Brazilin Inhibits α-Synuclein Fibrillogenesis, Disrupts Mature Fibrils, and Protects against Amyloid-Induced Cytotoxicity

Abstract

The inhibitory effect of brazilin against α-synuclein (α-syn) fibrillogenesis, disruption effect against mature fibrils, and the following cytotoxicity were examined by systematical biochemical, biophysical, cellular biological, and molecular simulation experiments. It is found that brazilin inhibited α-syn fibrillogenesis and disrupted the performed fibrils with a concentration-dependent manner. Moreover, cellular experimental data showed that brazilin effectively reduced the cytotoxicity induced by α-syn aggregates. Finally, molecular dynamics simulations were performed to explore the interactions between brazilin and α-syn pentamer. It is found that brazilin directly interacts with α-syn pentamer, and the hydrophobic interactions are favorable for brazilin binding with the α-syn pentamer, while the electrostatic part provides adverse effects. Three binding regions were identified to inhibit α-syn fibrillogenesis or disrupt the preformed aggregates. Furthermore, six important residues (i.e., G51, V52, A53, E61, V66, and K80) of α-syn were also identified. We expected that brazilin is an effective agent against α-syn fibrillogenesis and associated cytotoxicity.

Brazilin Removes Toxic alpha-Synuclein and Seeding Competent Assemblies from Parkinson Brain by Altering Conformational Equilibrium

“…Correspondingly, sequestering smaller amyloid species into larger aggregates removes α-syn toxicity [4,52]. This is in agreement with the reduced toxicity of α-syn fibrils after Brazilin treatment in neuronal model cells [51] which has been confirmed in primary neurons in our study (Figure 8). Our molecular modelling suggests binding of the Brazilin anions to hydrophobic pockets on the fibril surface, specifically in the vicinity of the positively charged residues K43 and K45 (Figure 9), which was also reported by Liu et al, that may promote lateral assembly of fibrils ( Figure 10).…”

“…The remodeled fibrils have a similar morphology to that of untreated α-syn; but appear in larger clusters (Figure 6c), which could explain their increased SDS-resistance and their decreased seeding capacity [4]. While Liu et al did not observe fibril clusters under their experimental conditions, Brazilin slightly altered the morphology preformed α-syn fibrils at 2:1 molar ratio, resulting in wider fibrils, which may be precursors of the fibril clusters we found at 10:1 molar ratio ( Figure 6) [51].…”

“…The similarities of Aβ and α-syn aggregation combined with Brazilin's structural similarities to other α-syn fibrillogenesis inhibitors as well as the inhibitory effects Brazilin has on Aβ, suggest that Brazilin could be a key candidate as a potential neuroprotective and therapeutic agent for PD. Very recently, it was confirmed that Brazilin inhibits α-syn fibril formation in vitro [51]. Here, we analyzed in detail the mechanism how Brazilin disrupts α-syn self-assembly and inactivates seeding competent α-syn assemblies derived from PD brain homogenates.…”

“…Brazilin inactivates seeding competent assemblies, both in vitro and when treating assemblies in the brain of PD patients (Figures 7 and 8). While reduction in ThT fluorescence and the absence of single fibrils in AFM and EM images would suggest fibril disassembly [47,51,64], increased insoluble high-molecular weight aggregates were observed by SDS-PAGE in the pellet fraction of ultracentrifugation experiments ( Figure 3). The remodeled fibrils have a similar morphology to that of untreated α-syn; but appear in larger clusters (Figure 6c), which could explain their increased SDS-resistance and their decreased seeding capacity [4].…”

The cytotoxicity of brazilin and derivatives might be due to an inhibition of the c-Src-kinase

“…Furthermore, a neuroprotective effect of 1 has been established in a human neuroblastoma model 13 . There are also some reports on the cytotoxic activity of brazilein but less for brazilin 11,[14][15][16][17][18][19][20][21] .…”