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The Flavin Reductase MsuE Is a Novel Nitroreductase that Can Efficiently Activate Two Promising Next-Generation Prodrugs for Gene-Directed Enzyme Prodrug Therapy.

Green LK, Storey MA, Williams EM, Patterson AV, Smaill JB, Copp JN, Ackerley DF - Cancers (Basel) (2013)

Bottom Line: Two promising hypoxia prodrugs for GDEPT are the dinitrobenzamide mustard PR-104A, and the nitrochloromethylbenzindoline prodrug nitro-CBI-DEI.However, MsuE exhibited comparable levels of activity with PR-104A and nitro-CBI-DEI, and is the first nitroreductase outside of the NfsA and NfsB enzyme families to do so.These in vitro findings suggest that MsuE is worthy of further evaluation in in vivo models of GDEPT.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington 6140, New Zealand. david.ackerley@vuw.ac.nz.

ABSTRACT
Bacterial nitroreductase enzymes that can efficiently catalyse the oxygen-independent reduction of prodrugs originally developed to target tumour hypoxia offer great potential for expanding the therapeutic range of these molecules to aerobic tumour regions, via the emerging cancer strategy of gene-directed enzyme prodrug therapy (GDEPT). Two promising hypoxia prodrugs for GDEPT are the dinitrobenzamide mustard PR-104A, and the nitrochloromethylbenzindoline prodrug nitro-CBI-DEI. We describe here use of a nitro-quenched fluorogenic probe to identify MsuE from Pseudomonas aeruginosa as a novel nitroreductase candidate for GDEPT. In SOS and bacteria-delivered enzyme prodrug cytotoxicity assays MsuE was less effective at activating CB1954 (a first-generation GDEPT prodrug) than the "gold standard" nitroreductases NfsA and NfsB from Escherichia coli. However, MsuE exhibited comparable levels of activity with PR-104A and nitro-CBI-DEI, and is the first nitroreductase outside of the NfsA and NfsB enzyme families to do so. These in vitro findings suggest that MsuE is worthy of further evaluation in in vivo models of GDEPT.

No MeSH data available.


Related in: MedlinePlus

Bacteria-delivered enzyme prodrug cytotoxicity assay. Calculated IC50 of HCT-116 cells post-incubation with E. coli 6KO cells (over-expressing either NfsA_Ec, NfsB_Ec, MsuE, or an empty pUCX control) across a 2-fold dilution series of (A) CB1954 (25 µM to 400 µM); or (B) nitro-CBI-DEI (0.06 µM to 15 µM). Percentage cell survival at each prodrug concentration was calculated relative to an unchallenged control by CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Data are the mean of three independent experiments, each performed in duplicate; and error bars are ±1 standard deviation. ** indicates p < 0.005 and *** p < 0.001 by one-way ANOVA with Dunnett comparison of test to control.
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cancers-05-00985-f004: Bacteria-delivered enzyme prodrug cytotoxicity assay. Calculated IC50 of HCT-116 cells post-incubation with E. coli 6KO cells (over-expressing either NfsA_Ec, NfsB_Ec, MsuE, or an empty pUCX control) across a 2-fold dilution series of (A) CB1954 (25 µM to 400 µM); or (B) nitro-CBI-DEI (0.06 µM to 15 µM). Percentage cell survival at each prodrug concentration was calculated relative to an unchallenged control by CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Data are the mean of three independent experiments, each performed in duplicate; and error bars are ±1 standard deviation. ** indicates p < 0.005 and *** p < 0.001 by one-way ANOVA with Dunnett comparison of test to control.

Mentions: In an effort to measure the relative abilities of MsuE, NfsA_Ec and NfsB_Ec to sensitise human colon carcinoma (HCT-116) cells to each prodrug we employed a bacteria-delivered enzyme cytotoxicity assay as previously described [13]. E. coli strains individually over-expressing each enzyme or an empty plasmid control were incubated in co-culture with replicate monolayers of HCT-116 cells, across a range of concentrations of each prodrug, after which IC50 values were measured (i.e., the concentration of prodrug required to reduce the viability of the HCT-116 cells to 50% relative to the no-prodrug control). In this assay system, independent experiments using CB1954 or nitro-CBI-DEI gave highly reproducible results for all strains. Despite MsuE exhibiting only limited activity with CB1954 in SOS assays (Section 2.3), HCT-116 cells co-cultured with the E. coli strain over-expressing this nitroreductase were 5.8-fold more sensitive to CB1954 than cells co-cultured with the empty plasmid control strain (Figure 4A). Consistent with the SOS assays, NfsA_Ec and NfsB_Ec were more effective than MsuE in sensitising HCT-116 cells to CB1954 (Figure 4A). With nitro-CBI-DEI however, MsuE was the most effective nitroreductase in sensitising HCT-116 cells to the administered prodrug (Figure 4B). The IC50 of 260 ± 40 nM measured in this assay system is comparable to that previously described for the most active nitro-CBI-DEI reductase yet to be identified (P. aeruginosa NfsB, 130 nM [17]); and substantially (although not quite significantly; p < 0.06) less than that measured for the best E. coli nitroreductase, NfsA_Ec (1.9 ± 1.2 µM; Figure 4B). Surprisingly, PR-104A was unable to exert a comparable effect in this assay, possibly due to the relatively reactive nature of the metabolites; irrespective of the conditions employed, concentration-dependent IC50 curves could not be obtained for any of the E. coli strains. Overall, based on the results of the SOS and bacteria-delivered enzyme cytotoxicity assays, MsuE appears to be a promising candidate for GDEPT, offering potential to repurpose next-generation prodrugs that have been independently developed as hypoxia activated therapeutics.


The Flavin Reductase MsuE Is a Novel Nitroreductase that Can Efficiently Activate Two Promising Next-Generation Prodrugs for Gene-Directed Enzyme Prodrug Therapy.

Green LK, Storey MA, Williams EM, Patterson AV, Smaill JB, Copp JN, Ackerley DF - Cancers (Basel) (2013)

Bacteria-delivered enzyme prodrug cytotoxicity assay. Calculated IC50 of HCT-116 cells post-incubation with E. coli 6KO cells (over-expressing either NfsA_Ec, NfsB_Ec, MsuE, or an empty pUCX control) across a 2-fold dilution series of (A) CB1954 (25 µM to 400 µM); or (B) nitro-CBI-DEI (0.06 µM to 15 µM). Percentage cell survival at each prodrug concentration was calculated relative to an unchallenged control by CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Data are the mean of three independent experiments, each performed in duplicate; and error bars are ±1 standard deviation. ** indicates p < 0.005 and *** p < 0.001 by one-way ANOVA with Dunnett comparison of test to control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3795375&req=5

cancers-05-00985-f004: Bacteria-delivered enzyme prodrug cytotoxicity assay. Calculated IC50 of HCT-116 cells post-incubation with E. coli 6KO cells (over-expressing either NfsA_Ec, NfsB_Ec, MsuE, or an empty pUCX control) across a 2-fold dilution series of (A) CB1954 (25 µM to 400 µM); or (B) nitro-CBI-DEI (0.06 µM to 15 µM). Percentage cell survival at each prodrug concentration was calculated relative to an unchallenged control by CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Data are the mean of three independent experiments, each performed in duplicate; and error bars are ±1 standard deviation. ** indicates p < 0.005 and *** p < 0.001 by one-way ANOVA with Dunnett comparison of test to control.
Mentions: In an effort to measure the relative abilities of MsuE, NfsA_Ec and NfsB_Ec to sensitise human colon carcinoma (HCT-116) cells to each prodrug we employed a bacteria-delivered enzyme cytotoxicity assay as previously described [13]. E. coli strains individually over-expressing each enzyme or an empty plasmid control were incubated in co-culture with replicate monolayers of HCT-116 cells, across a range of concentrations of each prodrug, after which IC50 values were measured (i.e., the concentration of prodrug required to reduce the viability of the HCT-116 cells to 50% relative to the no-prodrug control). In this assay system, independent experiments using CB1954 or nitro-CBI-DEI gave highly reproducible results for all strains. Despite MsuE exhibiting only limited activity with CB1954 in SOS assays (Section 2.3), HCT-116 cells co-cultured with the E. coli strain over-expressing this nitroreductase were 5.8-fold more sensitive to CB1954 than cells co-cultured with the empty plasmid control strain (Figure 4A). Consistent with the SOS assays, NfsA_Ec and NfsB_Ec were more effective than MsuE in sensitising HCT-116 cells to CB1954 (Figure 4A). With nitro-CBI-DEI however, MsuE was the most effective nitroreductase in sensitising HCT-116 cells to the administered prodrug (Figure 4B). The IC50 of 260 ± 40 nM measured in this assay system is comparable to that previously described for the most active nitro-CBI-DEI reductase yet to be identified (P. aeruginosa NfsB, 130 nM [17]); and substantially (although not quite significantly; p < 0.06) less than that measured for the best E. coli nitroreductase, NfsA_Ec (1.9 ± 1.2 µM; Figure 4B). Surprisingly, PR-104A was unable to exert a comparable effect in this assay, possibly due to the relatively reactive nature of the metabolites; irrespective of the conditions employed, concentration-dependent IC50 curves could not be obtained for any of the E. coli strains. Overall, based on the results of the SOS and bacteria-delivered enzyme cytotoxicity assays, MsuE appears to be a promising candidate for GDEPT, offering potential to repurpose next-generation prodrugs that have been independently developed as hypoxia activated therapeutics.

Bottom Line: Two promising hypoxia prodrugs for GDEPT are the dinitrobenzamide mustard PR-104A, and the nitrochloromethylbenzindoline prodrug nitro-CBI-DEI.However, MsuE exhibited comparable levels of activity with PR-104A and nitro-CBI-DEI, and is the first nitroreductase outside of the NfsA and NfsB enzyme families to do so.These in vitro findings suggest that MsuE is worthy of further evaluation in in vivo models of GDEPT.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington 6140, New Zealand. david.ackerley@vuw.ac.nz.

ABSTRACT
Bacterial nitroreductase enzymes that can efficiently catalyse the oxygen-independent reduction of prodrugs originally developed to target tumour hypoxia offer great potential for expanding the therapeutic range of these molecules to aerobic tumour regions, via the emerging cancer strategy of gene-directed enzyme prodrug therapy (GDEPT). Two promising hypoxia prodrugs for GDEPT are the dinitrobenzamide mustard PR-104A, and the nitrochloromethylbenzindoline prodrug nitro-CBI-DEI. We describe here use of a nitro-quenched fluorogenic probe to identify MsuE from Pseudomonas aeruginosa as a novel nitroreductase candidate for GDEPT. In SOS and bacteria-delivered enzyme prodrug cytotoxicity assays MsuE was less effective at activating CB1954 (a first-generation GDEPT prodrug) than the "gold standard" nitroreductases NfsA and NfsB from Escherichia coli. However, MsuE exhibited comparable levels of activity with PR-104A and nitro-CBI-DEI, and is the first nitroreductase outside of the NfsA and NfsB enzyme families to do so. These in vitro findings suggest that MsuE is worthy of further evaluation in in vivo models of GDEPT.

No MeSH data available.


Related in: MedlinePlus