Foldamers Reveal and Validate Novel Therapeutic Targets Associated with Toxic α-Synuclein Self-Assembly
Posted on: 5 June 2021
Preprint posted on 8 May 2021
Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-022-29724-4
A synthetic foldamer blocks the toxic aggregation of alpha-Synuclein, and reveals where alpha-Synuclein can be targeted in order to develop Parkinson’s disease treatment.
Selected by Joanna Zell
Categories: biochemistry, bioengineering, molecular biology, pharmacology and toxicology
Background
Alpha-Synuclein (αS) is a neuronal protein critical for synaptic vesicle traffic and neurotransmission. Self-aggregation of αS due to misfolding is the major hallmark of Parkinson’s disease (PD). Chemically blocking the aggregation of αS holds great promise for PD treatment. However, the molecular details of how αS aggregates is still unclear. Some chemicals have been discovered that slow αS aggregation, but little is known about their binding sites, which limits further development. It is known that the N-terminal of αS is involved in aggregation.
Foldamers are oligomers which are able to adopt a 3D structure, similar to how oligopeptides fold into proteins. This expansive 3D structure allows foldamers to interact strongly with proteins, if the right foldamer is synthesised! The little-known family of oligoquinoline foldamers are synthesised by consecutively ligating quinoline building blocks through amide bonds (peptide bonds). This gives a stepwise and infinitely modifiable synthesis, in which any number of side chains can be used.
In this preprint, Ahmed et al. screened a library of oligoquinoline foldamers for their ability to inhibit αS aggregation. The hit molecule, SK-129 (fig 1c), is able to rescue the toxic effects of αS aggregation, even showing an improvement in the motility of a C elegans PD model, comparable to healthy C elegans. The Kumar group has previously used oligoquinoline-based foldamers to modify the assembly of amyloid peptides (ref 21-26). Having found a molecule which binds extremely efficiently, the authors have been able to study αS aggregation with new atomic-level precision, with the hope of unravelling how to treat αS aggregation with druggable molecules.
Key findings
(In vitro) A library of oligoquinoline foldamers was screened for inhibition of αS aggregation in an assay based on Thioflavin T (ThT), which produces a strong fluorescent signal when interacting with amyloid protein (fig 1d). The best hit, SK-129, has 4 quinoline groups, with 2 hydrophobic and 2 carboxylic acid sidechains (fig. 1c). A fluorescently labelled SK-129 was also used to quantify the high binding with αS (Kd=0.7µM).
(In cellulo) Neuroblastoma cells treated with pre-aggregated αS show greatly reduced cell survival after 24h, which is rescued by co- (fig. 1h) and post-treatment with SK-129. HEK cells expressing fluorescent-labelled αS show large regions of aggregation when treated with exogenous pre-aggregated αS, in a prion-like mechanism of aggregation, and co-treatment with SK-129 rescues this toxic aggregation (fig 1i).
(Ex vivo) Human brain extracts of PD patients can induce prion-like seeding of αS aggregation in live rat hippocampal neurons, assessed by ThT, Lewis body biomarker antibodies and cell survival. SK-129 could almost completely rescue the effect of aggregation.
(In vivo) SK-129 treatment could rescue loss of motility in an in vivo C elegans PD model (NL5901 strain). NL5901 express fluorescent αS-YFP, and are characterised by frequent αS aggregation compared to WT (fig. 5c,d). They have weaker motility compared to WT (N2), and motility is rescued by SK-129 (fig. 5f).
(Structural studies) 1H- and 15N-NMR studies indicated the binding sites of SK-129, confirming the interaction with the N-terminus. SK-129 binding induces structural changes of residues 6-12, 15-23, 36-45 and 48-53 (fig. 3a). These residues were mutated in four αS variants and used to show that residues 36-45 and 48-53 are directly responsible for self-aggregation and seed-induced aggregation. Circular dichroism (CD) studies further clarified the structural changes occurring to αS when SK-129 binds. αS alone self-aggregates from random coil to β-sheet, but in the presence of SK-129, αS adopts an a-helical structure, indicating that SK-129 functions by maintaining a soluble and healthy a-helical structure.
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Figure 3. (a) Schematic of four mutants produced with corresponding residues deleted at sites involved in SK-129 binding. (k-o) TEM imaging of αS variants allowed to self-aggregate for 7 days. WT αS self-aggregates to fibrils (k) whilst αS2,3 and 4 remain as random coil (o). (p-t) CD spectra of αS variant before and after 7 days of aggregation. WT αS changes from random coil to β-sheet (p), whilst αS4 remains as a random coil (t). |
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What caught my eye in this preprint
This is a wonderful example of interdisciplinary work, where a molecule’s biological potential is discovered and taken right to in vivo testing and showing real phenotypical effects. The impact of this foldamer applied to the field of PD is simultaneously important for therapeutic advancements, and for fundamental studies into the aggregation process of a key protein. Every part of mechanism is investigated, from the creation of molecules to the phenotypic changes in C elegans.
Questions for authors
- How did the group come across this family of foldamers in relation to amyloid proteins? Do you believe that the quinoline groups are involved in π-π interactions with the protein, or just with themselves in order to fold onto itself?
- What is the relevance of the two aS mutants used, αSA30P and αSA53T?
- The C elegans PD model is defined as expressing WT αS-YFP, but how is it considered a PD model? The phenotypic motility difference is remarkable, but what are the genetic differences?
- Could you use this molecule in a mouse model? What is your next step?
- You show that the foldamer is cell permeable in C elegans. What do you expect for the pharmacokinetic properties of this molecule in mammalians?
Acknowledgements
All the figures used in this preLight are taken directly from Ahmed J et. al., 2021 under a CC BY-NC-ND 4.0 international license.
doi: https://doi.org/10.1242/prelights.29399
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