New compounds identified through in silico approaches reduce the α-synuclein expression by inhibiting prolyl oligopeptidase in vitro

Abstract

Prolyl oligopeptidase (POP) is a serine protease that is responsible for the maturation and degradation of short neuropeptides and peptide hormones. The inhibition of POP has been demonstrated in the treatment of α-synucleinopathies and several neurological conditions. Therefore, ligand-based and structure-based pharmacophore models were generated and validated in order to identify potent POP inhibitors. Pharmacophore-based and docking-based virtual screening of a drug-like database resulted in 20 compounds. The in vitro POP assays indicated that the top scoring compounds obtained from virtual screening, Hit 1 and Hit 2 inhibit POP activity at a wide range of concentrations from 0.1 to 10 µM. Moreover, treatment of the hit compounds significantly reduced the α-synuclein expression in SH-SY5Y human neuroblastoma cells, that is implicated in Parkinson's disease. Binding modes of Hit 1 and Hit 2 compounds were explored through molecular dynamics simulations. A detailed investigation of the binding interactions revealed that the hit compounds exhibited hydrogen bond interactions with important active site residues and greater electrostatic and hydrophobic interactions compared to those of the reference inhibitors. Finally, our findings indicated the potential of the identified compounds for the treatment of synucleinopathies and CNS related disorders.

Figure 1

The structure of the human prolyl oligopeptidase (PDB ID: 3DDU) with functional subunits designated by colour. (A) The protease catalytic domain is shown in red, the β-propeller domain is shown in blue and the bound inhibitor is shown in green. The active site residues are represented in a stick model, and the catalytic residues are highlighted in a pink colour. (B) The general scheme of the pharmacophores showing the complementary interaction sites (Sx) at the POP active site with portions (Px) of the substrate or peptidomimetic inhibitors. The dotted red lines represent the hydrogen bonds.

Figure 2

Schematic workflow of the virtual screening for identifying the prolyl oligopeptidase inhibitors.

Figure 3

Pharmacophore models of POP generated with distance constraints. (A) Common feature pharmacophore, Pharm-A; (B) Structure-based pharmacophore, Pharm-B.

Figure 4

Clustered structure-based pharmacophore model in the active site of POP. Hydrogen bond acceptors and hydrophobic features are shown in green and cyan colours, respectively. Active site residues are shown as stick models in grey, while the catalytic residues are shown in pink. The protein is represented by the grey line ribbons. The pharmacophore features were represented without location spheres for clarity.

Figure 5

The effect of hit compounds on enzyme activity determined by in vitro POP assay. All samples are divided into the following 11 groups: positive control, negative control, Reference inhibitor (Ref), Hit 1 and Hit 2 compounds at three different doses (0.1, 1 and 10 µM). The data are shown as the mean ± SEM of triplicates from three independent in vitro experiments. *p < 0.05, ***p < 0.001 and ns p > 0.05 compared to positive control.

Figure 6

The expression of α-synuclein in SH-SY5Y human neuroblastoma cells treated with the hit compounds. All samples were divided into the following six groups: control SH-SY5Y cells, overexpressing SH-SY5Y cells transfected with EGFP-alphasynuclein-A53T plasmid (α-syn), overexpressing SH-SY5Y cells treated with (A) Reference inhibitor, (B) Hit 1, and (C) Hit 2 compounds at four different doses (0.1, 1, 10, and 100 µM for 24 h). Western blots were stained with anti-α-synuclein, and anti-b-actin was used as a loading control. The data are shown as the mean ± SEM of triplicates from three independent in vitro experiments. The histogram represents the optical density-based quantification of α-synuclein/b-actin; **p < 0.01, ***p < 0.001 and ns p > 0.05.

Figure 7

A pictorial representation of binding modes of the reference inhibitors and hit compounds. (A) Inh 1 (B) Inh 2 (C) Hit 1 (D) Hit 2. The 2D structures of all compounds are shown as insets. The active site residues are represented by a grey stick model, while the catalytic residues are shown in pink. The protein is shown in line ribbons. Hydrogen bonds are displayed as dotted lines. Only polar hydrogen atoms are shown for clarity.