Pharmacophore-based discovery of 2-(phenylamino)aceto-hydrazides as potent eosinophil peroxidase (EPO) inhibitors

There is an increasing interest in developing novel eosinophil peroxidase (EPO) inhibitors, in order to provide new treatment strategies against chronic inflammatory and neurodegenerative diseases caused by eosinophilic disorder. Within this study, a ligand-based pharmacophore model for EPO inhibitors was generated and used for in silico screening of large 3 D molecular structure databases, containing more than 4 million compounds. Hits obtained were clustered and a total of 277 compounds were selected for biological assessment. A class of 2-(phenyl)amino-aceto-hydrazides with different substitution pattern on the aromatic ring was found to contain the most potent EPO inhibitors, exhibiting IC50 values down to 10 nM. The generated pharmacophore model therefore, represents a valuable tool for the selection of compounds for biological testing. The compounds identified as potent EPO inhibitors will serve to initiate a hit to lead and lead optimisation program for the development of new therapeutics against eosinophilic disorders

Kinetics and thermodynamics of halide and nitrite oxidation by mammalian heme peroxidases

The human heme peroxidases myeloperoxidase (MPO), eosinophil peroxidase (EPO) and lactoperoxidase (LPO) are able to oxidise (pseudo)halides and nitrite to reactive species that participate in host defence against foreign microorganisms as well as in immunomodulation and tissue degradation in certain pathologies. The heme in EPO and LPO is covalently linked to the apoprotein by two ester bonds, whereas in MPO it is additionally linked by a unique sulfonium ion bond to a methionine residue. As a consequence, the prosthetic group in MPO is significantly distorted from a planar conformation. These structural differences are reflected by distinct spectral and redox properties as well as reactivities toward chloride, bromide, iodide, thiocyanate and nitrite, which function as endogenous two- and one-electron donors for these enzymes in vivo. Standard reduction potentials at pH 7 have been determined for all redox couples involved in the halogenation and peroxidase cycle of MPO and LPO and partially of EPO. A detailed thermodynamic analysis of the formation of reactive halide species by MPO and EPO was also performed. Thus, for the first time, a comprehensive analysis of reactions catalysed by human heme peroxidases is presented that allows a better understanding of their role in physiological and pathophysiological processes

Disruption of the aspartate to heme ester linkage in human myeloperoxidase: Impact on ligand binding, redox chemistry and interconversion of redox intermediates

In human heme peroxidases the prosthetic group is covalentlyattached to the protein via two ester linkages between conservedglutamate and aspartate residues and modified methyl groupson pyrrole rings A and C. Here, monomeric recombinantmyeloperoxidase (MPO) and the variants D94V and D94N wereproduced in Chinese hamster ovary cell lines. Disruption of theAsp94 to heme ester bond decreased the one-electron reductionpotential E0 [Fe(III)/Fe(II)] from 1 to 55 mV at pH 7.0 and25 °C, whereas the kinetics of binding of low spin ligands and ofcompound I formation was unaffected. By contrast, in both variantsrates of compound I reduction by chloride and bromide(but not iodide and thiocyanate) were substantially decreasedcompared with the wild-type protein. Bimolecular rates of compoundII (but not compound I) reduction by ascorbate and tyrosinewere slightly diminished in D94V and D94N. The presentedbiochemical and biophysical data suggest that the Asp94 to hemelinkage is no precondition for the autocatalytic formation of theother two covalent links found in MPO. The findings are discussedwith respect to the known active site structure of MPOand its complexes with ligands