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.

Reference:
Gestwicki JE, Crabtree GR, Graef IA. Harnessing chaperones to generate small-molecule inhibitors of amyloid β aggregation. Science. 2004 Oct 29;306(5697):865-9. Abstract