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Discovery and evaluation of Escherichia coli nitroreductases that activate the anti-cancer prodrug CB1954
G A Prosser1, J N Copp, S P Syddall
1School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington, New Zealand.
Abstract:
Gene-directed enzyme prodrug therapy (GDEPT) aims to achieve highly selective tumor-cell killing through the use of tumor-tropic gene delivery vectors coupled with systemic administration of otherwise inert prodrugs. Nitroaromatic prodrugs such as CB1954 hold promise for GDEPT as they are readily reduced to potent DNA alkylating agents by bacterial nitroreductase enzymes (NTRs). Transfection with the nfsB gene from Escherichia coli can increase the sensitivity of tumor cells to CB1954 by greater than 1000-fold. However, poor catalytic efficiency limits the activation of CB1954 by NfsB at clinically relevant doses. A lack of flexible, high-throughput screening technology has hindered efforts to discover superior NTR candidates. Here we demonstrate how the SOS chromotest and complementary screening technologies can be used to evaluate novel enzymes that activate CB1954 and other bioreductive and/or genotoxic prodrugs. We identify the major E. coli NTR, NfsA, as 10-fold more efficient than NfsB in activating CB1954 as purified protein (k(cat)/K(m)) and when over-expressed in an E. coli nfsA(-)/nfsB(-) gene deleted strain. NfsA also confers sensitivity to CB1954 when expressed in HCT-116 human colon carcinoma cells, with similar efficiency to NfsB. In addition, we identify two novel E. coli NTRs, AzoR and NemA, that have not previously been characterized in the context of nitroaromatic prodrug activation.
Insights
Researchers identified a more efficient enzyme, NfsA, for activating the cancer drug CB1954 in gene-directed enzyme prodrug therapy (GDEPT). This discovery could improve cancer treatment by enhancing prodrug activation and tumor cell killing.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Therapy
Background:
- Gene-directed enzyme prodrug therapy (GDEPT) uses tumor-specific gene delivery and prodrugs for selective cancer cell killing.
- Nitroaromatic prodrugs like CB1954 are activated by bacterial nitroreductase enzymes (NTRs) into DNA-alkylating agents.
- Escherichia coli's nfsB gene enhances CB1954 sensitivity but has limited catalytic efficiency for clinical application.
Purpose of the Study:
- To develop and utilize screening technologies for identifying superior NTR candidates for GDEPT.
- To evaluate novel enzymes for their ability to activate CB1954 and other bioreductive prodrugs.
- To compare the efficiency of different E. coli NTRs in activating CB1954.
Main Methods:
- Employed the SOS chromotest and complementary screening technologies to assess novel NTRs.
- Purified and over-expressed E. coli NTRs in a gene-deleted strain for activity assessment.
- Expressed NTRs in HCT-116 human colon carcinoma cells to evaluate in vivo activation.
Main Results:
- Identified E. coli's major NTR, NfsA, as 10-fold more efficient than NfsB in activating CB1954.
- NfsA demonstrated significant CB1954 activation when over-expressed in a bacterial strain lacking nfsA and nfsB.
- NfsA conferred CB1954 sensitivity in HCT-116 cells, comparable to NfsB.
- Discovered two novel E. coli NTRs, AzoR and NemA, with potential for nitroaromatic prodrug activation.
Conclusions:
- NfsA is a superior NTR candidate compared to NfsB for CB1954 activation in GDEPT.
- The developed screening methods are effective for discovering and evaluating novel NTRs.
- AzoR and NemA represent new potential candidates for activating nitroaromatic prodrugs in cancer therapy.
