A cyclic peptide toolkit reveals mechanistic principles of peptidylarginine deiminase IV (PADI4) regulation

Peptidylarginine deiminase IV (PADI4) deregulation promotes the development of autoimmunity, cancer, atherosclerosis and age-related tissue fibrosis. Genetic or pharmacological PADI4 inhibition is therapeutically efficacious in mice, but no clinically relevant inhibitors currently exist. PADI4 additionally mediates immune responses and cellular reprogramming, although the full extent of its physiological roles is unexplored. Despite detailed molecular knowledge of PADI4 activation in vitro, we lack understanding of its regulation within cells, largely due to lack of appropriate systems and tools. Here, we developed and applied a set of potent and selective PADI4 modulators. Using the mRNA-display-based RaPID system, we screened >10to the 12 cyclic peptides for high-affinity, conformation-selective binders. We report PADI4\_3, an inhibitor specific for the active conformation of PADI4; PADI4\_7, an inert binder, which we functionalised for the isolation and study of cellular PADI4; and PADI4\_11, a first-in-class activator. Using a newly developed method for the quantification of cellular PADI4 activity, we show that PADI4\_3 and PADI4\_11 are effective in cells. Structural studies with PADI4\_11 reveal an allosteric binding mode that may reflect the mechanism that promotes cellular PADI4 activation. This work offers new understanding of PADI4 regulation and a toolkit for the study and modulation of PADI4 across physiological and pathophysiological contexts. ### Competing Interest Statement UK Patent application No. 230979.0, which has been filed in respect to methods that modulate cellular reprogramming through enhancing the activity of the PADI4 enzyme