Identification of compounds inhibiting prion replication and toxicity by removing PrPC from the cell surface.

The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.

Sodium hydroxide treatment effectively inhibits PrPCWD replication in farm soil.

Chronic wasting disease (CWD) agents are shed into biological samples, facilitating their horizontal transmission between cervid species. Once prions enter the environment, binding of PrPCWD by soil particles may maintain them near the soil surface, posing a challenge for decontamination. A 2 N sodium hydroxide (NaOH) or 2% sodium hypochlorite (NaClO) solution is traditionally recommended for prion decontamination of equipment and surfaces. Using protein misfolding cyclic amplification with beads and a bioassay with TgElk mice, we compared the effects of these disinfectants in CWD-contaminated soil for 1 or 16 h to those of controls of known infectious titres. Our results suggest that 2 N NaOH in a 1/5 farm soil volume provides a large decrease (>102-fold) in prion infectivity.

In Vitro Prion Amplification Methodology for Inhibitor Screening.

Prion (PrPC) is an endogenous protein found mainly in the nervous system, and its misfolded isoform (PrPSc) is associated with a group of neurodegenerative disorders known as transmissible spongiform encephalopathies, or simply prion diseases. The PrPSc isoform shows an intriguing ability to self-perpetuate, acting as template for PrPC misfolding and consequent aggregation. Aggregation in vitro and in vivo follows a fibrillation processes that is associated with neurodegeneration. Therefore, it is important to investigate and understand the molecular mechanisms involved in this process; such understanding also allows investigation of the action of possible candidate molecules to inhibit this process. Here, we highlight useful in vitro methodologies and analyses that were developed using PrP as a protein model but that, as other amyloid proteins also exhibit the same behavior, may be applied to understand other "prion-like" diseases such as Alzheimer's and Parkinson's disease.

Discovery of small molecules binding to the normal conformation of prion by combining virtual screening and multiple biological activity evaluation methods.

Conformational conversion of the normal cellular prion protein, PrPC, into the misfolded isoform, PrPSc, is considered to be a central event in the development of fatal neurodegenerative diseases. Stabilization of prion protein at the normal cellular form (PrPC) with small molecules is a rational and efficient strategy for treatment of prion related diseases. However, few compounds have been identified as potent prion inhibitors by binding to the normal conformation of prion. In this work, to rational screening of inhibitors capable of stabilizing cellular form of prion protein, multiple approaches combining docking-based virtual screening, steady-state fluorescence quenching, surface plasmon resonance and thioflavin T fluorescence assay were used to discover new compounds interrupting PrPC to PrPSc conversion. Compound 3253-0207 that can bind to PrPC with micromolar affinity and inhibit prion fibrillation was identified from small molecule databases. Molecular dynamics simulation indicated that compound 3253-0207 can bind to the hotspot residues in the binding pocket composed by ß1, ß2 and α2, which are significant structure moieties in conversion from PrPC to PrPSc.

Inhibition of Prion Propagation by 3,4-Dimethoxycinnamic Acid.

Neurodegenerative diseases are associated with accumulation of amyloid-type protein misfolding products. Prion protein (PrP) is known for its ability to aggregate into soluble oligomers that in turn associate into amyloid fibrils. Preventing the formation of these infective and neurotoxic entities represents a viable strategy to control prion diseases. Numerous attempts to find dietary compounds with anti-prion properties have been made; however, the most promising agent found so far was curcumin, which is poorly soluble and merely bioavailable. In the present work, we identify 3,4-dimethoxycinnamic acid (DMCA) which is a bioavailable coffee component as a perspective anti-prion compound. 3,4-Dimethoxycinnamic acid was found to bind potently to prion protein with a Kd of 405 nM. An in vitro study of DMCA effect on PrP oligomerization and fibrillization was undertaken using isothermal titration calorimetry (ITC), dynamic light scattering (DLS) and circular dichroism (CD) methodologies. We demonstrated that DMCA affects PrP oligomer formation reducing the oligomer content by 30-40%, and enhancing SH-SY5Y cell viability treated with prion oligomers. Molecular docking studies allowed to suggest a site where DMCA is able to bind stabilizing PrP tertiary structure. We suggest that DMCA is a perspective dietary compound for prophylaxis of neurodegenerative diseases that needs further research. Copyright © 2017 John Wiley & Sons, Ltd.

Phage M13 for the treatment of Alzheimer and Parkinson disease.

The studies of microbes have been instrumental in combatting infectious diseases, but they have also led to great insights into basic biological mechanism like DNA replication, transcription, and translation of mRNA. In particular, the studies of bacterial viruses, also called bacteriophage, have been quite useful to study specific cellular processes because of the ease to isolate their DNA, mRNA, and proteins. Here, I review the recent discovery of how properties of the filamentous phage M13 emerge as a novel approach to combat neurodegenerative diseases.

New approaches for the selection and evaluation of anti-prion organic compounds.

Transmissible spongiform encephalopathies (TSEs) are infectious neurodegenerative disorders for which symptomatic, curative, or prophylactic treatments are not available. TSEs arise as a consequence of the conversion of soluble cellular prion protein (PrP(C)) into the scrapie isoform (PrP(Sc)), which aggregates and accumulates in the central nervous system. Proposed drugs against TSEs range from small organic compounds to antibodies; various therapeutic strategies have been proposed, including blocking the conversion of PrP(C) to PrP(Sc), increasing PrP(Sc) clearance, and/or stabilizing PrP(C). While several compounds have been effective in vitro and in animal models, none have proven effective in clinical studies to date. Such lack of in vivo efficacy is attributable to high compound toxicity and the lack of permeability of the selected compounds across the blood-brain barrier. In this review, we discuss recent advances in the screening and evaluation of organic compounds for anti-prion activity using multiple approaches, including initial screening in prion-infected cell cultures, in silico prediction of pharmacokinetic and physicochemical properties, ex vivo evaluation of cellular toxicity, and in vitro assays using purified recombinant prion proteins. The main challenges for effective discrimination of candidate lead compounds as therapeutic agents for TSEs, and the disadvantages of each screening strategy are discussed. We propose that a combination of in vitro, ex vivo, and in silico approaches would be useful for the rapid identification of novel anti-prion drug candidates with suitable pharmacokinetic and pharmacodynamic properties that would support their use as drugs.

Convection-enhanced delivery of AAV2-PrPshRNA in prion-infected mice.

Prion disease is caused by a single pathogenic protein (PrPSc), an abnormal conformer of the normal cellular prion protein PrPC. Depletion of PrPC in prion knockout mice makes them resistant to prion disease. Thus, gene silencing of the Prnp gene is a promising effective therapeutic approach. Here, we examined adeno-associated virus vector type 2 encoding a short hairpin RNA targeting Prnp mRNA (AAV2-PrP-shRNA) to suppress PrPC expression both in vitro and in vivo. AAV2-PrP-shRNA treatment suppressed PrP levels and prevented dendritic degeneration in RML-infected brain aggregate cultures. Infusion of AAV2-PrP-shRNA-eGFP into the thalamus of CD-1 mice showed that eGFP was transported to the cerebral cortex via anterograde transport and the overall PrPC levels were reduced by ∼ 70% within 4 weeks. For therapeutic purposes, we treated RML-infected CD-1 mice with AAV2-PrP-shRNA beginning at 50 days post inoculation. Although AAV2-PrP-shRNA focally suppressed PrPSc formation in the thalamic infusion site by ∼ 75%, it did not suppress PrPSc formation efficiently in other regions of the brain. Survival of mice was not extended compared to the untreated controls. Global suppression of PrPC in the brain is required for successful therapy of prion diseases.

Prediction of antiprion activity of therapeutic agents with structure-activity models.

We have developed computational structure-activity models for the prediction of antiprion activity of compounds with known molecular structure. The aim is to apply the developed classification and predictive models in further drug design of antiprion therapeutics. The neural network models developed on the counter-propagation reinforcement learning strategy performed better than the linear regression models. The initial data set was composed of 461 compounds representing diverse groups of chemicals (derivatives of acridine, quinolone, Congo red, 2-aminopyridine-3,5-dicarbonitrile, styrylbenzoazole, 2,5-diamino-benzoquinone), which have been tested in comparable cell-screening assay studies for their activity against prion accumulation. Initially, we have designed a classification model for preliminary sorting of compounds into highly active, active, and inactive groups. Further, only the active compounds with IC50 less or equal to 10 µM were considered as the initial source of data. Altogether, 158 compounds were used to train the artificial neural network model for the estimation of the antiprion activity. The predictive ability of the model was significantly improved after selection of influential variables with genetic algorithm. The root- mean-squared error of the predicted pIC50 values for the external validation set (RMS EV) was slightly above 0.50 log units. A linear regression model, developed for the reasons of comparison, performed with a lower predictive ability (RMS EV 0.92 log units). The applicability domain of the models was assessed by a leverage and distance approach. The set of selected influential structural variables was further studied with the aim to get a better insight into the structural features of compounds potentially involved in disturbing of the prion-prion interactions.

Animal models for testing anti-prion drugs.

Prion diseases belong to a group of fatal infectious diseases with no effective therapies available. Throughout the last 35 years, less than 50 different drugs have been tested in different experimental animal models without hopeful results. An important limitation when searching for new drugs is the existence of appropriate models of the disease. The three different possible origins of prion diseases require the existence of different animal models for testing anti-prion compounds. Wild type, over-expressing transgenic mice and other more sophisticated animal models have been used to evaluate a diversity of compounds which some of them were previously tested in different in vitro experimental models. The complexity of prion diseases will require more pre-screening studies, reliable sporadic (or spontaneous) animal models and accurate chemical modifications of the selected compounds before having an effective therapy against human prion diseases. This review is intended to put on display the more relevant animal models that have been used in the search of new antiprion therapies and describe some possible procedures when handling chemical compounds presumed to have anti-prion activity prior to testing them in animal models.

Therapy in prion diseases.

In the last two decades, knowledge of the neurobiology of prion diseases or transmissible spongiform encephalopathies (TSE) has significantly advanced, but a successful therapy to stop or delay the progression of these disorders remains one of the most challenging goals of biomedical research. Several obstacles to this achievement are in common with other neurodegenerative disorders: difficulties to move from experimental level to clinical stage; appropriate timing of intervention; correct set up of clinical trial. Also in terms of molecular bases of disease, TSE and the other neurodegenerative disorders associated with protein misfolding such as Alzheimer, Parkinson and Huntington diseases, share a central pathogenic role of soluble small aggregates, named oligomers, considered the culprit of neuronal dysfunction: accordingly, these disorders could by termed oligomeropathies. However, the rapid progression of TSE, together with their clinical and molecular heterogeneity, make the therapeutic approach particularly problematic. The main target of the antiprion strategy has been the pathological form of the cellular prion protein (PrP(C)) termed PrP(Sc), invariably associated with the diseases. Several compounds have been found to affect PrP(Sc) formation or enhance its clearance in in vitro models, and prolong survival in experimental animals. However, few of them such as quinacrine and pentosan polysulfate have reached the clinical evaluation; more recently, we have conducted a clinical trial with doxycycline in patients with Creutzfeldt-Jakob disease without satisfactory results. In experimental conditions, active and passive immunization with antibodies against PrP and mucosal vaccination have shown to protect from peripheral infection. Other studies have proposed new potentially effective molecules targeting PrP oligomers. Furthermore, the possibility to interfere with PrP(C) to PrP(Sc) conversion by an active control of PrP(C) is another interesting approach emerging from experimental studies. However, in common with the other oligomeropathies, early diagnosis allowing to treat at risk population in a preclinical stage represent the more realistic perspective for efficient TSE therapy.

Synthesis and evaluation of 1-amino-6-halo-ß-carbolines as antimalarial and antiprion agents.

Malaria is one of the world's most devastating parasitic diseases, causing almost one million deaths each year. Growing resistance to classical antimalarial drugs, such as chloroquine, necessitates the discovery of new therapeutic agents for successful control of this global disease. Here, we report the synthesis of some 6-halo-ß-carbolines as analogues of the potent antimalarial natural product, manzamine A, retaining its heteroaromatic core whilst providing compounds with much improved synthetic accessibility. Two compounds displayed superior activity to chloroquine itself against a resistant Plasmodium falciparum strain, identifying them as promising leads for future development. Furthermore, in line with previous reports of similarities in antimalarial and antiprion effects of aminoaryl-based antimalarial agents, the 1-amino-ß-carboline libraries were also found to possess significant bioactivity against a prion-infected cell line.

Discovery of a class of diketopiperazines as antiprion compounds.

Prion diseases are fatal neurodegenerative and infectious disorders for which effective pharmacological tools are not yet available. This unmet challenge and the recently proposed interplay between prion diseases and Alzheimer's have led to a more urgent demand for new antiprion agents. Herein, we report the identification of a novel bifunctional diketopiperazine (DKP) derivative 1 d, which exhibits activity in the low micromolar range against prion replication in ScGT1 cells, while showing low cytotoxicity. Supported by properly addressed molecular modeling studies, we hypothesized that a planar conformation is the major determinant for activity in this class of compounds. Moreover, studies aimed at assessing the mechanism-of-action at the molecular level showed that 1 d might interact directly with recombinant prion protein (recPrP) to prevent its conversion to the pathogenic misfolded prion protein (PrP(Sc))-like form. This investigation suggests that DKP based antiprion compounds can serve as a promising lead scaffold in developing new drugs to combat prion diseases.

SPR biosensor as a tool for screening prion protein binders as potential antiprion leads.

Prion diseases, also called transmissible spongiform encephalopathies (TSEs), are a group of neurodegenerative disorders affecting animals and humans. No effective treatments are currently available for the diseases, vCJD in particular. It is believed that the formation of protease-resistant insoluble prion protein (PrP(Sc)), which is the main component of amyloidal deposits, from the cellular prion protein (PrP(C)), is essential for the progression of the disease. Therefore, both PrP(Sc) and PrP(C) are currently being used as potential drug targets.This protocol details an optimised experimental protocol to conduct an affinity screening of compound libraries by the immobilisation of PrP(C) using an SPR-based instrument, Biacore 3000.

Peptide NMHRYPNQ of the cellular prion protein (PrP(C)) inhibits aggregation and is a potential key for understanding prion-prion interactions.

Pathogenesis of transmissible spongiform encephalopathies is correlated with a conversion of the normal cellular form of the prion protein (PrP(C)) into the abnormal isoform (scrapie form of PrP). Contact of the normal PrP with its abnormal isoform, the scrapie form of PrP, induces the transformation. Knowledge of molecules that inhibit such contacts leads to an understanding of the mechanism of the aggregation, and these molecules may serve as leads for drugs against transmissible spongiform encephalopathies. Therefore, we screened a synthetic octapeptide library of the globular domain of the human PrP(C) for binding affinity to PrP(C). Two fragments with binding affinity, 149YYRENMHR156 and 153NMHRYPNQ160, were identified with K(d) values of 21 and 25 microM, respectively. A 10-fold excess of peptide 153NMHRYPNQ160 inhibits aggregation of the PrP by 99%. NMR and mass spectrometry showed that the binding region of the peptide 153NMHRYPNQ160 is located at helix 3 of the PrP.

Procedure for identification and characterization of drugs efficient against mammalian prion: from a yeast-based antiprion drug screening assay to in vivo mouse models.

Prion diseases are fatal and incurable infectious neurodegenerative disorders affecting humans and other mammals. Prions are composed essentially if not solely of PrP(Sc), a misfolded form of the host-encoded PrP protein. PrP(Sc) catalyzes the transconformation of the normal endogenous PrP (PrP(C)) into more PrP(Sc). Prion replication thus corresponds to the propagation of an altered folding state of PrP. Several prion proteins have also been identified in the simple model organism Saccharomyces cerevisiae. Yeast prion-based screening assays have allowed identification of drugs active against mammalian prions, thus revealing the existence of common prion propagation mechanisms conserved from yeast to human. To identify these conserved targets, antiprion compounds isolated in yeast can be used as baits in reverse screening strategies. Once identified, these targets could in turn lead to the development of mechanism-based cell-free antiprion screening assays. A reverse screening procedure has been performed for 6AP and GA, two antiprion compounds isolated using a yeast-based assay. Protein folding activity of the large ribosomal RNA was found to be a physical and a functional target of both 6AP and GA therefore suggesting that this activity of the ribosome may constitute a novel mechanism involved in prion propagation and, as a consequence, a new screening target.

Variety of antiprion compounds discovered through an in silico screen based on cellular-form prion protein structure: Correlation between antiprion activity and binding affinity.

Transmissible spongiform encephalopathies are associated with the conformational conversion of the prion protein from the cellular form (PrP(C)) to the scrapie form. This process could be disrupted by stabilizing the PrP(C) conformation, using a specific ligand identified as a chemical chaperone. To discover such compounds, we employed an in silico screen that was based on the nuclear magnetic resonance structure of PrP(C). In combination, we performed ex vivo screening using the Fukuoka-1 strain-infected neuronal mouse cell line at a compound concentration of 10 microM and surface plasmon resonance. Initially, we selected 590 compounds according to the calculated docked energy and finally discovered 24 efficient antiprion compounds, whose chemical structures are quite diverse. Surface plasmon resonance studies showed that the binding affinities of compounds for PrP(C) roughly correlated with the compounds' antiprion activities, indicating that the identification of chemical chaperones that bind to the PrP(C) structure and stabilize it is one efficient strategy for antiprion drug discovery. However, some compounds possessed antiprion activities with low affinities for PrP(C), indicating a mechanism involving additional modulation factors. We classified the compounds roughly into five categories: (i) binding and effective, (ii) low binding and effective, (iii) binding and not effective, (iv) low binding and not effective, and (v) acceleration. In conclusion, we found a spectrum of compounds, many of which are able to modulate the pathogenic conversion reaction. The appropriate categorization of these diverse compounds would facilitate antiprion drug discovery and help to elucidate the pathogenic conversion mechanism.

Antiprion properties of prion protein-derived cell-penetrating peptides.

In prion diseases, the cellular prion protein (PrP(C)) becomes misfolded into the pathogenic scrapie isoform (PrP(Sc)) responsible for prion infectivity. We show here that peptides derived from the prion protein N terminus have potent antiprion effects. These peptides are composed of a hydrophobic sequence followed by a basic segment. They are known to have cell-penetrating ability like regular cell-penetrating peptides (CPPs), short peptides that can penetrate cellular membranes. Healthy (GT1-1) and scrapie-infected (ScGT1-1) mouse neuronal hypothalamic cells were treated with various CPPs, including the prion protein-derived CPPs. Lysates were analyzed for altered protein levels of PrP(C) or PrP(Sc). Treatment with the prion protein-derived CPPs mouse mPrP(1-28) or bovine bPrP(1-30) significantly reduced PrP(Sc) levels in prion-infected cells but had no effect on PrP(C) levels in noninfected cells. Further, presence of prion protein-derived CPPs significantly prolonged the time before infection was manifested when infecting GT1-1 cells with scrapie. Treatment with other CPPs (penetratin, transportan-10, or poly-L-arginine) or prion protein-derived peptides lacking CPP function (mPrP(23-28,) mPrP(19-30,) or mPrP(23-50)) had no effect on PrP(Sc) levels. The results suggest a mechanism by which the signal sequence guides the prion protein-derived CPP into a cellular compartment, where the basic segment binds specifically to PrP(Sc) and disables formation of prions.

Synthesis and evaluation of a focused library of pyridine dicarbonitriles against prion disease.

We report the preparation and screening of a set of 55 pyridine dicarbonitriles as potential prion disease therapeutics. Use of microwave irradiation in an attempt to improve the synthesis typically led to only small enhancement in yields but gave cleaner reactions facilitating product isolation. The library was analysed for binding to human prion protein (huPrPC) by surface plasmon resonance and for inhibition of the formation of its partially protease resistant isoform PrPSc in mouse brain cells (SMB). A total of 26 compounds were found to bind to huPrPC whilst 12 showed discernable inhibition of PrPSc formation, five displaying EC(50)s in the range 2.5-9microwo compounds were found to reduce PrPSc levels to below 30% relative to an untreated control at 50nM.

Comparison of CR36, a new heparan mimetic, and pentosan polysulfate in the treatment of prion diseases.

Sulfated polyanions, including pentosan polysulfate (PPS) and heparan mimetics, number among the most effective drugs that have been used in experimental models of prion disease and are presumed to act in competition with endogenous heparan sulfate proteoglycans as co-receptors for prion protein (PrP) on the cell surface. PPS has been shown to prolong the survival of animals after intracerebral perfusion and is in limited use for the experimental treatment of human transmissible spongiform encephalopathies (TSEs). Here, PPS is compared with CR36, a new heparan mimetic. Ex vivo, CR36 was more efficient than PPS in reducing PrPres in scrapie-infected cell cultures and showed long-lasting activity. In vivo, CR36 showed none of the acute toxicity observed with PPS and reduced PrPres accumulation in spleens, but had only a marginal effect on the survival time of mice infected with bovine spongiform encephalopathy. In contrast, mice treated with PPS that survived the initial toxic mortality had no detectable PrPres in the spleens and lived 185 days longer than controls (+55%). These results show, once again, that anti-TSE drugs cannot be encouraged for human therapeutic trials solely on the basis of in vitro or ex vivo observations, but must first be subjected to in vivo animal studies.