Busting up Plaques—Small Molecules Aided by Protein Heavies
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If your plaque-busting molecule is too small to prevent amyloid formation, have it bring along a bigger buddy. This is a strategy that has shown some promise, as reported in the 29 October 2004 issue of Science. Isabella Graef and her colleagues at Stanford University in Stanford, California, reveal that Congo red, when tethered to a much larger chaperone protein, can prevent β-amyloid fibril formation and consequent neurotoxicity.
Small molecules such as Congo red can, in large enough quantities, reduce the aggregation of Aβ into plaques (see ARF related news story), as well as inhibit the aggregation of polyglutamine repeat proteins (see ARF related news story), but their effectiveness is limited by their size, argue first author Jason Gestwicki and colleagues. They outline several challenges for the small molecule trying to prevent the binding of two large proteins: "The binding energy that drives protein-protein contacts is typically distributed over a large area and, unlike enzymes, these surfaces lack a defined hotspot for pharmacological intervention," they write. Moreover, the inherent plasticity of the protein surfaces allows them to shrug off the annoyance of a small molecule as if it were "a grain of salt in a strip of Velcro," to borrow a metaphor that Graef used in presenting this work at the 9th International Conference on Alzheimer’s Disease and Related Disorders earlier this year in Philadelphia.
So the authors have set their sights on recruiting larger molecules to add the "steric bulk" that can prevent Aβ from forming large aggregates. Their choice was a family of chaperones, the FK506 binding proteins (FKBP), found in large supply in many cellular compartments and even the extracellular space. Gestwicki and colleagues tethered the amyloidophilic Congo red to a synthetic ligand for FKBP. The idea is that while the Congo red end of this small molecule seeks out and attaches itself to Aβ, the other end brings along a bulky FKBP. In theory, Aβ will have a much more difficult time ignoring this complex and binding to a fellow Aβ oligomer.
And in practice, this seems to have worked. The authors report that the compound completely suppressed fibrillization—as measured by either turbidity or thioflavin T fluorescence—in an incubation medium containing Aβ1-42 and FKBP. Congo red or FKBP by themselves had much less and no effect, respectively. Electron microscopy confirmed the absence of fibrils, but the authors did find that the compound was allowing the formation of small Aβ oligomers (28 +/- 5 nm in size). The fact that these were fairly uniform in size, and that this particular size of oligomer was not spotted when Aβ was incubated with Congo red alone, suggests to the authors that they have "interrupted fibril formation at a discrete step in the aggregation pathway."
Given recent evidence that smaller Aβ oligomers are toxic, the researchers wondered whether by creating a "ceiling" on the size of Aβ oligomers, they had simply trapped Aβ at a more dangerous size. This did not seem to be the case in cultured hippocampal neurons, however. The Congo red compound and its FKBP helper managed to reduce the in vitro toxicity of Aβ approximately fourfold, and helped prevent damage such as dystrophic neurites, nuclear fragmentation, and membrane blebbing.
In a final series of experiments, Gestwicki examined whether increasing the length of the tether between Congo red and the FKBP-binding ligand would give FKBP more freedom to float around and find an optimal spot at which to prevent binding of Aβ oligomers. They found that a longer tether allowed for even more potent prevention of fibril formation, as well as greater protection of neurons from Aβ toxicity.
"Whereas other inhibitors of Aβ aggregation, such as [Congo red] and short peptides, are active in the 2 to 10 [micro]M range, our best compound was effective at 50 nM," write the authors. That would be powerful medicine indeed. It will be interesting to see whether this approach can be translated to APP transgenic animals with success and without side effects. For example, is there any possible negative effect of tying up FKBP chaperones in this therapeutic mission, and could the Congo red-FKBP complex itself prove detrimental to organisms?—Hakon Heimer
References
News Citations
Further Reading
Primary Papers
- Gestwicki JE, Crabtree GR, Graef IA. Harnessing chaperones to generate small-molecule inhibitors of amyloid beta aggregation. Science. 2004 Oct 29;306(5697):865-9. PubMed.
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David Geffen School of Medicine at UCLA
In the 29 October issue of Science, Gestwicki, Crabtree, and Graef report results of a beautiful series of experiments testing the hypothesis that small-molecule inhibition of protein assembly can work, as long as the inhibitor isn't small! Gestwicki et al. synthesized a bifunctional compound containing one binding site for the amyloid β-protein (Aβ) and a second binding site for the chaperone FK506 binding protein (FKBP). The Aβ-binding moiety was the amyloidophilic dye Congo red (CR). The FKBP ligand was a synthetic ligand for FKBP, abbreviated SLF. The SLF-CR compound then was tested in a variety of assays to determine its effects. The assays included turbidometric and fluorescent (ThT) monitoring of fibril assembly and associated β-sheet formation, electron (EM) and atomic force (AFM) microscopic visualization of fibril morphology, light microscopic and immunofluorescent visualization of neuron morphology and TUNEL staining, MTT assays for cellular metabolism, and quantitative determination of oligomer distributions. Controls included Aβ, CR, FKBP, SLF, and SLF-CR tested singly and in the appropriate combinations.
Bottom line—the hypothesis holds.
By adding SLF-CR and the chaperone FKBP to Aβ fibril assembly reactions, fibril formation was blocked. Few, if any, fibrils could be observed by EM or AFM, and Aβ samples treated in this manner were no longer able to damage (MTT) or kill (TUNEL and morphology) primary cultures of hippocampal neurons. The inhibitory effect did not result from the complete prevention of peptide self-assembly because AFM revealed a population of "approximately uniform" particles of size 28 square nm. PICUP analysis, a photochemical cross-linking method which blocks the interconversion of monomer, oligomer, and higher-order species, thus allowing their quantitation by SDS-PAGE, showed that the SLF-CR/FKBP treatment produced an abundance of tetramers. The authors suggest that fibril formation might be "interrupted…at a discrete step." It is interesting that the area of the particles observed by AFM corresponds to particles of size similar to that of paranuclei, small oligomers of Aβ42 described previously using the same technique (Bitan et al., 2003). Whether paranuclei correspond to the fibril intermediates present at the "discrete step" at which the assembly path is blocked by SLF-CR/FKBP remains to be determined.
Having demonstrated the efficacy of the SLF-CR "lead compound," Gestwicki et al. did what any good medicinal chemists would do: They began to make systematic alterations in the compound's structure. The first site they examined was the linker connecting the two functional groups. Glycyl, butyl, and benzyl linkers produced successively more potent compounds that were active both in inhibiting fibril formation and neurotoxicity.
Even at this embryonic stage of drug development, the fact that IC50 values of 50 nm were obtained is quite encouraging. However, now comes the hard part. The strategy must work in the body. Here, the bifunctional compound and its targets, Aβ and the chaperone protein, must be colocalized. In addition, the site of colocalization must be relevant to the pathogenetic mechanism of Aβ-induced toxicity. Is the site in the ER, lipid rafts, or an extracellular compartment? Although toxicity was blocked in experiments on ex vivo neurons, will the effect be reproducible in the brain? An important related question is whether the small oligomers (tetramers) produced by the treatment are themselves toxic in vivo. Only future experiments will answer these questions.
References:
Bitan G, Kirkitadze MD, Lomakin A, Vollers SS, Benedek GB, Teplow DB. Amyloid beta -protein (Abeta) assembly: Abeta 40 and Abeta 42 oligomerize through distinct pathways. Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):330-5. PubMed.
Mass General Hospital/Harvard Medical School
This is an interesting paper that describes a clever approach for targeting amyloid-β and preventing further aggregation. It is particularly interesting that a relatively uniform Aβ oligomer results from treatment that prevents the formation of fibrils. This could help in understanding the natural history of aggregate formation.
It would be very interesting to try and develop similar analogues that would be useful clinically, but this would probably be quite difficult. The current "small molecules" are quite large, and probably will not enter the CNS. Nonetheless, the bifunctional model compound represents an interesting new approach to this problem.
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