Fig. 3: PAD-PF2 potently inhibits PAD1-4 isoforms in a Ca²⁺-dependent manner.

a PAD-PF2 (open circles) inhibits PAD4-mediated citrullination of peptide substrate Atto655-(Ser-Arg-Gly-Ala)₃ employing a fluorescence quenching (FQ) assay in the presence of 0.4 mM Ca²⁺. Data points and error bars represent the mean and SD of three independent experiments in duplicate. IC₅₀ = 42.7 nM, pIC₅₀ = 7.37 (0.03), mean (SD), n = 3 independent experiments. The enantiomer of PAD-PF2 (ent-PAD-PF2, open squares) was less potent. IC₅₀ = 26.0 µM, pIC₅₀ = 4.59 (0.45), mean (SD), n = 3 independent experiments. b PAD-PF2 induces a thermal stabilization of PAD2 (open circles, EC₅₀ = 2.04 µM) and PAD4 (open squares, EC₅₀ = 1.95 µM) protein. Individual data points shown for a duplicate determination. c–f Inhibition of the respective PAD isoform by PAD-PF2 determined using the GDH-enzyme coupled assay in the presence of 0.25 mM Ca²⁺. Individual data points shown for a duplicate determination along with the fitted IC₅₀ curves (line). c PAD1, IC₅₀ = 109 nM. d PAD2, IC₅₀ = 28.5 nM. e PAD3, IC₅₀ = 106 nM. f PAD4, IC₅₀ = 24.0 nM. g, h Calcium dependence of PAD2 or PAD4 inhibition by PAD-PF2. Inhibition was determined using the GDH enzyme-coupled assay. Data points from duplicate experiments performed at high (open squares) and low (open circles) Ca²⁺ concentrations are plotted together, and the fitted IC₅₀ curves are shown (line). g The determined IC₅₀ values for PAD2 inhibition were 27.9 nM and 3.09 µM (0.25 and 1.5 mM Ca²⁺, respectively). h The determined IC₅₀ values for PAD4 inhibition were 20.1 nM and 694 nM (0.25 and 1.5 mM Ca²⁺, respectively). Source data are provided as a Source Data file.