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...  Read more

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.

View all comments by David Teplow

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.

View all comments by Brian Bacskai