Design of Myeloperoxidase Inhibitors as new anti-inflammatory drugs: A in silico, in vitro and in vivo study

• Ano de defesa: 2022
• Autor(a) principal: Matos, Isaac de Araujo
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: Artrite de gotosa; Gout arthritis; Inflamação; Inflammation; Inhibitors; Inibidores; Mieloperoxidase; Myeloperoxidase; Triagem virtual; Virtual screening
• Link de acesso: https://www.teses.usp.br/teses/disponiveis/46/46131/tde-02122022-154640/

This thesis discusses the use of computational and experimental methods for the development of new myeloperoxidase (MPO) inhibitors. MPO is a key enzyme present majority in neutrophil cells. Using hydrogen peroxide, MPO oxidizes chloride generating hypochlorous acid, a strong microbicidal agent. Although MPO has a microbicidal role, its activity is associated to inflammation progression and tissue damage. Beside the oxidation of halides and pseudo-halides in a chlorinating cycle, MPO can oxidize other endogenous substrates, such as urate, ascorbate, serotonin and tyrosine through a peroxidatic cycle. The oxidation of substrates in the peroxidatic cycle produces free radicals and can trigger a free radical chain reaction. In this context, MPO inhibitors emerge as new candidates to anti-inflammatory drugs. To overcome the limitations of the current methodologies available in the search of new MPO inhibitors, a pipeline of several computational and experimental approaches was developed in this thesis. Initially, known potent MPO inhibitors were used to elaborate an inhibitor-like rule. This rule suggests that compounds that match this criterion are more likely to both inhibit MPO and be orally active. By applying this rule to the ZINC12 database, a sub-library of 6546 molecules was recovered. After molecular docking steps, this set was reduced to 242 molecules with good stereospecificity for the active site of MPO. In a first experimental validation, 10 compounds were tested and 6 inhibited MPO chlorinating activity. The most potent compound, ZINC9089086, reversibly inhibited the enzyme with a IC50 = 2.2 µM. It also inhibited hypochlorous acid production by cells. To confirm the predictability of the virtual screening methodology, 18 additional compounds were selected. Confirming the methodology robustness, 12 of these compounds inhibited the chlorinating activity of the enzyme, while 13 inhibited the peroxidatic one. The most potent compounds, RL6 and RL7, exhibited IC50 in the nanomolar range (270 nM and 560 nM, respectively). Both compounds decreased hypochlorous acid production and NETosis in dHL-60 cells and peripheral blood neutrophils. Mechanistic studies indicate that RL6 is a reversible non-oxidizable MPO inhibitor, while RL7 is an hypochlorous acid scavenger. To confirm that the MPO inhibitor-like rule do not only select MPO inhibitors but also in vivo active compounds, a murine model of gouty arthritis was used. Corroborating our hypothesis, all tested compounds (RL6, RL7, RL21 and ZINC9089086) inhibited paw edema when administered intraperitoneally and 3 compounds were also orally active (RL6, RL7 and RL21). Finally, the integration of computational and experimental methods has proven to be a golden approach for the discovery of in vivo active MPO inhibitors.