Skip to main content
SpringerLink
Log in

Directed evolution of the B. subtilis nitroreductase YfkO improves activation of the PET-capable probe SN33623 and CB1954 prodrug

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Akiva E, Copp JN, Tokuriki N, Babbitt PC (2017) Evolutionary and molecular foundations of multiple contemporary functions of the nitroreductase superfamily. Proc Natl Acad Sci USA 114:E9549–E9558

    Article  CAS  Google Scholar 

  • Anderson RF, Smaill JB, Patterson AV, Ashoorzadeh A et al (2014) Compounds and methods for selective imaging and/or ablation. PCT filing WO2014007650A1

  • Chung-Faye G, Palmer D, Anderson D, Clark J et al (2001) Virus-directed, enzyme prodrug therapy with nitroimidazole reductase: a phase I and pharmacokinetic study of its prodrug, CB1954. Clin Cancer Res 7:2662–2668

    CAS  PubMed  Google Scholar 

  • Copp JN, Williams EM, Rich MH, Patterson AV, Smaill JB, Ackerley DF (2014) Toward a high-throughput screening platform for directed evolution of enzymes that activate genotoxic prodrugs. Protein Eng Des Sel 27:399–403

    Article  CAS  Google Scholar 

  • Copp JN, Mowday AM, Williams EM, Guise CP, Ashoorzadeh A, Sharrock AV, Flanagan JU, Smaill JB, Patterson AV, Ackerley DF (2017) Engineering a multifunctional nitroreductase for improved activation of prodrugs and PET probes for cancer gene therapy. Cell Chem Biol 24:391–840

    Article  CAS  Google Scholar 

  • Copp JN, Pletzer D, Brown AS, van der Heijden J et al (2020) Mechanistic understanding enables the rational design of salicylanilide combination therapies for Gram-negative infections. mBio 11:e02068-20. https://doi.org/10.1128/mBio.02068-20

  • Düzgüneş N (2019) Origins of suicide gene therapy. Methods Mol Biol 1895:1–9

    Article  Google Scholar 

  • Hall KR, Robins KJ, Rich MH, Calcott MJ et al (2020) A giant leap in sequence space reveals the intracellular complexities of evolving a new function. BioRxiv. https://doi.org/10.1101/2020.05.27.118489

    Article  PubMed  PubMed Central  Google Scholar 

  • Onion D, Patel P, Pineda RG, James N, Mautner V (2009) Antivector and tumor immune responses following adenovirus-directed enzyme prodrug therapy for the treatment of prostate cancer. Hum Gene Ther 20:1249–1258

    Article  CAS  Google Scholar 

  • Palmer DH, Mautner V, Mirza D, Oliff S et al (2004) Virus-directed enzyme prodrug therapy: intratumoral administration of a replication-deficient adenovirus encoding nitroreductase to patients with resectable liver cancer. J Clin Oncol 22:1546–1552

    Article  CAS  Google Scholar 

  • Patel P, Young JG, Mautner V, Ashdown D et al (2009) A phase I/II clinical trial in localized prostate cancer of an adenovirus expressing nitroreductase with CB1954 [correction of CB1984]. Mol Ther 17:1292–1299

    Article  CAS  Google Scholar 

  • Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612

    Article  CAS  Google Scholar 

  • Prosser GA et al (2010) Discovery and evaluation of Escherichia coli nitroreductases that activate the anti-cancer prodrug CB1954. Biochem Pharmacol 79:678–687

    Article  CAS  Google Scholar 

  • Prosser GA, Copp JN, Mowday AM, Guise CP et al (2013) Creation and screening of a multi-family bacterial oxidoreductase library to discover novel nitroreductases that efficiently activate the bioreductive prodrugs CB1954 and PR-104A. Biochem Pharmacol 85:1091–1103

    Article  CAS  Google Scholar 

  • Rich MH, Sharrock AV, Hall KR, Ackerley DF, MacKichan JK (2018) Evaluation of NfsA-like nitroreductases from Neisseria meningitidis and Bartonella henselae for enzyme-prodrug therapy, targeted cellular ablation, and dinitrotoluene bioremediation. Biotechnol Lett 40(2):359–367

    Article  CAS  Google Scholar 

  • Sambrook JF, Russell D (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385

    Article  CAS  Google Scholar 

  • Sekar TV, Paulmurugan R (2016) Theranostic imaging of cancer gene therapy. Methods Mol Biol 1461:241–254

    Article  CAS  Google Scholar 

  • Vorobyeva AG, Stanton M, Godinat A, Lund KB et al (2015) Development of a bioluminescent nitroreductase probe for preclinical imaging. PLoS One 10:e0131037

    Article  Google Scholar 

  • Williams EM, Little RF, Mowday AM, Rich MH, Chan-Hyams JV, Copp JN, Smaill JB, Patterson AV, Ackerley DF (2015) Nitroreductase gene-directed enzyme prodrug therapy: insights and advances toward clinical utility. Biochem J 471:131–153

    Article  CAS  Google Scholar 

  • Williams EM, Rich MH, Mowday AM, Ashoorzadeh A et al (2019) Engineering Escherichia coli NfsB to activate a hypoxia-resistant analogue of the PET Probe EF5 to enable non-invasive imaging during enzyme prodrug therapy. Biochemistry 58:3700–3710

    Article  CAS  Google Scholar 

  • Yang C, Wang Q, Ding W (2019) Recent progress in the imaging detection of enzyme activities in vivo. RSC Adv 9:25285–25302

    Article  CAS  Google Scholar 

  • Zhao H, Zha W (2006) In vitro ‘sexual’ evolution through the PCR-based staggered extension process (StEP). Nat Protoc 1:1865–1871

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by the Genesis Oncology Trust [contract GOT-1252-RPG to DFA] and the Cancer Society of New Zealand [contract 13/01 to DFA, AVP, JBS]. MHR was supported by a Cancer Society of New Zealand Training Scholarship and Victoria University of Wellington PhD Scholarship.

Supplementary Information

Supplementary Figure 1—Structures of SN33623 and CB1954.

Supplementary Figure 2—SWISS-MODEL amino acid alignment of B. subtilis YfkO.

Supplementary Figure 3—Homology model of B. subtilis YfkO produced using SWISS-MODEL, with PDB structure 6WT2 as a template.

Supplementary Figure 4—FACS histograms depicting the distribution of GFP fluorescent events post-challenge of yfkO-expressing SOS-R4 cells with 5 µM SN33623.

Supplementary Table 1—IC50 measurements in strain SOS-R4 for the 21 most fluorescent clones recovered from the Round 1 FACS sort.

Author information

Author notes

  1. Michelle H. Rich

    Present address: BiOrbic, Bioeconomy Research Centre, University College Dublin, Belfield, Dublin 4, Ireland

Authors and Affiliations

  1. School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand

    Michelle H. Rich, Abigail V. Sharrock & David F. Ackerley

  2. Centre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand

    Abigail V. Sharrock & David F. Ackerley

  3. Auckland Cancer Society Research Centre, University of Auckland, Auckland, New Zealand

    Amir Ashoorzadeh, Adam V. Patterson & Jeff B. Smaill

  4. Maurice Wilkins Centre for Molecular Biodiscovery http://www.mauricewilkinscentre.org/

    Amir Ashoorzadeh, Adam V. Patterson, Jeff B. Smaill & David F. Ackerley

Authors
  1. Michelle H. Rich
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abigail V. Sharrock
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Amir Ashoorzadeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Adam V. Patterson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeff B. Smaill
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. David F. Ackerley
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David F. Ackerley.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 1765.3 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rich, M.H., Sharrock, A.V., Ashoorzadeh, A. et al. Directed evolution of the B. subtilis nitroreductase YfkO improves activation of the PET-capable probe SN33623 and CB1954 prodrug. Biotechnol Lett 43, 203–211 (2021). https://doi.org/10.1007/s10529-020-02992-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-020-02992-0

Keywords

Search

Navigation