With an approach that could revolutionize the development of anti-inflammatory agents, researchers at RIDC Redoxoma have discovered new inhibitors of myeloperoxidase (MPO), an enzyme involved in various inflammatory processes. The compounds were shown to be effective in HL-60 cells, human neutrophils, and an animal model of gouty arthritis.

The new method consists of an MPO inhibitor-like rule integrated with a structure-based virtual screening and was created by researcher Isaac de Araújo Matos, during his doctorate. “The importance of this work is the development of a method to search for MPO inhibitors, with a high success rate and a high chemical diversity of the discovered compounds. The original idea was to use a generic methodology, which was not tied to the structure of the compound, but would bring properties that would increase the probability of the set to be tested to have true MPO inhibitors”, said the researcher.

The study was carried out under the supervision of Professor Flavia Carla Meotti, from the Instituto de Química at Universidade de São Paulo (USP) and a member of the RIDC Redoxoma. The computational method was developed in collaboration with Professor Nivan Bezerra da Costa Júnior, from the Universidade Federal do Sergipe (UFS), and published in an article in the Journal of Chemical Information and Modeling, a publication of the American Chemical Society.

Myeloperoxidase

Myeloperoxidase (MPO) is a heme peroxidase found in large amounts in neutrophils, which are the most abundant leukocytes in the bloodstream and form the first line of defense against infections. The enzyme uses hydrogen peroxide to oxidize chloride to hypochlorous acid, a strong microbicidal agent.

However, despite fighting microorganisms, the inflammatory processes triggered by MPO are at the origin of several diseases, such as cystic fibrosis, asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, Alzheimer’s disease, and Parkinson’s disease. More recently, it has also been implicated in severe acute respiratory syndrome associated with pulmonary neutrophilic infiltrate in covid-19.

Although being a promising target for the development of new anti-inflammatory agents, so far no MPO inhibitor has been approved for clinical use. According to Matos, the inhibitors currently in clinical trials are potent because they are irreversible, and, as they are reactive molecules, it is difficult to select the reactivity just to inhibit MPO, increasing the risk of serious adverse effects. One of the advantages of the inhibitors discovered with the new methodology is that they are reversible. “We have proved that it is possible to develop a potent reversible inhibitor that is active in cells and animals,” he said.

New method

To create the new method, the researchers analyzed generic molecular properties, such as molecular mass variation, polarity, lipophilicity, and the number of rotatable bonds, of 143 previously described inhibitors, and added the Lipinski and Veber rules, used to assess the oral bioavailability of compounds. With that, they established a rule, which they called the MPO inhibitor-like rule. “So compounds that meet all these rules would, in theory, have a chance of inhibiting MPO and being orally active.”

With this rule, they made a virtual screening of more than 35 million molecules then available in the ZINC12 bank and obtained 6,546 molecules of interest. The ZINC database is a collection of chemical compounds specially prepared for virtual screening.

As the inhibitor-like rule is not structure-based, they needed a methodology to exclude compounds that could not bind to the active site of the enzyme. “Then we used the receptor-based virtual screening, or molecular docking, in which the 6,546 compounds were fitted into the active site of the enzyme. Several programs were used, the best parameters were tested for validity, half of the set was analyzed by visual inspection”, Matos says. In this step, 242 compounds were selected, and they underwent further docking and analysis. Thus, they identified ten candidates, of which six inhibited MPO, representing a success rate of 60%. The compounds showed high chemical diversity and none had been previously reported as an MPO inhibitor. “With that, we saw that the methodology worked,” Matos said.

The researcher explains that, unlike other works that use screening based on pharmacophore models, basically finding molecules similar to those used as a model, the method developed in this study finds compounds with high chemical diversity. “Diversity is key because a molecule sometimes inhibits the enzyme very well, but it is toxic. If you have multiple structures, you can select the best one. We want chemical diversity as much as possible.”

They then re-selected and tested 18 molecules from the same pool of 242 compounds, four of which were tested in an animal model of gouty arthritis. Gouty arthritis, or gout, is an inflammatory disease caused by the precipitation of urate crystals in the joints, affecting approximately 7% of the world population. The accumulation of crystals causes painful inflammatory flare-ups.

“In the animal model, we saw that when we administered the crystals to the paw the edema tripled and the MPO activity was also high. And when we treated them with MPO inhibitors, we saw a significant reduction in inflammation.” All four compounds had an anti-edematous effect, that is, they reduced the swelling of the animal’s paw, in addition to decreasing the activity of total peroxidase and pro-inflammatory cytokines when administered intraperitoneally and three of them showed the same effects when administered orally. According to the researcher, although there is evidence of the participation of the MPO in gout, there are still no studies showing inhibitors of this enzyme in the treatment of the disease.

Among the compounds tested, one of them proved to be more potent as an anti-inflammatory: the naphthalamide, which showed inhibitory potency in the nanomolar range. This compound has some similarities with ABAH, or benzoic acid 4-hydrazide, considered a classic MPO inhibitor, although it has important differences in structure and mechanism of action. The main one is the fact that ABAH is an irreversible inhibitor.

“Naphthalamide is a small, potent, and reversible molecule, which is not yet ready and can be easily optimized. As it is a new chemical and pharmacological class, we have patented naphthalamides as MPO inhibitors for the development of anti-inflammatory drugs”, explained Matos.

The drug discovery process is expensive, slow, and complex, and it is a risky investment from an economic point of view, with budgets of several million dollars and a high rate of disapproved compounds. According to Matos, computational chemistry can support the process of developing new drugs, especially in the early stages. “Virtual screening has proven to be a useful tool, accelerating hits identification and fostering collaborations in the modern drug discovery process.”

Data from research in an animal model of gouty arthritis are not yet published. Naphthalamide compounds are under patent application with the National Institute of Industrial Property (INPI). The article Integration of an Inhibitor-like Rule and Structure-based Virtual Screening for the Discovery of Novel Myeloperoxidase Inhibitors, by Isaac de Araújo Matos, Nivan Bezerra da Costa Júnior and Flavia Carla Meotti, can be read here.