Development and validation of a screening procedure for the assessment of inhibition using a recombinant enzyme.

Myeloperoxidase (MPO, E.C. 1.1.11.7) is a heme-containing enzyme that catalyses the synthesis of hypochlorous acid (HOCl) in the presence of hydrogen peroxide (H2O2) and chlorine anions. The production of HOCl is kept under strict control of neutrophils. However, in several pathological conditions, MPO is leaked in the extracellular fluid, which involves an over-production of reactive oxygen species like HOCl and promotes the damages caused by neutrophils. As a consequence, the inhibition of MPO by various agents has been investigated and a variety of molecules have been evaluated for this activity in different models. The present study aims to describe and validate a rapid screening method based on the taurine assay and using a recombinant MPO. After validation of the stock solutions used during the experiments, the amount of MPO for the completion of the reaction was measured and fixed to an optimal value. The inhibiting concentration at 50% of flufenamic acid (taken as a reference molecule) was then assessed in both a simple tube test and a microplate test and delivered similar results (1.3+/-0.2 microM vs 1.4+/-0.2 microM, P=0.2). Finally, different molecules able to inhibit MPO were evaluated in this rapid assay system providing results comparable to literature. The high throughput screening is undoubtedly a first line assessment method which affords the selection of inhibitors and permits to reduce the number of candidates for a further elucidation of the mechanism of MPO inhibition.