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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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HPLC analysis of time course and regiospecificity of CB1954 reduction by SKOV3 cells expressing NfsA or NfsB. (A, C, E) Time courses of CB1954 reduction and accumulation of CB1954 metabolites in extracellular medium (mean and s.e.m of three experiments; key in panel A also applies for panels C and E); no metabolites of CB1954 were detected using SKOV3-GFP cells (panel A). (B, D, F) HPLC traces of medium sampled after 2 h incubation. Peaks marked C and P correspond to CB1954 and phenol red; peaks marked 2 and 4 correspond to the 2-NHOH and 4-NHOH products and those marked 2′ and 4′ indicate the corresponding -NH2 metabolites. Panels A and B represent SKOV3-GFP cells (control); panels C and D represent SKOV3-NfsA cells; and panels E and F represent SKOV3-NfsB cells.
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fig4: HPLC analysis of time course and regiospecificity of CB1954 reduction by SKOV3 cells expressing NfsA or NfsB. (A, C, E) Time courses of CB1954 reduction and accumulation of CB1954 metabolites in extracellular medium (mean and s.e.m of three experiments; key in panel A also applies for panels C and E); no metabolites of CB1954 were detected using SKOV3-GFP cells (panel A). (B, D, F) HPLC traces of medium sampled after 2 h incubation. Peaks marked C and P correspond to CB1954 and phenol red; peaks marked 2 and 4 correspond to the 2-NHOH and 4-NHOH products and those marked 2′ and 4′ indicate the corresponding -NH2 metabolites. Panels A and B represent SKOV3-GFP cells (control); panels C and D represent SKOV3-NfsA cells; and panels E and F represent SKOV3-NfsB cells.

Mentions: To compare the prodrug activation products generated by the enzymes in the intracellular environment, the metabolites released to the medium by SKOV3 cells stably expressing NfsA or NfsB, or control cells (expressing GFP) were examined at different times after the addition of CB1954 (Figures 4A, C and E). Figures 4B, D and F show HPLC traces corresponding to the 2 h time point. The HPLC profiles show some peaks in addition to CB1954 (from media components) present in the supernatant of the control, GFP-expressing cells (Figure 4B). However, there was no reduction in the CB1954 peak during the incubation, and no metabolites of CB1954 were detected in the supernatant from SKOV3-GFP cells (Figure 4A). Two additional major peaks were produced in the medium from SKOV3-NfsA cells (Figure 4D), corresponding to the 2-NHOH and 2-NH2 derivatives of CB1954. As shown in the time course (Figure 4C), accumulation of the 2-NH2 species in supernatant of SKOV3-NfsA cells lags behind that of the 2-NHOH, as expected for a further reduction product from the hydroxylamine. As the amine metabolites of CB1954 are not detected after reduction by purified NfsA or NfsB, they may result from the action of cellular enzymes on the hydroxylamine products initially generated by NfsA or NfsB. Relatively little of the 4-NHOH and derived 4-NH2 species were detectable with SKOV3-NfsA cells; the 2-NO2 reduction products were in 25- to 40-fold excess over the products of 4-NO2 reduction (at 30 min to 2 h). In contrast, with cells expressing NfsB, the levels of 2-NO2 and 4-NO2 reduction products were more similar (only ∼1.7- to 1.8-fold excess of 2-NO2 metabolites, at 1–2 h) (Figures 4E, F). The departure from a 1 : 1 ratio of 2-NO2 and 4-NO2 reduction products released to the medium by SKOV3-NfsB cells may be attributable to greater reactivity of the 4-NHOH species inside the cell. Metabolite ratios were similar in a separate experiment using a more pharmacologically relevant CB1954 concentration (10 μM), which confirmed the preference of NfsA for the reduction of the 2-NO2 group (data not shown). Thus, in cells and with the purified enzyme, NfsA shows a strong bias towards reduction of the 2-NO2 group of CB1954.


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)

HPLC analysis of time course and regiospecificity of CB1954 reduction by SKOV3 cells expressing NfsA or NfsB. (A, C, E) Time courses of CB1954 reduction and accumulation of CB1954 metabolites in extracellular medium (mean and s.e.m of three experiments; key in panel A also applies for panels C and E); no metabolites of CB1954 were detected using SKOV3-GFP cells (panel A). (B, D, F) HPLC traces of medium sampled after 2 h incubation. Peaks marked C and P correspond to CB1954 and phenol red; peaks marked 2 and 4 correspond to the 2-NHOH and 4-NHOH products and those marked 2′ and 4′ indicate the corresponding -NH2 metabolites. Panels A and B represent SKOV3-GFP cells (control); panels C and D represent SKOV3-NfsA cells; and panels E and F represent SKOV3-NfsB cells.
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fig4: HPLC analysis of time course and regiospecificity of CB1954 reduction by SKOV3 cells expressing NfsA or NfsB. (A, C, E) Time courses of CB1954 reduction and accumulation of CB1954 metabolites in extracellular medium (mean and s.e.m of three experiments; key in panel A also applies for panels C and E); no metabolites of CB1954 were detected using SKOV3-GFP cells (panel A). (B, D, F) HPLC traces of medium sampled after 2 h incubation. Peaks marked C and P correspond to CB1954 and phenol red; peaks marked 2 and 4 correspond to the 2-NHOH and 4-NHOH products and those marked 2′ and 4′ indicate the corresponding -NH2 metabolites. Panels A and B represent SKOV3-GFP cells (control); panels C and D represent SKOV3-NfsA cells; and panels E and F represent SKOV3-NfsB cells.
Mentions: To compare the prodrug activation products generated by the enzymes in the intracellular environment, the metabolites released to the medium by SKOV3 cells stably expressing NfsA or NfsB, or control cells (expressing GFP) were examined at different times after the addition of CB1954 (Figures 4A, C and E). Figures 4B, D and F show HPLC traces corresponding to the 2 h time point. The HPLC profiles show some peaks in addition to CB1954 (from media components) present in the supernatant of the control, GFP-expressing cells (Figure 4B). However, there was no reduction in the CB1954 peak during the incubation, and no metabolites of CB1954 were detected in the supernatant from SKOV3-GFP cells (Figure 4A). Two additional major peaks were produced in the medium from SKOV3-NfsA cells (Figure 4D), corresponding to the 2-NHOH and 2-NH2 derivatives of CB1954. As shown in the time course (Figure 4C), accumulation of the 2-NH2 species in supernatant of SKOV3-NfsA cells lags behind that of the 2-NHOH, as expected for a further reduction product from the hydroxylamine. As the amine metabolites of CB1954 are not detected after reduction by purified NfsA or NfsB, they may result from the action of cellular enzymes on the hydroxylamine products initially generated by NfsA or NfsB. Relatively little of the 4-NHOH and derived 4-NH2 species were detectable with SKOV3-NfsA cells; the 2-NO2 reduction products were in 25- to 40-fold excess over the products of 4-NO2 reduction (at 30 min to 2 h). In contrast, with cells expressing NfsB, the levels of 2-NO2 and 4-NO2 reduction products were more similar (only ∼1.7- to 1.8-fold excess of 2-NO2 metabolites, at 1–2 h) (Figures 4E, F). The departure from a 1 : 1 ratio of 2-NO2 and 4-NO2 reduction products released to the medium by SKOV3-NfsB cells may be attributable to greater reactivity of the 4-NHOH species inside the cell. Metabolite ratios were similar in a separate experiment using a more pharmacologically relevant CB1954 concentration (10 μM), which confirmed the preference of NfsA for the reduction of the 2-NO2 group (data not shown). Thus, in cells and with the purified enzyme, NfsA shows a strong bias towards reduction of the 2-NO2 group of CB1954.

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