Partial peptide of α-synuclein modified with small-molecule inhibitors specifically inhibits amyloid fibrillation of α-synuclein

Abstract

We have previously reported that pyrroloquinoline quinone (PQQ) prevents the amyloid formation of α-synuclein, amyloid β(1-42) (Aβ(1-42)), and mouse prion protein. Moreover, PQQ-modified α-synuclein and a proteolytic fragment of the PQQ-modified α-synuclein are able to inhibit the amyloid formation of α-synuclein. Here, we identified the peptide sequences that play an important role as PQQ-modified specific peptide inhibitors of α-synuclein. We demonstrate that the PQQ-modified α-Syn(36-46) peptide, which is a partial sequence of α-synuclein, prevented α-synuclein amyloid fibril formation but did not inhibit Aβ(1-42) fibril formation. In addition, the α-synuclein partial peptide modified with other small-molecule inhibitors, Baicalein and epigallocatechin gallate (EGCG), prevented α-synuclein fibril formation. Currently reported quinone amyloid inhibitors do not have selectivity toward protein molecules. Therefore, our achievements provide a novel strategy for the development of targeted specific amyloid formation inhibitors: the combination of quinone compounds with specific peptide sequence from target proteins involved in amyloid formation.

Figure 1

Inhibitory effect of PQQ-modified α-Syn peptides on the fibril formation of α-Syn. The time course of amyloid fibril formation by α-Syn was determined by the TfT assay. The sigmoidal curve analysis was performed by PRI. The fibril formation of 50 μM α-Syn in the presence or absence of 25, 50, and 500 μM the α-Syn_3–13-PQQ (A), α-Syn_21–28-PQQ (B), and α-Syn_36–46-PQQ (C) were analyzed (n = 3).

Figure 2

Cytotoxicity evaluation of α-Syn119 aggregates incubated with α-Syn_36–46-PQQ. In the presence or absence of inhibitors, α-Syn119 samples were incubated for 18 h and then the cytotoxicity of the samples was analyzed by CC8 (A) and ATP assay (B). PQQ and α-Syn_36–46-PQQ showed lower cytotoxicity than that of α-Syn119 (p < 0.0014 and p < 0.0028 in CC8 assay, respectively and p < 0.001 and p < 0.0063 in ATP assay, respectively).

Figure 3

Inhibitory effect of α-Syn_36–46-PQQ on the fibril formation of Aβ_1–42. The time course of amyloid fibril formation of Aβ_1–42 was determined using the TfT assay. The sigmoidal curve analysis was performed by PRI. The fibril formation of 25 μM Aβ_1–42 in the presence of 25 μM unmodified α-Syn_36–46, 200 μM α-Syn_36–46-PQQ or 200 μM PQQ were analyzed (n = 3).

Figure 4

Inhibitory effect of α-Syn_36–46-Baicalein (A) and α-Syn_36–46-EGCG (B) on the fibril formation of α-Syn. The time course of amyloid fibril formation of α-Syn was determined using the TfT assay. The sigmoidal curve analysis was performed by PRI. The fibril formation of 50 μM α-Syn in the presence of 50 μM Baicalein, Baicalein-modified α-Syn peptides, EGCG or EGCG-modified α-Syn peptides were analyzed (n = 3).