Application of a microfluidic reactor for screening cancer prodrug activation using silica-immobilized nitrobenzene nitroreductase.

Abstract

The nitroreductase-catalyzed conversion of a strong electron-withdrawing nitro group to the corresponding electron-donating hydroxylamine is useful in a variety of biotechnological applications. Activation of prodrugs for cancer treatments or antibiotic therapy are the most common applications. Here, we show that a bacterial nitrobenzene nitroreductase (NbzA) from Pseudomonas pseudoalcaligenes JS45 activates the dinitrobenzamide cancer prodrug CB1954 and the proantibiotic nitrofurazone. NbzA was purified by affinity chromatography and screened for substrate specificity with respect to prodrug activation. To facilitate screening of alternate potential prodrugs, polyethyleneimine-mediated silica formation was used to immobilize NbzA with high immobilization yields and high loading capacities. Greater than 80% of the NbzA was immobilized, and enzyme activity was significantly more stable than NbzA in solution. The resulting silica-encapsulated NbzA was packed into a microfluidic microreactor that proved suitable for continuous operation using nitrobenzene, CB1954, and the proantibiotic nitrofurazone. The flow-through system provides a rapid and reproducible screening method for determining the NbzA-catalyzed activation of prodrugs and proantibiotics.

• Authors: Cécile Berne, Lorena Betancor, Heather R Luckarift, Jim C Spain
• Journal: Biomacromolecules (Biomacromolecules) Vol. 7 Issue 9 Pg. 2631-6 (Sep 2006) ISSN: 1525-7797 [Print] United States
• PMID: 16961327 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Antineoplastic Agents
• Aziridines
• Enzymes, Immobilized
• Nitrobenzenes
• Prodrugs
• Silicon Dioxide
• tretazicar
• Polyethyleneimine
• nitrobenzene
• aromatic NADH-dependent nitroreductase
• Nitroreductases

Topics

• Antineoplastic Agents (chemistry)
• Aziridines (pharmacology)
• Biotechnology (instrumentation, methods)
• Dose-Response Relationship, Drug
• Drug Design
• Drug Screening Assays, Antitumor
• Enzymes, Immobilized (chemistry)
• Humans
• Neoplasms (drug therapy)
• Nitrobenzenes (chemistry)
• Nitroreductases (chemistry)
• Polyethyleneimine (chemistry)
• Prodrugs (chemistry)
• Pseudomonas (enzymology)
• Silicon Dioxide (chemistry)
• Substrate Specificity