Oleksandr Brener

Oral treatment with the d-enantiomeric peptide D3 improves the pathology and behavior of Alzheimer's Disease transgenic mice.

Several lines of evidence suggest that the amyloid-β-peptide (Aβ) plays a central role in the pathogenesis of Alzheimers disease (AD). Not only Aβ fibrils but also small soluble Aβ oligomers in particular are suspected to be the major toxic species responsible for disease development and progression. The present study reports on in vitro and in vivo properties of the Aβ targeting d-enantiomeric amino acid peptide D3. We show that next to plaque load and inflammation reduction, oral application of the peptide improved the cognitive performance of AD transgenic mice. In addition, we provide in vitro data elucidating the potential mechanism underlying the observed in vivo activity of D3. These data suggest that D3 precipitates toxic Aβ species and converts them into nonamyloidogenic, nonfibrillar, and nontoxic aggregates without increasing the concentration of monomeric Aβ. Thus, D3 exerts an interesting and novel mechanism of action that abolishes toxic Aβ oligomers and thereby supports their decisive role in AD development and progression.

Combining Independent Drug Classes into Superior, Synergistically Acting Hybrid Molecules

Increasing the potency of synthesized drugs has been a stepwise process accomplished by progressively modifying the chemical scaffold of a single parent lead compound. To date, there has been no basis for thinking that the combination of pharmacological effects of independently acting drugs could be achieved beyond mere simultaneous administration. We reasoned that if the target molecule of two independent classes of drugs was the same, chemical synthesis of a hybrid compound where these drugs presented moieties within one molecule might yield synergistic effects; that is, a new quality might emerge that would be more than the sum of the singlemoiety compounds. Such multifunctional hybrid compounds that assign different functions to its different moieties to achieve a synergistic pharmacodynamic effect have successful predecessors in nature: for example, bleomycin is a natural compound with three different moieties acting in concert to cleave DNA.

Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2

The adaptive immunity of bacteria against foreign nucleic acids, mediated by CRISPR (clustered regularly interspaced short palindromic repeats), relies on the specific incorporation of short pieces of the invading foreign DNA into a special genomic locus, termed CRISPR array. The stored sequences (spacers) are subsequently used in the form of small RNAs (crRNAs) to interfere with the target nucleic acid. We explored the DNA-binding mechanism of the immunization protein Csn2 from the human pathogen Streptococcus agalactiae using different biochemical techniques, atomic force microscopic imaging and molecular dynamics simulations. The results demonstrate that the ring-shaped Csn2 tetramer binds DNA ends through its central hole and slides inward, likely by a screw motion along the helical path of the enclosed DNA. The presented data indicate an accessory function of Csn2 during integration of exogenous DNA by end-joining.

QIAD assay for quantitating a compound's efficacy in elimination of toxic Aβ oligomers.

Strong evidence exists for a central role of amyloid β-protein (Aβ) oligomers in the pathogenesis of Alzheimer’s disease. We have developed a fast, reliable and robust in vitro assay, termed QIAD, to quantify the effect of any compound on the Aβ aggregate size distribution. Applying QIAD, we studied the effect of homotaurine, scyllo-inositol, EGCG, the benzofuran derivative KMS88009, ZAβ3W, the D-enantiomeric peptide D3 and its tandem version D3D3 on Aβ aggregation. The predictive power of the assay for in vivo efficacy is demonstrated by comparing the oligomer elimination efficiency of D3 and D3D3 with their treatment effects in animal models of Alzheimer´s disease.

Amyloid Aggregation Inhibitory Mechanism of Arginine-rich D-peptides

It is widely believed that Alzheimers disease pathogenesis is driven by the production and deposition of the amyloid-β peptide (Aβ) in the brain. In this study, we employ a combination of in silico and in vitro approaches to investigate the inhibitory properties of selected arginine-rich D-enantiomeric peptides (D-peptides) against amyloid aggregation. The D-peptides include D3, a 12-residue peptide with anti-amyloid potencies demonstrated in vitro and in vivo, RD2, a scrambled sequence of D3, as well as truncated RD2 variants. Using a global optimization method together with binding free energy calculations followed by molecular dynamics simulations, we perform a detailed analysis of D-peptide binding to Aβ monomer and a fibrillar Aβ structure. Results obtained from both molecular simulations and surface plasmon resonance experiments reveal a strong binding of D3 and RD2 to Aβ, leading to a significant reduction in the amount of β structures in both monomer and fibril, which was also demonstrated in Thioflavin T assays. The binding of the D-peptides to Aβ is driven by electrostatic interactions, mostly involving the D-arginine residues and Glu11, Glu22 and Asp23 of Aβ. Furthermore, we show that the anti-amyloid activities of the D-peptides depend on the length and sequence of the Dpeptide, its ability to form multiple weak hydrophobic interactions with Aβ, as well as the Aβ oligomer size.

Progress towards structural understanding of infectious sheep PrP-amyloid

The still elusive structural difference of non-infectious and infectious amyloid of the mammalian prion protein (PrP) is a major pending milestone in understanding protein-mediated infectivity in neurodegenerative diseases. Preparations of PrP-amyloid proven to be infectious have never been investigated with a high-resolution technique. All available models to date have been based on low-resolution data. Here, we establish protocols for the preparation of infectious samples of full-length recombinant (rec) PrP-amyloid in NMR-sufficient amounts by spontaneous fibrillation and seeded fibril growth from brain extract. We link biological and structural data of infectious recPrP-amyloid, derived from bioassays, atomic force microscopy, and solid-state NMR spectroscopy. Our data indicate a semi-mobile N‑terminus, some residues with secondary chemical shifts typical of α‑helical secondary structure in the middle part between ∼115 to ∼155, and a distinct β‑sheet core C‑terminal of residue ∼155. These findings are not in agreement with all current models for PrP-amyloid. We also provide evidence that samples seeded from brain extract may not differ in the overall arrangement of secondary structure elements, but rather in the flexibility of protein segments outside the β-core region. Taken together, our protocols provide an essential basis for the high-resolution characterization of non-infectious and infectious PrP-amyloid in the near future.

The Aβ oligomer eliminating D -enantiomeric peptide RD2 improves cognition without changing plaque pathology

While amyloid-β protein (Aβ) aggregation into insoluble plaques is one of the pathological hallmarks of Alzheimer’s disease (AD), soluble oligomeric Aβ has been hypothesized to be responsible for synapse damage, neurodegeneration, learning, and memory deficits in AD. Here, we investigate the in vitro and in vivo efficacy of the d-enantiomeric peptide RD2, a rationally designed derivative of the previously described lead compound D3, which has been developed to efficiently eliminate toxic Aβ42 oligomers as a promising treatment strategy for AD. Besides the detailed in vitro characterization of RD2, we also report the results of a treatment study of APP/PS1 mice with RD2. After 28 days of treatment we observed enhancement of cognition and learning behaviour. Analysis on brain plaque load did not reveal significant changes, but a significant reduction of insoluble Aβ42. Our findings demonstrate that RD2 was significantly more efficient in Aβ oligomer elimination in vitro compared to D3. Enhanced cognition without reduction of plaque pathology in parallel suggests that synaptic malfunction due to Aβ oligomers rather than plaque pathology is decisive for disease development and progression. Thus, Aβ oligomer elimination by RD2 treatment may be also beneficial for AD patients.

Increase of Positive Net Charge and Conformational Rigidity Enhances the Efficacy of d-Enantiomeric Peptides Designed to Eliminate Cytotoxic Aβ Species

Alzheimers disease (AD) is a neurodegenerative disorder and the most common type of dementia. Until now, there is no curative therapy available. Previously, we selected the amyloid-beta (Aβ) targeting peptide D3 consisting of 12 d-enantiomeric amino acid residues by mirror image phage display as a potential drug candidate for the treatment of AD. In the current approach, we investigated the optimization potential of linear D3 with free C-terminus (D3COOH) by chemical modifications. First, the impact of the net charge was investigated and second, cyclization was introduced which is a well-known tool for the optimization of peptides for enhanced target affinity. Following this strategy, three D3 derivatives in addition to D3COOH were designed: C-terminally amidated linear D3 (D3CONH2), cyclic D3 (cD3), and cyclic D3 with an additional arginine residue (cD3r) to maintain the net charge of linear D3CONH2. These four compounds were compared to each other according to their binding affinities to Aβ(1-42), their efficacy to eliminate cytotoxic oligomers, and consequently their potency to neutralize Aβ(1-42) oligomer induced neurotoxicity. D3CONH2 and cD3r versions with equally increased net charge showed superior properties over D3COOH and cD3, respectively. The cyclic versions showed superior properties compared to their linear version with equal net charge, suggesting cD3r to be the most efficient compound among these four. Indeed, treatment of the transgenic AD mouse model Tg-SwDI with cD3r significantly enhanced spatial memory and cognition of these animals as revealed by water maze performance. Therefore, charge increase and cyclization imply suitable modification steps for an optimization approach of the Aβ targeting compound D3.

Immobilization of homogeneous monomeric, oligomeric and fibrillar Aβ species for reliable SPR measurements.

There is strong evidence that the amyloid-beta peptide (Aβ) plays a central role in the pathogenesis of Alzheimers disease (AD). In this context, a detailed quantitative description of the interactions with different Aβ species is essential for characterization of physiological and artificial ligands. However, the high aggregation propensity of Aβ in concert with its susceptibility to structural changes due to even slight changes in solution conditions has impeded surface plasmon resonance (SPR) studies with homogeneous Aβ conformer species. Here, we have adapted the experimental procedures to state-of-the-art techniques and established novel approaches to reliably overcome the aforementioned challenges. We show that the application of density gradient centrifugation (DGC) for sample purification and the use of a single chain variable fragment (scFv) of a monoclonal antibody directed against the amino-terminus of Aβ allows reliable SPR measurements and quality control of the immobilized Aβ aggregate species at any step throughout the experiment.

Large-Scale Oral Treatment Study with the Four Most Promising D3-Derivatives for the Treatment of Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is associated with the aggregation of the amyloid β protein (Aβ). Aβ oligomers are currently thought to be the major neurotoxic agent responsible for disease development and progression. Thus, their elimination is highly desirable for therapy development. Our therapeutic approach aims at specific and direct elimination of toxic Aβ oligomers by stabilizing Aβ monomers in an aggregation-incompetent conformation. We have proven that our lead compound “D3”, an all d-enantiomeric-peptide, specifically eliminates Aβ oligomers in vitro. In vivo, D3 enhances cognition and reduces plaque load in several transgenic AD mouse models. Here, we performed a large-scale oral proof of concept efficacy study, in which we directly compared four of the most promising D3-derivatives in transgenic mice expressing human amyloid precursor protein with Swedish and London mutations (APPSL), transgenic mice, to identify the most effective compound. RD2 and D3D3, both derived from D3 by rational design, were discovered to be the most effective derivatives in improving cognition in the Morris water maze. The performance of RD2- and D3D3-treated mice within the Morris water maze was significantly better than placebo-treated mice and, importantly, nearly as good as those of non-transgenic littermates, suggesting a complete reversal of the cognitive deficit of APPSL mice.