Discovery of a new class of inhibitors for the protein arginine deiminase type 4 (PAD4) by structure-based virtual screening

Abstract

Background Rheumatoid arthritis (RA) is an autoimmune disease with unknown etiology. Anticitrullinated protein autoantibody has been documented as a highly specific autoantibody associated with RA. Protein arginine deiminase type 4 (PAD4) is the enzyme responsible for catalyzing the conversion of peptidylarginine into peptidylcitrulline. PAD4 is a new therapeutic target for RA treatment. In order to search for inhibitors of PAD4, structure-based virtual screening was performed using LIDAEUS (Ligand discovery at Edinburgh university). Potential inhibitors were screened experimentally by inhibition assays. Results Twenty two of the top-ranked water-soluble compounds were selected for inhibitory screening against PAD4. Three compounds showed significant inhibition of PAD4 and their IC50 values were investigated. The structures of the three compounds show no resemblance with previously discovered PAD4 inhibitors, nor with existing drugs for RA treatment. Conclusion Three compounds were discovered as potential inhibitors of PAD4 by virtual screening. The compounds are commercially available and can be used as scaffolds to design more potent inhibitors against PAD4.

Novel furan‐containing peptide‐based inhibitors of protein arginine deiminase type IV (PAD4)

“…The binding of the designed peptides to PAD4 was compared to a synthetic substrate of PAD4, N ‐α‐benzoyl‐L‐arginine ethyl ester (BAEE). Basically, the citrulline colorimetric assay previously reported was performed for this purpose . PAD4 was expressed and purified as previously reported .…”

“…Basically, the citrulline colorimetric assay previously reported was performed for this purpose . PAD4 was expressed and purified as previously reported . Briefly, reaction buffer containing 100 m m Tris–HCl buffer, 10 m m calcium chloride, 10 m m DTT, and 0.067 μ m of PAD4 was preincubated for 2 min at 37°C.…”

“…The IC 50 value is the concentration of an inhibitor required to achieve 50% inhibition. The IC 50 values of compounds that showed significant inhibition against PAD4 were determined by preincubating various concentrations of the inhibitors in the reaction buffer as previously described . The enzymatic reaction was initiated by adding 1 m m BAEE, and the reaction was allowed to occur for 15 min.…”

“…AutoDock Tools (ADT) was utilized to set the size and the center of the grid box as well as to prepare the input.pdbqt files for the PAD4 structure (PDB: 2DEW) and the ligand. The grid box cavity was set as previously reported at 21.295, 41.956, 22.264 (in the dimensions of x , y , z ) with a grid size of 20 × 30 × 20 using 1.000 Å spacing . The predicted binding affinity (kcal/mol) was calculated based on the scoring function used in autodock vina .…”

“…Hence, there is a need for the design and synthesis of a drug that is aimed at treating the root cause of RA with minimal side‐effects. Our previous studies showed that the furan ring could be an important structural component for a PAD4 inhibitor. In this study, peptide‐based inhibitors were designed based on the amino acid sequence of one of the PAD4 natural substrates, nucleophosmin.…”