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E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954.

Vass SO, Jarrom D, Wilson WR, Hyde EI, Searle PF - Br. J. Cancer (2009)

Bottom Line: We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB.Although NfsB reduces either the 2-NO(2) or 4-NO(2) positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO(2) group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB.NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, UK.

ABSTRACT
Prodrug activation gene therapy is a developing approach to cancer treatment, whereby prodrug-activating enzymes are expressed in tumour cells. After administration of a non-toxic prodrug, its conversion to cytotoxic metabolites directly kills tumour cells expressing the activating enzyme, whereas the local spread of activated metabolites can kill nearby cells lacking the enzyme (bystander cell killing). One promising combination that has entered clinical trials uses the nitroreductase NfsB from Escherichia coli to activate the prodrug, CB1954, to a potent bifunctional alkylating agent. NfsA, the major E. coli nitroreductase, has greater activity with nitrofuran antibiotics, but it has not been compared in the past with NfsB for the activation of CB1954. We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB. Although NfsB reduces either the 2-NO(2) or 4-NO(2) positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO(2) group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB. NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.

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In vitro kinetics of CB1954 activation by purified NfsA and NfsB, using NADH or NADPH as cofactors. Graphs plot initial rate of CB1954 activation (v) normalised by enzyme concentration (E), using 50 μM NAD(P)H at a range of CB1954 concentrations, with Michaelis–Menten curves fitted to the data. (A) Kinetics of NfsA. (B) Kinetics of NfsB (solid lines); for comparison, the dotted line again shows the kinetics of NfsA using NADPH.
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fig2: In vitro kinetics of CB1954 activation by purified NfsA and NfsB, using NADH or NADPH as cofactors. Graphs plot initial rate of CB1954 activation (v) normalised by enzyme concentration (E), using 50 μM NAD(P)H at a range of CB1954 concentrations, with Michaelis–Menten curves fitted to the data. (A) Kinetics of NfsA. (B) Kinetics of NfsB (solid lines); for comparison, the dotted line again shows the kinetics of NfsA using NADPH.

Mentions: Figure 2 compares the rates of CB1954 activation by purified NfsA and NfsB as a function of CB1954 concentration, using either NADH or NADPH as a cofactor. In agreement with earlier work (Zenno et al, 1996), NfsA has a higher catalytic activity with NADPH than with NADH. At 50 μM cofactor, this results in ∼8-fold higher maximum rate of CB1954 activation with NADPH (Figure 2A). For NfsB, there is a small preference for NADH over NADPH, with a <2-fold difference in the rate of CB1954 activation for the two cofactors over the achievable solubility range of CB1954 (Figure 2B, solid lines). With its preferred cofactor NADPH, NfsA shows a much greater efficiency of CB1954 activation than NfsB, as shown by the dotted curve in Figure 2B. At the low substrate concentrations achievable in vivo (<10 μM), the rate of reaction is proportional to kcat/Km; for NfsA this is ∼25-fold higher than for NfsB (see Table 1).


E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954.

Vass SO, Jarrom D, Wilson WR, Hyde EI, Searle PF - Br. J. Cancer (2009)

In vitro kinetics of CB1954 activation by purified NfsA and NfsB, using NADH or NADPH as cofactors. Graphs plot initial rate of CB1954 activation (v) normalised by enzyme concentration (E), using 50 μM NAD(P)H at a range of CB1954 concentrations, with Michaelis–Menten curves fitted to the data. (A) Kinetics of NfsA. (B) Kinetics of NfsB (solid lines); for comparison, the dotted line again shows the kinetics of NfsA using NADPH.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2690450&req=5

fig2: In vitro kinetics of CB1954 activation by purified NfsA and NfsB, using NADH or NADPH as cofactors. Graphs plot initial rate of CB1954 activation (v) normalised by enzyme concentration (E), using 50 μM NAD(P)H at a range of CB1954 concentrations, with Michaelis–Menten curves fitted to the data. (A) Kinetics of NfsA. (B) Kinetics of NfsB (solid lines); for comparison, the dotted line again shows the kinetics of NfsA using NADPH.
Mentions: Figure 2 compares the rates of CB1954 activation by purified NfsA and NfsB as a function of CB1954 concentration, using either NADH or NADPH as a cofactor. In agreement with earlier work (Zenno et al, 1996), NfsA has a higher catalytic activity with NADPH than with NADH. At 50 μM cofactor, this results in ∼8-fold higher maximum rate of CB1954 activation with NADPH (Figure 2A). For NfsB, there is a small preference for NADH over NADPH, with a <2-fold difference in the rate of CB1954 activation for the two cofactors over the achievable solubility range of CB1954 (Figure 2B, solid lines). With its preferred cofactor NADPH, NfsA shows a much greater efficiency of CB1954 activation than NfsB, as shown by the dotted curve in Figure 2B. At the low substrate concentrations achievable in vivo (<10 μM), the rate of reaction is proportional to kcat/Km; for NfsA this is ∼25-fold higher than for NfsB (see Table 1).

Bottom Line: We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB.Although NfsB reduces either the 2-NO(2) or 4-NO(2) positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO(2) group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB.NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, UK.

ABSTRACT
Prodrug activation gene therapy is a developing approach to cancer treatment, whereby prodrug-activating enzymes are expressed in tumour cells. After administration of a non-toxic prodrug, its conversion to cytotoxic metabolites directly kills tumour cells expressing the activating enzyme, whereas the local spread of activated metabolites can kill nearby cells lacking the enzyme (bystander cell killing). One promising combination that has entered clinical trials uses the nitroreductase NfsB from Escherichia coli to activate the prodrug, CB1954, to a potent bifunctional alkylating agent. NfsA, the major E. coli nitroreductase, has greater activity with nitrofuran antibiotics, but it has not been compared in the past with NfsB for the activation of CB1954. We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB. Although NfsB reduces either the 2-NO(2) or 4-NO(2) positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO(2) group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB. NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.

Show MeSH
Related in: MedlinePlus