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

Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954 The GenBank accession number for the sequence reported in this paper is AF373598.

“…The k cat value of the novel protein is lower than that of the NTR and it is not yet known which of the kinetic parameters will prove to be crucial in an in vivo setting. Nevertheless, the k cat value of B. amyloliquefaciens YwrO is greater than that of the Walker enzyme, which is able to achieve efficient in vitro cytotoxicity using CB 1954 and NADH (Knox et al, 1988b). It may be that a critical concentration of the toxic product is needed in vitro, where a relatively low initial cell density will allow diffusion of the product away into the surrounding medium.…”

“…1) (Anlezark et al, 1995 ;Knox et al, 1988b). The difunctional alkylating agent formed upon reduction with these enzymes can cross-link DNA via a non-enzymic combination with cellular thiols .…”

“…Therefore, CB 1954 has potential applications in cancer therapy. CB 1954 is toxic to Walker rat carcinoma cells due to the presence of a form of NAD(P)H oxidoreductase, DT-diaphorase (DTD ; EC 1;6;99;2) (Knox et al, 1988b), in these cells. The enzyme DTD was originally defined as having the ability to use the cofactors di-and triphosphopyridine nucleotide (NADH and NADPH) equally well.…”

Professor Tom Connors and the development of novel cancer therapies by the Phase I/II Clinical Trials Committee of Cancer Research UK

“…Whilst chemically only a monofunctional alkylating agent (by virtue of its single aziridine function), CB1954 exhibited a dramatic curative and highly selective action against the Walker 256 rat tumour with the highest therapeutic index of any compound studied (TI ¼ 70) (Cobb et al, 1969;Connors and Melzack, 1971). It was subsequently shown that Walker cells could bioactivate CB1954 by aerobic reduction of the 4-nitro group to the corresponding hydroxylamine, which rapidly acylated to a bifunctional alkylating agent (Roberts et al, 1986;Knox et al, 1988bKnox et al, , 1991. The enzyme responsible for reducing CB1954 in rat tissues is DT-diaphorase (NQO1, NAD(P)H dehydrogenase (quinone) (Knox et al, 1988a); however, the human form of DT-diaphorase metabolises CB1954 much less efficiently than rat DT-diaphorase (Boland et al, 1991), and even cells that are high in human DT-diaphorase are insensitive to the drug (Boland et al, 1991;Mehta et al, 1999).…”

Studies relating to the synthesis, enzymatic reduction and cytotoxicity of a series of nitroaromatic prodrugs

“…[1][2][3] In rats, reductive metabolism of the 4-nitro-group by the enzyme NAD(P)H: quinone oxidoreductase 1 (NQO1, also known as DT-diaphorase) resulted in the formation of the corresponding 4-hydroxylamine derivative 2 that subsequently underwent acylation generating the cytotoxic species 3. 2,3 DNA alkylation then occurred through the acylated hydroxylamine-group (via a putative nitrenium species) and presumably the aziridine moiety, thus creating the DNA crosslinks.…”