Background
Foldamers are protein mimics that do not have a canonical peptide backbone but are able to form secondary structures such as α-helices and β-sheets. These molecules are critical for understanding the parameters of protein folding in terms of purely molecular features (e.g., hydrogen bonding and hydrophobic interactions) rather than features that may be peptide-specific (Goodman et al.,
2007, Zhang et al., 2012). There are distinct classes of foldamers. While the aliphatic foldamers have carbon chains that have variations to the natural α-peptide framework, there is also a class of aromatic foldamers, which include the arylamide foldamers, that contain aromatic rings within the repeating backbone motif (Fig II-17A) (Goodman et al., 2007). Despite having such a distinct configuration from the canonical α-peptide, aromatic foldamers are also able to adopt secondary folds such as the α-helix or β-sheet (Fig II-17B) (Gellman, 1998). Additionally,
Figure II-17 Foldamer framework
(A) The α-peptide is the canonical backbone used in natural peptides. The arylamide foldamers employ aromatic rings as spacers within the backbone.
(B) The polymeric arylamide chain is able to adopt folds as an aliphatic chain would. Shown is a crude example of an arylamide
foldamer stacking in the α-helix
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foldamers are able to oligomerize to form even more complicated quaternary structures (Goodman et al., 2007).
Because these foldamers are able to mimic protein structures, but are not susceptible to cellular proteolytic degradation pathways (Horne et al., 2009), these molecules may be an ideal treatment for interrupting protein aggregation associated with disease.
Certain foldamers inhibit Aβ42 fibrillization
The aim of these experiments was to establish the potential application of aromatic foldamers in combating Aβ42 amyloid formation. We collaborated with Professor William DeGrado, who selected and supplied the panel of arylamide foldamers used here (Fig II-18). The collection of molecules includes carbon side chain additions with various amine arrangements as well as intermittent addition of an ether group to the aromatic rings within the backbone.
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We performed the Aβ42 fibrillization experiment in the presence of equimolar foldamer and monitored amyloid formation by ThT. The foldamers had varying degrees ofsuccess in preventing Aβ42 fibril formation, with the impact ranging from no effect to almost complete inhibition (Fig II-19).
From this collection, we have pulled out three foldamers to highlight the distinct interactions that these molecules may have with Aβ42 (Fig II-20). Foldamer 344 (Fig II- 20, top panel) had no effect on Aβ42 fibrillization kinetics. Foldamer 615 is particularly interesting because it did not suppress fibril formation and the final amyloid load is equivalent to untreated Aβ42, but the foldamer delayed the lag phase preceding fibril formation (Fig II-20, middle panel). The precise mechanism of this interaction requires further characterization, but this finding may indicate that 615 is suppressing the oligomer formation that is required to initiate amyloid formation, but once an elongation- competent structure is formed, the foldamer has no effect on rapid fibril polymerization.
Figure II-19 Certain foldamers prevent Aβ42 fibrillization
Foldamers have varying degrees of success in preventing Aβ42 fibrillization. Monomeric Aβ42 (10 µM) and the indicated foldamer (10 µM) were agitated at 12.7 Hz at 37ºC for 4 h, with ThT monitoring fibril formation over time. Certain foldamers had a strong inhibitory effect while other had no impact on amyloidogenesis. n=6, Mean±SEM
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Additionally, we identified Foldamer 334 as a potent suppressor of Aβ42 fibril formation, which seems to completely inhibit amyloidogenesis (Fig II-20, bottom panel).While certain foldamers were capable of preventing Aβ42 fibrillization, all molecules tested were unable to reverse pre-formed fibrils (Fig II-21). Even the potent inhibitor of amyloid formation, 334, had no effect on eliminating existing Aβ42 fibrils. Therefore, foldamers must be interacting with Aβ42 along the amyloidogenesis pathway (e.g., capping fibril-templating structures) and have no ability to break apart the existing stable contacts common to the amyloid cross-β fold.
No foldamer was able to eliminate existing amyloid fibrils. Pre-formed Aβ42 fibrils (10 µM) were treated with the indicated foldamer (10 µM) at 37ºC for 16 h, with ThT monitoring changes in amyloid level. Each treatment mirrored the untreated Aβ42 group, with amyloid level slightly decreasing over time, but none
demonstrating dramatic
disassembly. The Aβ42 alone condition did have a slight initial increase in amyloid levels, and all Figure II-20 Foldamers have distinct
effects on Aβ42
Foldamers may interact with Aβ42 at different stages of amyloidogenesis. Graphs taken directly from Fig II-19. Foldamer 344 had no effect on Aβ42 fibril formation. 615 extended the lag phase that precedes rapid elongation, suggesting an interaction with obligate pre- amyloid oligomers. 334 was a very strong suppressor of amyloid formation.
Figure II-21 Foldamers do not disassemble Aβ42 fibrils
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of the foldamers prevented this, again consistent with previous results that the foldamers interfere with amyloid formation but are incapable of reversing the stable amyloid contacts once they exist.
Conclusions and Future Directions
These preliminary results suggest that foldamers are indeed a promising avenue to prevent Aβ42 fibril formation. Further experiments are needed to characterize the inhibitory effect and determine how the foldamers interfere with amyloidogenesis. The mechanism will be distinct from a small molecule, such as DAPH-1, which is able to break apart existing contacts in addition to preventing contact formation. This is another caveat of developing foldamers as a therapeutic; it must be administered prior to development of amyloid load. However, the idea that sequestration of toxic soluble species into fibrils is beneficial suggests that foldamers may be ideal to prevent growth of existing plaques without releasing toxic components by disassembling existing aggregates.
Further collections of foldamers might be tested for potency in preventing Aβ42 fibril formation, with the successful structures determined here guiding selection of new molecules. Moreover, a greater understanding of the foldamer state is needed to understand the interaction and predict successful analogs. Some of these foldamers associate into oligomeric complexes, and this may be critical to anti-Aβ42 function. These issues, as well as determining ideal stoichiometry, are currently being investigated in the Shorter lab.
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Overall conclusions: Developing novel antagonists of Aβ42 amyloidogenesis The novel treatments presented here introduce potential disease therapies that might be further developed to combat Aβ42 amyloid conformers. Hsp104, DAPH analongs, and arylamide foldamers each present a separate mode of action, which, in turn, may each be well suited for a specific disease environment. For example, our biochemical assays indicate that Hsp104 and successful DAPH analogs physically dismantle pre-formed Aβ42 fibrils, while successful foldamers could only prevent de novo amyloid formation. As such, foldamer application to an existing amyloid fibril population would be completely ineffective. However, one caveat of reversing Aβ42 fibrils is the potential release of toxic species from sequestration within these aggregates (Hardy and Selkoe, 2002, Koffie et al., 2009). Therefore, a combinatorial therapy might allow synergistic synthesis of these distinct activities. One can imagine that Hsp104 applied alongside small molecules or foldamers might be more effective at eliminating amyloid fibrils that Hsp104 alone because the Hsp104-remodeled Aβ42 monomers can be kept soluble by the other treatment molecules. Moreover, it has been reported that amyloid fibrils formed in the presence of an inhibitory small molecule alter their intermolecular contacts such that the molecule’s target site is altered, creating drug-resistant strains (Roberts et al., 2009). A combination of small molecules was able to overcome this imposed strain selection, but our hope is that even more diverse molecules may synergize further.
The results presented here are mostly preliminary in nature, and further investigations will ultimately determine the potential of each of these treatment molecules in combating Aβ42 amyloidogenesis.