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 assay-guided 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.

• Authors: Elsie M Williams, Michelle H Rich, Alexandra M Mowday, Amir Ashoorzadeh, Janine N Copp, Christopher P Guise, Robert F Anderson, Jack U Flanagan, Jeff B Smaill, Adam V Patterson, David F Ackerley
• Journal: Biochemistry (Biochemistry) Vol. 58 Issue 35 Pg. 3700-3710 (09 03 2019) ISSN: 1520-4995 [Electronic] United States
• PMID: 31403283 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antineoplastic Agents
• Escherichia coli Proteins
• Hydrocarbons, Fluorinated
• Imidazoles
• Nitroimidazoles
• Prodrugs
• SN33623
• Etanidazole
• 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
• NfsB protein, E coli
• Nitroreductases

Topics

• Antineoplastic Agents (therapeutic use)
• Biosensing Techniques (methods)
• Cell Hypoxia (physiology)
• Drug Monitoring (methods)
• Enzyme Activation
• Escherichia coli (enzymology, genetics)
• Escherichia coli Proteins (genetics, metabolism)
• Etanidazole (analogs & derivatives, chemistry, metabolism)
• Genetic Therapy (methods)
• HCT116 Cells
• Humans
• Hydrocarbons, Fluorinated (chemistry, metabolism)
• Imidazoles (pharmacology, therapeutic use)
• Molecular Imaging (methods)
• Neoplasms (drug therapy, metabolism, pathology)
• Nitroimidazoles (pharmacology, therapeutic use)
• Nitroreductases (genetics, metabolism)
• Positron-Emission Tomography (methods)
• Prodrugs (metabolism, therapeutic use)
• Protein Engineering