Vaccination with Prion Peptide-Displaying Polyomavirus-Like Particles Prolongs Incubation Time in Scrapie-Infected Mice.

Prion diseases like scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle or Creutzfeldt-Jakob disease (CJD) in humans are fatal neurodegenerative diseases characterized by the conformational conversion of the normal, mainly α-helical cellular prion protein (PrPC) into the abnormal ß-sheet rich infectious isoform PrPSc. Various therapeutic or prophylactic approaches have been conducted, but no approved therapeutic treatment is available so far. Immunisation against prions is hampered by the self-tolerance to PrPC in mammalian species. One strategy to avoid this tolerance is presenting PrP variants in virus-like particles (VLPs). Therefore, we vaccinated C57/BL6 mice with nine prion peptide variants presented by hamster polyomavirus capsid protein VP1/VP2-derived VLPs. Mice were subsequently challenged intraperitoneally with the murine RML prion strain. Importantly, one group exhibited significantly increased mean survival time of 240 days post-inoculation compared with 202 days of the control group. These data show that immunisation with VLPs presenting PrP peptides may represent a promising strategy for an effective vaccination against transmissible spongiform encephalitis agents.

Piperazine derivatives inhibit PrP/PrP(res) propagation in vitro and in vivo.

Prion diseases are fatal neurodegenerative disorders, which are not curable and no effective treatment exists so far. The major neuropathological change in diseased brains is the conversion of the normal cellular form of the prion protein PrPc(C) into a disease-associated isoform PrP(Sc). PrP(Sc) accumulates into multimeres and fibrillar aggregates, which leads to the formation of amyloid plaques. Increasing evidence indicates a fundamental role of PrP(Sc) species and its aggregation in the pathogenesis of prion diseases, which initiates the pathological cascade and leads to neurodegeneration accompanied by spongiform changes. In search of compounds that have the potential to interfere with PrP(Sc) formation and propagation, we used a cell based assay for the screening of potential aggregation inhibitors. The assay deals with a permanently prion infected cell line that was adapted for a high-throughput screening of a compound library composed of 10,000 compounds (DIVERset 2, ChemBridge). We could detect six different classes of highly potent inhibitors of PrP(Sc) propagation in vitro and identified piperazine derivatives as a new inhibitory lead structure, which increased incubation time of scrapie infected mice.

Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson's disease.

In neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and prion diseases, deposits of aggregated disease-specific proteins are found. Oligomeric aggregates are presumed to be the key neurotoxic agent. Here we describe the novel oligomer modulator anle138b [3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole], an aggregation inhibitor we developed based on a systematic high-throughput screening campaign combined with medicinal chemistry optimization. In vitro, anle138b blocked the formation of pathological aggregates of prion protein (PrP(Sc)) and of α-synuclein (α-syn), which is deposited in PD and other synucleinopathies such as dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Notably, anle138b strongly inhibited all prion strains tested including BSE-derived and human prions. Anle138b showed structure-dependent binding to pathological aggregates and strongly inhibited formation of pathological oligomers in vitro and in vivo both for prion protein and α-synuclein. Both in mouse models of prion disease and in three different PD mouse models, anle138b strongly inhibited oligomer accumulation, neuronal degeneration, and disease progression in vivo. Anle138b had no detectable toxicity at therapeutic doses and an excellent oral bioavailability and blood-brain-barrier penetration. Our findings indicate that oligomer modulators provide a new approach for disease-modifying therapy in these diseases, for which only symptomatic treatment is available so far. Moreover, our findings suggest that pathological oligomers in neurodegenerative diseases share structural features, although the main protein component is disease-specific, indicating that compounds such as anle138b that modulate oligomer formation by targeting structure-dependent epitopes can have a broad spectrum of activity in the treatment of different protein aggregation diseases.

A Medicinal Herb Scutellaria lateriflora Inhibits PrP Replication in vitro and Delays the Onset of Prion Disease in Mice.

Transmissible spongiform encephalopathies (TSE) are characterized by the misfolding of the host encoded prion protein (PrP(C)) into a pathogenic isoform (PrP(Sc)) which leads to the accumulation of ß-sheet-rich fibrils and subsequent loss of neurons and synaptic functions. Although many compounds have been identified which inhibit accumulation or dissolve fibrils and aggregates in vitro there is no therapeutic treatment to stop these progressive neurodegenerative diseases. Here we describe the effects of the traditional medicinal herb Scutellaria lateriflora (S. lateriflora) and its natural compounds, the flavonoids baicalein and baicalin, on the development of prion disease using in vitro and in vivo models. S. lateriflora extract as well as both constituents reduced the PrP(res) accumulation in scrapie-infected cell cultures and cell-free conversion assays and lead to the destabilization of pre-existing PrP(Sc) fibrils. Moreover, tea prepared from S. lateriflora, prolonged significantly the incubation time of scrapie-infected mice upon oral treatment. Therefore S. lateriflora extracts as well as the individual compounds can be considered as promising candidates for the development of new therapeutic drugs against TSEs and other neurodegenerative diseases like Alzheimer's and Parkinson's disease.

From high-throughput cell culture screening to mouse model: identification of new inhibitor classes against prion disease.

Transmissible spongiform encephalopathies (TSE) or prion diseases belong to a category of fatal and so far untreatable neurodegenerative conditions. All prion diseases are characterized by both degeneration in the central nervous system (CNS) in humans and animals and the deposition and accumulation of Proteinase K-resistant prion protein (PrP(res)). Until now, no pharmaceutical product has been available to cure these diseases or to alleviate their associated symptoms. Here, a cell-culture screening system is described that allows for the large-scale analysis of the PrP(res) inhibitory potential of a library of compounds and the identification of structural motifs leading potent compounds able to cause PrP(res) clearance at the cellular level. Based on different scrapie-infected cell lines, 10,000 substances were tested, out of which 530 potential inhibitors were identified. After re-screening and validation using a series of dilutions, 14 compounds were identified as the most effective. These 14 compounds were then used for therapeutic studies in a mouse bioassay to test and verify their in vivo potency. Two compounds exhibited therapeutic potential in the mouse model by significantly extending the survival time of intracerebrally infected mice, when treated 90 days after infection with scrapie.

Diphenylpyrazole-derived compounds increase survival time of mice after prion infection.

Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative disorders that can be transmitted by natural infection or inoculation. TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. The emergence of a variant form of CJD (vCJD), which has been associated with BSE, produced strong pressure to search for effective treatments with new drugs. Up to now, however, TSEs have proved incurable, although many efforts have been made both in vitro and in vivo to search for potent therapeutic and prophylactic compounds. For this purpose, we analyzed a compound library consisting of 10,000 compounds with a cell-based high-throughput screening assay dealing with scrapie-infected scrapie mouse brain and ScN(2)A cells and identified a new class of inhibitors consisting of 3,5-diphenylpyrazole (DPP) derivatives. The most effective DPP derivative showed half-maximal inhibition of PrP(Sc) formation at concentrations (IC(50)) of 0.6 and 1.2 µM, respectively. This compound was subsequently subjected to a number of animal experiments using scrapie-infected wild-type C57BL/6 and transgenic Tga20 mice. The DPP derivative induced a significant increase of incubation time both in therapeutic and prophylactic experiments. The onset of the prion disease was delayed by 37 days after intraperitoneal and 42 days after oral application, respectively. In summary, we demonstrate a high in vitro efficiency of DPP derivatives against prion infections that was substantiated in vivo for one of these compounds. These results indicate that the novel class of DPP compounds should comprise excellent candidates for future therapeutic studies.