Diseases of aberrant protein folding and assembly are a tremendous threat to global public health. Over 35-million people suffer from AD alone, and the numbers of patients are increasing steeply threatening to create a global financial crisis.40
Effective inhibition or modulation of the aberrant assembly process therefore is an appealing strategy for preventing and/or treating these diseases. Achieving this goal, however, is complicated due to the metastable nature of amyloidogenic protein oligomers and the unfavorable characteristics of amyloid fibrils as targets 41,42
. Though amyloidogenic proteins comprise distinct amino acid sequences, amyloid fibrils2
and oligomers of amyloidogenic proteins38
each share a great deal of structural similarity, which largely is sequence-independent. It is therefore not surprising that inhibitors have been found to arrest the aggregation of more than one protein.43,44
However, to our knowledge, this is the first study in which compounds were sought using a rational approach with the goal to obtain agents that inhibit general amyloid protein assembly and toxicity in a process-specific manner.
Based on the idea of targeting an amino acid residue that participates in all types of the key interactions involved in the nucleation, oligomerization, and elongation processes, we used Lys-specific MTs and found a lead compound, CLR01, which is capable of disrupting the aggregation and toxicity of multiple disease-related proteins. CLR01 was found to inhibit formation of amyloid fibrils by proteins related to AD (Aβ and tau, ), PD (α-synuclein)28
, T2D (IAPP), and several other amyloid-related diseases (). In most cases, the inhibition of aggregation correlated with inhibition of toxicity of these proteins in cell culture ( and and ), suggesting that both were mediated by CLR01 binding to exposed Lys residues and preventing their intra- and intermolecular interactions. Importantly, our toxicity experiments were designed to assess inhibition of the harmful action of toxic oligomers rather than the disruption of the oligomers themselves. Thus, proteins were incubated under conditions that promote oligomerization and CLR01 was added to this preparation immediately before addition to the cells. The data suggest that CLR01 indeed inhibits the toxicity of the oligomers, either by rapidly modulating them into non-toxic structures or by preventing their interaction with their cellular targets.
An exception to the list of proteins inhibited by CLR01 was PrP(106–126), which contains two Lys residues at the N-terminus, away from the long amyloidogenic C-terminal sequence, providing little opportunity for CLR01 binding to interfere with the aggregation. Thus, despite modulating the aggregation of PrP(106–126) into predominantly non-fibrillar structures, CLR01 had little effect on PrP(106–126) aggregation kinetics as measured by turbidity () and only partially attenuated PrP(106–126)-induced toxicity at ≥10-fold excess (). This result, however, does not exclude the possibility that CLR01 may inhibit the conversion of full-length human PrP, which contains 10 Lys residues corresponding to 4.8% of the sequence, from the soluble, cellular form, PrPC, to the insoluble, toxic, and infectious scrapie form, PrPSc. We also could not study the inhibition of lysozyme aggregation by CLR01 because in vitro lysozyme aggregation requires very low pH and high concentration, which are incompatible with CLR01 solubility. However, in vivo lysozyme aggregates at physiologic pH and substantially lower concentrations and its aggregation may be inhibited by CLR01.
The observation that CLR01 inhibited IAPP aggregation and toxicity was surprising because IAPP has a single N-terminal Lys, away from the sequences known to be involved in the amyloid core.45
Moreover, CLR01 appeared to inhibit IAPP aggregation effectively, even at substoichiometric concentrations. However, unlike with most of the other proteins examined, for which the concentration required for inhibition of aggregation was similar to that needed for inhibition of toxicity, in the case of IAPP, high excess CLR01 was needed for inhibition of toxicity. This discrepancy between the stoichiometries of inhibition of aggregation and toxicity suggests that interactions other than CLR01 binding to Lys1 in IAPP may mediate the inhibition. Subsequent studies support this hypothesis and will be reported elsewhere in the near future39
. How inhibitors of protein aggregation bind to their targets and what structures form as a result of inhibitor binding are difficult questions to answer. Here, we used Aβ as a model protein for elucidating the structural details of CLR01 binding and its effect on the resulting assemblies. We found that CLR01 stabilized oligomeric Aβ structures that were similar in size () to those forming in the absence of CLR01. Nevertheless, unlike the Aβ oligomers formed in the absence of inhibitors, those that formed in the presence of CLR01 did not bind A11 () and were not toxic to differentiated PC-12 cells. These observations suggest that CLR01 modulates the structure of early Aβ assemblies in a subtle way that cannot be detected easily by gross morphological or spectroscopic methods, such as EM or DLS, but is sufficient to render the resulting assemblies non-toxic and prevent their further aggregation. Notably, stabilization of non-toxic oligomers appears to be a general mechanism for inhibitors of Aβ assembly and toxicity, including scyllo
and other polyphenols,48
and peptides derived from the C-terminus of Aβ42.49,50
If this mechanism is found to be applicable to other amyloidogenic proteins, the data imply that maintaining the culprit proteins soluble and non-toxic in vivo
may be sufficient to allow their degradation by natural cellular clearance mechanisms, as demonstrated recently for α-synuclein.28
The moderate binding affinity of CLR01 for Lys residues16
and NMR titration experiments (Supplementary Table S1
) suggest that the binding is highly labile (high on–off rate). Our data support the hypothesis that this labile binding is sufficient for interfering with the weak molecular interactions that lead to formation of oligomers or nuclei. We speculate that at the same time, the moderate-affinity binding of CLR01 may not interfere with normal cellular function until concentrations substantially higher than those needed for inhibition are used (). This putative mechanism needs further validation and is beyond the scope of the present study. In this context, it is important to remember that solvent-exposed Lys residues commonly are utilized for attachment of various tags (e.g., biotin, fluorescent dyes) without interfering with biological activity of stably folded proteins. It is therefore plausible that labile binding of CLR01 to these proteins would not affect their bioactivity.
Lys-specific MTs represent a novel, process-specific approach to preventing pathologic protein assembly and therefore are promising drug candidates for treatment and/or prevention of amyloid-related diseases. Recent investigation has demonstrated that CLR01 rescued Aβ-induced decrease in dendritic spine morphology and number, miniature excitatory post-synaptic currents, and long-term potentiation, and significantly reduced Aβ plaque load in the brain of AD transgenic mice.51
These data and rescue of α-synuclein-induced toxicity in a zebrafish model28
suggest that CLR01 also is an effective inhibitor of amyloidogenic proteins’ toxicity in vivo