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Engineering Escherichia coli NfsB to activate a hypoxia-resistant analog of the PET probe EF5 to enable non-invasive imaging during enzyme-prodrug therapy.

Gene directed enzyme prodrug therapy (GDEPT) uses tumor-tropic vectors to deliver prodrug-converting enzymes such as nitroreductases specifically to the tumor environment. The nitroreductase NfsB from E. coli (NfsB_Ec) has received particular focus for GDEPT and over the past 25 years has been the subject of several engineering studies seeking to improve catalysis of prodrug substrates. To facilitate clinical development there is also a need to enable effective non-invasive imaging capabilities. SN33623, a 5-nitroimidazole analog of the 2-nitroimidazole hypoxia probe EF5, offers prospects for PET imaging exogenously-delivered nitroreductases without generating confounding background due to tumor hypoxia. However, we show here that SN33623 is a poor substrate for NfsB_Ec. To address this, we used assay-guided sequence and structure analysis to identify two conserved residues that block SN33623 activation in NfsB_Ec and close homologs. Introduction of the rational substitutions F70A/F108Y into NfsB_Ec conferred high levels of SN33623 activity and enabled specific labelling of E. coli expressing the engineered enzyme. Serendipitously, the F70A/F108Y substitutions also substantially improved activity with the anti-cancer prodrug CB1954 and the 5-nitroimidazole antibiotic prodrug metronidazole, which is a potential biosafety agent for targeted ablation of nitroreductase-expressing vectors.