Coli Nitroreductase Research Articles

Synthesis, Antimicrobial, and Anticancer Activities of Novel Nitrofuran Derivatives

Keeping in view the varying therapeutic attributes of 5-nitrofuran and isatin derivatives, novel 5-nitrofuran‒isatin molecular hybrids (2, 5–7) were synthesized by standard protocols, characterized by various spectroscopic techniques, and eventually evaluated against a group of pathogenic bacteria and fungi. Greater potency against Methicillin-resistant Staphylococcus aureus (MRSA) was exhibited by hybrids 5 and 6 with minimum inhibitory concentration values of 8 and 1 μg/mL, respectively. Cytotoxicity against both human embryonic kidney cells (HEK-293) and human red blood cells (RBCs) was investigated for the hybrids in hand. All hybrids appeared to have good safety; all of them were devoid of cytotoxicity, and none displayed hemolytic activity at the highest test concentration (CC50 and HI10 > 32 μg/mL). To support the postulation that these hybrids would be analogous to drugs containing the 5-nitrofurn ring system, molecular docking was carried out to streamline the binding affinity of the investigated hybrids towards the E. coli nitroreductase (NTR). Compared to the standard drug nitrofurazone, hybrid 6 demonstrated a higher affinity and better binding interactions with the NTR binding pocket. Therefore, it could be concluded that 6 displays its antibacterial action through a mechanism similar to that of nitrofurazone. Nonetheless, further wet investigations are to be conducted to confirm this finding. Encouraged by the well-established anticancer activity of isatin derivatives, 2, 5–7 were assessed for their potential antitumor activity, and they well demonstrated potent inhibitory activity against the human colon cancer cell line HCT 116 (IC50 = 1.62–8.8 μM) with isatin hybrid 3 being the best (IC50 = 1.62 μM). Thus, it is herein reported that these 5-nitrofuran‒isatin molecular hybrids could represent an ideal starting point for future studies to develop potent antimicrobial agents.

Molecular Docking Study of Novel Nitrofuran Derivatives As Urinary Tract Anti Infectives

Modern drug design commonly uses molecular docking to understand drug-receptor interaction. For docking tests against 1YLU, an E. coli nitro-reductase, substituted furan derivatives were used in the current work. The study's primary goal is to dock the chosen nitrofuran derivatives onto the protein and compare the results to those of nitrofurantoin as a standard drug. PyRx and the discovery studio visualizer (DSV) application were used to carry out the molecular docking analysis, and E. coli nitro-reductase (1YLU) was retrieved from the protein data bank (PDB) website. All seven of the nitrofuran compounds docking scores were discovered to range between -5.9 and -8.8 Kcal/mol. Compound 2a received the highest binding score, with a score of -8.80 Kcal/mol.At the protein active site, compound 2a interacts with amino acids such as glutamic acid (GLU 165), arginine (ARG 10 & 207), serine (SER 39 & 12), glutamine (GLN 142), and lysine (LYS205).Numerous nitrofuran substituted compounds have been identified for the activity and the nitrofuran derivatives have been discovered to exhibit urinary tract anti-infective activities.

The 3D-structure, kinetics and dynamics of the E. coli nitroreductase NfsA with NADP+ provide glimpses of its catalytic mechanism.

Nitroreductases activate nitroaromatic antibiotics and cancer prodrugs to cytotoxic hydroxylamines and reduce quinones to quinols. Using steady-state and stopped-flow kinetics, we show that the Escherichia coli nitroreductase NfsA is 20-50 fold more active with NADPH than with NADH and that product release may be rate-limiting. The crystal structure of NfsA with NADP+ shows that a mobile loop forms a phosphate-binding pocket. The nicotinamide ring and nicotinamide ribose are mobile, as confirmed in molecular dynamics (MD) simulations. We present a model of NADPH bound to NfsA. Only one NADP+ is seen bound to the NfsA dimers, and MD simulations show that binding of a second NADP(H) cofactor is unfavourable, suggesting that NfsA and other members of this protein superfamily may have a half-of-sites mechanism.

Analogs of nitrofuran antibiotics are potent GroEL/ES inhibitor pro-drugs

In two previous studies, we identified compound 1 as a moderate GroEL/ES inhibitor with weak to moderate antibacterial activity against Gram-positive and Gram-negative bacteria including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, and SM101 Escherichia coli (which has a compromised lipopolysaccharide biosynthetic pathway making bacteria more permeable to drugs). Extending from those studies, we developed two series of analogs with key substructures resembling those of known antibacterials, nitroxoline (hydroxyquinoline moiety) and nifuroxazide/nitrofurantoin (bis-cyclic-N-acylhydrazone scaffolds). Through biochemical and cell-based assays, we identified potent GroEL/ES inhibitors that selectively blocked E. faecium, S. aureus, and E. coli proliferation with low cytotoxicity to human colon and intestine cells in vitro. Initially, only the hydroxyquinoline-bearing analogs were found to be potent inhibitors in our GroEL/ES-mediated substrate refolding assays; however, subsequent testing in the presence of an E. coli nitroreductase (NfsB) in situ indicated that metabolites of the nitrofuran-bearing analogs were potent GroEL/ES inhibitor pro-drugs. Consequently, this study has identified a new target of nitrofuran-containing drugs, and is the first reported instance of such a unique class of GroEL/ES chaperonin inhibitors. The intriguing results presented herein provide impetus for expanded studies to validate inhibitor mechanisms and optimize this antibacterial class using the respective GroEL/ES chaperonin systems and nitroreductases from E. coli and the ESKAPE bacteria.

Cellular pharmacology of evofosfamide (TH-302): A critical re-evaluation of its bystander effects

Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug with proven efficacy against hypoxic cells in preclinical tumour models. TH-302 is designed to release the DNA crosslinking agent bromo-isophosphoramide mustard (Br-IPM) when reduced in hypoxic tissue. Br-IPM is considered to diffuse locally from hypoxic regions, eliciting additional tumour cell killing, but the latter ‘bystander effect’ has not been demonstrated directly. Previous studies with multicellular co-cultures that included cells expressing the E. coli nitroreductase NfsA as a model TH-302 reductase have provided clear evidence of a bystander effect (which we confirm in the present study). However, NfsA is an oxygen-insensitive two-electron reductase that is not expected to generate the nitro radical intermediate that has been demonstrated to fragment to release Br-IPM. Here, we use mass spectrometry methods to characterise TH-302 metabolites generated by one-electron reduction (steady-state radiolysis by ionising radiation and cellular metabolism under hypoxia, including HCT116 cells that overexpress P450 oxidoreductase, POR) or by NfsA expressed in HCT116 cells under oxic conditions, and investigate the stability and cytotoxicity of these products. Br-IPM is shown to have very low cytotoxic potency when added to extracellular culture medium and to be rapidly converted to other hydrophilic products including dichloro-isophosphoramide mustard (IPM). Only traces of Br-IPM or IPM were detected in the extracellular medium when generated by cellular metabolism of TH-302. We identify, in NfsA-expressing cells, the hydroxylamine metabolite of TH-302, and downstream products resulting from rearrangement or hydration of the imidazole ring, and demonstrate that formation of these candidate bystander effect mediators is suppressed by hypoxia. This characterisation of the cellular pharmacology of TH-302 implies that bystander effects from hypoxic activation of TH-302 are unlikely to contribute to its anticancer activity.

PH-Sensitive PEGylated liposomes for delivery of an acidic dinitrobenzamide mustard prodrug: Pathways of internalization, cellular trafficking and cytotoxicity to cancer cells

This paper aims to develop and evaluate a pH-sensitive PEGylated liposomal (pPSL) system for tumor-targeted intracellular delivery of SN25860, a weakly acidic, poorly water-soluble dinitrobenzamide mustard prodrug which is activated by the E. coli nitroreductase nfB. pPSL and non pH-sensitive liposomes (nPSL), as reference, were formulated by thin-film hydration; an active drug loading method was developed with the aid of solubilizers. Cytotoxicity was evaluated in an nfsB-transfected EMT6 mouse mammary carcinoma cell line. Cellular uptake of liposomes was evaluated by both high performance liquid chromatography and flow cytometry. Intracellular trafficking was visualised by confocal microscopy. High drug loading (7.0±0.2% w/w) was achieved after systematic optimization of drug loading conditions. pPSL-SN25860 demonstrated a 21 and 24- fold increase in antiproliferative potency compared to nPSL-SN25860 and free drug, respectively. Cells treated with pPSL had a 1.6–2.5- fold increase in intracellular drug concentration compared to nPSL. This trend was consistent with flow cytometry results. Cells treated with chlorpromazine demonstrated reduced uptake of both nPSL (40%) and pPSL (46%), indicating clathrin-mediated endocytosis was the major pathway. Confocal microscopy showed that pPSL had not only undergone faster and greater endocytosis than nPSL but was also homogeneously distributed in the cytosol and nuclei suggesting endosome escape, in contrast to nPSL.

Optimization of silicon dioxide surface functionalization protocol for designing the receptor layer of a biosensor for detecting explosives

The development of the receptor layer of the biosensor for detecting explosive compounds is described. The covalent modification has been chosen for immobilizing E. coli nitroreductase on the gate oxide of the ion-sensitive field effect transistor (ISFET) that is comprised of silicon dioxide. The self-assembled monolayer technique has been used for immobilization. This method assumes the usage of different silanes and spacer molecules for activating the surface of SiO2. Two different immobilization strategies have been compared, one using asymmetric spacers (3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) and 4-(4-maleimidophenyl)butyric acid N-hydroxysuccinimide ester (SMPB)) and another using a symmetric glutaric dialdehyde linker both accompanied by appropriate silanes. For the first method, the dependence of functionalization efficiency on silane concentration has been studied. The sufficient density of enzyme molecules on the surface of SiO2 has been achieved at a concentration of silane of 0.0015%. The type of asymmetric linker has no influence on immobilization efficiency. The method implying glutaric dialdehyde results in higher activity of the immobilized enzyme. For this method, the immobilization procedure has been optimized. The method has been adapted for immobilization of E. coli nitroreductase inside the channel of a microfluidic system on the surface of ISFET. For this purpose, (3-aminopropyl)triethoxysilane (APTES) has been changed to the corresponding silatrane, and the concentration of the enzyme has been increased to 30 μg/mL. The optimized procedure has been successfully used to develop a biosensor for detecting explosives.

Nitroreductase from Bacillus licheniformis: A stable enzyme for prodrug activation

5-Aziridinyl-2,4-dinitrobenzamide (CB1954) has potential applications in enzyme/prodrug targeted anti-cancer therapies since it can be activated by nitroreductases to form a cytotoxic, bifunctional hydroxylamine derivative. A nitroreductase that can activate CB1954 has been previously isolated from Escherichia coli, but its usefulness is limited by its poor stability and low catalytic efficiency for CB1954. We now report the identification and characterization of a nitroreductase enzyme from the thermophilic bacterium Bacillus licheniformis. Although there is only 28% amino acid sequence identity between this enzyme and the previously isolated E. coli nitroreductase, the two enzymes have a number of characteristics in common. Both enzymes have been shown to reduce both CB1954 and menadione in the presence of NADH and NADPH. However, whereas E. coli nitroreductase produces equimolar amounts of the 2- and 4- hydroxylamine derivative of CB1954, the B. licheniformis enzyme produces only the desired 4-hydroxylamine derivative. It has a preference for NADPH as cosubstrate, and is also active with a range of CB1954 derivatives as substrate and reduced pyridinium cofactor analogues. Moreover, the enzyme is much more thermostable than the E. coli nitroreductase and shows maximum activity at 30 °C. These characteristics suggest that the B. licheniformis nitroreductase may be a possible candidate enzyme for enzyme/prodrug therapies due to its bacterial origin, the high activity observed with CB1954 and its enhanced stability.

Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase

In order to generate a zebrafish model of β cell regeneration, we have expressed an Escherichia coli gene called nfsB in the β cells of embryonic zebrafish. This bacterial gene encodes a nitroreductase (NTR) enzyme, which can convert prodrugs such as metronidazole (Met) to cytotoxins. By fusing nfsB to mCherry, we can simultaneously render β cells susceptible to prodrug and visualize Met dependent cell ablation. We show that the neighboring α and δ cells are unaffected by prodrug treatment and that ablation is β cell specific. Following drug removal and 36 h of recovery, β cells regenerate. Using ptf1a morphants, it is clear that this β cell recovery occurs independently of the presence of the exocrine pancreas. Also, by using photoconvertible Kaede to cell lineage trace and BrdU incorporation to label proliferation, we investigate mechanisms for β regeneration. Therefore, we have developed a unique resource for the study of β cell regeneration in a living vertebrate organism, which will provide the opportunity to conduct large-scale screens for pharmacological and genetic modifiers of β cell regeneration.

Design, Synthesis, and Biological Evaluation of Cyclic and Acyclic Nitrobenzylphosphoramide Mustards for E. coli Nitroreductase Activation

In efforts to obtain anticancer prodrugs for antibody-directed or gene-directed enzyme prodrug therapy using E. coli nitroreductase, a series of nitrobenzylphosphoramide mustards were designed and synthesized incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive activation. All analogues were good substrates of E. coli nitroreductase with half-lives between 2.9 and 11.9 min at pH 7.0 and 37 degrees C. Isomers of the 4-nitrophenylcyclophosphamide analogues 3 and 5 with a benzylic oxygen para to the nitro group showed potent selective cytotoxicity in nitroreductase (NTR) expressing cells, while analogues 4 and 6 with a benzylic nitrogen para to the nitro group showed little selective cytotoxicity despite their good substrate activity. These results suggest that good substrate activity and the benzylic oxygen are both required for reductive activation of 4-nitrophenylcyclophosphamide analogues by E. coli nitroreductase. Isomers of analogue 3 showed 23,000-29,000x selective cytotoxicity toward NTR-expressing V79 cells with an IC(50) as low as 27 nM. They are about as active as and 3-4x more selective than 5-aziridinyl-2,4-dinitrobenzamide (CB1954). The acyclic 4-nitrobenzylphosphoramide mustard ((+/-)-7) was found to be the most active and most selective compound for activation by NTR with 170,000x selective cytotoxicity toward NTR-expressing V79 cells and an IC(50) of 0.4 nM. Compound (+/-)-7also exhibited good bystander effect compared to 5-aziridinyl-2,4-dinitrobenzamide. The low IC(50), high selectivity, and good bystander effects of nitrobenzylphosphoramide mustards in NTR-expressing cells suggest that they could be used in combination with E. coli nitroreductase in enzyme prodrug therapy.

2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT.

An important feature of gene-directed enzyme-prodrug therapy is that prodrug activation can provide diffusible cytotoxic metabolites capable of generating a local bystander effect in tumours. Activation of the aziridinyl dinitrobenzamide CB 1954 by E. coli nitroreductase (NTR) provides a bystander effect assumed to be due to the potently cytotoxic 4-hydroxylamine metabolite. We show that there are four cytotoxic extracellular metabolites of CB 1954 in cultures of NTR-expressing tumour cells (the 2- and 4-hydroxylamines and their corresponding amines). The 4-hydroxylamine is the most cytotoxic in DNA crosslink repair defective cells, but the 2-amino derivative (CB 10-236) is of similar potency to the 4-hydroxylamine in human tumour cell lines. Importantly, CB 10-236 has much superior diffusion properties to the 4-hydroxylamine in multicellular layers grown from the SiHa human cervical carcinoma cell line. These results suggest that the 2-amine, not the 4-hydroxylamine, is the major bystander metabolite when CB 1954 is activated by NTR in tumours. The corresponding dinitrobenzamide nitrogen mustard SN 23862 is reduced by NTR to form a single extracellular metabolite (also the 2-amine), which has superior cytotoxic potency and diffusion properties to the CB 1954 metabolites. These results are consistent with the reported high bystander efficiency of SN 23862 as an NTR prodrug in multicellular layers and tumour xenografts.

Prodrugs in Genetic Chemoradiotherapy

Improvements in the radiotherapeutic management of solid tumors through the concurrent use of gene therapy is a realistic possibility. Of the broad array of candidate genes that have been evaluated, those encoding prodrug-activating enzymes are particularly appealing since they directly complement ongoing clinical chemoradiation regimes. Gene-Directed Enzyme-Prodrug Therapy (GDEPT) only requires a fraction of the target cells to be genetically modified, providing that the resultant cytotoxic prodrug metabolites redistribute efficiently (the bystander effect). This transfer of cytotoxicity to neighboring non-targeted cancer cells is central to the success of any gene therapy strategy, irrespective of the therapeutic gene employed. In the context of genetic chemoradiotherapy, efficient prodrug metabolite diffusion will be a prerequisite for efficient radiosensitization. Some, but not all GDEPT approaches have been analysed in combination with radiotherapy. Examples of prodrugs of clinically established chemotherapeutic agents currently used in conjunction with radiotherapy include: 5-fluorocytosine (5FC), cyclophosphamide (CPA), irinotecan (CPT-11), gemcitabine (dFdC), capecitabine, mitomycin C (MMC) and AQ4N. Other GDEPT paradigms, such as ganciclovir (GCV) and Herpes Simplex thymidine kinase (HSV-tk), dinitrobenzamide (DNB) mustard or aziridinyl analogs and the E. coli nitroreductase (NTR), CMDA or ZP2767P with Pseudomonas aeruginosa carboxypeptidase G2 (CPG2), and indole-3-acetic acid (IAA) activated by horseradish peroxidase (HRP) have no clinically established chemotherapeutic counterpart. Each prodrug is discussed in this review in the context of GDEPT, with a particular attention to translational research and clinical utility in combination with radiotherapy.

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 °C and were good substrates of E. coli nitroreductase with half lives between 7 and 24 min at pH 7.0 and 37 °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.

Inducible ablation of adipocytes in adult transgenic mice expressing the E. coli nitroreductase gene.

We describe the use of an enzyme prodrug system based on E. coli nitroreductase (NTR) to achieve the specific ablation of adipose tissue. Transgenic mice expressing the NTR gene specifically in the adipose tissue were generated using the adipocyte specific promoter aP2. After treatment with the prodrug CB1954 these mice showed extensive cell depletion in all fat depots; this was directly correlated to both the dose of prodrug and the levels of NTR expression. Higher doses of CB1954 resulted in complete disappearance of visible adipose stores in some transgenic mice. These mice exhibited an impaired ability to thermoregulate body temperature. Lower doses of CB1954 resulted in a partial reduction of the adipose tissue leaving non-expressing cells that escape ablation. These animals show normal levels of blood glucose and triglycerides but have reduced leptin levels. After 30 days they were able to regenerate the fat depots and leptin levels returned to normal but, interestingly, no NTR-expressing cells were detectable. The present model provides a new approach to manipulate the number of adipocytes at different stages of mouse development and provides a new system for the study of fat metabolism especially in abnormal conditions such as obesity and its modulation through manipulation of the target cell population.

E. coli Nitroreductase/CB1954: In Vitro Studies into a Potential System for Feline Cancer Gene Therapy

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.

Sensitisation of human ovarian cancer cells to killing by the prodrug CB1954 following retroviral or adenoviral transfer of the E. coli nitroreductase gene.

The enzyme nitroreductase from E. coli can reduce the relatively non-toxic prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) to the 2- or 4-hydroxylamino derivatives; the latter is a potent cytotoxic agent, which following further non-enzymatic reaction with thioesters, leads to the generation of interstrand crosslinks in DNA [1–4] (Figure 1a). These cannot be efficiently repaired by the cell, and lead to cell death. Nitroreductase and CB1954 are therefore of interest as an enzyme-prodrug combination for “virus directed enzyme-prodrug therapy” (VDEPT) of cancer [5, 6]. We describe here the use of retroviral and adenoviral vectors to deliver the gene to human ovarian cancer cells in vitro, and their consequent sensitisation to CB1954.

Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment.

A fundamental obstacle in gene therapy for cancer treatment is the specific delivery of an anticancer gene product to a solid tumor. Although several strategies exist to control gene expression once a vector is directly introduced into a tumor, as yet no systemic delivery system exists that specifically targets solid tumors. Nonpathogenic, obligate anaerobic bacteria of the genus Clostridium have been used experimentally as anticancer agents because of their selective growth in the hypoxic regions of solid tumors after systemic application. In this report we further describe a novel approach to cancer gene therapy in which genetically engineered clostridia are used as tumor-specific vectors for the delivery of antitumor genes. We have introduced into a strain of C. beijerinckii the gene for an E. coli nitroreductase known to activate the nontoxic prodrug CB 1954 to a toxic anticancer drug. Nitroreductase produced by these clostridia enhanced the killing of tumor cells in vitro by CB 1954, by a factor of 22. To demonstrate the specificity of this approach for tumor targeting, we intravenously injected the inactive spore form of C. beijerinckii, which upon transition to a reproductive state will express the E. coli nitroreductase gene. Nitroreductase activity was detectable in 10 of 10 tumors during the first 5 days after intravenous injection of inactive clostridial spores, indicating a rapid transition from spore to reproductive state. Tumors harboring clostridial spores which did not possess the E. coli nitroreductase gene were devoid of nitroreductase activity. Most importantly, E. coli nitroreductase protein was not found in a large survey of normal mouse tissues following intravenous injection of nitroreductase containing clostridia, strongly suggesting that obligate anaerobic bacteria such as clostridia can be utilized as highly specific gene delivery vectors for cancer therapy.

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 ic 50 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 k cat and ic 50 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 k cat compared with SN 23862 ( ic 50 ratios 214 and 135; k cat values of 75 and 26.4 sec −1, respectively). An analogue (SN 25507) incorporating both these structural changes had an enhanced k cat, 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.

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

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).

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 (quinne), 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.