Michelle H Rich1,2, Abigail V Sharrock1,3, Amir Ashoorzadeh4,5, Adam V Patterson4,5, Jeff B Smaill4,5, David F Ackerley6,7,8. 1. School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand. 2. BiOrbic, Bioeconomy Research Centre, University College Dublin, Belfield, Dublin 4, Ireland. 3. Centre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand. 4. Auckland Cancer Society Research Centre, University of Auckland, Auckland, New Zealand. 5. Maurice Wilkins Centre for Molecular Biodiscovery. 6. School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand. david.ackerley@vuw.ac.nz. 7. Centre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand. david.ackerley@vuw.ac.nz. 8. Maurice Wilkins Centre for Molecular Biodiscovery, . david.ackerley@vuw.ac.nz.
Abstract
OBJECTIVES: To use directed evolution to improve YfkO-mediated reduction of the 5-nitroimidazole PET-capable probe SN33623 without impairing conversion of the anti-cancer prodrug CB1954. RESULTS: Two iterations of error-prone PCR, purifying selection, and FACS sorting in a DNA damage quantifying GFP reporter strain were used to identify three YfkO variants able to sensitize E. coli host cells to at least 2.4-fold lower concentrations of SN33623 than the native enzyme. Two of these variants were able to be purified in a functional form, and in vitro assays revealed these were twofold and fourfold improved in kcat/KM with SN33623 over wild type YfkO. Serendipitously, the more-active variant was also nearly fourfold improved in kcat/KM versus wild type YfkO in converting CB1954 to a genotoxic drug. CONCLUSIONS: The enhanced activation of the PET imaging probe SN33623 and CB1954 prodrug exhibited by the lead evolved variant of YfkO offers prospects for improved enzyme-prodrug therapy.
OBJECTIVES: To use directed evolution to improve YfkO-mediated reduction of the 5-nitroimidazole PET-capable probe SN33623 without impairing conversion of the anti-cancer prodrug CB1954. RESULTS: Two iterations of error-prone PCR, purifying selection, and FACS sorting in a DNA damage quantifying GFP reporter strain were used to identify three YfkO variants able to sensitize E. coli host cells to at least 2.4-fold lower concentrations of SN33623 than the native enzyme. Two of these variants were able to be purified in a functional form, and in vitro assays revealed these were twofold and fourfold improved in kcat/KM with SN33623 over wild type YfkO. Serendipitously, the more-active variant was also nearly fourfold improved in kcat/KM versus wild type YfkO in converting CB1954 to a genotoxic drug. CONCLUSIONS: The enhanced activation of the PET imaging probe SN33623 and CB1954 prodrug exhibited by the lead evolved variant of YfkO offers prospects for improved enzyme-prodrug therapy.
Entities:
Keywords:
Cancer gene therapy; Error-prone PCR; GDEPT; Niclosamide; Nitroimidazole; Nitroreductase
Authors: G Chung-Faye; D Palmer; D Anderson; J Clark; M Downes; J Baddeley; S Hussain; P I Murray; P Searle; L Seymour; P A Harris; D Ferry; D J Kerr Journal: Clin Cancer Res Date: 2001-09 Impact factor: 12.531
Authors: Xin Hu; Jonathan H Shrimp; Hui Guo; Miao Xu; Catherine Z Chen; Wei Zhu; Alexey Zakharov; Sankalp Jain; Paul Shinn; Anton Simeonov; Matthew D Hall; Min Shen Journal: bioRxiv Date: 2021-03-17