Initially, the synthetic peptides were incubated with PADI4 and PADI4_C645A and samples analysed by intact-MS. This confirmed all peptides were binding at Cys645, in the active site of PADI4, without any additional sites of reaction (Figure ). Next, to determine their potency, IC50 values were determined using an established PADI4 activity assay, the Colour Developing Reagent (COLDER) assay, using N-a-benzoyl-L-arginine ethyl ester (BAEE) as the substrate. All peptides showed inhibitory activity against PADI4, both with and without onehour of preincubation between PADI4 and peptide prior to initiating the assay through addition of BAEE (Figure ). With preincubation, cP4_15 was the most potent peptide with an IC50 of 52 nM, a slight improvement over PADI4_3_H4(2) (IC50 = 61 nM). The least potent peptide, cP4_2, had an IC50 of 870 nM. The same trend in IC50 values was observed without preincubation of the peptides with PADI4, however, the IC50 values were much higher (Table ). This time-dependent improvement in IC50 is indicative of covalent inhibition. Therefore, to characterise the covalent behaviour further, kinetic parameters were determined using a modified COLDER assay design. Varied concentrations of each peptide were incubated with PADI4 and BAEE and the reactions quenched at 15-minute time intervals. Apparent IC50 values were calculated at each time point. This allowed an IC50 vs time correlation to be determined and fitted to the Krippendorff Equation which allows determination of KI and kinact values (Figure , Table ). The kinact/KI values determined were up to 10-fold higher than those previously reported for PADI4 covalent inhibitors. Table : Sequences of peptides synthesised after the first selection, and the rationally designed PADI4_3_H4(2). Where (2) is the FAO warhead and y is chloroacetyl-D-tyrosine which is cyclised with the cysteine residue in each peptide. Their corresponding IC50 values from COLDER assay with or without 1 h preincubation of peptide and PADI4 are also shown. Data shows mean ± SEM of two independent replicates.