Virtual Screening and In vitro Evaluation Identify Methotrexate and Testosterone are Inhibitors of Protein Arginine Deiminase 4

Background: Peptidyl arginine deiminases (PAD) are proteins that modify arginine residues to citrulline. In humans there are five isoforms PAD1-4 and PAD6. PAD4 is involved in several human diseases such as Alzheimer, breast cancer, rheumatoid arthritis and therefore has been recognized as a potential pharmacological target. Methods: In this work, 3175 molecules retrieved from ZINC12 Food and Drug Administration (FDA) catalog database were assayed through a structure-based virtual screening to identify potential PAD4 binders. Three were confirmed through enzyme inhibition studies. Flow cytometry analysis were used to check for drug cytotoxic effects on blood cells from three healthy donors. Results: Molecular docking in a wild-type PAD4 structure showed the top 20 FDA molecules with the best binding energies. Those molecules were distributed in clusters of mixed binding energies but with similar chemical structures. These patterns along with the FDA information were used to aid in the rational selection of methotrexate, testosterone and leucovorin compounds for in vitro evaluations. Time course fluorescent enzyme kinetics confirmed that methotrexate (0.01–1 mmol) and testosterone enanthate (49.9–12.5 mmol) inhibit PAD4 at similar levels to the known inhibitor BB-Cl-amidine (8.8 mmol), except leucovorin. The chosen concentrations lack cytotoxic effects on blood cells. Conclusion: These findings encourage repositioning campaigns in the quest of PAD4 inhibitors. © 2022

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