Stabilization of Monomeric Tau Protein by All D-Enantiomeric Peptide Ligands as Therapeutic Strategy for Alzheimer's Disease and Other Tauopathies.

Alzheimer's disease and other tauopathies are the world's leading causes of dementia and memory loss. These diseases are thought to be caused by the misfolding and aggregation of the intracellular tau protein, ultimately leading to neurodegeneration. The tau protein is involved in a multitude of different neurodegenerative diseases. During the onset of tauopathies, tau undergoes structural changes and posttranslational modifications and aggregates into amyloid fibrils that are able to spread with a prion-like behavior. Up to now, there is no therapeutic agent which effectively controls or reverses the disease. Most of the therapeutics that were developed and underwent clinical trials targeted misfolded or aggregated forms of tau. In the current manuscript, we present the selection and characterization of two all D-enantiomeric peptides that bind monomeric tau protein with a low nanomolar KD, stabilize tau in its monomeric intrinsically disordered conformation, and stop the conversion of monomers into aggregates. We show that the effect of the two all D-enantiomeric peptides is strong enough to stop ongoing tau aggregation in vitro and is able to significantly reduce tau fibril assembly in cell culture. Both compounds may serve as new lead components for the development of therapeutic agents against Alzheimer's disease and other tauopathies.

Structural details of amyloid ß oligomers in complex with human prion protein as revealed by solid-state MAS NMR spectroscopy.

Human PrP (huPrP) is a high-affinity receptor for oligomeric amyloid ß (Aß) protein aggregates. Binding of Aß oligomers to membrane-anchored huPrP has been suggested to trigger neurotoxic cell signaling in Alzheimer's disease, while an N-terminal soluble fragment of huPrP can sequester Aß oligomers and reduce their toxicity. Synthetic oligomeric Aß species are known to be heterogeneous, dynamic, and transient, rendering their structural investigation particularly challenging. Here, using huPrP to preserve Aß oligomers by coprecipitating them into large heteroassemblies, we investigated the conformations of Aß(1-42) oligomers and huPrP in the complex by solid-state MAS NMR spectroscopy. The disordered N-terminal region of huPrP becomes immobilized in the complex and therefore visible in dipolar spectra without adopting chemical shifts characteristic of a regular secondary structure. Most of the well-defined C-terminal part of huPrP is part of the rigid complex, and solid-state NMR spectra suggest a loss in regular secondary structure in the two C-terminal α-helices. For Aß(1-42) oligomers in complex with huPrP, secondary chemical shifts reveal substantial ß-strand content. Importantly, not all Aß(1-42) molecules within the complex have identical conformations. Comparison with the chemical shifts of synthetic Aß fibrils suggests that the Aß oligomer preparation represents a heterogeneous mixture of ß-strand-rich assemblies, of which some have the potential to evolve and elongate into different fibril polymorphs, reflecting a general propensity of Aß to adopt variable ß-strand-rich conformers. Taken together, our results reveal structural changes in huPrP upon binding to Aß oligomers that suggest a role of the C terminus of huPrP in cell signaling. Trapping Aß(1-42) oligomers by binding to huPrP has proved to be a useful tool for studying the structure of these highly heterogeneous ß-strand-rich assemblies.

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 ß protein (Aß). Aß 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 Aß oligomers by stabilizing Aß monomers in an aggregation-incompetent conformation. We have proven that our lead compound "D3", an all d-enantiomeric-peptide, specifically eliminates Aß 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.

Design of D-Amino Acids SARS-CoV-2 Main Protease Inhibitors Using the Cationic Peptide from Rattlesnake Venom as a Scaffold.

The C30 endopeptidase (3C-like protease; 3CLpro) is essential for the life cycle of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) since it plays a pivotal role in viral replication and transcription and, hence, is a promising drug target. Molecules isolated from animals, insects, plants, or microorganisms can serve as a scaffold for the design of novel biopharmaceutical products. Crotamine, a small cationic peptide from the venom of the rattlesnake Crotalus durissus terrificus, has been the focus of many studies since it exhibits activities such as analgesic, in vitro antibacterial, and hemolytic activities. The crotamine derivative L-peptides (L-CDP) that inhibit the 3CL protease in the low µM range were examined since they are susceptible to proteolytic degradation; we explored the utility of their D-enantiomers form. Comparative uptake inhibition analysis showed D-CDP as a promising prototype for a D-peptide-based drug. We also found that the D-peptides can impair SARS-CoV-2 replication in vivo, probably targeting the viral protease 3CLpro.

Ligand-Induced Stabilization of the Native Human Superoxide Dismutase 1.

A common characteristic of familial (fALS) and sporadic amyotrophic lateral sclerosis (sALS) is the accumulation of aberrant proteinaceous species in the motor neurons and spinal cord of ALS patients-including aggregates of the human superoxide dismutase 1 (hSOD1). hSOD1 is an enzyme that occurs as a stable dimeric protein with several post-translational modifications such as the formation of an intramolecular disulfide bond and the acquisition of metal cofactors that are essential for enzyme activity and further contribute to protein stability. Some mutations and/or destabilizing factors promote hSOD1 misfolding, causing neuronal death. Aggregates containing misfolded wild-type hSOD1 have been found in the spinal cords of sALS as well as in non-hSOD1 fALS patients, leading to the hypothesis that hSOD1 misfolding is a common part of the ALS pathomechanism. Therefore, stabilizing the native conformation of SOD1 may be a promising approach to prevent the formation of toxic hSOD1 species and thus ALS pathogenesis. Here, we present the 16-mer peptide S1VL-21 that interferes with hSOD1 aggregation. S1VL-21 was identified by phage display selection with the native conformation of hSOD1 as a target. Several methods such as microscale thermophoresis (MST) measurements, aggregation assays, and cell viability assays revealed that S1VL-21 has a micromolar binding affinity to native hSOD1 and considerably reduces the formation of hSOD1 aggregates. This present work therefore provides the first important data on a potential lead compound for hSOD1-related drug development for ALS therapy.

Oral absorption enhancement of the amyloid-ß oligomer eliminating compound RD2 by conjugation with folic acid.

Amyloid-ß (Aß) plays a central role in the development and progression of Alzheimer's disease (AD) with Aß oligomers representing the most toxic species. The all-d-enantiomeric peptide RD2, which recently successfully completed clinical phase I, specifically eliminates Aß oligomers in vitro as well as in vivo and improves cognitive deficits in various transgenic AD mouse models even after oral administration. To further enhance the oral absorption of RD2, folic acid has been conjugated to the d-peptide promoting an endocytosis-mediated uptake via a folate receptor located in the intestine. Two different conjugation strategies were selected to obtain prodrugs with folic acid being cleaved after intestinal absorption releasing unmodified RD2 in order to enable RD2's unaltered systemic efficacy. Both conjugates remained stable in simulated gastrointestinal fluids. But only one of them was suitable as prodrug as it was cleaved to RD2 in vitro in human blood plasma and liver microsomes and in vivo in mice after intravenous injection leading to a systemic release of RD2. Furthermore, the conjugate's permeability in vitro and after oral administration in mice was strongly enhanced compared to unconjugated RD2 demonstrating the prodrug's functionality. However, the conjugate seemed to have impaired the mice's wellbeing shortly after oral administration possibly resulting from strain-specific hypersensitivity to folic acid. Nevertheless, we assume that the prodrug is actually non-toxic, especially in lower concentrations as verified by a cell viability test. Furthermore, lower dosages can be applied with unaltered efficacy due to its enhanced oral absorption.

Metabolic resistance of the D-peptide RD2 developed for direct elimination of amyloid-ß oligomers.

Alzheimer's disease (AD) is a neurodegenerative disorder leading to dementia. Aggregation of the amyloid-ß peptide (Aß) plays an important role in the disease, with Aß oligomers representing the most toxic species. Previously, we have developed the Aß oligomer eliminating therapeutic compound RD2 consisting solely of D-enantiomeric amino acid residues. RD2 has been described to have an oral bioavailability of more than 75% and to improve cognition in transgenic Alzheimer's disease mouse models after oral administration. In the present study, we further examined the stability of RD2 in simulated gastrointestinal fluids, blood plasma and liver microsomes. In addition, we have examined whether RD2 is a substrate for the human D-amino acid oxidase (hDAAO). Furthermore, metabolite profiles of RD2 incubated in human, rodent and non-rodent liver microsomes were compared across species to search for human-specific metabolites that might possibly constitute a threat when applying the compound in humans. RD2 was remarkably resistant against metabolization in all investigated media and not converted by hDAAO. Moreover, RD2 did not influence the activity of any of the tested enzymes. In conclusion, the high stability and the absence of relevant human-specific metabolites support RD2 to be safe for oral administration in humans.

In Vitro Potency and Preclinical Pharmacokinetic Comparison of All-D-Enantiomeric Peptides Developed for the Treatment of Alzheimer's Disease.

Diffusible amyloid-ß (Aß) oligomers are currently presumed to be the most cytotoxic Aß assembly and held responsible to trigger the pathogenesis of Alzheimer's disease (AD). Thus, Aß oligomers are a prominent target in AD drug development. Previously, we reported on our solely D-enantiomeric peptide D3 and its derivatives as AD drug candidates. Here, we compare one of the most promising D3 derivatives, ANK6, with its tandem version (tANK6), and its head-to-tail cyclized isoform (cANK6r). In vitro tests investigating the D-peptides' potencies to inhibit Aß aggregation, eliminate Aß oligomers, and reduce Aß-induced cytotoxicity revealed that all three D-peptides efficiently target Aß. Subsequent preclinical pharmacokinetic studies of the three all-D-peptides in wildtype mice showed promising blood-brain barrier permeability with cANK6r yielding the highest levels in brain. The peptides' potencies to lower Aß toxicity and their remarkable brain/plasma ratios make them promising AD drug candidates.

Comparison of blood-brain barrier penetration efficiencies between linear and cyclic all-d-enantiomeric peptides developed for the treatment of Alzheimer's disease.

Alzheimer's disease (AD), until now, is an incurable progressive neurodegenerative disease. To target toxic amyloid ß oligomers in AD patients' brains and to convert them into non-toxic aggregation-incompetent species, we designed peptides consisting solely of d-enantiomeric amino acid residues. The original lead compound was named D3 and several D3 derivatives were designed to enhance beneficial properties. Here, we compare four d-peptides concerning their efficiencies to pass the blood-brain barrier (BBB). We demonstrate that the d-peptides' concentrations in murine brain directly correlate with concentrations in cerebrospinal fluid. The cyclic d-enantiomeric peptide cRD2D3 is characterized by the highest efficiency to pass the BBB. For in total three cyclic peptides we show that administration of cyclic peptides resulted in up to tenfold higher peak concentrations in brain as compared to their linear equivalents which have partially been characterized before (Jiang et al., 2015; Leithold et al., 2016a). These results suggest that cyclic peptides pass the murine BBB more efficiently than their linear equivalents. cRD2D3's proteolytic stability, oral bioavailability, long duration of action and its favorable brain/plasma ratio reveal that it may become a suitable drug for long-term AD-treatment from a pharmacokinetic point of view.

Aß oligomer eliminating compounds interfere successfully with pEAß(3-42) induced motor neurodegenerative phenotype in transgenic mice.

Currently, there are no causative or disease modifying treatments available for Alzheimer's disease (AD). Previously, it has been shown that D3, a small, fully d-enantiomeric peptide is able to eliminate low molecular weight Aß oligomers in vitro, enhance cognition and reduce plaque load in AD transgenic mice. To further characterise the therapeutic potential of D3 towards N-terminally truncated and pyroglutamated Aß (pEAß(3-42)) we tested D3 and its head-to-tail tandem derivative D3D3 both in vitro and in vivo in the new mouse model TBA2.1. These mice produce human pEAß(3-42) leading to a strong, early onset motor neurodegenerative phenotype. In the present study, we were able to demonstrate 1) strong binding affinity of both D3 and D3D3 to pEAß(3-42) in comparison to Aß(1-42) and 2) increased affinity of the tandem derivative D3D3 in comparison to D3. Subsequently we tested the therapeutic potentials of both peptides in the TBA2.1 animal model. Truly therapeutic, non-preventive treatment with D3 and D3D3 clearly slowed the progression of the neurodegenerative TBA2.1 phenotype, indicating the strong therapeutic potential of both peptides against pEAß(3-42) induced neurodegeneration.

Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization.

HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Studies have shown that the association of Nef with the inner leaflet of the plasma membrane and with endocytic and perinuclear vesicles is essential for most activities of Nef. Using purified recombinant proteins in pull-down assays and by co-immunoprecipitation assays we demonstrate that Nef binds directly and specifically to all GABARAP family members, but not to LC3 family members. Based on nuclear magnetic resonance (NMR) experiments we showed that Nef binds to GABARAP via two surface exposed hydrophobic pockets. S53 and F62 of GABARAP were identified as key residues for the interaction with Nef. During live-cell fluorescence microscopy an accumulation of Nef and all GABARAP family members in vesicular structures throughout the cytoplasm and at the plasma membrane was observed. This plasma membrane accumulation was significantly reduced after knocking down GABARAP, GABARAPL1 and GABARAPL2 with respective siRNAs. We identified GABARAPs as the first known direct interaction partners of Nef that are essential for its plasma membrane localization.

Fibril structure of amyloid-ß(1-42) by cryo-electron microscopy.

Amyloids are implicated in neurodegenerative diseases. Fibrillar aggregates of the amyloid-ß protein (Aß) are the main component of the senile plaques found in brains of Alzheimer's disease patients. We present the structure of an Aß(1-42) fibril composed of two intertwined protofilaments determined by cryo-electron microscopy (cryo-EM) to 4.0-angstrom resolution, complemented by solid-state nuclear magnetic resonance experiments. The backbone of all 42 residues and nearly all side chains are well resolved in the EM density map, including the entire N terminus, which is part of the cross-ß structure resulting in an overall "LS"-shaped topology of individual subunits. The dimer interface protects the hydrophobic C termini from the solvent. The characteristic staggering of the nonplanar subunits results in markedly different fibril ends, termed "groove" and "ridge," leading to different binding pathways on both fibril ends, which has implications for fibril growth.

Optimization of d-Peptides for Aß Monomer Binding Specificity Enhances Their Potential to Eliminate Toxic Aß Oligomers.

Amyloid-beta (Aß) oligomers are thought to be causative for the development and progression of Alzheimer's disease (AD). Starting from the Aß oligomer eliminating d-enantiomeric peptide D3, we developed and applied a two-step procedure based on peptide microarrays to identify D3 derivatives with increased binding affinity and specificity for monomeric Aß(1-42) to further enhance the Aß oligomer elimination efficacy. Out of more than 1000 D3 derivatives, we selected seven novel d-peptides, named ANK1 to ANK7, and characterized them in more detail in vitro. All ANK peptides bound to monomeric Aß(1-42), eliminated Aß(1-42) oligomers, inhibited Aß(1-42) fibril formation, and reduced Aß(1-42)-induced cytotoxicity more efficiently than D3. Additionally, ANK6 completely inhibited the prion-like propagation of preformed Aß(1-42) seeds and showed a nonsignificant tendency for improving memory performance of tg-APPSwDI mice after i.p. application for 4 weeks. This supports the hypothesis that stabilization of Aß monomers and thereby induced elimination of Aß oligomers is a suitable therapeutic strategy.

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

While amyloid-ß protein (Aß) aggregation into insoluble plaques is one of the pathological hallmarks of Alzheimer's disease (AD), soluble oligomeric Aß 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 Aß42 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 Aß42. Our findings demonstrate that RD2 was significantly more efficient in Aß oligomer elimination in vitro compared to D3. Enhanced cognition without reduction of plaque pathology in parallel suggests that synaptic malfunction due to Aß oligomers rather than plaque pathology is decisive for disease development and progression. Thus, Aß oligomer elimination by RD2 treatment may be also beneficial for AD patients.

Correction: Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity.

[This corrects the article DOI: 10.1371/journal.pone.0147470.].

Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity.

Alzheimer´s disease is the most prominent type of dementia and currently no causative treatment is available. According to recent studies, oligomeric species of the amyloid beta (Aß) peptide appear to be the most toxic Aß assemblies. Aß monomers, however, may be not toxic per se and may even have a neuroprotective role. Here we describe a competitive mirror image phage display procedure that allowed us to identify preferentially Aß1-42 monomer binding and thereby stabilizing peptides, which destabilize and thereby eliminate toxic oligomer species. One of the peptides, called Mosd1 (monomer specific d-peptide 1), was characterized in more detail. Mosd1 abolished oligomers from a mixture of Aß1-42 species, reduced Aß1-42 toxicity in cell culture, and restored the physiological phenotype in neuronal cells stably transfected with the gene coding for human amyloid precursor protein.

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

Alzheimer's 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 (Aß) 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 Aß(1-42), their efficacy to eliminate cytotoxic oligomers, and consequently their potency to neutralize Aß(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 Aß targeting compound D3.

Optimization of the All-D Peptide D3 for Aß Oligomer Elimination.

The aggregation of amyloid-ß (Aß) is postulated to be the crucial event in Alzheimer's disease (AD). In particular, small neurotoxic Aß oligomers are considered to be responsible for the development and progression of AD. Therefore, elimination of thesis oligomers represents a potential causal therapy of AD. Starting from the well-characterized d-enantiomeric peptide D3, we identified D3 derivatives that bind monomeric Aß. The underlying hypothesis is that ligands bind monomeric Aß and stabilize these species within the various equilibria with Aß assemblies, leading ultimately to the elimination of Aß oligomers. One of the hereby identified d-peptides, DB3, and a head-to-tail tandem of DB3, DB3DB3, were studied in detail. Both peptides were found to: (i) inhibit the formation of Thioflavin T-positive fibrils; (ii) bind to Aß monomers with micromolar affinities; (iii) eliminate Aß oligomers; (iv) reduce Aß-induced cytotoxicity; and (v) disassemble preformed Aß aggregates. The beneficial effects of DB3 were improved by DB3DB3, which showed highly enhanced efficacy. Our approach yielded Aß monomer-stabilizing ligands that can be investigated as a suitable therapeutic strategy against AD.

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

Strong evidence exists for a central role of amyloid ß-protein (Aß) 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 Aß aggregate size distribution. Applying QIAD, we studied the effect of homotaurine, scyllo-inositol, EGCG, the benzofuran derivative KMS88009, ZAß3W, the D-enantiomeric peptide D3 and its tandem version D3D3 on Aß 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.