Richard J. Knox

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.

Walker tumour cells in vivo or in vitro are exceptionally sensitive to the monofunctional alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) (Cobb LM et al., Biochem Pharmacol 18: 1519-1527, 1969). CB 1954 forms DNA interstrand crosslinks in a time-dependent manner in Walker tumour cells but not in non-toxically affected Chinese hamster V79 cells [(Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986)]. However, co-culturing Chinese hamster V79 cells with Walker cells in the presence of CB 1954 renders the hamster cells sensitive to CB 1954 and leads to the formation of interstrand crosslinks in their DNA, findings indicative of the formation by Walker cells of a diffusible toxic metabolite of CB 1954. A flavoprotein, of molecular weight 33.5 kDa as estimated by SDS-polyacrylamide gel electrophoresis, has been isolated from Walker cells and identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, EC 1.6.99.2). This enzyme, in the presence of NADH or NADPH, catalyses the aerobic reduction of CB 1954 to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. This new compound can form interstrand crosslinks in the DNA of Chinese hamster V79 cells to which it is also highly toxic.

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)☆

A nitroreductase enzyme has been isolated from Walker 256 rat carcinoma cells which can convert 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to a cytotoxic DNA interstrand crosslinking agent by reduction of its 4-nitro group to the corresponding hydroxylamino species (Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986; Knox RJ et al., Biochem Pharmacol 37: 4661-4669, 1988). The enzyme has now been identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, menadione reductase (NMOR), phylloquinone reductase, quinone reductase, EC 1.6.99.2) by comparison of partial protein sequences, coenzymes, substrate and inhibitor specificities, and spectroscopic data. 2-Phenyl-5(4)-aminoimidazole-4(5)-carboxamide and 5(4)-aminoimidazole-4(5)-carboxamide were shown to be inhibitors of the isolated Walker cell enzyme. This observation could explain the reported antagonistic action of the aminoimidazole carboxamides to the antitumour effects of CB 1954.

The bioactivation of 5-(aziridin-1-yl)2,4-dinitrobenzamide (CB1954). II: A comparison of an Escherichia coli nitroreductase and walker DT diaphorase

A nitroreductase enzyme that has been isolated from Escherichia coli B is capable of bioactivating CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] to a cytotoxic agent, a property shared with the mammalian enzyme Walker DT diaphorase [NAD(P)H dehydrogenase (quinone), EC 1.6.99.2] as isolated from Walker cells. In contrast to Walker DT diaphorase, which can only reduce the 4-nitro group of CB1954, the E. coli nitroreductase can reduce either (but not both) nitro groups of CB1954 to the corresponding hydroxylamino species. The two hydroxylamino species are formed in equal proportions and at the same rates. CB1954 is reduced much more rapidly by the E. coli nitroreductase than by Walker DT diaphorase. If the reduction of CB1954 was carried out in the presence of V79 cells (which are insensitive to CB1954) a large cytotoxic effect was evident. This cytotoxicity was only observed under conditions in which the E. coli nitroreductase or Walker DT diaphorase reduced the drug. It is proposed that E. coli B nitroreductase would be a suitable enzyme for antibody-directed enzyme prodrug therapy (ADEPT) in combination with CB1954.

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)

DT diaphorase (NAD(P)H dehydrogenase (quinone), EC 1.6.99.2) isolated from Walker 256 rat carcinoma cells can convert CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) to a cytotoxic DNA interstrand cross-linking agent. This is achieved by reduction of the 4-nitro group of CB 1954 to produce the hydroxylamino species, a bioactivation which accounts for the much greater sensitivity of Walker cells to CB 1954 when compared with other cells which are unable to carry out this reduction (Knox et al., Biochem Pharmacol 37: 4661-4669 and 4671-4677, 1988). As predicted from their measured DT diaphorase activities a number of rat hepatoma and hepatocyte cell lines were also shown to be sensitive to CB 1954. However, no CB 1954-sensitive cell lines of human origin were found, although levels of DT diaphorase similar to those in the sensitive rat cells were present in these cells. The human cells were as sensitive as rat cells to the active form of CB 1954 (5-(aziridin-1-yl)-4-hydroxyla mino-2-nitrobenzamide). DT diaphorase, purified to homogeneity from human Hep G2 cells, did metabolize CB 1954 to this 4-hydroxylamino product, but the rate of CB 1954 reduction and thus production of the cytotoxic product, was much lower than that of purified Walker enzyme (ratio of Kcat = 6.4). In addition, CB 1954 could be considered an inhibitor of, rather than a substrate for, the human form of DT diaphorase. The purified rat and human DT diaphorases possessed otherwise similar biochemical and molecular properties. These findings explain the decreased sensitivity towards CB 1954 of human cell lines when compared to rat cell lines.

The bioactivation of CB 1954 and its use as a prodrug in antibody-directed enzyme prodrug therapy (ADEPT)

Walker cellsin vivo orin vitro are exceptionally sensitive to the monofunctional alkylating agent CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The basis of the sensitivity is that CB 1954 forms DNA interstrand crosslinks in Walker cells but not in insensitive cells. Crosslink formation is due to the aerobic reduction of CB 1954 to form 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide by the enzyme DT diaphorase. The 4-hydroxylamine can not crosslink DNA directly but requires further activation by a non-enzymatic reaction with a thioester (such as acetyl coenzyme A). As predicted from their measured DT diaphorase activities, a number of rat hepatoma and hepatocyte cell lines are also sensitive to CB 1954. However, no CB 1954-sensitive tumours or cell lines of human origin have been found. This is because the rate of reduction of CB 1954 by the human form of DT diaphorase is much lower than that of the Walker enzyme (ratio of kcat= 6.4). To overcome this intrinsic resistance of human cells towards CB 1954 a number of strategies have been developed. First, analogues have been developed that are more rapidly reduced by the human form of CB 1954. Second, the cytotoxicity of CB 1954 can be potentiated by reduced pyridinium compounds. Third, a CB 1954 activating enzyme can be targeted to human tumours by conjugating it to an antibody (ADEPT). A nitroreductase enzyme has been isolated fromE. coli that can bioactivate CB 1954 much more rapidly than Walker DT diaphorase and is very suitable for ADEPT. Thus CB 1954 may have a role in the therapy of human tumours.

Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase

A nitroreductase isolated and purified from Escherichia coli B has been demonstrated to have potential applications in ADEPT (antibody-directed enzyme prodrug therapy) by its ability in vitro to reduce dinitrobenzamides (e.g. 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bischloroethylamino analogue, SN 23862) to form cytotoxic derivatives. In contrast to CB 1954, in which either nitro group is reducible to the corresponding hydroxylamine, SN 23862 is reduced by the nitroreductase to form only the 2-hydroxylamine. This hydroxylamine can react with S-acetylthiocholine to form a species capable of producing interstrand crosslinks in naked DNA. In terms of ADEPT, SN 23862 has a potential advantage over CB 1954 in that it is not reduced by mammalian DT diaphorases. Therefore, a series of compounds related to SN 23862 has been synthesized, and evaluated as potential prodrugs both by determination of kinetic parameters and by ratio of IC50 against UV4 cells when incubated in the presence of prodrug, with and without the E. coli enzyme and cofactor (NADH). Results from the two studies were generally in good agreement in that compounds showing no increase in cytotoxicity in presence of enzyme and cofactor were not substrates for the enzyme. None of the analogues were activated by DT diaphorase isolated from Walker 256 carcinoma cells. For those compounds which were substrates for the E. coli nitroreductase, there was a positive correlation between kcat and IC50 ratio. Two compounds showed advantageous properties: SN 25261 (with a dihydroxypropylcarboxamide ring substituent) which has a more than 10-fold greater aqueous solubility than SN 23862 whilst retaining similar kinetic characteristics and cytotoxic potency; and SN 25084, where a change in the position of the carboxamide group relative to the mustard resulted in an increased cytotoxicity ratio and kcat compared with SN 23862 (IC50 ratios 214 and 135; kcat values of 75 and 26.4 sec-1, respectively). An analogue (SN 25507) incorporating both these structural changes had an enhanced kcat of 576 sec-1. This study elucidates some of the structural requirements of the enzyme and aids identification of further directions in the search for suitable prodrugs for an ADEPT nitroreductase system.

Bioactivation of CB 1954: Reaction of the active 4-hydroxylamino derivative with thioesters to form the ultimate DNA-DNA interstrand crosslinking species

5-(Aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is the active form of CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). This hydroxylamine is formed by the bioreduction of CB 1954 by the enzyme DT diaphorase and accounts for the highly selective cytotoxicity of this compound. The reason why the hydroxylamine derivative is so cytotoxic is that, in contrast to CB 1954, it can react difunctionally as characterized by the formation of DNA-DNA interstrand crosslinks in cells treated by this agent. However, although the 4-hydroxylamine compound can produce these crosslinks in cells it cannot crosslink naked DNA (Knox et al., Biochem Pharmacol 37: 4661-4669, 1988). We show here that 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide can become a species capable of binding to DNA and producing interstrand crosslinks, by a direct, non-enzymatic reaction with either acetyl coenzyme A, butyl and propyl coenzyme A or S-acetylthiocholine. Coenzyme A itself cannot produce these effects. The major product of the reaction between the 4-hydroxylamine and thioesters was identified as 4-amino-5-(aziridin-1-yl)-2-nitrobenzamide. However, this compound is not capable of producing the above effects and the major DNA reactive species was a minor product of the reaction. It is proposed that the ultimate, DNA reactive, derivative of CB 1954 is 4-(N-acetoxy)-5-(aziridin-1-yl)-2-nitrobenzamide.

Nitrobenzocyclophosphamides as potential prodrugs for bioreductive activation: Synthesis, stability, enzymatic reduction, and antiproliferative activity in cell culture

In efforts to obtain potential anticancer prodrugs for gene-directed enzyme prodrug therapy using Eschericia coli nitroreductase, a series of four benzocyclophosphamide analogues were designed and synthesized incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive activation. All four analogues were found to be stable in phosphate buffer at pH 7.4 and 37 degrees C and were good substrates of E. coli nitroreductase with half lives between 7 and 24 min at pH 7.0 and 37 degrees C. However, only two analogues 6a and 6c, both with a benzylic oxygen in the phosphorinane ring para to the nitro group, showed a modest 33-36-fold enhanced cytotoxicity in E. coli nitroreductase-expressing cells. These results suggest that good substrate activity and the para benzylic oxygen are required for activation by E. coli nitroreductase. Compounds 6a and 6c represent a new structure type for reductive activation and a lead for further modification in the development of better analogues with improved selective toxicity to be used in gene-directed enzyme prodrug therapy.

Structure-function studies of DT-diaphorase (NQO1) and NRH:quinone oxidoreductase (NQO2)

DT-diaphorase, also referred to as NQO1 or NAD(P)H: quinone acceptor oxidoreductase, is a flavoprotein that catalyzes the two-electron reduction of quinones and quinonoid compounds to hydroquinones, using either NADH or NADPH as the electron donor. NRH (dihydronicotinamide riboside): quinone oxidoreductase, also referred to as NQO2, has a high nucleotide sequence identity to DT-diaphorase and is considered to be an isozyme of DT-diaphorase. These enzymes transfer two electrons to a quinone, resulting in the formation of a hydroquinone product without the accumulation of a dissociated semiquinone. Steady and rapid-reaction kinetic experiments have been performed to determine the reaction mechanism of DT-diaphorase. Furthermore, chimeric and site-directed mutagenesis experiments have been performed to determine the molecular basis of the catalytic differences between the two isozymes and to identify the critical amino acid residues that interact with various inhibitors of the enzymes. In addition, functional studies of a natural occurring mutant Pro-187 to Ser (P187S) have been carried out. Results obtained from these investigations are summarized and discussed.

CB 1954 (2,4-dinitro-5-aziridinyl benzamide) becomes a DNA interstrand crosslinking agent in Walker tumour cells

Walker tumour cells were shown to be uniquely sensitive to CB 1954 when compared with other cells in vitro. CB 1954 forms DNA-DNA interstrand crosslinks in a time-dependent manner in Walker tumour but not Chinese hamster cells. The absence of interstrand crosslinks in hamster cells was not due to a lack of uptake of drug but rather to a failure to convert (probably by bioreduction) CB 1954 to the required reactive difunctional intermediate.