Screening and identification of peptidyl arginine deiminase 4 inhibitors from herbal plants extracts and purified natural products by a trypsin assisted sensitive immunoassay based on streptavidin magnetic beads

Peptidyl arginine deiminase 4 (PAD4) plays a critical role in many autoimmune diseases including rheumatoid arthritis. Herein, a trypsin assisted highly immunoassay method was established to determine PAD4 activity and screen potent inhibitors from herbal plants extracts and purified natural products. The method was applied to determine endogenous PAD4 activity in both cell and tissue lysates, as well as the inhibitory effects of 20 herbal plants and 50 purified natural products. The Cinnamomi ramulus extract showed strongest inhibitory potency with IC50 value lower than 5 mu g/mL. Meanwhile, pyrroloquinoline quinone (PQQ), widely used as a dietary supplement, was discovered as a promising PAD4 inhibitor with an IC50 value lower than 4 mu M. The inhibition kinetic analysis, drug affinity response target stability (DARTS) and molecular docking were performed to confirm the interaction between PQQ and PAD4. This method has great potential for researchers to monitor activities and discover potential inhibitors of PAD4.