Nicole Moguilevsky

Inhibition of the myeloperoxidase chlorinating activity by non-steroidal anti-inflammatory drugs investigated with a human recombinant enzyme.

Non-steroidal anti-inflammatory drugs (NSAIDs) were investigated for their ability to affect the chlorinating activity of human myeloperoxidase and to scavenge HOCl, the main myeloperoxidase system product. Fourteen drugs representative of various NSAIDs families were tested with the chlorination of taurine used as a detection system. All were unable to inhibit taurine chlorination in a system without myeloperoxidase. In contrast, most of them induced a dose-dependent inhibition of the taurine chlorination mediated by a myeloperoxidase/H2O2/Cl- system. This took place at variable drug concentrations and IC50 were calculated. The inhibitory effect was therefore due to a direct interaction with the enzyme rather than to HOCl scavenging. A spectroscopic method used to measure the myeloperoxidase compound II lifetime in presence of the different drugs showed that all the drugs, which inhibited chlorination activity were able to induce accumulation of compound II. The extent of chlorinating activity inhibition (IC50) was inversely related to the duration of the block of enzyme in compound II form. This further demonstrates that myeloperoxidase is an interesting target for anti-inflammatory therapy. The recombinant myeloperoxidase used for the first time in this kind of study was as convenient for pharmacological purposes as the purified one.

A transient kinetic study on the reactivity of recombinant unprocessed monomeric myeloperoxidase.

Spectral and kinetic features of the redox intermediates of human recombinant unprocessed monomeric myeloperoxidase (recMPO), purified from an engineered Chinese hamster ovary cell line, were studied by the multi‐mixing stopped‐flow technique. Both the ferric protein and compounds I and II showed essentially the same kinetic behavior as the mature dimeric protein (MPO) isolated from polymorphonuclear leukocytes. Firstly, hydrogen peroxide mediated both oxidation of ferric recMPO to compound I (1.9×107 M−1 s−1, pH 7 and 15°C) and reduction of compound I to compound II (3.0×104 M−1 s−1, pH 7 and 15°C). With chloride, bromide, iodide and thiocyanate compound I was reduced back to the ferric enzyme (3.6×104 M−1 s−1, 1.4×106 M−1 s−1, 1.4×107 M−1 s−1 and 1.4×107 M−1 s−1, respectively), whereas the endogenous one‐electron donor ascorbate mediated transformation of compound I to compound II (2.3×105 M−1 s−1) and of compound II back to the resting enzyme (5.0×103 M−1 s−1). Comparing the data of this study with those known from the mature enzyme strongly suggests that the processing of the precursor enzyme (recMPO) into the mature form occurs without structural changes at the active site and that the subunits in the mature dimeric enzyme work independently.