Discovery of pentagalloylglucose in Moutan Cortex as a highly potent inhibitor of human peptidyl arginine deiminase 4 (PAD4) by UHPLC-MS/MS.

Peptidyl arginine deiminase 4 (PAD4) is an enzyme playing essential role in many biological processes especially immune responses, and is a highly attractive therapeutic target. However, the number of PAD4 inhibitors is limited, and there is no inhibitor in clinical trial. This study aims to identify natural compounds holding high inhibitory effect on PAD4 activity from traditional herbal medicine. Of the thirty-six extracts of twelve medical plants, the dry root bark of Paeonia × suffruticosa Andrews (also known as Moutan Cortex) extracts showed highest inhibition effect on PAD4 activity. Further experiment was conducted with different batches of Moutan Cortex to confirm this result. In addition, we also evaluated the inhibitory activity of different parts of the Paeonia × suffruticosa Andrews, including flower ball, flower, leaf, pollen and seed meal. The results substantiated the hypothesis that Paeonia × suffruticosa Andrews contains compounds with high PAD4 inhibitory effect. Therefore, a practical bioactivity-guided fractionation coupling with a chemical profiling strategy was used to identify the fractions from Moutan Cortex extract with strong PAD4 inhibition activity, and the major constituents in these bioactive fractions were further characterized by LC-MS/MS. Among all the identified compounds, pentagalloylglucose (PGG) showed the most potent inhibition effect on PAD4 activity, with an IC50 value of 4.50 µM. The inhibition kinetic analysis, drug affinity response target stability (DARTS) and molecular docking experiments were performed to confirm the interaction between PGG and PAD4. The findings indicated that PGG functions as a mixed model PAD4 inhibitor. In summary, our results showed that PGG in Moutan Cortex is a highly potent PAD4 inhibitor, and PGG can be used as a promising lead compound for the development of effective PAD4 inhibitors.