Developing of inhibitors of human myeloperoxidase

The human heme peroxidases myeloperoxidase (MPO), eosinophil peroxidase (EPO) and lactoperoxidase (LPO) are part of the antimicrobial defense system. They can catalyze halogenation, nitrosylation and oxidation of biological substrates and thus also contribute to oxidative tissue injury and inflammation. MPO and EPO are present in phagocytes at high concentration and their level strongly correlates with infections and inflammatory states. In addition, human peroxidases are associated to the pathogenesis of many brain, circulation and intestinal diseases. Thus, it is strongly desired to understand structure-function relationships in order to develop efficient inhibitors that will dampen inflammation without impairing their immunological roles. It was the aim of this project to develop mechanism-based inhibitors, which needs understanding of structural and functional differences within the mammalian peroxidase superfamily. In the present project the spectral, kinetic and redox properties of the short-living intermediates (ferrous proteins, compounds I, II & III) of MPO, EPO and LPO have been investigated. The group developed a new method for the determination of redox properties of all short-living intermediates involved in the halogenation and peroxidase cycle of MPO and EPO. Furthermore, the sequence and kinetics of reactions of interconversion of ferrous MPO and compound III as well as ferrous MPO, compound II and compound III have been elucidated. The obtained data together with those from comparative stopped-flow studies clearly demonstrated that an important point to diminish chlorination activity of MPO (without attacking the necessary physiological halogenation activity of EPO, LPO or thyroid peroxidase) is the high standard reduction potential of the redox couple compound I/compound II of MPO (E` = 1.35 V). Based on these kinetic and thermodynamic data the best so far known inhibitors of MPO, namely 5-chlorotryptamine and 5-fluorotryptamine (50% inhibition of chlorination activity at pH 7 at 0.7 M inhibitor concentration) were developed. These inhibitors are easily oxidized by MPO compound I (but not by EPO or LPO compound I) at 1 x 107 M -1 s -1 , but not by MPO compound II. As a consequence the enzyme is effectively driven out of the chlorination cycle. Ferrous MPO as target of inactivation was also investigated, but anaerobic stopped-flow studies clearly demonstrated, that both hydrogen peroxide or molecular oxygen transform it directly to compound II or compound III at reasonable rates, thus protecting ferrous MPO rather than participating in heme destruction as was one working hypothesis in the beginning.