The characterisation and application of bacterial nitroreductase enzymes

<p>Cancer is an increasing global concern, with the number of people diagnosed growing rapidly each year. Gene directed enzyme prodrug therapy (GDEPT) is emerging as a front-runner of new technologies that seek to combat the growing number of cases. One developing approach to GDEPT involves the use of bacterial nitroreductase enzymes to reduce prodrug substrates, which, upon reduction to their active form, are toxic to cancer cells through DNA crosslinking. Nitroreductases have the ability to activate a variety of nitro-quenched compounds, not only anti-cancer prodrugs, but also nil bystander antibiotics and masked fluorophores, through the reduction of strongly electron-withdrawing nitro substituents on aromatic rings. My research initially sought to exploit this capability by partnering nitroreductases with nil bystander antibiotics for targeted cell ablation, as a component of a larger gene directed enzyme prodrug therapy project. This has potential to provide important safety features for removal of viral and bacterial vectors following anti-cancer gene therapy. From this, the main focus evolved into utilising nitroreductase enzymes for targeted cell ablation for applications in developmental and regenerative biology. This exploited the ability of nitroreductases to activate nil bystander antibiotics in partnership with masked fluorophores for imaging purposes. It has previously been shown that antibiotics can be applied to a nitroreductase under control of a tissue-specific promoter in a transgenic model organism, enabling controlled ablation of that tissue at precise stages of development. However, direct imaging of the nitroreductase location and activity, by application of masked fluorophore probes prior to ablation, has not previously been explored. During the course of this work, several promising combinations of nitroreductases that exhibit opposing specificities for certain combinations of masked fluorophores and nil-bystander antibiotics were identified through screening in bacterial systems. In general, these results were found to translate effectively into eukaryotic cell lines. Pairs of nitroreductases that have opposite specificities for two different antibiotic substrates offer potential for the multiplexed ablation of either (or both) of two different labelled tissues in the same transgenic model organism, according to the substrate(s) administered to that organism. Throughout this screening process, a nitroaromatic substrate (niclosamide) was identified that is, uniquely, initially toxic to Escherichia coli but becomes non-toxic upon reduction of the nitro substituent. Using niclosamide, a novel strategy with potential for identification of new nitroreductases, as well as selection-based directed evolution to improve desired activities, was explored.</p>

The characterisation and application of bacterial nitroreductase enzymes

<p>Cancer is an increasing global concern, with the number of people diagnosed growing rapidly each year. Gene directed enzyme prodrug therapy (GDEPT) is emerging as a front-runner of new technologies that seek to combat the growing number of cases. One developing approach to GDEPT involves the use of bacterial nitroreductase enzymes to reduce prodrug substrates, which, upon reduction to their active form, are toxic to cancer cells through DNA crosslinking. Nitroreductases have the ability to activate a variety of nitro-quenched compounds, not only anti-cancer prodrugs, but also nil bystander antibiotics and masked fluorophores, through the reduction of strongly electron-withdrawing nitro substituents on aromatic rings. My research initially sought to exploit this capability by partnering nitroreductases with nil bystander antibiotics for targeted cell ablation, as a component of a larger gene directed enzyme prodrug therapy project. This has potential to provide important safety features for removal of viral and bacterial vectors following anti-cancer gene therapy. From this, the main focus evolved into utilising nitroreductase enzymes for targeted cell ablation for applications in developmental and regenerative biology. This exploited the ability of nitroreductases to activate nil bystander antibiotics in partnership with masked fluorophores for imaging purposes. It has previously been shown that antibiotics can be applied to a nitroreductase under control of a tissue-specific promoter in a transgenic model organism, enabling controlled ablation of that tissue at precise stages of development. However, direct imaging of the nitroreductase location and activity, by application of masked fluorophore probes prior to ablation, has not previously been explored. During the course of this work, several promising combinations of nitroreductases that exhibit opposing specificities for certain combinations of masked fluorophores and nil-bystander antibiotics were identified through screening in bacterial systems. In general, these results were found to translate effectively into eukaryotic cell lines. Pairs of nitroreductases that have opposite specificities for two different antibiotic substrates offer potential for the multiplexed ablation of either (or both) of two different labelled tissues in the same transgenic model organism, according to the substrate(s) administered to that organism. Throughout this screening process, a nitroaromatic substrate (niclosamide) was identified that is, uniquely, initially toxic to Escherichia coli but becomes non-toxic upon reduction of the nitro substituent. Using niclosamide, a novel strategy with potential for identification of new nitroreductases, as well as selection-based directed evolution to improve desired activities, was explored.</p>

Characterisation and optimisation of nitroreductase-prodrug combinations for bacterial-directed enzyme-prodrug therapy

<p>Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses (VDEPT) and bacteria (BDEPT) to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to a prodrug. Bacterial nitroreductases, which are able to activate a range of anti-cancer nitroaromatic prodrugs to genotoxic metabolites, are of particular interest for GDEPT. The bystander effect is crucial to the success of GDEPT. The bystander effect is a measure of how efficiently activated prodrug metabolites are transferred from gene-expressing cells to neighbouring tissues. This promotes more extensive tumour cell killing. The bystander effect has been quantified for multiple nitroaromatic prodrugs in mixed multilayer human cell cultures. Although this is a good model for VDEPT it cannot simulate the ability of these prodrug metabolites to exit the bacterial vectors relevant to BDEPT. Prior to this work there was an unmet need for an in vitro method of quantifying the bystander effect as it occurs in BDEPT, i.e. a bacterial model of cell-to-cell transfer of activated prodrug metabolites. This thesis presents a method for measuring the bacterial bystander effect in vitro in a microplate based assay that was validated by flow cytometry. In this assay two Escherichia coli strains are grown 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 activators to the recipients. Using this method, the bacterial bystander effect of the clinically relevant prodrugs, metronidazole, CB1954, nitro-CBI-DEI, PR-104A and SN27686, was assessed. Consistent with the bystander efficiencies 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. In contrast with observations in human cell multilayers, the PR-104A and SN27686 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, I further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products. The outcomes of this investigation highlighted the importance of evaluating enzyme-prodrug combinations in models relevant to the intended GDEPT vector, as there can evidently be profound differences in efficacy in different settings. These findings motivated an investigation into the influence of the bystander effect on certain screening strategies used for directed evolution of nitroreductases. It was observed that the bacterial bystander effect can occur during fluorescence activated cell sorting (FACS) of a nitroreductase variant library and negatively impact the recovery of more active variants. Significantly fewer nfsA-expressing cells were recovered from FACS when using CB1954 and nitro-CBI-DEI, when the bystander effect was given time to occur, as compared to controls in which the bystander effect was given no time to occur. In contrast, at the preferred challenge concentrations the mustard prodrugs PR-104A and SN27686 did not yield significantly lower proportions of nfsA-expressing cells under bystander condition. A subsequent investigation compared the evolutionary outcomes arising from screening a nitroreductase variant library using FACS, in which the bystander effect can occur, in parallel to a manual pre-selection method of individual clones for detoxification of structurally divergent nitroaromatic antibiotics. Overall the results of this investigation were inconclusive after just a single round of selection, but there is some evidence that the FACS strategy was more effective than niclosamide/chloramphenicol pre-selection in enriching for superior CB1954-reducing variants. Finally, a panel of nitroreductase candidates was evaluated with the next generation prodrugs PR-104A and SN36506 for possible Clostridia-DEPT development. It was found that the Vibrio vulnificus NfsB F70A/F108Y variant displayed the highest catalytic efficiency with PR-104A reported thus far compared to any other nitroreductase, and was the only NfsB family nitroreductase to exhibit any activity with SN36506 at the purified protein level. At the time this research was performed only NfsB family nitroreductases had been successfully expressed in C. sporogenes by our collaborators, hence the V. vulnificus NfsB F70A/F108Y variant was selected as a promising lead enzyme for further development.</p>

Characterisation and optimisation of nitroreductase-prodrug combinations for bacterial-directed enzyme-prodrug therapy

<p>Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses (VDEPT) and bacteria (BDEPT) to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to a prodrug. Bacterial nitroreductases, which are able to activate a range of anti-cancer nitroaromatic prodrugs to genotoxic metabolites, are of particular interest for GDEPT. The bystander effect is crucial to the success of GDEPT. The bystander effect is a measure of how efficiently activated prodrug metabolites are transferred from gene-expressing cells to neighbouring tissues. This promotes more extensive tumour cell killing. The bystander effect has been quantified for multiple nitroaromatic prodrugs in mixed multilayer human cell cultures. Although this is a good model for VDEPT it cannot simulate the ability of these prodrug metabolites to exit the bacterial vectors relevant to BDEPT. Prior to this work there was an unmet need for an in vitro method of quantifying the bystander effect as it occurs in BDEPT, i.e. a bacterial model of cell-to-cell transfer of activated prodrug metabolites. This thesis presents a method for measuring the bacterial bystander effect in vitro in a microplate based assay that was validated by flow cytometry. In this assay two Escherichia coli strains are grown 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 activators to the recipients. Using this method, the bacterial bystander effect of the clinically relevant prodrugs, metronidazole, CB1954, nitro-CBI-DEI, PR-104A and SN27686, was assessed. Consistent with the bystander efficiencies 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. In contrast with observations in human cell multilayers, the PR-104A and SN27686 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, I further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products. The outcomes of this investigation highlighted the importance of evaluating enzyme-prodrug combinations in models relevant to the intended GDEPT vector, as there can evidently be profound differences in efficacy in different settings. These findings motivated an investigation into the influence of the bystander effect on certain screening strategies used for directed evolution of nitroreductases. It was observed that the bacterial bystander effect can occur during fluorescence activated cell sorting (FACS) of a nitroreductase variant library and negatively impact the recovery of more active variants. Significantly fewer nfsA-expressing cells were recovered from FACS when using CB1954 and nitro-CBI-DEI, when the bystander effect was given time to occur, as compared to controls in which the bystander effect was given no time to occur. In contrast, at the preferred challenge concentrations the mustard prodrugs PR-104A and SN27686 did not yield significantly lower proportions of nfsA-expressing cells under bystander condition. A subsequent investigation compared the evolutionary outcomes arising from screening a nitroreductase variant library using FACS, in which the bystander effect can occur, in parallel to a manual pre-selection method of individual clones for detoxification of structurally divergent nitroaromatic antibiotics. Overall the results of this investigation were inconclusive after just a single round of selection, but there is some evidence that the FACS strategy was more effective than niclosamide/chloramphenicol pre-selection in enriching for superior CB1954-reducing variants. Finally, a panel of nitroreductase candidates was evaluated with the next generation prodrugs PR-104A and SN36506 for possible Clostridia-DEPT development. It was found that the Vibrio vulnificus NfsB F70A/F108Y variant displayed the highest catalytic efficiency with PR-104A reported thus far compared to any other nitroreductase, and was the only NfsB family nitroreductase to exhibit any activity with SN36506 at the purified protein level. At the time this research was performed only NfsB family nitroreductases had been successfully expressed in C. sporogenes by our collaborators, hence the V. vulnificus NfsB F70A/F108Y variant was selected as a promising lead enzyme for further development.</p>

In Vitro and in Vivo Characterisation of P. Aeruginosa Oxidoreductase Enzymes in Pathogenesis and Therapy

<p>Pseudomonas aeruginosa, an increasingly multi-drug resistant human pathogen, is now one of the top three causes of opportunistic infection and there is much interest in identifying novel therapeutic targets for treatment. As a bacterial pathogen, P. aeruginosa encounters innate immune system defences and must continue to adapt to its defence strategies to accommodate the ever-changing environment. Though P. aeruginosa virulence determinants have been heavily characterised over the last several decades, most recent work acknowledges the complex interaction between the human host and the pathogen as an on-going dialogue of virulence factors adapting to the continuum that is the immune response. A major challenge that P. aeruginosa must overcome are reactive oxygen species (ROS) that are released at all stages of infection. Based on previous work which demonstrated a role for soluble nitro- and quinone oxidoreductase (NQOR) enzymes in protecting a related bacterium (Pseudomonas putida) from oxidative stress, we hypothesized that P. aeruginosa would similarly utilize NQORs to withstand ROS. This thesis seeks to understand the role of ROS-protecting enzymes in pathogenesis as well as their potential applications in a therapeutic context. Several NQORs of P. aeruginosa were identified to possess biochemical characteristics consistent with the enzymatic capacity to indirectly reduce reactive species like H₂O₂. However, when individual genes encoding NQORs were deleted from P. aeruginosa, no apparent H₂O₂ sensitivity was seen. In contrast, when candidate genes were over-expressed, certain NQOR enzymes conferred the ability to tolerate H₂O₂ challenge at low concentrations; indicating that these NQORs may play a protective role whose effects are masked in vitro by genetic redundancy as well as a highly active endogenous catalase. By developing a novel in vivo cell culture infection model, the survival of P. aeruginosa post exposure to immunocompetent murine macrophages was also assessed. This not only demonstrated that several putative NQORs were activated in the presence of macrophages but also that an in vivo modelling system is likely to be more appropriate for discovering virulence determinants. In a different aspect of this study it was investigated whether the reductive capacity of the P. aeruginosa-derived NQORs might hold potential for gene-directed enzyme-prodrug therapy (GDEPT). Prodrugs, such as 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) or the nitro-chloromethyl benzindoline SN 26438, are nontoxic in their native form, but become highly toxic upon reduction of their nitro functional groups. The P. aeruginosa NQORs, were tested to identify enzymes capable of efficient activation of CB1954 or SN 26438. Although none of these enzymes exhibited greater activity with CB1954 than the “best in class” Eschericha coli enzymes NfsA or NfsB, the P. aeruginosa NfsB orthologue (PA5190) demonstrated greater than 20-fold improved activity over NfsB from Escherichia coli in its ability to sensitise human cells to SN 26438. This finding offers promise for development of PA5190 and SN 26438 as a novel enzyme-prodrug paradigm for GDEPT.</p>

In Vitro and in Vivo Characterisation of P. Aeruginosa Oxidoreductase Enzymes in Pathogenesis and Therapy

<p>Pseudomonas aeruginosa, an increasingly multi-drug resistant human pathogen, is now one of the top three causes of opportunistic infection and there is much interest in identifying novel therapeutic targets for treatment. As a bacterial pathogen, P. aeruginosa encounters innate immune system defences and must continue to adapt to its defence strategies to accommodate the ever-changing environment. Though P. aeruginosa virulence determinants have been heavily characterised over the last several decades, most recent work acknowledges the complex interaction between the human host and the pathogen as an on-going dialogue of virulence factors adapting to the continuum that is the immune response. A major challenge that P. aeruginosa must overcome are reactive oxygen species (ROS) that are released at all stages of infection. Based on previous work which demonstrated a role for soluble nitro- and quinone oxidoreductase (NQOR) enzymes in protecting a related bacterium (Pseudomonas putida) from oxidative stress, we hypothesized that P. aeruginosa would similarly utilize NQORs to withstand ROS. This thesis seeks to understand the role of ROS-protecting enzymes in pathogenesis as well as their potential applications in a therapeutic context. Several NQORs of P. aeruginosa were identified to possess biochemical characteristics consistent with the enzymatic capacity to indirectly reduce reactive species like H₂O₂. However, when individual genes encoding NQORs were deleted from P. aeruginosa, no apparent H₂O₂ sensitivity was seen. In contrast, when candidate genes were over-expressed, certain NQOR enzymes conferred the ability to tolerate H₂O₂ challenge at low concentrations; indicating that these NQORs may play a protective role whose effects are masked in vitro by genetic redundancy as well as a highly active endogenous catalase. By developing a novel in vivo cell culture infection model, the survival of P. aeruginosa post exposure to immunocompetent murine macrophages was also assessed. This not only demonstrated that several putative NQORs were activated in the presence of macrophages but also that an in vivo modelling system is likely to be more appropriate for discovering virulence determinants. In a different aspect of this study it was investigated whether the reductive capacity of the P. aeruginosa-derived NQORs might hold potential for gene-directed enzyme-prodrug therapy (GDEPT). Prodrugs, such as 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) or the nitro-chloromethyl benzindoline SN 26438, are nontoxic in their native form, but become highly toxic upon reduction of their nitro functional groups. The P. aeruginosa NQORs, were tested to identify enzymes capable of efficient activation of CB1954 or SN 26438. Although none of these enzymes exhibited greater activity with CB1954 than the “best in class” Eschericha coli enzymes NfsA or NfsB, the P. aeruginosa NfsB orthologue (PA5190) demonstrated greater than 20-fold improved activity over NfsB from Escherichia coli in its ability to sensitise human cells to SN 26438. This finding offers promise for development of PA5190 and SN 26438 as a novel enzyme-prodrug paradigm for GDEPT.</p>

Enzyme prodrug therapy: cytotoxic potential of paracetamol turnover with recombinant horseradish peroxidase

Targeted cancer treatment is a promising, less invasive alternative to chemotherapy as it is precisely directed against tumor cells whilst leaving healthy tissue unaffected. The plant-derived enzyme horseradish peroxidase (HRP) can be used for enzyme prodrug cancer therapy with indole-3-acetic acid or the analgesic paracetamol (acetaminophen). Oxidation of paracetamol by HRP in the presence of hydrogen peroxide leads to N-acetyl-p-benzoquinone imine and polymer formation via a radical reaction mechanism. N-acetyl-p-benzoquinone imine binds to DNA and proteins, resulting in severe cytotoxicity. However, plant HRP is not suitable for this application since the foreign glycosylation pattern is recognized by the human immune system, causing rapid clearance from the body. Furthermore, plant-derived HRP is a mixture of isoenzymes with a heterogeneous composition. Here, we investigated the reaction of paracetamol with defined recombinant HRP variants produced in E. coli, as well as plant HRP, and found that they are equally effective in paracetamol oxidation at a concentration ≥ 400 µM. At low paracetamol concentrations, however, recombinant HRP seems to be more efficient in paracetamol oxidation. Yet upon treatment of HCT-116 colon carcinoma and FaDu squamous carcinoma cells with HRP–paracetamol no cytotoxic effect was observed, neither in the presence nor absence of hydrogen peroxide. Graphic abstract

Dendrimers as Non-Viral Vectors in Gene-Directed Enzyme Prodrug Therapy

Gene-directed enzyme prodrug therapy (GDEPT) has been intensively studied as a promising new strategy of prodrug delivery, with its main advantages being represented by an enhanced efficacy and a reduced off-target toxicity of the active drug. In recent years, numerous therapeutic systems based on GDEPT strategy have entered clinical trials. In order to deliver the desired gene at a specific site of action, this therapeutic approach uses vectors divided in two major categories, viral vectors and non-viral vectors, with the latter being represented by chemical delivery agents. There is considerable interest in the development of non-viral vectors due to their decreased immunogenicity, higher specificity, ease of synthesis and greater flexibility for subsequent modulations. Dendrimers used as delivery vehicles offer many advantages, such as: nanoscale size, precise molecular weight, increased solubility, high load capacity, high bioavailability and low immunogenicity. The aim of the present work was to provide a comprehensive overview of the recent advances regarding the use of dendrimers as non-viral carriers in the GDEPT therapy.

Application of Gene-Directed Enzyme Prodrug Therapy in Cancer Treatment

Gene-directed enzyme prodrug therapy (GDEPT) is an advanced cancer therapy that has potential use against localized and metastasized cancer. This strategy aims to improve the limitations of chemotherapy and existing cancer treatments by specific gene delivery, which allows the conversion of systemically administered nontoxic prodrugs to active chemotherapeutic drugs inside the target tumor cells, thereby resulting in a significant therapeutic index by introducing high concentrations of cytotoxic compounds to the tumor cells while limiting the systemic toxicity. The main attraction of GDEPT is by expanding the toxicity to adjacent non-expressing target cancer cells through local and distal bystander effects, leading to tumor regression. This review focused on the application of the six main GDEPT systems for treating cancer, including herpes simplex virus thymidine kinase (HSV-TK) with ganciclovir (GCV), cytosine deaminase (CD) from bacteria or yeast with 5-fluorocytosine (5-FC), E. coli nitroreductase (NfsB) with 5-(aziridin-1-yl)-2,4- initrobenzamide (CB1954), hepatic cytochrome P4l50 (CYP450) with cyclophosphamide (CPA), purine nucleoside phosphorylase (PNP) from E. coli with 6-methylpurine deoxyriboside (MEP), and bacterial carboxypeptidase G2 (CPG2) with 4-[(2-chloroethyl)(2-mesloxyethyl)amino] benzoyl-L-glutamic acid (CMDA). In each system, the mechanism of action, clinical trials for the past decades, limitations, and areas that need improvement are discussed.