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

© 2018 Elsevier Inc. 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.

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

10.26686/wgtn.17148002.v1 ◽

2021 ◽

Author(s):

◽

Jasmine Chan-Hyams

Keyword(s):

Human Cell ◽

Bacterial Cell ◽

Bystander Effect ◽

Unmet Need ◽

Cell Transfer ◽

Bacterial Strains ◽

Enzyme Prodrug Therapy ◽

Cell To Cell Transfer ◽

Nitro Reduction

<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>

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

10.26686/wgtn.17148002 ◽

2021 ◽

Author(s):

◽

Jasmine Chan-Hyams

Keyword(s):

Human Cell ◽

Bacterial Cell ◽

Bystander Effect ◽

Unmet Need ◽

Cell Transfer ◽

Bacterial Strains ◽

Enzyme Prodrug Therapy ◽

Cell To Cell Transfer ◽

Nitro Reduction

<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>

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Discovery and Characterization of Chemical Compounds That Inhibit the Function of Aspartyl-tRNA Synthetase from Pseudomonas aeruginosa

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555217744559 ◽

2017 ◽

Vol 23 (3) ◽

pp. 294-301 ◽

Cited By ~ 2

Author(s):

Araceli Corona ◽

Stephanie O. Palmer ◽

Regina Zamacona ◽

Benjamin Mendez ◽

Frank B. Dean ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Aspartic Acid ◽

Cell Cultures ◽

Human Cell ◽

Efflux Pump ◽

Chemical Compounds ◽

Opportunistic Pathogen ◽

Trna Synthetase ◽

Bacterial Strains ◽

Human Cell Cultures

Pseudomonas aeruginosa, an opportunistic pathogen, is highly susceptible to developing resistance to multiple antibiotics. The gene encoding aspartyl-tRNA synthetase (AspRS) from P. aeruginosa was cloned and the resulting protein characterized. AspRS was kinetically evaluated, and the KM values for aspartic acid, ATP, and tRNA were 170, 495, and 0.5 μM, respectively. AspRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen 1690 chemical compounds, resulting in the identification of two inhibitory compounds, BT02A02 and BT02C05. The minimum inhibitory concentrations (MICs) were determined against nine clinically relevant bacterial strains, including efflux pump mutant and hypersensitive strains of P. aeruginosa. The compounds displayed broad-spectrum antibacterial activity and inhibited growth of the efflux and hypersensitive strains with MICs of 16 μg/mL. Growth of wild-type strains were unaffected, indicating that efflux was likely responsible for this lack of activity. BT02A02 did not inhibit growth of human cell cultures at any concentration. However, BT02C05 did inhibit human cell cultures with a cytotoxicity concentration (CC50) of 61.6 μg/mL. The compounds did not compete with either aspartic acid or ATP for binding AspRS, indicating that the mechanism of action of the compound occurs outside the active site of aminoacylation.

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Protocol for evaluating the abilities of diverse nitroaromatic prodrug metabolites to exit a model Gram negative bacterial vector

10.26686/wgtn.12444101.v1 ◽

2020 ◽

Author(s):

JVE Chan-Hyams ◽

David Ackerley

Keyword(s):

Escherichia Coli ◽

Flow Cytometry ◽

Bystander Effect ◽

Recipient Strain ◽

Converting Enzyme ◽

Microtitre Plate ◽

Enzyme Prodrug Therapy ◽

Gram Negative ◽

E Coli ◽

Bacterial Vector

© 2020 The Authors Bacterial-directed enzyme-prodrug therapy (BDEPT) uses tumour-tropic bacteria armed with a genetically-encoded prodrug-converting enzyme to sensitise tumours to a systemically-administered prodrug. A strong bystander effect (i.e., efficient bacteria-to-tumour transfer of activated prodrug metabolites) is critical to maximise tumour cell killing and avoid bacterial self-sterilisation. To investigate the bystander effect in bacteria we developed a sensitive screen that utilised two Escherichia coli strains grown in co-culture. The first of these was an activator strain that overexpressed the E. coli nitroreductase NfsA, and the second was a nitroreductase null recipient strain bearing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by genotoxic prodrug metabolites can only occur following their transfer from the activator to the recipient cells. This can be monitored both in fluorescence based microtitre plate assays and by flow-cytometry, enabling modelling of the abilities of diverse nitroaromatic prodrug metabolites to exit a Gram negative vector.

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Protocol for evaluating the abilities of diverse nitroaromatic prodrug metabolites to exit a model Gram negative bacterial vector

10.26686/wgtn.12444101 ◽

2020 ◽

Author(s):

JVE Chan-Hyams ◽

David Ackerley

Keyword(s):

Escherichia Coli ◽

Flow Cytometry ◽

Bystander Effect ◽

Recipient Strain ◽

Converting Enzyme ◽

Microtitre Plate ◽

Enzyme Prodrug Therapy ◽

Gram Negative ◽

E Coli ◽

Bacterial Vector

© 2020 The Authors Bacterial-directed enzyme-prodrug therapy (BDEPT) uses tumour-tropic bacteria armed with a genetically-encoded prodrug-converting enzyme to sensitise tumours to a systemically-administered prodrug. A strong bystander effect (i.e., efficient bacteria-to-tumour transfer of activated prodrug metabolites) is critical to maximise tumour cell killing and avoid bacterial self-sterilisation. To investigate the bystander effect in bacteria we developed a sensitive screen that utilised two Escherichia coli strains grown in co-culture. The first of these was an activator strain that overexpressed the E. coli nitroreductase NfsA, and the second was a nitroreductase null recipient strain bearing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by genotoxic prodrug metabolites can only occur following their transfer from the activator to the recipient cells. This can be monitored both in fluorescence based microtitre plate assays and by flow-cytometry, enabling modelling of the abilities of diverse nitroaromatic prodrug metabolites to exit a Gram negative vector.

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SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL EVALUATION OF 3-FORMYL INDOLE BASED SCHIFF BASES

Journal of Biomedical and Pharmaceutical Research ◽

10.32553/jbpr.v7i6.561 ◽

2018 ◽

Vol 7 (6) ◽

Author(s):

Singh Gurvinder ◽

Singh Prabhsimran ◽

Dhawan R. K.

Keyword(s):

Antimicrobial Activity ◽

Spectral Analysis ◽

Schiff Bases ◽

Antimicrobial Agents ◽

Biological Evaluation ◽

Fungal Strain ◽

Bacterial Strains ◽

Standard Drug ◽

Gram Negative ◽

E Coli

In order to develop new antimicrobial agents, a series of 3-formyl indole based Schiff bases were synthesized by reacting 3-formyl indole(indole-3-carboxaldehyde) with substituted aniline taking ethanol as solvent. The reaction was carried in the presence of small amount of p-toluene sulphonic acid as catalyst.All the synthesized compounds were characterized by IR, 1H-NMR spectral analysis. All the synthesized compounds were evaluated for antimicrobial activity against two gram positive bacterial strains (B. subtilisand S. aureus) and two gram negative bacterial strains (P. aeruginosaand E. coli) and one fungal strain (C. albicans). All the synthesized compounds were found to have moderate to good antimicrobial activity. The standard drug amoxicillin, fluconazole were used for antimicrobial activity. Among the synthesized compounds, the maximum antimicrobial activity was shown by compounds GS04, GS07, GS08 and GS10.

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