Discovery and evaluation of Escherichia coli nitroreductases that activate the anti-cancer prodrug CB1954.
Biochem Pharmacol
; 79(5): 678-87, 2010 Mar 01.
Article
en En
| MEDLINE
| ID: mdl-19852945
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.
Texto completo:
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Base de datos:
MEDLINE
Asunto principal:
Aziridinas
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Nitrorreductasas
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Profármacos
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Proteínas de Escherichia coli
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Escherichia coli
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Antineoplásicos
Límite:
Humans
Idioma:
En
Revista:
Biochem Pharmacol
Año:
2010
Tipo del documento:
Article
País de afiliación:
Nueva Zelanda