Alternating anti-prion regimens reduce combination drug resistance but do not further extend survival in scrapie-infected mice.

Prion diseases are fatal and infectious neurodegenerative diseases in humans and other mammals caused by templated misfolding of the endogenous prion protein (PrP). Although there is currently no vaccine or therapy against prion disease, several classes of small-molecule compounds have been shown to increase disease-free incubation time in prion-infected mice. An apparent obstacle to effective anti-prion therapy is the emergence of drug-resistant strains during static therapy with either single compounds or multi-drug combination regimens. Here, we treated scrapie-infected mice with dynamic regimens that alternate between different classes of anti-prion drugs. The results show that alternating regimens containing various combinations of Anle138b, IND24 and IND116135 reduce the incidence of combination drug resistance, but do not significantly increase long-term disease-free survival compared to monotherapy. Furthermore, the alternating regimens induced regional vacuolation profiles resembling those generated by a single component of the alternating regimen, suggesting the emergence of strain dominance.

[Effects of SGI-1027 on Formation and Elimination of PrP

Recently, SGI-1027, a well-known inhibitor of DNA-methyl transferases (DNMTs), was reported to effectively reduce formation of pathogenic PrP^(Sc) in prion-infected cells. Herein, we confirm the elimination of PrP^(Sc) in chronic wasting disease (CWD) prion-infected neurons by SGI-1027, and pinpoint the binding region of human prion protein to SGI-1027. SGI-1027 is broadly functional against various prion disease types, including human prions. Previously, the inhibitory effects of SGI-1027 on DNMT function is well tested in various cell culture models. While neither treatment with a DNMTs enhancer S-adenosyl-L-methionine (SAM), nor with their inhibitor, 5-azacytidine, prevented PrP^(Sc) propagation, SGI-1027 did. Our study suggest that the anti-prion effects of SGI-1027 are a result of its direct interaction with PrP^(C), which effectively interferes with the pathogenic conformational change of PrP^(C) to PrP^(Sc). We conclude that SGI-1027 driven suppression of pathogenic PrP^(Sc) is independent of DNMT.

Sephin1 Reduces Prion Infection in Prion-Infected Cells and Animal Model.

Prion diseases are fatal infectious neurodegenerative disorders in human and animals caused by misfolding of the cellular prion protein (PrPC) into the infectious isoform PrPSc. These diseases have the potential to transmit within or between species, and no cure is available to date. Targeting the unfolded protein response (UPR) as an anti-prion therapeutic approach has been widely reported for prion diseases. Here, we describe the anti-prion effect of the chemical compound Sephin1 which has been shown to protect in mouse models of protein misfolding diseases including amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) by selectively inhibiting the stress-induced regulatory subunit of protein phosphatase 1, thus prolonging eIF2α phosphorylation. We show here that Sephin1 dose and time dependently reduced PrPSc in different neuronal cell lines which were persistently infected with various prion strains. In addition, prion seeding activity was reduced in Sephin1-treated cells. Importantly, we found that Sephin1 significantly overcame the endoplasmic reticulum (ER) stress induced in treated cells, as measured by lower expression of stress-induced aberrant prion protein. In a mouse model of prion infection, intraperitoneal treatment with Sephin1 significantly prolonged survival of prion-infected mice. When combining Sephin1 with the neuroprotective drug metformin, the survival of prion-infected mice was also prolonged. These results suggest that Sephin1 could be a potential anti-prion drug selectively targeting one component of the UPR pathway.

Cellulose ether treatment in vivo generates chronic wasting disease prions with reduced protease resistance and delayed disease progression.

Chronic wasting disease (CWD) is a prion disease of free-ranging and farmed cervids that is highly contagious because of extensive prion shedding and prion persistence in the environment. Previously, cellulose ether compounds (CEs) have been shown to significantly extend the survival of mice inoculated with mouse-adapted prion strains. In this study, we used CEs, TC-5RW, and 60SH-50, in vitro and in vivo to assess their efficacy to interfere with CWD prion propagation. In vitro, CEs inhibited CWD prion amplification in a dose-dependent manner. Transgenic mice over-expressing elk PrPC (tgElk) were injected subcutaneously with a single dose of either of the CEs, followed by intracerebral inoculation with different CWD isolates from white tailed deer, mule deer, or elk. All treated groups showed a prolonged survival of up to more than 30 % when compared to the control group regardless of the CWD isolate used for infection. The extended survival in the treated groups correlated with reduced proteinase K resistance of prions. Remarkably, passage of brain homogenates from treated or untreated animals in tgElk mice resulted in a prolonged life span of mice inoculated with homogenates from CE-treated mice (of + 17%) even in the absence of further treatment. Besides the delayed disease onset upon passage in TgElk mice, the reduced proteinase K resistance was maintained but less pronounced. Therefore, these compounds can be very useful in limiting the spread of CWD in captive and wild-ranging cervids.

Inactivation Methods for Prions.

Incidences of iatrogenic Creutzfeldt-Jakob disease (iCJD) are caused by transplantation of prion-contaminated hormones, cornea and dura mater as well as contact with prion- contaminated medical devices, such as stereotactic electrodes, used in neurosurgery. Because prions are highly resistant and difficult to inactivate, prion contamination is a severe risk when medical instruments are reused after surgical procedures involving suspicious and confirmed cases of patients with prion diseases. Therefore, when high-risk procedures such as cerebral surgery, craniotomy surgery, orthopaedic spinal surgery and ophthalmic surgery are performed for high-risk patients or individuals with prion diseases, it is neces- sary to appropriately treat the medical devices using scientifically proven prion inactivation methods. In this chapter, we introduce fundamental aspects of prion inactivation methods, looking specifically at the practical issues involved in their implementation.

Anti-prion drug screening system in Saccharomyces cerevisiae based on an artificial [LEU2+] prion.

Proteinaceous infectious particles causing mammalian transmissible spongiform encephalopathies or prions are being extensively studied. However due to their hazardous nature, the initial screening of potential anti-prion drugs is often made in a yeast-based screening system utilizing a well-characterized [PSI+] prion (amyloid formed by the translation termination factor Sup35p). In the [PSI+] prion screening system (white/red colony assay), the prion phenotype yields white colonies while addition of an anti-prion drug will yield red colonies. However, this system has some limitations. It is difficult to quantify the effectiveness of the anti-prion compound, the diffusion of the studied compound may affect the result, and the deficiency of glutathione in cells may prevent the formation of red pigment in cured cells. Therefore, alternative yeast prion screening systems are still needed. This article aims to present an alternative yeast-based system to evaluate anti-prion activity of chemical compounds. The method that was used is based on an artificial [LEU2+] prion created by fusing Leu2p with the prion-forming domain of Sup35p in Saccharomyces cerevisiae. Phenotypic analysis and semi-denaturating detergent agarose gel electrophoresis (SDD-AGE) confirmed the presence of the artificial [LEU2+] prion in yeast cells. This screening system verified the anti-prion activity of 3 drugs that were found to have been active in the white/red colony assay, while one compound (6-chlorotacrine) that was active in the white/red colony assay was found to be inactive in the [LEU2+] system. This new system also appears to be more sensitive than the white/red colony assay.

Anti-prion Butenolides and Diphenylpropanones from the Australian Ascidian Polycarpa procera.

A library of 500 Australian marine invertebrate extracts was screened for anti-prion activity using a yeast-based assay, and this resulted in an extract from the ascidian Polycarpa procera showing potent activity. Purification of this extract led to the isolation of six new butenolide metabolites, the procerolides 1-4 and two related diphenylpropanones, the procerones 5 and 6, as the bioactive components. The structures of 1-6 were elucidated from the analysis of 1D/2D NMR and MS data, and their absolute configurations determined from comparison of experimental and computed ECD data. Compounds 1-6 were tested for anti-prion activity in a yeast-based assay, and 1 and 5 displayed potent bioactivity (EC50 of 23 and 29 µM, respectively) comparable to the potently active anti-prion compound guanabenz. The procerolides and procerones are the first anti-prion compounds to be reported from ascidians, indicating that ascidians may be an untapped source of new lead anti-prion compounds.

Application of PMCA to screen for prion infection in a human cell line used to produce biological therapeutics.

Advances in biotechnology have led to the development of a number of biological therapies for the treatment of diverse human diseases. Since these products may contain or are made using human or animal (e.g. cattle) derived materials, it is crucial to test their safety by ensuring the absence of infectious agents; specifically prions, which are highly resilient to elimination and produce fatal diseases in humans. Many cases of iatrogenic Creutzfeldt-Jakob disease have been caused by the use of biological materials (e.g. human growth hormone) contaminated with prions. For this reason, it is important to screen cells and biological materials for the presence of prions. Here we show the utility of the Protein Misfolding Cyclic Amplification (PMCA) technology as a screening tool for the presence of human (vCJD) and bovine (BSE) prions in a human cell therapy product candidate. First, we demonstrated the sensitivity of PMCA to detect a single cell infected with prions. For these experiments, we used RKM7 cells chronically infected with murine RML prions. Serial dilutions of an infected cell culture showed that PMCA enabled prion amplification from a sample comprised of only one cell. Next, we determined that PMCA performance was robust and uncompromised by the spiking of large quantities of uninfected cells into the reaction. Finally, to demonstrate the practical application of this technology, we analyzed a human cell line being developed for therapeutic use and found it to be PMCA-negative for vCJD and BSE prions. Our findings demonstrate that the PMCA technology has unparalleled sensitivity and specificity for the detection of prions, making it an ideal quality control procedure in the production of biological therapeutics.

Decrease of Protease-Resistant PrPSc Level in ScN2a Cells by Polyornithine and Polyhistidine.

Based on previous studies reporting the anti-prion activity of poly-L-lysine and poly-L-arginine, we investigated cationic poly-L-ornithine (PLO), poly-L-histidine (PLH), anionic poly-L-glutamic acid (PLE) and uncharged poly-L-threonine (PLT) in cultured cells chronically infected by prions to determine their anti-prion efficacy. While PLE and PLT did not alter the level of PrPSc, PLO and PLH exhibited potent PrPSc inhibition in ScN2a cells. These results suggest that the anti-prion activity of poly-basic amino acids is correlated with the cationicity of their functional groups. Comparison of anti-prion activity of PLO and PLH proposes that the anti-prion activity of poly-basic amino acids is associated with their acidic cellular compartments.

Toward Therapy of Human Prion Diseases.

Three decades after the discovery of prions as the cause of Creutzfeldt-Jakob disease and other transmissible spongiform encephalopathies, we are still nowhere close to finding an effective therapy. Numerous pharmacological interventions have attempted to target various stages of disease progression, yet none has significantly ameliorated the course of disease. We still lack a mechanistic understanding of how the prions damage the brain, and this situation results in a dearth of validated pharmacological targets. In this review, we discuss the attempts to interfere with the replication of prions and to enhance their clearance. We also trace some of the possibilities to identify novel targets that may arise with increasing insights into prion biology.

Biological Safety of a Highly Purified 10% Liquid Intravenous Immunoglobulin Preparation from Human Plasma.

BACKGROUND: A highly purified 10% liquid intravenous immunoglobulin, IQYMUNE®, has been developed using an innovative manufacturing process including an affinity chromatography step for the removal of anti-A and anti-B hemagglutinins. OBJECTIVES: The pathogen (viruses and prions) clearance efficacy of the manufacturing process and its robustness for critical steps were investigated. METHODS: The manufacturing process of IQYMUNE® includes two dedicated complementary virus reduction steps: solvent/detergent (S/D) treatment and 20 nm nanofiltration as well as two contributing steps, namely caprylic acid fractionation and anion-exchange chromatography. The clearance capacity and robustness of these steps were evaluated with a wide range of viruses (enveloped and non-enveloped) and with a model of human transmissible spongiform encephalopathies (TSEs). RESULTS: The IQYMUNE® manufacturing process demonstrated a high and robust virus removal capacity with global reduction factors (RFs) of relevant and model viruses: ≥14.8 log10 for human immunodeficiency virus type 1 (HIV-1), ≥16.9 log10 for bovine viral diarrhoea virus (BVDV)/Sindbis virus, ≥15.7 log10 for pseudorabies virus (PRV), ≥12.8 log10 for encephalomyocarditis virus (EMCV) and 11.0 log10 for porcine parvovirus (PPV). The process also exhibited a high removal capacity for the TSE agent with an overall RF of ≥12.9 log10 due to the complementary actions of the caprylic acid fractionation, anion-exchange chromatography and nanofiltration steps. CONCLUSION: Data from virus and prion clearance studies fully support the high safety profile of IQYMUNE®, with a minimal reduction of 11 log10 for the smallest and most resistant non-enveloped virus, PPV, and more than 12 log10 for the TSE agent.

De novo [PSI +] prion formation involves multiple pathways to form infectious oligomers.

Prion and other neurodegenerative diseases are associated with misfolded protein assemblies called amyloid. Research has begun to uncover common mechanisms underlying transmission of amyloids, yet how amyloids form in vivo is still unclear. Here, we take advantage of the yeast prion, [PSI +], to uncover the early steps of amyloid formation in vivo. [PSI +] is the prion form of the Sup35 protein. While [PSI +] formation is quite rare, the prion can be greatly induced by overexpression of the prion domain of the Sup35 protein. This de novo induction of [PSI +] shows the appearance of fluorescent cytoplasmic rings when the prion domain is fused with GFP. Our current work shows that de novo induction is more complex than previously thought. Using 4D live cell imaging, we observed that fluorescent structures are formed by four different pathways to yield [PSI +] cells. Biochemical analysis of de novo induced cultures indicates that newly formed SDS resistant oligomers change in size over time and lysates made from de novo induced cultures are able to convert [psi -] cells to [PSI +] cells. Taken together, our findings suggest that newly formed prion oligomers are infectious.

Developing Therapeutics for PrP Prion Diseases.

The prototypical PrP prion diseases are invariably fatal, and the search for agents to treat them spans more than 30 years, with limited success. However, in the last few years, the application of high-throughput screening, medicinal chemistry, and pharmacokinetic optimization has led to important advances. The PrP prion inoculation paradigm provides a robust assay for testing therapeutic efficacy, and a dozen compounds have been reported that lead to meaningful extension in survival of prion-infected mice. Here, we review the history and recent progress in the field, focusing on studies validated in animal models. Based on screens in cells infected with mouse-passaged PrP prions, orally available compounds were generated that double or even triple the survival of mice infected with the same prion strain. Unfortunately, no compounds have yet shown efficacy against human prions. Nevertheless, the speed of the recent advances brings hope that an effective therapeutic can be developed. A successful treatment for any neurodegenerative disease would be a major achievement, and the growing understanding that the more common neurodegenerative diseases, including Alzheimer's and Parkinson's, progress by an analogous prion mechanism serves to highlight the importance of antiprion therapeutics.

Reactivity of 9-aminoacridine drug quinacrine with glutathione limits its antiprion activity.

Quinacrine-the drug based on 9-aminoacridine-failed in clinical trials for prion diseases, whereas it was active in in vitro studies. We hypothesize that aromatic nucleophilic substitution at C9 could be contributing factor responsible for this failure because of the transfer of acridine moiety from quinacrine to abundant glutathione. Here, we described the semi-large-scale synthesis of the acridinylated glutathione and the consequences of its formation on biological and biophysical activities. The acridinylated glutathione is one order of magnitude weaker prion protein binder than the parent quinacrine. Moreover, according to log DpH 7.4 , the glutathione conjugate is two orders of magnitude more hydrophilic than quinacrine. Its higher hydrophilicity and higher dsDNA binding potency will significantly decrease its bioavailability in membrane-like environment. The glutathione deactivates quinacrine not only directly but also decreases its bioavailability. Furthermore, the conjugate can spontaneously decompose to practically insoluble acridone, which is precipitated out from the living systems.

Mechanisms of prion-induced neurodegeneration.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative disorders characterised by long incubation period, short clinical duration, and transmissibility to susceptible species. Neuronal loss, spongiform changes, gliosis and the accumulation in the brain of the misfolded version of a membrane-bound cellular prion protein (PrP(C)), termed PrP(TSE), are diagnostic markers of these diseases. Compelling evidence links protein misfolding and its accumulation with neurodegenerative changes. Accordingly, several mechanisms of prion-mediated neurotoxicity have been proposed. In this paper, we provide an overview of the recent knowledge on the mechanisms of neuropathogenesis, the neurotoxic PrP species and the possible therapeutic approaches to treat these devastating disorders.

[Development and validation of an analytical method to quantify residues of cleaning products able to inactivate prion]. / Développement et validation d'une méthode de dosage des traces de détergents inactivants totaux du prion.

OBJECTIVES: In this study, a novel analytical method to quantify prion inactivating detergent in rinsing waters coming from the washer-disinfector of a hospital sterilization unit has been developed. The final aim was to obtain an easy and functional method in a routine hospital process which does not need the cleaning product manufacturer services. METHODS: An ICP-MS method based on the potassium dosage of the washer-disinfector's rinsing waters was developed. Potassium hydroxide is present on the composition of the three prion inactivating detergent currently on the French market. The detergent used in this study was the Actanios LDI(®) (Anios laboratories). A Passing and Bablok regression compares concentrations measured with this developed method and with the HPLC-UV manufacturer method. RESULTS: According to results obtained, the developed method is easy to use in a routine hospital process. The Passing and Bablok regression showed that there is no statistical difference between the two analytical methods during the second rinsing step. Besides, both methods were linear on the third rinsing step, with a 1.5ppm difference between the concentrations measured for each method. CONCLUSIONS: This study shows that the ICP-MS method developed is nonspecific for the detergent, but specific for the potassium element which is present in all prion inactivating detergent currently on the French market. This method should be functional for all the prion inactivating detergent containing potassium, if the sensibility of the method is sufficient when the potassium concentration is very low in the prion inactivating detergent formulation.

Rapid chemical decontamination of infectious CJD and scrapie particles parallels treatments known to disrupt microbes and biofilms.

Neurodegenerative human CJD and sheep scrapie are diseases caused by several different transmissible encephalopathy (TSE) agents. These infectious agents provoke innate immune responses in the brain, including late-onset abnormal prion protein (PrP-res) amyloid. Agent particles that lack detectable PrP sequences by deep proteomic analysis are highly infectious. Yet these agents, and their unusual resistance to denaturation, are often evaluated by PrP amyloid disruption. To reexamine the intrinsic resistance of TSE agents to denaturation, a paradigm for less resistant viruses and microbes, we developed a rapid and reproducible high yield agent isolation procedure from cultured cells that minimized PrP amyloid and other cellular proteins. Monotypic neuronal GT1 cells infected with the FU-CJD or 22L scrapie agents do not have complex brain changes that can camouflage infectious particles and prevent their disruption, and there are only 2 reports on infectious titers of any human CJD strain treated with chemical denaturants. Infectious titers of both CJD and scrapie were reduced by >4 logs with Thiourea-urea, a treatment not previously tested. A mere 5 min exposure to 4M GdnHCl at 22°C reduced infectivity by >5 logs. Infectious 22L particles were significantly more sensitive to denaturation than FU-CJD particles. A protocol using sonication with these chemical treatments may effectively decontaminate complicated instruments, such as duodenoscopes that harbor additional virulent microbes and biofilms associated with recent iatrogenic infections.

Neuroprotective effect of cellular prion protein (PrPC) is related with activation of alpha7 nicotinic acetylcholine receptor (α7nAchR)-mediated autophagy flux.

Activation of the alpha7 nicotinic acetylcholine receptor (α7nAchR) is regulated by prion protein (PrPC) expression and has a neuroprotective effect by modulating autophagic flux. In this study, we hypothesized that PrPC may regulate α7nAchR activation and that may prevent prion-related neurodegenerative diseases by regulating autophagic flux. PrP(106-126) treatment decreased α7nAchR expression and activation of autophagic flux. In addition, the α7nAchR activator PNU-282987 enhanced autophagic flux and protected neuron cells against PrP(106-126)-induced apoptosis. However, activation of autophagy and the protective effects of PNU-282987 were inhibited in PrPC knockout hippocampal neuron cells. In addition, PrPC knockout hippocampal neuron cells showed decreased α7nAchR expression levels. Adenoviral overexpression of PrPC in PrPC knockout hippocampal neuron cells resulted in activation of autophagic flux and inhibition of prion peptide-mediated cell death via α7nAchR activation. This is the first report demonstrating that activation of α7nAchR-mediated autophagic flux is regulated by PrPC, and that activation of α7nAchR regulated by PrPC expression may play a pivotal role in protection of neuron cells against prion peptide-induced neuron cell death by autophagy. These results suggest that α7nAchR-mediated autophagic flux may be involved in the pathogenesis of prion-related diseases and may be a therapeutic target for prion-related neurodegenerative diseases.

[The influence of dipole modifiers on the channel-forming activity of amyloid and amyloid-like peptides in lipid bilayers].

We have studied the steady-state transmembrane current induced by amyloid and amyloid-like peptides in lipid bilayers in the presence of dipole modifiers. It has been shown that the addition of dipole modifier, phloretin, to the membrane bathing solutions leads to an increase in the multichannel activity of amyloid beta-peptide fragment 25-35, [Gly35]-amyloid beta-peptide fragment 25--35, prion protein fragment 106-126 and amyloid-like peptides myr-BASP1 (1--13), myr-BASP1(1--19) and GAP-43(1--40). We have found that the effect of phloretin is not the result of dipole potential changes due to adsorption of this modifier on the membrane. Using the various fragments of amyloid beta-peptide, presenilin, prion protein and neuronal proteins BASP1 and GAP-43 allowes to conclude that the steady-state peptide-induced transmembrane current in the case of addition of phloretin is due to the electrostatic interaction between the positively charged channel-forming agents and negatively charged dipole modifier. The results obtained by electron microscopy have demonstrated that this interaction increases degree of peptide oligomerization.

Heparinase I-specific disaccharide unit of heparin is a key structure but insufficient for exerting anti-prion activity in prion-infected cells.

Glycosaminoglycans reportedly play important roles in prion formation, but because of their structural complexity, the chemical structures affecting prion formation have not been fully evaluated. Here, we compared two types of low molecular weight heparins and found that heparinase I-sensitive structures influenced anti-prion activity in prion-infected cells. Surface plasmon resonance analyses showed significant binding of a representative heparinase I substrate disaccharide unit, GlcNS6S-IdoA2S, to recombinant prion protein (PrP) fragments, such as full-length PrP23-231 and N-terminal domain PrP23-89, but not to PrP89-230. This binding was competitively inhibited by heparin or pentosan polysulfate, but not by Cu(2+). These PrP binding profiles of the disaccharide unit are consistent with those previously reported for heparin. However, synthetic compounds comprising disaccharide unit alone or its multimers exhibited no anti-prion activity in prion-infected cells. Consequently, the findings suggest that the heparin disaccharide unit that binds to the N-terminal region of PrP is a key structure, but it is insufficient for exerting anti-prion activity.