The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)—I: Purification and properties of a nitroreductase enzyme from Escherichia coli—A potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT)

doi:10.1016/0006-2952(92)90671-5

Biochemical Pharmacology

Volume 44, Issue 12, 15 December 1992, Pages 2289-2295

https://doi.org/10.1016/0006-2952(92)90671-5 Get rights and content

Abstract

A nitroreductase enzyme has been isolated from Escherichia coli B. This enzyme is an FMN-containing flavoprotein with a molecular mass of 24 kDa and requires either NADH or NADPH as a cofactor. Partial protein sequence analysis showed extensive homology with the “classical nitroreductase” of Salmonella typhimurium and a nitroreductase induced in Enterobacter cloacae. In common with the Salmonella enzyme, the E. coli B enzyme is capable of reducing nitrofurazone. The E. coli nitroreductase is also capable of reducing the anti-tumour agent CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], a property shared with the mammalian enzyme DT diaphorase [NAD(P)H dehydrogenase (quinone)] as isolated species. Both enzymes also share the properties of being able to reduce quinones and are both inhibited by dicoumarol. The nitroreductase is a more active against CB1954 (k_cat=360 min⁻) han Walker DT diaphorase (K_cat = 4 min⁻¹) and also has a lower K_m for NADH (6 vs 75 μM).

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A sensitive method for the detection of mutagenic nitroarenes: construction of nitroreductase-overproducing derivatives of Salmonella typhimurium strains TA98 and TA100

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Chemical screening reveals Ronidazole is a superior prodrug to Metronidazole for nitroreductase-induced cell ablation system in zebrafish larvae
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The Metronidazole (MTZ)/nitroreductase (NTR)-mediated cell ablation system is the most commonly used chemical-genetic cell ablation method in zebrafish. This system can specifically ablate target cells under spatial and temporal control. The MTZ/NTR system has become a widely used cell ablation system in biological, developmental, and functional studies. However, the inadequate cell-ablation ability of some cell types and the side effects of high concentration MTZ impede extensive applications of the MTZ/NTR system. In the present study, the US drug collection library was searched to extend the NTR system. Six MTZ analogs were found, and the cell-ablation ability of these analogs was tested in zebrafish larvae. The results revealed that two of the NTR substrates, Furazolidone and Ronidazole, ablated target cells more efficiently than MTZ at lower concentrations. Furthermore, the working concentration of Ronidazole, but not Furazolidone and MTZ, did not affect axonal bridge formation during spinal cord regeneration. Our results, taken together, indicate that Ronidazole is a superior prodrug to MTZ for the NTR system, especially for the study of neuron regeneration in zebrafish larvae.
Interactions of the antioxidant enzymes NAD(P)H: Quinone oxidoreductase 1 (NQO1) and NRH: Quinone oxidoreductase 2 (NQO2) with pharmacological agents, endogenous biochemicals and environmental contaminants
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Citation Excerpt :
However, metabolic activation is necessary to activate it into a potent cytotoxic anticancer agent. NQO1 (DT-diaphorase at the time) was initially found by virtue of its nitroreductase activity to carry out reductive activation of CB1954 (Fig. 4) [51,52]. CB1954 active metabolites (hydroxylamine or nitroso) are cytotoxic since they alkylate DNA and are capable of forming DNA-DNA interstrand crosslinks [153–155].
NAD(P)H: Quinone Oxidoreductase 1 (NQO1) is an antioxidant enzyme that catalyzes the two-electron reduction of several different classes of quinone-like compounds (quinones, quinone imines, nitroaromatics, and azo dyes). One-electron reduction of quinone or quinone-like metabolites is considered to generate semiquinones to initiate redox cycling that is responsible for the generation of reactive oxygen species and oxidative stress and may contribute to the initiation of adverse drug reactions and adverse health effects. On the other hand, the two-electron reduction of quinoid compounds appears important for drug activation (bioreductive activation) via chemical rearrangement or autoxidation. Two-electron reduction decreases quinone levels and opportunities for the generation of reactive species that can deplete intracellular thiol pools. Also, studies have shown that induction or depletion (knockout) of NQO1 were associated with decreased or increased susceptibilities to oxidative stress, respectively. Moreover, another member of the quinone reductase family, NRH: Quinone Oxidoreductase 2 (NQO2), has a significant functional and structural similarity with NQO1. The activity of both antioxidant enzymes, NQO1 and NQO2, becomes critically important when other detoxification pathways are exhausted. Therefore, this article summarizes the interactions of NQO1 and NQO2 with different pharmacological agents, endogenous biochemicals, and environmental contaminants that would be useful in the development of therapeutic approaches to reduce the adverse drug reactions as well as protection against quinone-induced oxidative damage. Also, future directions and areas of further study for NQO1 and NQO2 are discussed.
Metagenomic bioprospecting of novel oxygen insensitive nitroreductase for degradation of nitro aromatic compounds
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Nitroreductases are enzymes that catalyse the reduction of nitro/nitroaromatic compounds. We have employed functional metagenomics, a culture independent technique, to isolate a novel nitroreductase. The metagenomic DNA was isolated directly from pharmaceutical industry effluent (rich in nitrocompounds), purified and size selected. Sequence unbiased fosmid library was constructed using pCC2FOS fosmid vector and subsequently transformed into E.coli EPI300- T1 cells by λ-phage expression. The host cells harbouring the metagenomic DNA were selected for oxygen insensitive nitroreductases, total of 26 colonies which showed positive for nitroreductase expression were selected for invitro assay. The 6 clones which showed maximum activity were selected, sequence of fosmid DNA was determined using Illumina Nextseq platform and reads were assembled. The BLAST analysis of assembled sequence detected the presence of a nitroreductase gene. The metagenome derived oxygen insensitive nitroreductase (NR1) was further cloned and expressed in expression host E.coli BL21. The optimal reaction temperature and pH for the activity of NR1 enzyme was found to be 40 °C and 8.0 respectively. NR1 is found to prefer NADPH over NADH as a cofactor for its reductase activity. Metal ions and enzyme inhibitors are found to have no effect on NR1 activity. Reductase activity of NR1 against various nitro/nitroaromatic compounds were tested and found to have maximum activity against 2,4-6- trinitrotoluene and 4-nitrophenol. NR1 was immobilized on Celite (545), the immobilized enzyme retained more than 50% of its initial activity for 20 cycles of reuse. The isolated nitroreductase needs to be further characterised for the potential application in degradation of nitrocompounds.
Evaluating the abilities of diverse nitroaromatic prodrug metabolites to exit a model Gram negative vector for bacterial-directed enzyme-prodrug therapy
2018, Biochemical Pharmacology
Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses and bacteria to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to the prodrug. Nitroreductases, able to activate a range of promising nitroaromatic prodrugs to genotoxic metabolites, are of great interest for GDEPT. The bystander effect (cell-to-cell transfer of activated prodrug metabolites) has been quantified for some nitroaromatic prodrugs in mixed multilayer human cell cultures, however while these provide a good model for viral DEPT (VDEPT) they do not inform on the ability of these prodrug metabolites to exit bacterial vectors (relevant to bacterial-DEPT (BDEPT)). To investigate this we grew two Escherichia coli strains in co-culture; an activator strain expressing the nitroreductase E. coli NfsA and a recipient strain containing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by reduced prodrug metabolites can only occur following their transfer from the activator to the recipient cells. We used this to investigate five clinically relevant prodrugs: metronidazole, CB1954, nitro-CBI-DEI, and two dinitrobenzamide mustard prodrug analogues, PR-104A and SN27686. Consistent with the bystander efficiencies previously measured in human cell multilayers, reduced metronidazole exhibited little bacterial cell-to-cell transfer, whereas nitro-CBI-DEI was passed very efficiently from activator to recipient cells post-reduction. However, in contrast with observations in human cell multilayers, the nitrogen mustard prodrug metabolites were not effectively passed between the two bacterial strains, whereas reduced CB1954 was transferred efficiently. Using nitroreductase enzymes that exhibit different biases for the 2- versus 4-nitro substituents of CB1954, we further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products, consistent with their relative bystander efficiencies in human cell culture. Overall, our data suggest that prodrugs may differ in their suitability for VDEPT versus BDEPT applications and emphasise the importance of evaluating an enzyme-prodrug partnership in an appropriate context for the intended vector.
Rational design of an AKR1C3-resistant analog of PR-104 for enzyme-prodrug therapy
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The clinical stage anti-cancer agent PR-104 has potential utility as a cytotoxic prodrug for exogenous bacterial nitroreductases expressed from replicating vector platforms. However substrate selectivity is compromised due to metabolism by the human one- and two-electron oxidoreductases cytochrome P450 oxidoreductase (POR) and aldo-keto reductase 1C3 (AKR1C3). Using rational drug design we developed a novel mono-nitro analog of PR-104A that is essentially free of this off-target activity in vitro and in vivo. Unlike PR-104A, there was no biologically relevant cytotoxicity in cells engineered to express AKR1C3 or POR, under aerobic or anoxic conditions, respectively. We screened this inert prodrug analog, SN34507, against a type I bacterial nitroreductase library and identified E. coli NfsA as an efficient bioactivator using a DNA damage response assay and recombinant enzyme kinetics. Expression of E. coli NfsA in human colorectal cancer cells led to selective cytotoxicity to SN34507 that was associated with cell cycle arrest and generated a robust ‘bystander effect’ at tissue-like cell densities when only 3% of cells were NfsA positive. Anti-tumor activity of SN35539, the phosphate pre-prodrug of SN34507, was established in ‘mixed’ tumors harboring a minority of NfsA-positive cells and demonstrated marked tumor control following heterogeneous suicide gene expression. These experiments demonstrate that off-target metabolism of PR-104 can be avoided and identify the suicide gene/prodrug partnership of E. coli NfsA/SN35539 as a promising combination for development in armed vectors.

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Biochemical Pharmacology

Abstract

References (22)

2,4-Dinitro-5-ethyleneiminobenzamide (CB1954); a potent and selective inhibitor of the growth of the Walker carcinoma 256

Chem Pharmacol

CB1954 (2,4-dinitro-5-aziridinyl benzamide) becomes a DNA interstrand crosslinking agent in Walker tumour cells

Biochem Biophys Res Commun

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 (CB1954) by a nitroreductase enzyme in Walker carcinoma cells

Biochem Pharmacol

The nitroreductase enzyme in Walker cells that activates 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) 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

DT-diaphorase and cancer chemotherapy

Biochem Pharmacol

The differential response of resistant and sensitive strains of Escherichia coli to the cytotoxic effects of 5-aziridino-2,4-dinitrobenzamide (CB1954)

Chem Biol Interact

Caffeine, aminoimidazolecarboxamide and dicoumarol, inhibitors of NAD(P)H dehydrogenase (quinone) (DT diaphorase), prevent both the cytotoxicity and DNA interstrand crosslinking produced by 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) in Walker cells

Biochem Pharmacol

Characterization of FMN-dependent NADH-quinone reductase induced by menadione in Escherichia coli

Biochim Biophys Acta

Cloning, nucleotide sequence and expression of the nitroreductase gene from Enterobacter cloacae

J Biol Chem

Mutagenicity and genotoxicity of nitroarenes. All nitro-containing chemicals were not created equal

Mutat Res

A sensitive method for the detection of mutagenic nitroarenes: construction of nitroreductase-overproducing derivatives of Salmonella typhimurium strains TA98 and TA100

Mutat Res

Cited by (197)

All-in-one IQ toggle switches with high versatilities for fine-tuning of transgene expression in mammalian cells and tissues

Chemical screening reveals Ronidazole is a superior prodrug to Metronidazole for nitroreductase-induced cell ablation system in zebrafish larvae

Interactions of the antioxidant enzymes NAD(P)H: Quinone oxidoreductase 1 (NQO1) and NRH: Quinone oxidoreductase 2 (NQO2) with pharmacological agents, endogenous biochemicals and environmental contaminants

Metagenomic bioprospecting of novel oxygen insensitive nitroreductase for degradation of nitro aromatic compounds

Evaluating the abilities of diverse nitroaromatic prodrug metabolites to exit a model Gram negative vector for bacterial-directed enzyme-prodrug therapy

Rational design of an AKR1C3-resistant analog of PR-104 for enzyme-prodrug therapy

Research paperThe bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)—I: Purification and properties of a nitroreductase enzyme from Escherichia coli—A potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT)

Abstract

Chem Pharmacol

Biochem Biophys Res Commun

Biochem Pharmacol

Biochem Pharmacol

Biochem Pharmacol

Chem Biol Interact

Biochem Pharmacol

Biochim Biophys Acta

J Biol Chem

Mutat Res

Mutat Res

Research paper
The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)—I: Purification and properties of a nitroreductase enzyme from Escherichia coli—A potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT)