Comparison of three amyloid assembly inhibitors: the sugar scyllo-inositol, the polyphenol epigallocatechin gallate, and the molecular tweezer CLR01

Abstract

Many compounds have been tested as inhibitors or modulators of amyloid β-protein (Aβ) assembly in hope that they would lead to effective, disease-modifying therapy for Alzheimer's disease (AD). These compounds typically were either designed to break apart β-sheets or selected empirically. Two such compounds, the natural inositol derivative scyllo-inositol and the green-tea-derived flavonoid epigallocatechin gallate (EGCG), currently are in clinical trials. Similar to most of the compounds tested thus far, the mechanism of action of scyllo-inositol and EGCG is not understood. Recently, we discovered a novel family of assembly modulators, Lys-specific molecular tweezers, which act by binding specifically to Lys residues and modulate the self-assembly of amyloid proteins, including Aβ, into formation of nontoxic oligomers by a process-specific mechanism (Sinha, S., Lopes, D. H., Du, Z., Pang, E. S., Shanmugam, A., Lomakin, A., Talbiersky, P., Tennstaedt, A., McDaniel, K., Bakshi, R., Kuo, P. Y., Ehrmann, M., Benedek, G. B., Loo, J. A., Klarner, F. G., Schrader, T., Wang, C., and Bitan, G. (2011) Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins. J. Am. Chem. Soc.133, 16958-16969). Here, we compared side-by-side the capability of scyllo-inositol, EGCG, and the molecular tweezer CLR01 to inhibit Aβ aggregation and toxicity. We found that EGCG and CLR01 had comparable activity whereas scyllo-inositol was a weaker inhibitor. Exploration of the binding of EGCG and CLR01 to Aβ using heteronuclear solution-state NMR showed that whereas CLR01 bound to the two Lys and single Arg residues in Aβ monomers, only weak, nonspecific binding was detected for EGCG, leaving the binding mode of the latter unresolved.

Figure 1

Schematic structures of EGCG, scyllo-inositol, and CLR01.

Figure 2

Inhibition of Aβ42 β-sheet formation. 10 μM Aβ42 was incubated at room temperature with mechanical agitation in the absence or presence of each inhibitor and β-sheet formation was measured using the ThT assay. (A) The effect of scyllo-inositol, EGCG and CLR01 was measured at 1:10 Aβ/inhibitor concentration ratio. (B) The effect of EGCG and CLR01 was measured at 1:1, 1:3, or 1:10 Aβ/inhibitor concentration ratio. The data are presented as mean ± SEM of three independent experiments.

Figure 3

Inhibition of Aβ42 oligomerization. Aβ oligomerization in the presence or absence of scyllo-inositol, EGCG, or CLR01 was probed using dot blots with polyclonal antibody A11. Identical membranes were probed using monoclonal antibody 6E10 as a loading control.

Figure 4

Inhibition of Aβ42-induced cell death in different cell types. 10 μM Aβ42 was added to differentiated PC-12 cells, primary rat hippocampal neurons, or primary rat hippocampal neurons mixed with glial cells in the absence or presence of 10-fold excess of each inhibitor. Cells were incubated with the peptide/inhibitor mixtures for 48 h, and cell death was measured using the LDH release assay. The data are presented as mean ± SEM for three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the Aβ42 in each group.

Figure 5

¹⁵N–¹H HSQC spectra of Aβ40:EGCG or Aβ40:CLR01 mixtures. (A) ¹⁵N–¹H spectra of 60 μM Aβ40 in the presence of 240 μM EGCG. (B) ¹⁵N–¹H spectra of 60 μM Aβ40 in the presence of 240 μM CLR01. (C) Degree of chemical shift change in individual backbone-amide protons and side-chain amide/guanidine protons along the sequence of Aβ40 upon addition of increasing concentrations of EGCG or CLR01.