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

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.

Figure 1

Sensitisation of Escherichia coli to CB1954 by the expression of NfsA or NfsB. Log phase cultures of E. coli UT5600 stably lysogenised with bacteriophage λ-vectors expressing NfsA or NfsB, or empty vector as control, were diluted and plated on agar containing 0, 50, 100, 200 or 300 μM CB1954. Colonies were counted after 24 h, and expressed as a percentage (%) of the number obtained from the same liquid culture plated in the absence of a prodrug (mean and range of duplicates).

Figure 2

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.

Figure 3

Sensitisation of human ovarian carcinoma cells to CB1954 by NfsA or NfsB. (A) SKOV3 cells were incubated in a medium containing 50 μMCB1954, 200 μM NADH or NADPH and a range of concentrations of purified NfsA or NfsB proteins. The components were mixed at the start of a 4-h incubation period at 37°C, after which the medium was changed. Cell viability was determined 2 days later using the MTT assay. One representative experiment of three is shown. (B) To confirm the enzyme concentrations used in panel A, 200 and 50 ng of the purified proteins were analysed by SDS-PAGE and stained with Coomassie blue. (C) SKOV3 cells were infected using 0, 30, 100 or 300 p.f.u. per cell of adenovirus vectors CTL102 or AdSV042, expressing NfsB or NfsA, respectively. After 2 days, they were exposed to a range of CB1954 concentrations for 4 h, before determination of cell viability using MTT assay 2 days later. (The values used are representative of at least three experiments.)

Figure 4

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.

Figure 5

Bystander effect with NfsA vs NfsB. (A) ‘Activator cells’, that is, SKOV3 cells stably expressing either NfsA or NfsB, parental SKOV3 cells and cell mixtures containing 50, 25, 12.5, 6.25, 3.12 and 1.56% of the activator cells mixed with SKOV3 target cells, were exposed to a range of CB1954 concentrations, and their subsequent survival determined using the MTT assay; symbols show the mean and range of duplicate wells. For clarity, only dose-response curves for 25 and 6.25% activator cells are shown. (B) The IC₅₀s (±95% CI), derived from the MTT assay cell survival curves in panel A are plotted against percentage (%) activator cells in the mixture. The horizontal lines indicate the IC₅₀s of parental SKOV3 cells (upper line), pure cultures of SKOV3-NfsA and SKOV3-NfsB (lower line), and the prodrug concentration midway between these on a log scale.