title: Engineering a Multifunctional Nitroreductase for Improved Activation of Prodrugs and PET Probes for Cancer Gene Therapy.
abstract: Gene-directed enzyme-prodrug therapy (GDEPT) is a promising anti-cancer strategy. However, inadequate prodrugs, inefficient prodrug activation, and a lack of non-invasive imaging capabilities have hindered clinical progression. To address these issues, we used a high-throughput Escherichia coli platform to evolve the multifunctional nitroreductase E. coli NfsA for improved activation of a promising next-generation prodrug, PR-104A, as well as clinically relevant nitro-masked positron emission tomography-imaging probes EF5 and HX4, thereby addressing a critical and unmet need for non-invasive bioimaging in nitroreductase GDEPT. The evolved variant performed better in E. coli than in human cells, suggesting optimal usefulness in bacterial rather than viral GDEPT vectors, and highlighting the influence of intracellular environs on enzyme function and the shaping of promiscuous enzyme activities within the "black box" of in vivo evolution. We provide evidence that the dominant contribution to improved PR-104A activity was enhanced affinity for the prodrug over-competing intracellular substrates.
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand; Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand.
authors.forename: Janine N
authors.initials: JN
authors.lastname: Copp
authors.affiliation: Auckland Cancer Society Research Centre, School of Medical Sciences, University of Auckland, Auckland 1023, New Zealand.
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Mowday
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand; Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand.
authors.forename: Elsie M
authors.initials: EM
authors.lastname: Williams
authors.affiliation: Auckland Cancer Society Research Centre, School of Medical Sciences, University of Auckland, Auckland 1023, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Guise
authors.affiliation: Auckland Cancer Society Research Centre, School of Medical Sciences, University of Auckland, Auckland 1023, New Zealand.
authors.forename: Amir
authors.initials: A
authors.lastname: Ashoorzadeh
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand.
authors.forename: Abigail V
authors.initials: AV
authors.lastname: Sharrock
authors.affiliation: Auckland Cancer Society Research Centre, School of Medical Sciences, University of Auckland, Auckland 1023, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand.
authors.forename: Jack U
authors.initials: JU
authors.lastname: Flanagan
authors.affiliation: Auckland Cancer Society Research Centre, School of Medical Sciences, University of Auckland, Auckland 1023, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand.
authors.forename: Jeff B
authors.initials: JB
authors.lastname: Smaill
authors.affiliation: Auckland Cancer Society Research Centre, School of Medical Sciences, University of Auckland, Auckland 1023, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand.
authors.forename: Adam V
authors.initials: AV
authors.lastname: Patterson
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand; Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand. Electronic address: david.ackerley@vuw.ac.nz.
authors.forename: David F
authors.initials: DF
authors.lastname: Ackerley
mesh_terms: D001665:Binding Sites; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017341:Etanidazole / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D006845:Hydrocarbons, Fluorinated / Q000737:chemistry / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism; D020128:Inhibitory Concentration 50; D008795:Metronidazole / Q000737:chemistry / Q000378:metabolism; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000175:diagnosis / Q000473:pathology / Q000628:therapy*; D009588:Nitrogen Mustard Compounds / Q000737:chemistry / Q000378:metabolism*; D009601:Nitroreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049268:Positron-Emission Tomography; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014230:Triazoles / Q000737:chemistry / Q000378:metabolism; D005466:Fluorocarbons
chemical_list: D029968:Escherichia coli Proteins; D017341:Etanidazole; D006845:Hydrocarbons, Fluorinated; D007093:Imidazoles; D008795:Metronidazole; D009588:Nitrogen Mustard Compounds; D009601:Nitroreductases; D011355:Prodrugs; D014230:Triazoles; C562168:HX4 compound; C524170:PR-104A; C096011:2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide; D005466:Fluorocarbons
keywords: BDEPT; GDEPT; PET imaging; PR-104A; SOS response; directed evolution; metabolic interference; nitroimidazole; nitroreductase; substrate promiscuity