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Comparative Analysis of Enzyme and Pathway Engineering Strategies for 5FC-Mediated Suicide Gene Therapy Applications

Bacterial and yeast encoded cytosine deaminases (bCD and yCD, respectively) are widely investigated suicide enzymes used in combination with the prodrug 5-fluorocytosine (5FC) to achieve localized cytotoxicity. Yet characteristics such as poor turnover rates of 5FC (bCD) and enzyme thermolability (y...

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Autores principales: Johnson, Adam J., Ardiani, Andressa, Sanchez-Bonilla, Marilyn, Black, Margaret E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3139007/
https://www.ncbi.nlm.nih.gov/pubmed/21394105
http://dx.doi.org/10.1038/cgt.2011.6
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author Johnson, Adam J.
Ardiani, Andressa
Sanchez-Bonilla, Marilyn
Black, Margaret E.
author_facet Johnson, Adam J.
Ardiani, Andressa
Sanchez-Bonilla, Marilyn
Black, Margaret E.
author_sort Johnson, Adam J.
collection PubMed
description Bacterial and yeast encoded cytosine deaminases (bCD and yCD, respectively) are widely investigated suicide enzymes used in combination with the prodrug 5-fluorocytosine (5FC) to achieve localized cytotoxicity. Yet characteristics such as poor turnover rates of 5FC (bCD) and enzyme thermolability (yCD) preclude their full therapeutic potential. We previously applied regio-specific random mutagenesis and computational design to create novel bCD and yCD variants with altered substrate preference (bCD(1525)) or increased thermostability (yCD(double), yCD(triple)) to aid in overcoming these limitations. Others have utilized pathway engineering in which the microbial enzyme uracil phosphoribosyltransferase (UPRT) is fused with its respective CD, creating bCD/bUPRT or yCD/yUPRT. In this study, we evaluated whether the overlay of CD mutants onto their respective CD/UPRT fusion construct would further enhance 5FC activation, cancer cell prodrug sensitivity and bystander activity in vitro and in vivo. We show that all mutant fusion enzymes allowed for significant reductions in IC(50) values relative to their mutant CD counterparts. However, in vivo the CD mutants displayed enhanced tumor growth inhibition capacity relative to the mutant fusions, with bCD(1525) displaying the greatest tumor growth inhibition and bystander activity. In summary, mutant bCD(1525) appears to be the most effective of all bacterial or yeast CD or CD/UPRT enzymes examined and as such is likely to be the best choice to significantly improve the clinical outcome of CD/5FC suicide gene therapy applications.
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spelling pubmed-31390072012-02-01 Comparative Analysis of Enzyme and Pathway Engineering Strategies for 5FC-Mediated Suicide Gene Therapy Applications Johnson, Adam J. Ardiani, Andressa Sanchez-Bonilla, Marilyn Black, Margaret E. Cancer Gene Ther Article Bacterial and yeast encoded cytosine deaminases (bCD and yCD, respectively) are widely investigated suicide enzymes used in combination with the prodrug 5-fluorocytosine (5FC) to achieve localized cytotoxicity. Yet characteristics such as poor turnover rates of 5FC (bCD) and enzyme thermolability (yCD) preclude their full therapeutic potential. We previously applied regio-specific random mutagenesis and computational design to create novel bCD and yCD variants with altered substrate preference (bCD(1525)) or increased thermostability (yCD(double), yCD(triple)) to aid in overcoming these limitations. Others have utilized pathway engineering in which the microbial enzyme uracil phosphoribosyltransferase (UPRT) is fused with its respective CD, creating bCD/bUPRT or yCD/yUPRT. In this study, we evaluated whether the overlay of CD mutants onto their respective CD/UPRT fusion construct would further enhance 5FC activation, cancer cell prodrug sensitivity and bystander activity in vitro and in vivo. We show that all mutant fusion enzymes allowed for significant reductions in IC(50) values relative to their mutant CD counterparts. However, in vivo the CD mutants displayed enhanced tumor growth inhibition capacity relative to the mutant fusions, with bCD(1525) displaying the greatest tumor growth inhibition and bystander activity. In summary, mutant bCD(1525) appears to be the most effective of all bacterial or yeast CD or CD/UPRT enzymes examined and as such is likely to be the best choice to significantly improve the clinical outcome of CD/5FC suicide gene therapy applications. 2011-03-11 2011-08 /pmc/articles/PMC3139007/ /pubmed/21394105 http://dx.doi.org/10.1038/cgt.2011.6 Text en Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms
spellingShingle Article
Johnson, Adam J.
Ardiani, Andressa
Sanchez-Bonilla, Marilyn
Black, Margaret E.
Comparative Analysis of Enzyme and Pathway Engineering Strategies for 5FC-Mediated Suicide Gene Therapy Applications
title Comparative Analysis of Enzyme and Pathway Engineering Strategies for 5FC-Mediated Suicide Gene Therapy Applications
title_full Comparative Analysis of Enzyme and Pathway Engineering Strategies for 5FC-Mediated Suicide Gene Therapy Applications
title_fullStr Comparative Analysis of Enzyme and Pathway Engineering Strategies for 5FC-Mediated Suicide Gene Therapy Applications
title_full_unstemmed Comparative Analysis of Enzyme and Pathway Engineering Strategies for 5FC-Mediated Suicide Gene Therapy Applications
title_short Comparative Analysis of Enzyme and Pathway Engineering Strategies for 5FC-Mediated Suicide Gene Therapy Applications
title_sort comparative analysis of enzyme and pathway engineering strategies for 5fc-mediated suicide gene therapy applications
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3139007/
https://www.ncbi.nlm.nih.gov/pubmed/21394105
http://dx.doi.org/10.1038/cgt.2011.6
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