US EPA

4239870 

Journal Article 

Engineering a Multifunctional Nitroreductase for Improved Activation of Prodrugs and PET Probes for Cancer Gene Therapy 

Copp, JN; Mowday, AM; Williams, EM; Guise, CP; Ashoorzadeh, A; Sharrock, AV; Flanagan, JU; Smaill, JB; Patterson, AV; Ackerley, DF 

2017 

1 

Cell Chemical Biology
ISSN: 2451-9456
EISSN: 2451-9448 

CELL PRESS 

CAMBRIDGE 

24 

3 

391-403 

English 

28262557 

10.1016/j.chembiol.2017.02.005 

WOS:000397424600016 

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.