Engineering Escherichia coli NfsB To Activate a Hypoxia-Resistant Analogue of the PET Probe EF5 To Enable Non-Invasive Imaging during Enzyme Prodrug Therapy

Abstract

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 Escherichia coli (NfsB_Ec) has been a particular focal point 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 analogue of 2-nitroimidazole hypoxia probe EF5, has potential 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 assayguided sequence and structure analysis to identify two conserved residues that block SN33623 activation in NfsB_Ec and close homologues. Introduction of the rational substitutions F70A and F108Y into NfsB_Ec conferred high levels of SN33623 activity and enabled specific labeling of E. coli expressing the engineered enzyme. Serendipitously, the F70A and F108Y substitutions also substantially improved activity with the anticancer prodrug CB1954 and the 5-nitroimidazole antibiotic prodrug metronidazole, which is a potential biosafety agent for targeted ablation of nitroreductase-expressing vectors.

NTR 2.0: a rationally-engineered prodrug converting enzyme with substantially enhanced efficacy for targeted cell ablation

“…This was a promising finding, as loss-of-activity and gain-of-activity rational mutagenesis previously demonstrated that the residues F70 and F108 impair 5nitroimidazole activity in NfsB_Ec 23 , and these residues are highly conserved in the NfsB_Ec-like enzymes (Supplementary Fig. S1).…”

“…S1). We hypothesized that introducing F70A/F108Y substitutions into NfsB_Vv and the other NfsB_Ec-like enzymes (Y70A/F108Y for NfsB_Pp) would enhance MTZ activity, as was previously found for NfsB_Ec 23 . This proved true in all cases, with host cells expressing the substituted variants being substantially more sensitive to MTZ.…”

“…NfsB_Ec has received near-exclusive attention as a prodrug-converting NTR for both cancer gene therapy and targeted cell ablation applications 1 . However, recent evidence suggests "NTR 1.0" is a relatively inefficient NTR compared to orthologous enzymes 23,33,34 . In particular, we showed that active site residues F70 and F108 impede activity with the 5-nitroimidazole PET probe SN33623 and that rational substitution of these residues yielded generic improvements in 5-nitroimidazole reduction 23 .…”

“…However, recent evidence suggests "NTR 1.0" is a relatively inefficient NTR compared to orthologous enzymes 23,33,34 . In particular, we showed that active site residues F70 and F108 impede activity with the 5-nitroimidazole PET probe SN33623 and that rational substitution of these residues yielded generic improvements in 5-nitroimidazole reduction 23 . Despite also containing F70 and F108 residues, the naturally occurring orthologous enzyme NfsB_Vv was nearly as efficient in activating MTZ as the rationally-engineered NfsB_Ec F70A/F108Y variant (Fig.…”

“…We previously compiled an E. coli gene library of 11 nfsB orthologs and used a DNA damage screen in E. coli host cells to monitor activation of SN33623, a PET imaging probe that shares a 5-nitroimidazole core structure with MTZ 23 . This same library was used here to evaluate ablation efficacy at higher SN33623 doses; relative growth of replicate E. coli cultures was assessed across a dilution series to establish EC50 values.…”