Peptide ligand screening of alpha-synuclein aggregation modulators by in silico panning

Abstract

Background: alpha-Synuclein is a Parkinson's-disease-related protein. It forms aggregates in vivo, and these aggregates cause cell cytotoxicity. Aggregation inhibitors are expected to reduce alpha-synuclein cytotoxicity, and an aggregation accelerator has recently been reported to reduce alpha-synuclein cytotoxicity. Therefore, amyloid aggregation modulating ligands are expected to serve as therapeutic medicines.

Results: We screened peptide ligands against alpha-synuclein by in silico panning, a method which we have proposed previously. In this study, we selected as the target a very hydrophobic region known as the amyloid-core-forming region. Since this region cannot be dissolved in water, it is difficult to carry out the in vitro screening of its peptide ligand. We carried out 6 rounds of in silico panning using a genetic algorithm and a docking simulation. After the in silico panning, we evaluated the top peptides screened in silico by in vitro assay. These peptides were capable of binding to alpha-synuclein.

Conclusion: We demonstrated that it is possible to screen alpha-synuclein-binding peptides by in silico panning. The screened peptides bind to alpha-synuclein, thus affecting the aggregation of alpha-synuclein.

Figure 1

Scheme of in silico panning. In silico panning consists of two steps: a docking simulation and a genetic algorithm. The genetic algorithm consists of selection, crossover and point mutation. This figure shows one example of a genetic algorithm.

Figure 2

Distribution of the docking energy. The ranks of the peptide ligands in each generation are shown on the X- and Y-axes. The Z-axis shows the docking energies, which were calculated as follows: (a) docking total energy = electrostatic energies + van der Waals energies + conformational energy of the ligand; (b) interaction energy = total docking energy - conformational energy of the ligand. At the 1^st to the 3^rd generation, the peptides were evaluated by the total docking energy (a); at the 4^th to the 6^th generation, the peptides were evaluated by the interaction energy (b).

Figure 3

Time course of α-synuclein fibril formation. The Y-axis shows the normalized fluorescence intensity of Thioflavin T at 482 nm. 100% is the final fluorescence intensity of α-synuclein without peptide. The samples contained the following: α-synuclein (140 μM) without peptide, (Circle); QSTQ, (Black diamond); GSQQ, (Empty diamond); SQTQ (Black square); AQTQ, (Empty square). All peptide concentrations were 700 μM.

Figure 4

Light scattering assay. The Y-axis shows the normalized optical density at 330 nm. 100% is the optical density at 330 nm of α-synuclein without peptide.