Virtual cofactors for an Escherichia coli nitroreductase enzyme: Relevance to reductively activated prodrugs in antibody directed enzyme prodrug therapy (ADEPT)

doi:10.1016/0006-2952(95)00077-D

Biochemical Pharmacology

Volume 49, Issue 11, 26 May 1995, Pages 1641-1647

https://doi.org/10.1016/0006-2952(95)00077-D Get rights and content

Abstract

A nitroreductase enzyme has been isolated from Escherichia coli that has the unusual property of being equally capable of using either NADH or NADPH as a cofactor for the reduction of its substrates which include menadione as well as 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954). This property is shared with the mammalian enzyme, DT diaphorase. The nitroreductase can, like DT diaphorase, also use simple reduced pyridinium compounds as virtual cofactors. The intact NAD(P)H molecule is not required and the simplest quaternary (and therefore reducible) derivative of nicotinamide, 1-methylnicotinamide (reduced), is as effective as NAD(P)H in its ability to act as an electron donor for the nitroreductase. The structure-activity relationship is not identical to that of DT diaphorase and nicotinic acid riboside (reduced) is selective, being active only for the nitroreductase. Irrespective of the virtual cofactor used, the nitroreductase formed the same reduction products of CB 1954 (the 2-and 4-hydroxylamino derivatives in equal proportions). Nicotinic acid riboside (reduced), unlike NADH, was stable to metabolism by serum enzymes and had a plasma half-life of seven minutes in the mouse after an i.v. bolus administration. NADH had an unmeasurably short half-life. Nicotinic acid riboside (reduced) could also be produced in vivo by administration of nicotinic acid 5′-O-benzoyl riboside (reduced). These results demonstrate that the requirement for a cofactor need not be a limitation in the use of reductive enzymes in antibody directed enzyme prodrug therapy (ADEPT). It is proposed that the E. coli nitroreductase would be a suitable enzyme for ADEPT in combination with CB 1954 and a synthetic, enzyme-selective, virtual cofactor such as nicotinic acid riboside (reduced).

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Biochem Pharmacol

(1992)

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Controlling the flow of carbon and reducing power in biological systems is a central theme in metabolic engineering. Often, trade-offs in pushing carbon flux through targeted pathways while operating in conditions agreeable to the host are required due to the central pools of the shared native redox cofactors NAD(P)/H. Noncanonical redox cofactors (NRCs) have emerged as promising tools to transform how engineers develop biotransformation systems. These new-to-Nature redox cofactors have been demonstrated to function orthogonally to the endogenous cofactors, support pathway thermodynamics optimization, and achieve product scopes previously difficult to reach due to endogenous pathway crosstalk. This review will discuss the development of NRC infrastructures, comprising NRC pools, cofactor reduction sources, and cofactor oxidation sinks, the (pool–source–sink) infrastructure.
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Application of NCBs in reductive reactions using flavin-dependent enzymes has proven to be extremely successful during the last few years. Initial attempts were already made in the 1990s by Friedlos and Knox using two flavin mononucleotide (FMN)-dependent enzymes, DT diaphorase and nitroreductase from Escherichia coli to convert semi-synthetic analogues as well as methyl-1,4-dihydronicotinamide (MNAH) with reasonable catalytic efficiency [21,22]. Within the last decade, a remarkably high number of reports indicates the extensive effort, which was devoted to investigating these exciting yellow enzymes in combination with NCBs [19,23–26,27••,28•,29–31,32•,33••,34•].
Nicotinamide cofactor biomimetics (NCBs) belong to a class of compounds that, as the name suggests, mimic the structures and functions of natural nicotinamide cofactors, namely nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate and their corresponding reduced forms. The first set of NCBs was discovered in the 1930s; these were initially used to study the chemical properties of this class of cofactors as well as understand nicotinamide binding of oxidoreductases. Since then, various NCBs, enzymes, and recycling systems have evolved and lately, new NCBs have been developed and used to run biocatalytic reactions.
EORTC-related new drug discovery and development activities: Role of the Pharmacology and Molecular Mechanisms Group
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Several investigators within the SPG and PAMM worked on this concept with nitroreductase as the best described example. Nitroreductase from E. coli can activate the prodrug CB1954 to a bifunctional DNA cross-linking drug enzyme, a concept which was developed at the ICR.11,12 Other prodrugs were developed to improve delivery and uptake by cancer cells, since a frequently observed resistance mechanism for nucleoside analogs is decreased uptake.
The EORTC Pharmacology and Molecular Mechanism Group (PAMM) focuses on applied research to translate basic/fundamental research discoveries in cancer biology into new drug discovery and development. PAMM provides a unique platform on the pharmacology, pharmacokinetics, pharmacodynamics of drug effects, molecular mechanisms of anticancer agents, and drug-related molecular pathology. For these purposes the group stimulates the interaction between basic scientists and clinicians in order to perform translational research on the pharmacology and molecular mechanisms of anticancer agents in Europe. The group has extensive expertise in various disciplines of pharmacology and has developed standards for studies performed in conjunction with clinical trials equivalent to those of good laboratory practice (GLP). The group serves as master organization for other EORTC (sub-)committees in the maximal interest of these groups and of the EORTC as a whole. PAMM merged with Preclinical Therapeutics Models Group (PTMG) in 2000 and with the Screening and Pharmacology Group (SPG) in 2003. The latter group continued as the Drug Discovery Committee within PAMM. The groups have always been involved in the development of anticancer agents, evolving from platinum analogs, anthracyclines, nitrosoureas, antifolates in the 1980's, to drugs derived from natural sources (trabectedin, taxanes) in the 1990's, and anti-signaling drugs, DNA alkylators, in the last decade. Several of these drugs have been registered. Mechanistic studies focused on drug activation/inactivation, target (DNA, receptors) in relation to efficacy and toxicity such as with several antimetabolites (5-fluorouracil, methotrexate), topoisomerase inhibitors (irinotecan), tyrosine kinase inhibitors (imatinib), acridones (C-1311), etc. The group recently included pharmacogenetics in the identification of genetic polymorphisms in order to use this information for personalized therapy.
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Citation Excerpt :
As well as being able to reduce CB 1954, NTR shares some other biochemical properties with NQO1. Like NQO1, NTR is also a quinone reductase (Table I) and utilizes either NADH or NADPH and other reduced nicotinamide analogs as cofactors.60,61 However, it is a much smaller protein (24 kD) than NQO1 (33.5 kD), and there is no obvious sequence homology between the two enzymes.60
This chapter discusses the clinical applications of quinone reductase–mediated nitro-reduction. The quinone reductase enzyme NQO1 catalyzes the reduction of quinines to their hydroquinone form. NQO1 has a number of unusual properties. It can use either NADPH or NADH with equal activity as a cofactor, and it has been shown that simple reduced nicotinamide derivatives can also act as cofactors for this enzyme. This chapter examines the clinical applications of the nitroreductase activity of three quinone reductase enzymes, NQO1, E. coli B nitroreductase and, in particular, NQO2. The nitroreductase properties of these enzymes are particularly relevant to the treatment of cancer, and the rationale for this approach is described in the chapter. With NQO2, an endogenous human enzyme has been discovered with significant nitroreductase activity that can bioactivate a proven anti-tumor prodrug designated CB 1954. Prodrugs activated by nitro-reduction in cancer therapy are described also described in the chapter. The chapter discusses the use of NQO1 as a nitroreductase. The chapter describes the functional characterization of truncated NQO1 and NQO1/NQO2 chimeric enzyme preparations. Bioactivation of CB 1954 by NQO2, and biodistribution of NQO2 in humans is also explained in the chapter.
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2001, Veterinary Journal
Investigations were carried out to identify a suitable prodrug activating system for feline gene therapy with the eventual aim of treating feline thyroid disease and feline neoplasia. The E. coli nitroreductase (NTR)/CB1954 prodrug activating system was evaluated in vitro in feline cells by transient transfection with a nitroreductase expressing construct and subsequent treatment with the prodrug CB1954. The feline cells successfully expressed E. coli nitroreductase, which was able to activate the prodrug CB1954 resulting in cytotoxicity to both transformed and adjacent cells (a bystander effect) in vitro. In the absence of nitroreductase, CB1954 was non-toxic to feline cells. In addition, the nitroreductase gene was expressed in rat thyroid cells under the control of the cell type specific feline thyroglobulin promoter. This paper demonstrates that the E. coli nitroreductase/CB1954 system may be suitable for in vivo feline gene therapy, and further investigations are warranted.
Free radical mechanisms in anti-cancer drug research
2001, Studies in Physical and Theoretical Chemistry
This chapter discusses free radicals derived from xenobiotic molecules, especially drugs of interest in cancer therapy. The chapter focuses on chemical properties and chemical mechanisms in anti-cancer drug research, with an overview of redox characteristics of drugs and radicals. A compilation of reduction potentials of one-electron couples involving free radicals in aqueous solution includes an introductory outline of the quantitative basis for expressing redox properties by means of reduction potentials. In studies of free radicals of drugs, the desire is to produce selected drug radicals, usually involving one-electron oxidation or reduction, which arc putative intermediates in drug activation or metabolism. The redox properties of likely activating enzymes are thus of interest, but selectivity of reaction is the most important. Thus oxidizing drugs by •OH radicals is seldom useful, because the sites of reaction with typical drugs will be multiple. More selective oxidants are used, such as the secondary radicals derived from halide or pseudohalide oxidation by •OH. Reducing radicals are less of a problem, because the radicals from scavenging •OH by formate or 2-propanol usually reduce oxidants to produce the same radicals as cellular reduction systems.

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Biochemical Pharmacology

Abstract

References (25)

Ablation of human choriocarcinoma xenografts in nude mice by antibodydirected enzyme prodrug therapy (ADEPT) with three novel compounds

Eur J Cancer

A novel targeted delivery system utilizing a cephalosporin-oncolytic prodrug activated by an antibody β-lactamase conjugate for the treatment of cancer

Bioorganic Med Chem Lett

Cephalosporin nitrogen mustard carbamate prodrugs for “ADEPT”

Tetrahedron Lett

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

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

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

Biochem Pharmacol

The properties of total adducts and interstrand crosslinks in the DNA of cells treated with CB 1954. Exceptional frequency and stability of the crosslink

Biochem Pharmacol

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

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)

Biochem Pharmacol

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

Biochem Pharmacol

Metabolism of NAD(P)H by blood components. Relevance to bioreductively activated prodrugs in a targeted enzyme therapy system

Biochem Pharmacol

Identification of novel reduced pyridinium derivatives as synthetic co-factors for the enzyme DT diaphorase (NAD(P)H dehydrogenase (quinone), EC 1.6.99.2)

Biochem Pharmacol

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E. coli nitroreductase/CB1954: In vitro studies into a potential system for feline cancer gene therapy

Free radical mechanisms in anti-cancer drug research

Research paperVirtual cofactors for an Escherichia coli nitroreductase enzyme: Relevance to reductively activated prodrugs in antibody directed enzyme prodrug therapy (ADEPT)

Abstract

Eur J Cancer

Bioorganic Med Chem Lett

Tetrahedron Lett

Biochem Pharmacol

Biochem Pharmacol

Biochem Pharmacol

Biochem Pharmacol

Biochem Pharmacol

Biochem Pharmacol

Biochem Pharmacol

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Biochem Pharmacol

Research paper
Virtual cofactors for an Escherichia coli nitroreductase enzyme: Relevance to reductively activated prodrugs in antibody directed enzyme prodrug therapy (ADEPT)