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.

Impact of the A2V Mutation on the Heterozygous and Homozygous Aß1-40 Dimer Structures from Atomistic Simulations.

The A2V mutation was reported to protect from Alzheimer's disease in its heterozygous form and cause an early Alzheimer's disease type dementia in its homozygous form. Experiments showed that the aggregation rate follows the order A2V > WT (wild-type) > A2V-WT. To understand the impact of this mutation, we carried out replica exchange molecular dynamics simulations of Aß1-40 WT-A2V and A2V-A2V dimers and compared to the WT dimer. Our atomistic simulations reveal that the mean secondary structure remains constant, but there are substantial differences in the intramolecular and intermolecular conformations upon single and double A2V mutation. Upon single mutation, the intrinsic disorder is reduced, the intermolecular potential energies are reduced, the population of intramolecular three-stranded ß-sheets is increased, and the number of all α dimer topologies is decreased. Taken together, these results offer an explanation for the reduced aggregation rate of the Aß1-40 A2V-WT peptides and the protective effect of A2V in heterozygotes.

Structures of the Alzheimer's Wild-Type Aß1-40 Dimer from Atomistic Simulations.

We have studied the dimer of amyloid beta peptide Aß of 40 residues by means of all-atom replica exchange molecular dynamics. The Aß-dimers have been found to be the smallest toxic species in Alzheimer's disease, but their inherent flexibilities have precluded structural characterization by experimental methods. Though the 24-µs-scale simulation reveals a mean secondary structure of 18% ß-strand and 10% α helix, we find transient configurations with an unstructured N-terminus and multiple ß-hairpins spanning residues 17-21 and 30-36, but the antiparallel and perpendicular peptide orientations are preferred over the parallel organization. Short-lived conformational states also consist of all α topologies, and one compact peptide with ß-sheet structure stabilized by a rather extended peptide with α-helical content. Overall, this first all-atom study provides insights into the equilibrium structure of the Aß1-40 dimer in aqueous solution, opening a new avenue for a comprehensive understanding of the impact of pathogenic and protective mutations in early-stage Alzheimer's disease on a molecular level.

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.

Folding Atomistic Proteins in Explicit Solvent Using Simulated Tempering.

Following a previous report on a coarse-grained protein model in implicit solvent, we applied simulated tempering (ST) with on-the-fly Helmholtz free energy (weight factors) determination to the folding or aggregation of seven proteins with the CHARMM, OPLS, and AMBER protein, and the SPC and TIP3P water force fields. For efficiency and reliability, we also performed replica exchange molecular dynamics (REMD) simulations on the alanine di- and deca-peptide, and the dimer of the Aß16-22 Alzheimer's fragment, and used experimental data and previous simulation results on the chignolin, beta3s, Trp-cage, and WW domain peptides of 10-37 amino acids. The sampling with ST is found to be more efficient than with REMD for a much lower CPU cost. Starting from unfolded or extended conformations, the WW domain and the Trp-cage peptide fold to their NMR structures with a backbone RMSD of 2.0 and 1 Å. Remarkably, the ST simulation explores transient non-native topologies for Trp-cage that have been rarely discussed by other simulations. Our ST simulations also show that the CHARMM22* force field has limitations in describing accurately the beta3s peptide. Taken together, these results open the door to the study of the configurations of single proteins, protein aggregates, and any molecular systems at atomic details in explicit solvent using a single normal CPU. They also demonstrate that our ST scheme can be used with any force field ranging from quantum mechanics to coarse-grain and atomistic.

Kinetics of amyloid aggregation: a study of the GNNQQNY prion sequence.

The small amyloid-forming GNNQQNY fragment of the prion sequence has been the subject of extensive experimental and numerical studies over the last few years. Using unbiased molecular dynamics with the OPEP coarse-grained potential, we focus here on the onset of aggregation in a 20-mer system. With a total of 16.9 µs of simulations at 280 K and 300 K, we show that the GNNQQNY aggregation follows the classical nucleation theory (CNT) in that the number of monomers in the aggregate is a very reliable descriptor of aggregation. We find that the critical nucleus size in this finite-size system is between 4 and 5 monomers at 280 K and 5 and 6 at 300 K, in overall agreement with experiment. The kinetics of growth cannot be fully accounted for by the CNT, however. For example, we observe considerable rearrangements after the nucleus is formed, as the system attempts to optimize its organization. We also clearly identify two large families of structures that are selected at the onset of aggregation demonstrating the presence of well-defined polymorphism, a signature of amyloid growth, already in the 20-mer aggregate.

A multiscale approach to characterize the early aggregation steps of the amyloid-forming peptide GNNQQNY from the yeast prion sup-35.

The self-organization of peptides into amyloidogenic oligomers is one of the key events for a wide range of molecular and degenerative diseases. Atomic-resolution characterization of the mechanisms responsible for the aggregation process and the resulting structures is thus a necessary step to improve our understanding of the determinants of these pathologies. To address this issue, we combine the accelerated sampling properties of replica exchange molecular dynamics simulations based on the OPEP coarse-grained potential with the atomic resolution description of interactions provided by all-atom MD simulations, and investigate the oligomerization process of the GNNQQNY for three system sizes: 3-mers, 12-mers and 20-mers. Results for our integrated simulations show a rich variety of structural arrangements for aggregates of all sizes. Elongated fibril-like structures can form transiently in the 20-mer case, but they are not stable and easily interconvert in more globular and disordered forms. Our extensive characterization of the intermediate structures and their physico-chemical determinants points to a high degree of polymorphism for the GNNQQNY sequence that can be reflected at the macroscopic scale. Detailed mechanisms and structures that underlie amyloid aggregation are also provided.