A foldamer-dendrimer conjugate neutralizes synaptotoxic ß-amyloid oligomers.

BACKGROUND AND AIMS: Unnatural self-organizing biomimetic polymers (foldamers) emerged as promising materials for biomolecule recognition and inhibition. Our goal was to construct multivalent foldamer-dendrimer conjugates which wrap the synaptotoxic ß-amyloid (Aß) oligomers with high affinity through their helical foldamer tentacles. Oligomeric Aß species play pivotal role in Alzheimer's disease, therefore recognition and direct inhibition of this undruggable target is a great current challenge. METHODS AND RESULTS: Short helical ß-peptide foldamers with designed secondary structures and side chain chemistry patterns were applied as potential recognition segments and their binding to the target was tested with NMR methods (saturation transfer difference and transferred-nuclear Overhauser effect). Helices exhibiting binding in the µM region were coupled to a tetravalent G0-PAMAM dendrimer. In vitro biophysical (isothermal titration calorimetry, dynamic light scattering, transmission electron microscopy and size-exclusion chromatography) and biochemical tests (ELISA and dot blot) indicated the tight binding between the foldamer conjugates and the Aß oligomers. Moreover, a selective low nM interaction with the low molecular weight fraction of the Aß oligomers was found. Ex vivo electrophysiological experiments revealed that the new material rescues the long-term potentiation from the toxic Aß oligomers in mouse hippocampal slices at submicromolar concentration. CONCLUSIONS: The combination of the foldamer methodology, the fragment-based approach and the multivalent design offers a pathway to unnatural protein mimetics that are capable of specific molecular recognition, and has already resulted in an inhibitor for an extremely difficult target.

Interactions of pathological hallmark proteins: tubulin polymerization promoting protein/p25, beta-amyloid, and alpha-synuclein.

The disordered tubulin polymerization promoting protein (TPPP/p25) was found to be co-enriched in neuronal and glial inclusions with α-synuclein in Parkinson disease and multiple system atrophy, respectively; however, co-occurrence of α-synuclein with ß-amyloid (Aß) in human brain inclusions has been recently reported, suggesting the existence of mixed type pathologies that could result in obstacles in the correct diagnosis and treatment. Here we identified TPPP/p25 as an interacting partner of the soluble Aß oligomers as major risk factors for Alzheimer disease using ProtoArray human protein microarray. The interactions of oligomeric Aß with proteins involved in the etiology of neurological disorders were characterized by ELISA, surface plasmon resonance, pelleting experiments, and tubulin polymerization assay. We showed that the Aß(42) tightly bound to TPPP/p25 (K(d) = 85 nm) and caused aberrant protein aggregation by inhibiting the physiologically relevant TPPP/p25-derived microtubule assembly. The pair-wise interactions of Aß(42), α-synuclein, and tubulin were found to be relatively weak; however, these three components formed soluble ternary complex exclusively in the absence of TPPP/p25. The aggregation-facilitating activity of TPPP/p25 and its interaction with Aß was monitored by electron microscopy with purified proteins by pelleting experiments with cell-free extracts as well as by confocal microscopy with CHO cells expressing TPPP/p25 or amyloid. The finding that the interaction of TPPP/p25 with Aß can produce pathological-like aggregates is tightly coupled with unusual pathology of the Alzheimer disease revealed previously; that is, partial co-localization of Aß and TPPP/p25 in the case of diffuse Lewy body disease with Alzheimer disease.

Protein array based interactome analysis of amyloid-ß indicates an inhibition of protein translation.

Oligomeric amyloid-ß is currently of interest in amyloid-ß mediated toxicity and the pathogenesis of Alzheimer's disease. Mapping the amyloid-ß interaction partners could help to discover novel pathways in disease pathogenesis. To discover the amyloid-ß interaction partners, we applied a protein array with more than 8100 unique recombinantly expressed human proteins. We identified 324 proteins as potential interactors of oligomeric amyloid-ß. The Gene Ontology functional analysis of these proteins showed that oligomeric amyloid-ß bound to multiple proteins with diverse functions both from extra and intracellular localizations. This undiscriminating binding phenotype indicates that multiple protein interactions mediate the toxicity of the oligomeric amyloid-ß. The most highly impacted cellular system was the protein translation machinery. Oligomeric amyloid-ß could bind to altogether 24 proteins involved in translation initiation and elongation. The binding of amyloid-ß to purified rat hippocampal ribosomes validated the protein array results. More importantly, in vitro translation assays showed that the oligomeric amyloid-ß had a concentration dependent inhibitory activity on translation. Our results indicate that the inhibited protein synthesis is one of the pathways that can be involved in the amyloid-beta induced neurotoxicity.

Two pyridine derivatives as potential Cu(II) and Zn(II) chelators in therapy for Alzheimer's disease.

Two pyridine derivatives, DMAP and ENDIP, have been investigated as possible metal chelators in the therapy of Alzheimer's disease. Their complex formation with Cu(ii) and Zn(ii) were characterised in detail. In the case of ENDIP a high stability tetradentate ML complex is formed at physiological pH both with Cu(ii) and Zn(ii). DMAP was found to be a weaker metal binder. At physiological pH, it forms a bidentate ML complex with Zn(ii) and MLH(-1) and ML(2) complexes with Cu(ii), depending on the metal ion to ligand ratio. Fluorescence spectroscopy and dynamic light scattering measurements proved that ENDIP effectively competes with aggregated amyloid-beta peptides (Abeta) for both Cu(ii) and Zn(ii) and thus is able to prevent the metal ion-induced amyloid aggregation and to resolubilise amyloid precipitates.

Controlled in situ preparation of A beta(1-42) oligomers from the isopeptide "iso-A beta(1-42)", physicochemical and biological characterization.

Beta-amyloid (A beta) peptides play a crucial role in the pathology of the neurodegeneration in Alzheimer's disease (AD). Biological experiments (both in vitro and animal model studies of AD) require synthetic A beta peptides of standard quality, aggregation grade, neurotoxicity and water solubility. The synthesis of A beta peptides has been difficult, owing to their hydrophobic character, poor solubility and high tendency for aggregation. Recently an isopeptide precursor (iso-A beta(1-42)) was synthesized by Fmoc-chemistry and transformed at neutral pH to A beta(1-42) by O-->N acyl migration in a short period of time. We prepared the same precursor peptide using Boc-chemistry and studied the transformation to A beta(1-42) by acyl migration. The peptide conformation and aggregation processes were studied by several methods (circular dichroism, atomic force and transmission electron microscopy, dynamic light scattering). The biological activity of the synthetic A beta(1-42) was measured by ex vivo (long-term potentiation studies in rat hippocampal slices) and in vivo experiments (spatial learning of rats). It was proven that O-->N acyl migration of the precursor isopeptide results in a water soluble oligomeric mixture of neurotoxic A beta(1-42). These oligomers are formed in situ just before the biological experiments and their aggregation grade could be standardized.