1-Piperidine Propionic Acid as an Allosteric Inhibitor of Protease Activated Receptor-2

Abstract

In the last decades, studies on the inflammatory signaling pathways in multiple pathological contexts have revealed new targets for novel therapies. Among the family of G-protein-coupled Proteases Activated Receptors, PAR2 was identified as a driver of the inflammatory cascade in many pathologies, ranging from autoimmune disease to cancer metastasis. For this reason, many efforts have been focused on the development of potential antagonists of PAR2 activity. This work focuses on a small molecule, 1-Piperidine Propionic Acid (1-PPA), previously described to be active against inflammatory processes, but whose target is still unknown. Stabilization effects observed by cellular thermal shift assay coupled to in-silico investigations, including molecular docking and molecular dynamics simulations, suggested that 1-PPA binds PAR2 in an allosteric pocket of the receptor inactive conformation. Functional studies revealed the antagonist effects on MAPKs signaling and on platelet aggregation, processes mediated by PAR family members, including PAR2. Since the allosteric pocket binding 1-PPA is highly conserved in all the members of the PAR family, the evidence reported here suggests that 1-PPA could represent a promising new small molecule targeting PARs with antagonistic activity.

Keywords: 1-piperidinepropionic acid; G protein-coupled receptors; Protease Activated Receptor 2; allosteric modulator; molecular dynamics.

Figure 1

CETSA assay on PAR2 in absence and presence of 1-PPA. (a) PAR2 thermal denaturation performed on live cells shows a decrease in soluble protein as the temperature increases. Data of protein content were initially normalized on Vinculin content and then on the untreated sample (38 °C); (b) 1-PPA at increasing concentration exerts a solubilizing effect on PAR2 even at temperatures higher than the determined T_agg. No effects are exerted on the Vinculin, which remains overall constant for each 1-PPA concentration. Therefore, data were normalized on Vinculin levels and then normalized on the highest concentration of 1-PPA (125 µM).

Figure 2

1-PPA binding site in PAR2 based on molecular dynamics. (a) wtPAR2 model of the transmembrane domain (Val61-Arg362) is in white. Among the 7TMD, 1-PPA and AZ8838 are represented in purple and light green, respectively; (b) Cut-through representation of the binding pockets, PAR2 surface is showing in gray while 1-PPA and AZ8838 are indicated by the arrows; (c) Top view of the allosteric pocket containing 1-PPA (in purple); residues interacting with it are shown as white sticks and are indicated with Ballesteros–Weinstein numeration. Hydrogen bonds formed with D121 and S124 are shown as black dashed lines. Na⁺ ion is shown in green; (d) Schematic representation of 1-piperidinepropionic acid structure and molecular weight (MW).

Figure 3

Molecular dynamics analysis. (a) Chemical structure of the compounds and sliced view of wtPAR2 model shows that three pockets, identified in the initial state (S0) in yellow, unite in a single funnel in the final state (S1), in purple; (b) Sliced view of wtPAR2 model, in white, shows the engulfment of 1-PPA (purple) after the simulations, in the allosteric site the by the allosteric pocket 2; (c) Cross-correlation matrix confronts the stabilizing effects performed by each compound during MDs; (d) RMSF plot representing the residue mobility, dotted lines shows the portion of the ECL2 while continuous lines box the ICL2 and 3; (e) RMSD variation during simulation time; (f) Distance variation of each compound in respect to the initial position shows that AZ8838 remains in the orthosteric pocket, while negative control Ro5-4864 and 1-PPA migrate.

Figure 4

Sequence alignment of PAR family members. ClustalO alignment of sequences from Protease Activated Receptor family members. Purple boxes highlight the main residues involved in the interaction between PAR2 and 1-PPA. Lined boxes refer to residues that are fully conserved in the other proteins in the same position, while pale blue boxes identify residues conserved for their side chain properties. Gray boxes indicate residues, in the binding site, that completely diverge compared to PAR2.

Figure 5

1-PPA antagonizes PARs activation. (a) Western Blot (WB) following the activation of PAR2 in HepG2 cell line; (b) Phosphorylation levels of Erk1/2 after treatment with SLIGKV-NH₂ peptide; (c) WB comparing the activation of PAR2 in HepG2 cell line in presence of different concentrations of 1-PPA. (d) Phosphorylation levels of Erk1/2 in PAR2-activated HepG2 cells in the presence of different concentrations of 1-PPA. (e) TRAP test plot showing the reduction of the platelet aggregation level during time compared to the reference level of untreated platelets. (f) Histograms representing the percentage of platelet aggregation in presence of different concentrations of 1-PPA.