Lysine-Specific Molecular Tweezers Are Broad-Spectrum Inhibitors of Assembly and Toxicity of Amyloid Proteins

Abstract

Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly therefore is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic and electrostatic interactions important for nucleation, oligomerization, and fibril elongation. Importantly, CLR01 shows no toxicity at concentrations substantially higher than those needed for inhibition. We used amyloid β-protein (Aβ) to further explore the binding site(s) of CLR01 and the impact of its binding on the assembly process. Mass-spectrometry and solution-state NMR demonstrated binding of CLR01 to the Lys residues in Aβ at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aβ oligomers but were non-toxic and were not recognized by the oligomer-specific antibody A11. Thus, CLR01 binds already at the monomer stage and modulates the assembly reaction into formation of non-toxic structures. The data suggest that molecular tweezers are unique, process-specific inhibitors of aberrant protein aggregation and toxicity, which hold promise for developing disease-modifying therapy for amyloidoses.

Comparison of Three Amyloid Assembly Inhibitors: The Sugar scyllo-Inositol, the Polyphenol Epigallocatechin Gallate, and the Molecular Tweezer CLR01

“…Previously, we showed that CLR01 bound to Aβ40 at distinct sites, Lys16, Lys28, and to a low extent Arg5, 16 consistent with its putative mechanism of action. Because EGCG showed similar or stronger inhibitory effects, we asked whether it bound to similar sites on Aβ.…”

“…At low CLR01 concentrations, these changes occurred predominantly around the three cationic bindings sites, and as the concentration of CLR01 increased, gradually the entire spectrum was affected ( Figure 5B), likely due to Aβ selfassembly into nontoxic oligomers. 16 In contrast, only slight resonance perturbation was found in Aβ40:EGCG spectra at ratios up to 1:4 compared to Aβ40 alone ( Figure 5A and B). The resonances affected the most were in the regions Aβ(11− 15), Aβ(16−23), and Aβ(31−33), yet due to the low magnitude of the perturbation these data are difficult to interpret.…”

“…respectively, 16 only weak interaction of EGCG with Aβ40 monomers was observed at concentration ratios up to 1:4, respectively ( Figure 5). At this concentration ratio, the highest ratio studied, the maximum chemical shift induced by EGCG was ∼8 times smaller than the equivalent chemical shift changes in the Aβ40 spectrum in the presence of a similar concentration of CLR01 ( Figure 5B).…”

“…22 Relative to other inhibitors, most of which have been found empirically, the putative mechanism of action of CLR01 is quite well understood. CLR01 binds to Lys residues with micromolar affinity 16,23 and interferes with a combination of hydrophobic and electrostatic interactions that are important in the self-assembly of most amyloidogenic proteins, including Aβ 24−26 and tau. 27−30 This mode of action is a novel, processspecific mechanism plausibly applicable to most amyloidogenic proteins.…”

“…Structural investigation using electron capture dissociation mass spectrometry and solution-state NMR has confirmed that CLR01 indeed binds to the two Lys residues and to a lesser extent to the single Arg residue, already in Aβ monomers. 16 Here, we asked how the activity of the artificial Lys-receptor, CLR01, which was explored as an inhibitor of amyloid proteins' assembly based on a mechanistic rationale, compared with those of the natural compounds, scyllo-inositol and EGCG. On the basis of the distinct structures of these three compounds (Figure 1), it is unlikely that they have a similar mode of interaction with Aβ.…”