Mechanisms Underlying Myeloperoxidase Inhibition: Evaluating the Efficacy and Safety of Anti-inflammatory MPO Inhibitors Designed to Limit the Generation of Reactive Oxygen and Nitrogen Species
Type of DegreeDissertation
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Myeloperoxidase (MPO), a heme-containing peroxidase, plays a critical role in the innate immune response by producing hypochlorous acid. MPO-mediated oxidation is also associated with chronic obstructive pulmonary disease (COPD), rheumatoid arthritis (RA), atherosclerosis, and other inflammation associated diseases. Therefore, identification of potent and selective MPO inhibitors can diminish the excessive formation of MPO-derived oxidants and ameliorate oxidative stress during inflammation. We applied the fluorogenic peroxidase substrate 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) in steady-state and transient kinetic studies of MPO function. Using initial kinetic parameters for the MPO system, we characterized under the same conditions a number of gold standards for MPO inhibition, namely 4-amino benzoic acid hydrazide (4-ABAH), isoniazid and NaN3 before expanding our focus to isomers of 4-ABAH and benzoic acid hydrazide analogs. We determined that in the presence of hydrogen peroxide that 4-ABAH and its isomer 2-ABAH are both slow-tight binding inhibitors of MPO requiring at least two steps, whereas NaN3 and isoniazid-based inhibition has a single observable step. We also determined that MPO inhibition by benzoic acid hydrazide and 4-(trifluoromethyl) benzoic acid hydrazide was due to hydrolysis of the ester bond between MPO heavy chain Glu 242 residue and the heme pyrrole A ring, freeing the light chain and heme b fragment from the larger remaining MPO heavy chain. In addition, we probed the structure and function relationship behind this ester bond cleavage using a panel of BAH analogs to gain insight into the constraints imposed by the MPO active site and channel leading to the buried protoporphyrin IX ring. We showed the evidence that Cy5-hydrazide, a fluorescent analog of BAH, cleaved MPO into the complex of LC with heme and the MPO HC, and the HC was labeled with Cy5-hydrazide. This new mechanism would essentially indicate that the benzoic acid hydrazide analogs impart inhibition through initial ejection of the heme catalytic moiety without prior loss of the active site iron. We found an oral Janus kinase (JAK) inhibitor tofacitinib reversibly inhibited MPO peroxidase activity and was docked into the active site moiety of MPO. A stopped-flow rapid kinetic study and an MPO fluorescence endpoint assay were used to determine the Ki and Kd of tofacitinib on MPO inhibition, respectively. Analysis of tofacitinib/BAH-treated MPO by SDS-PAGE and in-gel luminescence imaging revealed that tofacitinib prevented the cleavage of MPO by BAH in the presence of H2O2, which indicates that it may compete for the active site of MPO with BAH. In addition, we also found that tofacitinib could prevent the Soret peak blue shift of MPO heme signature caused by BAH using spectral analysis of MPO with tofacitinib/BAH. These results collectively demonstrate that tofacitinib is a reversible inhibitor of MPO, which further provides new avenues for future drug development efforts that aim to diminish the formation of peroxidase-derived oxidants in chronic inflammatory diseases.