Nitroreductases for use in enzyme prodrug therapy

Abstract

The monofunctional prodrug, 5-aziridin-2,4-dinitrobenzamide (CB1954), can be activated via nitroreductase enzymes to form a cytotoxic, bifunctional hydroxylamine derivative. As a result of this, CB1954 has potential applications in enzyme/prodrug targeted anti-cancer therapies.

A nitroreductase that can activate CB1954 has previously been isolated from Escherichia coli. However, the use of this enzyme in targeted anti-cancer therapy is limited by its stability and the poor catalytic efficiency of this enzyme for CB1954. In addition, the reaction catalysed by E. coli nitroreductase yields two products, the active 4-hydroxylamine and also a relatively non-toxic 2-hydroxylamine product.

This thesis reports the successful identification, isolation and characterisation of two novel nitroreductases, from Bacillus licheniformis and Aeropyrum pemix and comparison of these enzymes with the previously isolated E. coli nitroreductase. Both of the enzymes isolated showed good potential as candidates for use in targeted anti-cancer therapies in combination with the prodrug CB1954. They are both of microbial origin, showed high specific activity with CB1954 and are stable at 37°C. In addition, the nitroreductase isolated from B. licheniformis was shown to produce only the biologically active 4-hydroxylamine product from the reduction of the prodrug CB1954.

Based on its high temperature optimum and thermal stability the nitroreductase from A. pemix may also be suited for a role in the reduction of nitro-groups and quinones in industrial biocatalytic processes. The use of nitroreductases in biocatalytic processes would be limited by the requirement for a reduced nicotinamide cofactor and therefore, an attempt was made to develop a nicotinamide cofactor regeneration system using nitroreductase and dihydroxyacetone, a reagent suspected of having the ability to reduce NAD(P)⁺ to NAD(P)H.