9-Methylfascaplysin Is a More Potent Aβ Aggregation Inhibitor than the Marine-Derived Alkaloid, Fascaplysin, and Produces Nanomolar Neuroprotective Effects in SH-SY5Y Cells

Abstract

β-Amyloid (Aβ) is regarded as an important pathogenic target for Alzheimer's disease (AD), the most prevalent neurodegenerative disease. Aβ can assemble into oligomers and fibrils, and produce neurotoxicity. Therefore, Aβ aggregation inhibitors may have anti-AD therapeutic efficacies. It was found, here, that the marine-derived alkaloid, fascaplysin, inhibits Aβ fibrillization in vitro. Moreover, the new analogue, 9-methylfascaplysin, was designed and synthesized from 5-methyltryptamine. Interestingly, 9-methylfascaplysin is a more potent inhibitor of Aβ fibril formation than fascaplysin. Incubation of 9-methylfascaplysin with Aβ directly reduced Aβ oligomer formation. Molecular dynamics simulations revealed that 9-methylfascaplysin might interact with negatively charged residues of Aβ₄₂ with polar binding energy. Hydrogen bonds and π⁻π interactions between the key amino acid residues of Aβ₄₂ and 9-methylfascaplysin were also suggested. Most importantly, compared with the typical Aβ oligomer, Aβ modified by nanomolar 9-methylfascaplysin produced less neuronal toxicity in SH-SY5Y cells. 9-Methylfascaplysin appears to be one of the most potent marine-derived compounds that produces anti-Aβ neuroprotective effects. Given previous reports that fascaplysin inhibits acetylcholinesterase and induces P-glycoprotein, the current study results suggest that fascaplysin derivatives can be developed as novel anti-AD drugs that possibly act via inhibition of Aβ aggregation along with other target mechanisms.

Keywords: Alzheimer’s disease; Aβ; fascaplysin; oligomer; β-carboline.

Figure 1

Synthesis of fascaplysin (3a) and 9-methylfascaplysin (3b). Reaction conditions: (a) o-halo acetophenone, I₂/H₂O₂, DMSO, reflux; (b) 220–230 °C, 20–80 min.

Figure 2

9-Methylfascaplysin can inhibit the formation of Aβ₄₂ fibril in a concentration-dependent manner. Aβ₄₂ monomers (10 μM) were incubated with different concentrations of 9-methylfascaplysin (0.3–30 μM), fascaplysin (3–10 μM), or curcumin (3–10 μM). The results were analyzed by Thioflavin-T (ThT) assay. The data shown represent the mean ± SEM of three separate experiments; ** p < 0.01 versus the control group (one-way ANOVA and Tukey’s test).

Figure 3

9-Methylfascaplysin inhibits the aggregation of Aβ₄₂ oligomer in a concentration-dependent manner. (A) A 20 μM Aβ₄₂ monomer solutions, with or without different concentrations of 9-methylfascaplysin (1–10 μM), were continuously vibrated for 48 h. The supernatant was spotted on the membrane after centrifuging the solution. The two membranes were incubated with A11 and 6E10 antibodies, respectively. (B) The optical density of dots was quantified by ImageJ. The data shown represent the mean ± SEM, ** p < 0.01 versus the control group (one-way ANOVA and Tukey’s test).

Figure 4

9-Methylfascaplysin can change the morphology of the Aβ₄₂ oligomer. Typical Aβ₄₂ monomers and 10 μM 9-methylfascaplysin-modified Aβ₄₂ monomers were separately incubated for two days to form oligomers. After centrifuging for 15 min, the supernatant was observed by TEM.

Figure 5

9-Methylfascaplysin can protect against the toxic of Aβ₄₂ oligomers in SH-SY5Y cells. High concentration Aβ₄₂ monomer was diluted to 1.5 μM with Milli-Q water or different concentrations of 9-methylfascaplysin (1–100 nM). The solutions were vibrated for 24 h, and samples were further added to the wells in 96-well plates. After incubating for 24 h, the MTT assay was used to analyze cell viability. Data reported are the mean ± SEM, ^## p < 0.01 versus the control group, ** p < 0.01 versus the Aβ oligomer group (one-way ANOVA and Tukey’s test).

Figure 6

Nanomolar 9-methylfascaplysin reduces Aβ₄₂-induced neurotoxicity in SH-SY5Y cells. (A) Aβ₄₂ oligomer and 100 nM 9-methylfascaplysin-modified Aβ oligomer were added to 6-well plates. FDA/PI double staining was used to demonstrate cell viability. (B) Quantitative analysis of the FDA/PI double staining. The results represent the mean ± SEM of three separate experiments; ^## p < 0.01 versus the control group, ** p < 0.01 versus the Aβ oligomer group (one-way ANOVA and Tukey’s test).

Figure 7

Suggested interactions between Aβ₄₂ and 9-methylfascaplysin in MD simulations. (A) The initial structures of 9-methylfascaplysin (upper) and Aβ₄₂ monomer (lower). (B) The typical conformation of the Aβ–9-methylfascaplysin complex. Aβ₄₂ is displayed as a new cartoon model, with the purple region representing the α-helix. 9-Methylfascaplysin is represented using a licorice model. (C) The contact numbers of each residue of 9-methylfascaplysin with Aβ₄₂ as calculated using the MD trajectories of the final 95–100 ns.

Figure 8

Suggested hydrogen bonding interactions between Aβ₄₂ and 9-methylfascaplysin. (A)Time-dependent plot of the number of the hydrogen bonds between Aβ₄₂ and 9-methylfascaplysin. (B,C) The typical snapshot of hydrogen bonding between Aβ₄₂ and 9-methylfascaplysin. The hydrogen bonding is represented by an orange dotted line.

Figure 9

Typical temporal snapshots in the simulation shows the π–π interactions suggested to occur between Aβ₄₂ and 9-methylfascaplysin. The aromatic residues (A) F4, (B)Y10, and (C) F20 interact with 9-methylfascaplysin via π–π interactions.