Why Is Research on Amyloid-ß Failing to Give New Drugs for Alzheimer's Disease?

The two hallmarks of Alzheimer's disease (AD) are the presence of neurofibrillary tangles (NFT) made of aggregates of the hyperphosphorylated tau protein and of amyloid plaques composed of amyloid-ß (Aß) peptides, primarily Aß1-40 and Aß1-42. Targeting the production, aggregation, and toxicity of Aß with small molecule drugs or antibodies is an active area of AD research due to the general acceptance of the amyloid cascade hypothesis, but thus far all drugs targeting Aß have failed. From a review of the recent literature and our own experience based on in vitro, in silico, and in vivo studies, we present some reasons to explain this repetitive failure.

Combined experimental and simulation studies suggest a revised mode of action of the anti-Alzheimer disease drug NQ-Trp.

Inhibition of the aggregation of the monomeric peptide ß-amyloid (Aß) into oligomers is a widely studied therapeutic approach in Alzheimer's disease (AD). Many small molecules have been reported to work in this way, including 1,4-naphthoquinon-2-yl-L-tryptophan (NQ-Trp). NQ-Trp has been reported to inhibit aggregation, to rescue cells from Aß toxicity, and showed complete phenotypic recovery in an in vivo AD model. In this work we investigated its molecular mechanism by using a combined approach of experimental and theoretical studies, and obtained converging results. NQ-Trp is a relatively weak inhibitor and the fluorescence data obtained by employing the fluorophore widely used to monitor aggregation into fibrils can be misinterpreted due to the inner filter effect. Simulations and NMR experiments showed that NQ-Trp has no specific "binding site"-type interaction with mono- and dimeric Aß, which could explain its low inhibitory efficiency. This suggests that the reported anti-AD activity of NQ-Trp-type molecules in in vivo models has to involve another mechanism. This study has revealed the potential pitfalls in the development of aggregation inhibitors for amyloidogenic peptides, which are of general interest for all the molecules studied in the context of inhibiting the formation of toxic aggregates.

Molecular structure of the NQTrp inhibitor with the Alzheimer Aß1-28 monomer.

The self-assembly of the amyloid-ß (Aß) peptide of various amino acid lengths into senile plaques is one hallmark of Alzheimer's disease pathology. In the past decade, many small molecules, including NQTrp, have been identified to reduce aggregation and toxicity. However, due to the heterogeneity of the conformational ensemble of Aß with drugs, we lack detailed structures of the transient complexes. Following our previous simulation of the monomer of Aß1-28, here we characterize the equilibrium ensemble of the Aß1-28 monomer with NQTrp by means of extensive atomistic replica exchange molecular dynamics simulations using a force field known to fold diverse proteins correctly. While the secondary structure content and the intrinsic disorder of the whole peptides are very similar and the lifetimes of the salt-bridges remain constant, the population of ß-hairpin is reduced by a factor of 1.5 and the population of α-helix in the region 17-24 is increased by a factor of two upon NQTrp binding. These two factors, which impact the free energy barrier for nucleation, provide a first explanation for the reported reduced Aß1-40/1-42 aggregation kinetics in the presence of NQTrp. Backbone and side-chain interactions of Aß with NQTrp may also inhibit Aß-Aß contacts. The fraction of free Aß1-28 monomer is, however, on the order of 20-25% at 17.5 mM, and this shows that the affinity of NQTrp is low and hence its inhibitory activity is not very strong. This inhibitor can be improved to reduce the formation of dimer, a critical step in aggregation and toxicity.

Role of metal ions in the self-assembly of the Alzheimer's amyloid-ß peptide.

Aggregation of amyloid-ß (Aß) by self-assembly into oligomers or amyloids is a central event in Alzheimer's disease. Coordination of transition-metal ions, mainly copper and zinc, to Aß occurs in vivo and modulates the aggregation process. A survey of the impact of Cu(II) and Zn(II) on the aggregation of Aß reveals some general trends: (i) Zn(II) and Cu(II) at high micromolar concentrations and/or in a large superstoichiometric ratio compared to Aß have a tendency to promote amorphous aggregations (precipitation) over the ordered formation of fibrillar amyloids by self-assembly; (ii) metal ions affect the kinetics of Aß aggregations, with the most significant impact on the nucleation phase; (iii) the impact is metal-specific; (iv) Cu(II) and Zn(II) affect the concentrations and/or the types of aggregation intermediates formed; (v) the binding of metal ions changes both the structure and the charge of Aß. The decrease in the overall charge at physiological pH increases the overall driving force for aggregation but may favor more precipitation over fibrillation, whereas the induced structural changes seem more relevant for the amyloid formation.

Engineered bacteriorhodopsin: a molecular scale potential switch.

Bacteriorhodopsin, BR, is a natural, photoresponsive, biomolecule that has potential application in data storage, imaging and sensing. Being membrane-bound, however, it is coupled with metallic electronic surfaces only with some difficulty. We report herein a facile method to generate uniformly orientated, anchored and active monolayers of BR on metallic electrodes. In the present study, the cytoplasmic side of the BR is equipped with an engineered cysteine to achieve largely lipid-free, orientation-specific, highly stable, covalent immobilization on gold surfaces. By using non-invasive Kelvin probe force microscopy, it is possible to measure the light-induced proton accumulation at the extracellular protein surface at truly molecular scales. The intimate probe-BR interaction possible on lipid removal facilitates the detection of photoinduced surface potential switching substantially larger ((20.4 ± 7.5) mV) with functional single delipidated mutant BR trimers than for the wild-type protein. The proton pumping detected is also notably highly unidirectional with the orientated protein.

Enhanced photocurrent in engineered bacteriorhodopsin monolayer.

The integration of the transmembrane protein bacteriorhodopsin (BR) with man-made electrode surfaces has attracted a great deal of interest for some two decades or more and holds significant promise from the perspective of derived photoresponse or energy capture interfaces. Here we demonstrate that a novel and strategically engineered cysteine site (M163C) can be used to intimately and effectively couple delipidated BR to supporting metallic electrode surfaces. By virtue of the combined effects of the greater surface molecular density afforded by delipidation, and the vicinity of the electrostatic changes associated with proton pumping to the transducing metallic continuum, the resulting films generate a considerably greater photocurrent density on wavelength-selective illumination than previously achievable with monolayers of BR. Given the uniquely photoresponsive, wavelength-selective, and photostable characteristics of this protein, the work has implications for utilization in solar energy capture and photodetector devices.

Molecular scale conductance photoswitching in engineered bacteriorhodopsin.

Bacteriorhodopsin (BR) is a robust light-driven proton pump embedded in the purple membrane of the extremophilic archae Halobacterium salinarium . Its photoactivity remains in the dry state, making BR of significant interest for nanotechnological use. Here, in a novel configuration, BR was depleted from most of its endogenous lipids and covalently and asymmetrically anchored onto a gold electrode through a strategically located and highly responsive cysteine mutation; BR has no indigenous cysteines. Chemisorption on gold was characterized by surface plasmon resonance, reductive striping voltammetry, ellipsometry, and atomic force microscopy (AFM). For the first time, the conductance of isolated protein trimers, intimately probed by conducting AFM, was reproducibly and reversibly switched under wavelength-specific conditions (mean resistance of 39 ± 12 MΩ under illumination, 137 ± 18 MΩ in the dark), demonstrating a surface stability that is relevant to potential nanodevice applications.

Deciphering the structure, growth and assembly of amyloid-like fibrils using high-speed atomic force microscopy.

Formation of fibrillar structures of proteins that deposit into aggregates has been suggested to play a key role in various neurodegenerative diseases. However mechanisms and dynamics of fibrillization remains to be elucidated. We have previously established that lithostathine, a protein overexpressed in the pre-clinical stages of Alzheimer's disease and present in the pathognomonic lesions associated with this disease, form fibrillar aggregates after its N-terminal truncation. In this paper we visualized, using high-speed atomic force microscopy (HS-AFM), growth and assembly of lithostathine protofibrils under physiological conditions with a time resolution of one image/s. Real-time imaging highlighted a very high velocity of elongation. Formation of fibrils via protofibril lateral association and stacking was also monitored revealing a zipper-like mechanism of association. We also demonstrate that, like other amyloid ß peptides, two lithostathine protofibrils can associate to form helical fibrils. Another striking finding is the propensity of the end of a growing protofibril or fibril to associate with the edge of a second fibril, forming false branching point. Taken together this study provides new clues about fibrillization mechanism of amyloid proteins.