedlos, F., Coles, B., Southan, C. & Roberts, J. J. ( 1988a; ). The nitroreductase enzyme in Walker cells that activates 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is a form of NAD(P)H dehydrogenase (quinone) (EC 1 . 6 . 99 . 2). Biochem Pharmacol 37, 4671-4677.[CrossRef]
    [Google Scholar]
  20. Knox, R. J., Friedlos, F., Jarman, M. & Roberts, J. J. ( 1988b; ). A new cytotoxic, DNA interstrand crosslinking agent, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, is formed from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by a nitroreductase enzyme in Walker carcinoma cells. Biochem Pharmacol 37, 4661-4669.[CrossRef]
    [Google Scholar]
  21. Knox, R. J., Friedlos, F., Marchbank , T. & Roberts, J. J. ( 1991; ). Bioactivation of CB 1954: reaction of the active 4-hydroxylamino derivative with thioesters to form the ultimate DNA–DNA interstrand crosslinking species. Biochem Pharmacol 42, 1691-1697.[CrossRef]
    [Google Scholar]
  22. Knox, R. J., Friedlos, F., Sherwood, R. F., Melton, R. G. & Anlezark, G. M. ( 1992; ). The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) – II. A comparison of an Escherichia coli nitroreductase and Walker DT diaphorase. Biochem Pharmacol 44, 2297-2301.[CrossRef]
    [Google Scholar]
  23. Knox, R. J., Friedlos, F., Jarman, M., Davies, L. C., Goddard, P., Anlezark, G. M., Melton, R. G. & Sherwood, R. F. ( 1995; ). Virtual cofactors for an Escherichia coli nitroreductase enzyme: relevance to reductively activated prodrugs in antibody directed enzyme prodrug therapy (ADEPT). Biochem Pharmacol 49, 1641-1647.[CrossRef]
    [Google Scholar]
  24. Kunst, F., Ogasawara, N., Moszer, I. & 148 other authors ( 1997; ). The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390, 249–256.[CrossRef]
    [Google Scholar]
  25. Latham, J. P., Searle, P. F., Mautner, V. & James, N. D. ( 2000; ). Prostate-specific antigen promoter/enhancer driven gene therapy for prostate cancer: construction and testing of a tissue-specific adenovirus vector. Cancer Res 60, 334-341.
    [Google Scholar]
  26. Lemmon, M. J., van Zijl, P., Fox, M. E., Mauchline, M. L., Giaccia, A. J., Minton, N. P. & Brown, J. M. ( 1997; ). Anaerobic bacteria as a gene delivery system that is controlled by the tumour microenvironment. Gene Ther 4, 791-796.[CrossRef]
    [Google Scholar]
  27. McNeish, I. A., Green, N. K., Gilligan, M. G., Ford, M. J., Mautner, V., Young, L. S., Kerr, D. J. & Searle, P. F. ( 1998; ). Virus directed enzyme prodrug therapy for ovarian and pancreatic cancer using retrovirally delivered E. coli nitroreductase and CB1954. Gene Ther 5, 1061-1069.[CrossRef]
    [Google Scholar]
  28. Mauger, A. B., Burke, P. J., Somani, H. H., Friedlos, F. & Knox, R. J. ( 1994; ). Self-immolative prodrugs: candidates for antibody-directed enzyme prodrug therapy in conjunction with a nitroreductase enzyme. J Med Chem 37, 3452-3458.[CrossRef]
    [Google Scholar]
  29. Minton, N. P., Mauchline, M. L., Lemmon, M. J., Brehm, J. K., Fox, M., Michael, N. P., Giaccia, A. & Brown, J. M. ( 1995; ). Chemotherapeutic tumour targeting using clostridial spores. FEMS Microbiol Rev 17, 357-364.[CrossRef]
    [Google Scholar]
  30. Palmer, B. D., Wilson, W. R., Cliffe, S. & Denny, W. A. ( 1992; ). Hypoxia-selective antitumour agents. 5. Synthesis of water-soluble nitroaniline mustards with selective cytotoxicity for hypoxic mammalian cells. J Med Chem 35, 3214-3222.[CrossRef]
    [Google Scholar]
  31. Roberts, J. J., Freidlos, F. & Knox, R. J. ( 1986; ). CB 1954 (2,4-dinitro-5-aziridinyl benzamide) becomes a DNA interstrand crosslinking agent in Walker tumour cells. Biochem Biophys Res Commun 140, 1073-1078.[CrossRef]
    [Google Scholar]
  32. Rosenkranz, E. J., McCoy, E. C., Mermelstein, R. & Rosenkranz, H. S. ( 1982; ). Evidence for the existence of distinct nitroreductases in Salmonella typhimurium: roles in mutagenesis. Carcinogenesis 3, 121-123.[CrossRef]
    [Google Scholar]
  33. Rubinstein, L. V., Shoemaker, R. H., Paull, K. D., Simon, R. M., Tosini, S., Skehan, P., Scudiero, D. A., Monks, A. & Boyd, M. R. ( 1990; ). Comparison of in vitro anti-cancer-drug-screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumour cell lines. J Natl Cancer Inst 82, 113-118.
    [Google Scholar]
  34. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  35. Santini, M. T. & Rainaldi, G. ( 1999; ). Three-dimensional spheroid model in tumour biology. Pathobiology 67, 148-157.[CrossRef]
    [Google Scholar]
  36. Searle, P. F., Weedon, S. J., McNeish, I. A., Gilligan, M. G., Ford, M. J., Friedlos, F., Springer, C. J., Young, L. S. & Kerr, D. J. ( 1998; ). Sensitisation of human ovarian cancer cells to killing by the prodrug CB1954 following retroviral or adenoviral transfer of the E. coli nitroreductase gene. Adv Exp Med Biol 451, 107-113.
    [Google Scholar]
  37. Siim, B. G., Denny, W. A. & Wilson, W. R. ( 1997; ). Nitro reduction as an electronic switch for bioreductive drug activation. Oncol Res 9, 357-369.
    [Google Scholar]
  38. Whelan, S. M., Elmore, M. J., Bodsworth, N. J., Atkinson, T. & Minton, N. P. ( 1992; ). The complete amino acid sequence of the Clostridium botulinum type-E neurotoxin, derived by nucleotide-sequence analysis of the encoding gene. Eur J Biochem 204, 657-667.[CrossRef]
    [Google Scholar]
  39. Wu, K. B., Knox, R., Sun, X. Z., Joseph, P., Jaiswal, A. K., Zhang, D., Deng, P. S. & Chen, S. ( 1997; ). Catalytic properties of NAD(P)H:quinone oxidoreductase-2 (NQ02), a dihydronicotinamide riboside dependent oxidoreductase. Arch Biochem Biophys 347, 221-228.[CrossRef]
    [Google Scholar]
  40. Zenno, S., Koike, H., Tanokura, M. & Saigo, K. ( 1996; ). Gene cloning, purification, and characterization of NfsB, a minor oxygen-insensitive nitroreductase from Escherichia coli, similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri. J Biochem 120, 736-744.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-1-297
Loading
/content/journal/micro/10.1099/00221287-148-1-297
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error