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.

A d-enantiomeric peptide interferes with heteroassociation of amyloid-ß oligomers and prion protein.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects millions of people worldwide. One AD hallmark is the aggregation of ß-amyloid (Aß) into soluble oligomers and insoluble fibrils. Several studies have reported that oligomers rather than fibrils are the most toxic species in AD progression. Aß oligomers bind with high affinity to membrane-associated prion protein (PrP), leading to toxic signaling across the cell membrane, which makes the Aß-PrP interaction an attractive therapeutic target. Here, probing this interaction in more detail, we found that both full-length, soluble human (hu) PrP(23-230) and huPrP(23-144), lacking the globular C-terminal domain, bind to Aß oligomers to form large complexes above the megadalton size range. Following purification by sucrose density-gradient ultracentrifugation, the Aß and huPrP contents in these heteroassemblies were quantified by reversed-phase HPLC. The Aß:PrP molar ratio in these assemblies exhibited some limited variation depending on the molar ratio of the initial mixture. Specifically, a molar ratio of about four Aß to one huPrP in the presence of an excess of huPrP(23-230) or huPrP(23-144) suggested that four Aß units are required to form one huPrP-binding site. Of note, an Aß-binding all-d-enantiomeric peptide, RD2D3, competed with huPrP for Aß oligomers and interfered with Aß-PrP heteroassembly in a concentration-dependent manner. Our results highlight the importance of multivalent epitopes on Aß oligomers for Aß-PrP interactions and have yielded an all-d-peptide-based, therapeutically promising agent that competes with PrP for these interactions.

Deceleration of the neurodegenerative phenotype in pyroglutamate-Aß accumulating transgenic mice by oral treatment with the Aß oligomer eliminating compound RD2.

Alzheimer's disease, a multifactorial incurable disorder, is mainly characterised by progressive neurodegeneration, extracellular accumulation of amyloid-ß protein (Aß), and intracellular aggregation of hyperphosphorylated tau protein. During the last years, Aß oligomers have been claimed to be the disease causing agent. Consequently, development of compounds that are able to disrupt already existing Aß oligomers is highly desirable. We developed d-enantiomeric peptides, consisting solely of d-enantiomeric amino acid residues, for the direct and specific elimination of toxic Aß oligomers. The drug candidate RD2 did show high oligomer elimination efficacy in vitro and the in vivo efficacy of RD2 was demonstrated in treatment studies by enhanced cognition in transgenic mouse models of amyloidosis. Here, we report on the in vitro and in vivo efficacy of the compound towards pyroglutamate-Aß, a particular aggressive Aß species. Using the transgenic TBA2.1 mouse model, which develops pyroglutamate-Aß(3-42) induced neurodegeneration, we are able to show that oral RD2 treatment resulted in a significant deceleration of the progression of the phenotype. The in vivo efficacy against this highly toxic Aß species further validates RD2 as a drug candidate for the therapeutic use in humans.

Do We Need Anti-Prion Compounds to Treat Alzheimer's Disease?

BACKGROUND: While phase III clinical trials for the treatment of Alzheimer's disease (AD) keep failing regardless of the target, more and more data suggest that the toxic protein assemblies of amyloid-beta protein (Aß) and tubulin binding protein (TAU) behave like prions. Irrespective of the question of whether AD is theoretically or practically contagious, the presence of a self-replicating toxic etiologic agent in the brains of AD patients must have decisive consequences for drug development programs and clinical trial designs. OBJECTIVES: We intend to challenge the hypothesis that the underlying etiologic agent of AD is behaving prion-like. We want to discuss whether the outcome of clinical trials could have been predicted based on this hypothesis, and whether compounds that directly disassemble the toxic prion could be more beneficial for AD treatment. METHOD: We collected publicly accessible pre-clinical efficacy data of Aß targeting compounds that failed or still are in phase III clinical trials. We describe the desired properties of an anti-prion compound and compare it the properties of past and current phase III drug candidates. RESULTS: We could not find convincing and reproducible pre-clinical efficacy data of past and current phase III drug candidates on cognition other than in preventive treatment settings. The desired properties of an anti-Aß-prionic compound are fulfilled by the drug candidate RD2, which has been developed to directly disassemble toxic Aß oligomers. CONCLUSION: RD2 is the first anti-prion drug candidate. It is able to enhance cognition and impede neurodegeneration in three different transgenic AD mouse models, even under truly non-preventive conditions and even when applied orally. In addition, it is safe in humans.

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.

Oral Treatment with d-RD2RD2 Impedes Early Disease Mechanisms in SOD1*G93A Transgenic Mice but Does Not Prolong Survival.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting upper and lower motor neurons, thus, progressing to complete muscle loss until the patient dies from respiratory arrest. The disease is not curable, and patients die approximately 2-5 years after diagnosis. Studying the underlying disease mechanisms to get access to new treatment options is, therefore, essential for patients' benefit. However, so far, only three drugs that alleviate the symptoms have been approved by the U.S. Food and Drug Administration (FDA). A new drug candidate for the treatment of ALS is the all-d-enantiomeric peptide RD2RD2. In this study, we investigated the therapeutic effect of RD2RD2 in two setups. First, we analyzed disease progression and survival in 7 week-old B6.Cg-Tg(SOD1*G93A)1Gur/J mice. Second, we confirmed the result of the survival analysis in the B6SJL-Tg(SOD1*G93A)1Gur/J mouse line. Shortly before disease onset, the mice were treated daily with an oral dose of 50 mg/kg body weight. Treatment with RD2RD2 led to a delayed disease onset and reduced motor phenotype as shown using the SHIRPA test, the splay reflex test, and the pole test, but did not affect survival. In conclusion, RD2RD2 has the ability to delay the onset of symptoms.

Aß oligomer concentration in mouse and human brain and its drug-induced reduction ex vivo.

The elimination of amyloid beta (Aß) oligomers is a promising strategy for therapeutic drug development of Alzheimer's disease (AD). AD mouse models that develop Aß pathology have been used to demonstrate in vivo efficacy of compounds that later failed in clinical development. Here, we analyze the concentration and size distribution of Aß oligomers in different transgenic mouse models of AD and in human brain samples by surface-based fluorescence intensity distribution analysis (sFIDA), a highly sensitive method for detecting and quantitating protein aggregates. We demonstrate dose- and time-dependent oligomer elimination by the compound RD2 in mouse and human AD brain homogenates as sources of native Aß oligomers. Such ex vivo target engagement analyses with mouse- and human-brain-derived oligomers have the potential to enhance the translational value from pre-clinical proof-of-concept studies to clinical trials.

Aß Oligomer Elimination Restores Cognition in Transgenic Alzheimer's Mice with Full-blown Pathology.

Oligomers of the amyloid-ß (Aß) protein are suspected to be responsible for the development and progression of Alzheimer's disease. Thus, the development of compounds that are able to eliminate already formed toxic Aß oligomers is very desirable. Here, we describe the in vivo efficacy of the compound RD2, which was developed to directly and specifically eliminate toxic Aß oligomers. In a truly therapeutic, rather than a preventive study, oral treatment with RD2 was able to reverse cognitive deficits and significantly reduce Aß pathology in old-aged transgenic Alzheimer's Disease mice with full-blown pathology and behavioral deficits. For the first time, we demonstrate the in vivo target engagement of RD2 by showing a significant reduction of Aß oligomers in the brains of RD2-treated mice compared to placebo-treated mice. The correlation of Aß elimination in vivo and the reversal of cognitive deficits in old-aged transgenic mice support the hypothesis that Aß oligomers are relevant not only for disease development and progression, but also offer a promising target for the causal treatment of Alzheimer's disease.

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.

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.

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.