Computer-aided repositioning and functional in vitro assessment of novel PAD4 inhibitors.

Peptidyl arginine deiminase 4 (PAD4) is a protein that catalyzes both normal and abnormal citrullination of interacting protein partners, affecting gene regulation and being associated with diseases such as Alzheimer's, cancer, and rheumatoid arthritis (RA). As a result, PAD4 has emerged as a potential therapeutic target; however, no inhibitors have been approved to date. In this study, the REFRAME and ZINC15 drug databases were virtually screened. The approach used molecular docking and dynamics simulation techniques to identify compounds with high-predicted binding affinity to PAD4 (PDB ID: 4DKT). Selected hits from this virtual screening underwent in vitro assays using fixed concentrations derived from the docking score to evaluate their ability to inhibit PAD4 activity, and their effect on neutrophil extracellular trap (NET) release was assessed using an ex vivo human neutrophil model. Computational analyses identified amodiaquine, folic acid, and pyroxamide as stable PAD4 binders. In vitro inhibition assays revealed that amodiaquine (5.0 μM to 1.0 nM) and pyroxamide (0.1 μM) were more potent inhibitors than the reference PAD4 inhibitor BBCla (8.8 μM), while folic acid showed a non-significant trend toward inhibition. Cytotoxicity assays confirmed that all compounds were non-toxic at the tested concentrations, except for amodiaquine at 50 μM. NETosis assays demonstrated that the three selected compounds altered chromatin decondensation and cellular morphology similarly to BBCla, although not uniformly across all cells. Overall, amodiaquine, folic acid, and pyroxamide were identified as PAD4 inhibitors through combined virtual and experimental approaches, supporting their potential as therapeutic candidates for PAD4-related diseases and warranting further investigation.