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.

Figure 1

Post-translational conversion of peptidylarginine into peptidylcitrulline catalyzed by protein arginine deminase (PAD) in the presence of Ca²⁺.

Figure 2

Structure of protein arginine deiminase type 4 [PDB:1WDA]. (a) Ribbon representation of PAD4; α-helix is illustrated in red and β-sheet is illustrated in yellow. (b) Surface representation of PAD4 with benzoyl-l-arginine amide (BAA) inside the enzyme binding site.

Figure 3

Percentage of remaining activity of PAD4 in the presence of 22 compounds. Twenty two compounds were selected from virtual screening results based on their solubility and AutoDock binding affinities. The concentration of each compounds was 100 μM.

Figure 4

Concentration - respond curves of compound 9 (a), 10 (b) and 59 (c) against PAD4.

Figure 5

Binding conformations of compounds 9 (a), 10 (b), and 59 (c) inside the PAD4 binding pocket. Hydrogen bonds are drawn as dashed lines, hydrophobic interactions are drawn as green lines. Residues that form hydrogen bond and hydrophobic interactions with each compound are shown. Figures were plotted using PoseView.

Figure 6

Binding conformations of benzoyl-L-arginine amide or BAA (PDB:2DW5] (a) and F-amidine [PDB:1WDA] (b) inside the PAD4 binding pocket. Hydrogen bonds are drawn as dashed lines, hydrophobic interactions are drawn as green lines. Residues that form hydrogen bond and hydrophobic interactions with each compound are shown. Figures were plotted using PoseView.

Figure 7

Binding pocket of PAD4 with two accessible tunnels labeled as "front door" and "back door" [PDB:1WDA]. Benzoyl-L-arginine amide (BAE) is seen occupying the "main door" with the amino acid residues that interact with BAE are marked yellow. The "back door" is visible with the residues that interact with compounds 9, 10, and 59 are marked red.

Figure 8

Correlation plot between AutoDock binding affinity and the inhibitory activity of the compounds.

Figure 9

Binding pose of compound 16 (SPH1-109-603) inside the PAD4 binding pocket. The binding conformation of this compound is seen occupying both "front door" and "back door". Hypothetically, this compound was thought having the highest inhibitory activity. However, quick screening result proved that the compound does not have any inhibitory activity against PAD4.