Adaptation of the protein misfolding cyclic amplification (PMCA) technique for the screening of anti-prion compounds.

Prion diseases result from the misfolding of the physiological prion protein (PrPC) to a pathogenic conformation (PrPSc). Compelling evidence indicates that prevention and/or reduction of PrPSc replication are promising therapeutic strategies against prion diseases. However, the existence of different PrPSc conformations (or strains) associated with disease represents a major problem when identifying anti-prion compounds. Efforts to identify strain-specific anti-prion molecules are limited by the lack of biologically relevant high-throughput screening platforms to interrogate compound libraries. Here, we describe adaptations to the protein misfolding cyclic amplification (PMCA) technology (able to faithfully replicate PrPSc strains) that increase its throughput to facilitate the screening of anti-prion molecules. The optimized PMCA platform includes a reduction in sample and reagents, as well as incubation/sonication cycles required to efficiently replicate and detect rodent-adapted and cervid PrPSc strains. The visualization of PMCA products was performed via dot blots, a method that contributed to reduced processing times. These technical changes allowed us to evaluate small molecules with previously reported anti-prion activity. This proof-of-principle screening was evaluated for six rodent-adapted prion strains. Our data show that these compounds targeted either none, all or some PrPSc strains at variable concentrations, demonstrating that this PMCA system is suitable to test compound libraries for putative anti-prion molecules targeting specific PrPSc strains. Further analyses of a small compound library against deer prions demonstrate the potential of this new PMCA format to identify strain-specific anti-prion molecules. The data presented here demonstrate the use of the PMCA technique in the selection of prion strain-specific anti-prion compounds.

Sigma Receptor Ligands Are Potent Antiprion Compounds that Act Independently of Sigma Receptor Binding.

Prion diseases are invariably fatal neurodegenerative diseases of humans and other animals for which there are no effective treatment options. Previous work from our laboratory identified phenethylpiperidines as a novel class of anti-prion compounds. While working to identify the molecular target(s) of these molecules, we unexpectedly discovered ten novel antiprion compounds based on their known ability to bind to the sigma receptors, σ1R and σ2R, which are currently being tested as therapeutic or diagnostic targets for cancer and neuropsychiatric disorders. Surprisingly, however, knockout of the respective genes encoding σ1R and σ2R (Sigmar1 and Tmem97) in prion-infected N2a cells did not alter the antiprion activity of these compounds, demonstrating that these receptors are not the direct targets responsible for the antiprion effects of their ligands. Further investigation of the most potent molecules established that they are efficacious against multiple prion strains and protect against downstream prion-mediated synaptotoxicity. While the precise details of the mechanism of action of these molecules remain to be determined, the present work forms the basis for further investigation of these compounds in preclinical studies. Given the therapeutic utility of several of the tested compounds, including rimcazole and haloperidol for neuropsychiatric conditions, (+)-pentazocine for neuropathic pain, and the ongoing clinical trials of SA 4503 and ANAVEX2-73 for ischemic stroke and Alzheimer's disease, respectively, this work has immediate implications for the treatment of human prion disease.

Improvement of anti-prion efficacy with stearoxy conjugation of hydroxypropyl methylcellulose in prion-infected mice.

Prion diseases are fatal transmissible neurodegenerative disorders. Among known anti-prions, hydroxypropyl methylcellulose compounds (HPMCs) are unique in their chemical structure and action. They have several excellent anti-prion properties but the effectiveness depends on the prion-infected mouse model. In the present study, we investigated the effects of stearoxy-modified HPMCs on prion-infected cells and mice. Stearoxy modification improved the anti-prion efficacy of HPMCs in prion-infected cells and significantly prolonged the incubation period in a lower HPMC-responding mouse model. However, stearoxy modification showed no improvement over nonmodified HPMCs in an HPMC-responding mouse model. These results offer a new line of inquiry for use with prion-infected mice that do not respond well to HPMCs.

Anti-prion drugs do not improve survival in novel knock-in models of inherited prion disease.

Prion diseases uniquely manifest in three distinct forms: inherited, sporadic, and infectious. Wild-type prions are responsible for the sporadic and infectious versions, while mutant prions cause inherited variants like fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). Although some drugs can prolong prion incubation times up to four-fold in rodent models of infectious prion diseases, no effective treatments for FFI and fCJD have been found. In this study, we evaluated the efficacy of various anti-prion drugs on newly-developed knock-in mouse models for FFI and fCJD. These models express bank vole prion protein (PrP) with the pathogenic D178N and E200K mutations. We applied various drug regimens known to be highly effective against wild-type prions in vivo as well as a brain-penetrant compound that inhibits mutant PrPSc propagation in vitro. None of the regimens tested (Anle138b, IND24, Anle138b + IND24, cellulose ether, and PSCMA) significantly extended disease-free survival or prevented mutant PrPSc accumulation in either knock-in mouse model, despite their ability to induce strain adaptation of mutant prions. Our results show that anti-prion drugs originally developed to treat infectious prion diseases do not necessarily work for inherited prion diseases, and that the recombinant sPMCA is not a reliable platform for identifying compounds that target mutant prions. This work underscores the need to develop therapies and validate screening assays specifically for mutant prions, as well as anti-prion strategies that are not strain-dependent.

Αnti-prion effects of anthocyanins.

Prion diseases, also known as Transmissible Spongiform Encephalopathies (TSEs), are protein-based neurodegenerative disorders (NDs) affecting humans and animals. They are characterized by the conformational conversion of the normal cellular prion protein, PrPC, into the pathogenic isoform, PrPSc. Prion diseases are invariably fatal and despite ongoing research, no effective prophylactic or therapeutic avenues are currently available. Anthocyanins (ACNs) are unique flavonoid compounds and interest in their use as potential neuroprotective and/or therapeutic agents against NDs, has increased significantly in recent years. Therefore, we investigated the potential anti-oxidant and anti-prion effects of Oenin and Myrtillin, two of the most common anthocyanins, using the most accepted in the field overexpressing PrPScin vitro model and a cell free protein aggregation model. Our results, indicate both anthocyanins as strong anti-oxidant compounds, upregulating the expression of genes involved in the anti-oxidant response, and reducing the levels of Reactive Oxygen Species (ROS), produced due to pathogenic prion infection, through the activation of the Keap1-Nrf2 pathway. Importantly, they showcased remarkable anti-prion potential, as they not only caused the clearance of pathogenic PrPSc aggregates, but also completely inhibited the formation of PrPSc fibrils in the Cerebrospinal Fluid (CSF) of patients with Creutzfeldt-Jakob disease (CJD). Therefore, Oenin and Myrtillin possess pleiotropic effects, suggesting their potential use as promising preventive and/or therapeutic agents in prion diseases and possibly in the spectrum of neurodegenerative proteinopathies.

Targeting Neuroinflammation by Pharmacologic Downregulation of Inflammatory Pathways Is Neuroprotective in Protein Misfolding Disorders.

Neuroinflammation plays a crucial role in the development of neurodegenerative protein misfolding disorders. This category of progressive diseases includes, but is not limited to, Alzheimer's disease, Parkinson's disease, and prion diseases. Shared pathogenesis involves the accumulation of misfolded proteins, chronic neuroinflammation, and synaptic dysfunction, ultimately leading to irreversible neuronal loss, measurable cognitive deficits, and death. Presently, there are few to no effective treatments to halt the advancement of neurodegenerative diseases. We hypothesized that directly targeting neuroinflammation by downregulating the transcription factor, NF-κB, and the inflammasome protein, NLRP3, would be neuroprotective. To achieve this, we used a cocktail of RNA targeting therapeutics (SB_NI_112) shown to be brain-penetrant, nontoxic, and effective inhibitors of both NF-κB and NLRP3. We utilized a mouse-adapted prion strain as a model for neurodegenerative diseases to assess the aggregation of misfolded proteins, glial inflammation, neuronal loss, cognitive deficits, and lifespan. Prion-diseased mice were treated either intraperitoneally or intranasally with SB_NI_112. Behavioral and cognitive deficits were significantly protected by this combination of NF-κB and NLRP3 downregulators. Treatment reduced glial inflammation, protected against neuronal loss, prevented spongiotic change, rescued cognitive deficits, and significantly lengthened the lifespan of prion-diseased mice. We have identified a nontoxic, systemic pharmacologic that downregulates NF-κB and NLRP3, prevents neuronal death, and slows the progression of neurodegenerative diseases. Though mouse models do not always predict human patient success and the study was limited due to sample size and number of dosing methods utilized, these findings serve as a proof of principle for continued translation of the therapeutic SB_NI_112 for prion disease and other neurodegenerative diseases. Based on the success in a murine prion model, we will continue testing SB_NI_112 in a variety of neurodegenerative disease models, including Alzheimer's disease and Parkinson's disease.

Trazodone rescues dysregulated synaptic and mitochondrial nascent proteomes in prion neurodegeneration.

The unfolded protein response (UPR) is rapidly gaining momentum as a therapeutic target for protein misfolding neurodegenerative diseases, in which its overactivation results in sustained translational repression leading to synapse loss and neurodegeneration. In mouse models of these disorders, from Alzheimer's to prion disease, modulation of the pathway-including by the licensed drug, trazodone-restores global protein synthesis rates with profound neuroprotective effects. However, the precise nature of the translational impairment, in particular the specific proteins affected in disease, and their response to therapeutic UPR modulation are poorly understood. We used non-canonical amino acid tagging (NCAT) to measure de novo protein synthesis in the brains of prion-diseased mice with and without trazodone treatment, in both whole hippocampus and cell-specifically. During disease the predominant nascent proteome changes occur in synaptic, cytoskeletal and mitochondrial proteins in both hippocampal neurons and astrocytes. Remarkably, trazodone treatment for just 2 weeks largely restored the whole disease nascent proteome in the hippocampus to that of healthy, uninfected mice, predominantly with recovery of proteins involved in synaptic and mitochondrial function. In parallel, trazodone treatment restored the disease-associated decline in synapses and mitochondria and their function to wild-type levels. In conclusion, this study increases our understanding of how translational repression contributes to neurodegeneration through synaptic and mitochondrial toxicity via depletion of key proteins essential for their function. Further, it provides new insights into the neuroprotective mechanisms of trazodone through reversal of this toxicity, relevant for the treatment of neurodegenerative diseases via translational modulation.

Combination of Styrylbenzoazole Compound and Hydroxypropyl Methylcellulose Enhances Therapeutic Effect in Prion-Infected Mice.

Prion diseases are fatal transmissible neurodegenerative disorders. Tremendous efforts have been made for prion diseases; however, no effective treatment is available. Several anti-prion compounds have a preference for which prion strains or prion-infected animal models to target. Styrylbenzoazole compound called cpd-B is effective in RML prion-infected mice but less so in 263K prion-infected mice, whereas hydroxypropyl methylcellulose is effective in 263K prion-infected mice but less so in RML prion-infected mice. In the present study, we developed a combination therapy of cpd-B and hydroxypropyl methylcellulose expecting synergistic effects in both RML prion-infected mice and 263K prion-infected mice. A single subcutaneous administration of this combination had substantially a synergistic effect in RML prion-infected mice but had no additive effect in 263K prion-infected mice. These results showed that the effect of cpd-B was enhanced by hydroxypropyl methylcellulose. The complementary nature of the two compounds in efficacy against prion strains, chemical properties, pharmacokinetics, and physical properties appears to have contributed to the effective combination therapy. Our results pave the way for the strategy of new anti-prion agents.

In Vitro and In Vivo Evidence towards Fibronectin's Protective Effects against Prion Infection.

A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrPSc. Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrPSc is tightly associated with proteins found in the systemic extracellular matrix, mostly fibronectin (FN). The interaction of PrPSc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich extracellular matrix while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrPSc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues.

Synthesis and anti-prion aggregation activity of acylthiosemicarbazide analogues.

Prions are infectious protein particles known to cause prion diseases. The biochemical entity of the pathogen is the misfolded prion protein (PrPSc) that forms insoluble amyloids to impair brain function. PrPSc interacts with the non-pathogenic, cellular prion protein (PrPC) and facilitates conversion into a nascent misfolded isoform. Several small molecules have been reported to inhibit the aggregation of PrPSc but no pharmacological intervention was well established thus far. We, here, report that acylthiosemicarbazides inhibit the prion aggregation. Compounds 7x and 7y showed almost perfect inhibition (EC50 = 5 µM) in prion aggregation formation assay. The activity was further confirmed by atomic force microscopy, semi-denaturing detergent agarose gel electrophoresis and real-time quaking induced conversion assay (EC50 = 0.9 and 2.8 µM, respectively). These compounds also disaggregated pre-existing aggregates in vitro and one of them decreased the level of PrPSc in cultured cells with permanent prion infection, suggesting their potential as a treatment platform. In conclusion, hydroxy-2-naphthoylthiosemicarbazides can be an excellent scaffold for the discovery of anti-prion therapeutics.

Therapeutic Trial of anle138b in Mouse Models of Genetic Prion Disease.

Phenotypic screening has yielded small-molecule inhibitors of prion replication that are effective in vivo against certain prion strains but not others. Here, we sought to test the small molecule anle138b in multiple mouse models of prion disease. In mice inoculated with the RML strain of prions, anle138b doubled survival and durably suppressed astrogliosis measured by live-animal bioluminescence imaging. In knock-in mouse models of the D178N and E200K mutations that cause genetic prion disease, however, we were unable to identify a clear, quantifiable disease endpoint against which to measure therapeutic efficacy. Among untreated animals, the mutations did not impact overall survival, and bioluminescence remained low out to >20 months of age. Vacuolization and PrP deposition were observed in some brain regions in a subset of mutant animals but appeared to be unable to carry the weight of a primary endpoint in a therapeutic study. We conclude that not all animal models of prion disease are suited to well-powered therapeutic efficacy studies, and care should be taken in choosing the models that will support drug development programs. IMPORTANCE There is an urgent need to develop drugs for prion disease, a currently untreatable neurodegenerative disease. In this effort, there is a debate over which animal models can best support a drug development program. While the study of prion disease benefits from excellent animal models because prions naturally afflict many different mammals, different models have different capabilities and limitations. Here, we conducted a therapeutic efficacy study of the drug candidate anle138b in mouse models with two of the most common mutations that cause genetic prion disease. In a more typical model where prions are injected directly into the brain, we found anle138b to be effective. In the genetic models, however, the animals never reached a clear, measurable point of disease onset. We conclude that not all prion disease animal models are ideally suited to drug efficacy studies, and well-defined, quantitative disease metrics should be a priority.

Bifunctional carbazole derivatives for simultaneous therapy and fluorescence imaging in prion disease murine cell models.

Prion diseases are characterized by the self-assembly of pathogenic misfolded scrapie isoforms (PrPSc) of the cellular prion protein (PrPC). In an effort to achieve a theranostic profile, symmetrical bifunctional carbazole derivatives were designed as fluorescent rigid analogues of GN8, a pharmacological chaperone that stabilizes the native PrPC conformation and prevents its pathogenic conversion. A focused library was synthesized via a four-step route, and a representative member was confirmed to have native fluorescence, including a band in the near-infrared region. After a cytotoxicity study, compounds were tested on the RML-infected ScGT1 neuronal cell line, by monitoring the levels of protease-resistant PrPSc. Small dialkylamino groups at the ends of the molecule were found to be optimal in terms of therapeutic index, and the bis-(dimethylaminoacetamido)carbazole derivative 2b was selected for further characterization. It showed activity in two cell lines infected with the mouse-adapted RML strain (ScGT1 and ScN2a). Unlike GN8, 2b did not affect PrPC levels, which represents a potential advantage in terms of toxicity. Amyloid Seeding Assay (ASA) experiments showed the capacity of 2b to delay the aggregation of recombinant mouse PrP. Its ability to interfere with the amplification of the scrapie RML strain by Protein Misfolding Cyclic Amplification (PMCA) was shown to be higher than that of GN8, although 2b did not inhibit the amplification of human vCJD prion. Fluorescent staining of PrPSc aggregates by 2b was confirmed in living cells. 2b emerges as an initial hit compound for further medicinal chemistry optimization towards strain-independent anti-prion compounds.

Therapeutic development of polymers for prion disease.

Prion diseases, also known as transmissible spongiform encephalopathies, are caused by the accumulation of abnormal isoforms of the prion protein (scrapie isoform of the prion protein, PrPSc) in the central nervous system. Many compounds with anti-prion activities have been found using in silico screening, in vitro models, persistently prion-infected cell models, and prion-infected rodent models. Some of these compounds include several types of polymers. Although the inhibition or removal of PrPSc production is the main target of therapy, the unique features of prions, namely protein aggregation and assembly accompanied by steric structural transformation, may require different strategies for the development of anti-prion drugs than those for conventional therapeutics targeting enzyme inhibition, agonist ligands, or modulation of signaling. In this paper, we first overview the history of the application of polymers to prion disease research. Next, we describe the characteristics of each type of polymer with anti-prion activity. Finally, we discuss the common features of these polymers. Although drug delivery of these polymers to the brain is a challenge, they are useful not only as leads for therapeutic drugs but also as tools to explore the structure of PrPSc and are indispensable for prion disease research.

Therapeutic strategies for identifying small molecules against prion diseases.

Prion diseases are fatal neurodegenerative disorders, for which there are no effective therapeutic and diagnostic agents. The main pathological hallmark has been identified as conformational changes of the cellular isoform prion protein (PrPC) to a misfolded isoform of the prion protein (PrPSc). Targeting PrPC and its conversion to PrPSc is still the central dogma in prion drug discovery, particularly in in silico and in vitro screening endeavors, leading to the identification of many small molecules with therapeutic potential. Nonetheless, multiple pathological targets are critically involved in the intricate pathogenesis of prion diseases. In this context, multi-target-directed ligands (MTDLs) emerge as valuable therapeutic approach for their potential to effectively counteract the complex etiopathogenesis by simultaneously modulating multiple targets. In addition, diagnosis occurs late in the disease process, and consequently a successful therapeutic intervention cannot be provided. In this respect, small molecule theranostics, which combine imaging and therapeutic properties, showed tremendous potential to cure and diagnose in vivo prion diseases. Herein, we review the major advances in prion drug discovery, from anti-prion small molecules identified by means of in silico and in vitro screening approaches to two rational strategies, namely MTDLs and theranostics, that have led to the identification of novel compounds with an expanded anti-prion profile.

Identification of a Cardiac Glycoside Exhibiting Favorable Brain Bioavailability and Potency for Reducing Levels of the Cellular Prion Protein.

Several strands of investigation have established that a reduction in the levels of the cellular prion protein (PrPC) is a promising avenue for the treatment of prion diseases. We recently described an indirect approach for reducing PrPC levels that targets Na,K-ATPases (NKAs) with cardiac glycosides (CGs), causing cells to respond with the degradation of these pumps and nearby molecules, including PrPC. Because the therapeutic window of widely used CGs is narrow and their brain bioavailability is low, we set out to identify a CG with improved pharmacological properties for this indication. Starting with the CG known as oleandrin, we combined in silico modeling of CG binding poses within human NKA folds, CG structure-activity relationship (SAR) data, and predicted blood-brain barrier (BBB) penetrance scores to identify CG derivatives with improved characteristics. Focusing on C4'-dehydro-oleandrin as a chemically accessible shortlisted CG derivative, we show that it reaches four times higher levels in the brain than in the heart one day after subcutaneous administration, exhibits promising pharmacological properties, and suppresses steady-state PrPC levels by 84% in immortalized human cells that have been differentiated to acquire neural or astrocytic characteristics. Finally, we validate that the mechanism of action of this approach for reducing cell surface PrPC levels requires C4'-dehydro-oleandrin to engage with its cognate binding pocket within the NKA α subunit. The improved brain bioavailability of C4'-dehydro-oleandrin, combined with its relatively low toxicity, make this compound an attractive lead for brain CG indications and recommends its further exploration for the treatment of prion diseases.

Prion therapeutics: Lessons from the past.

Prion diseases are a group of incurable zoonotic neurodegenerative diseases (NDDs) in humans and other animals caused by the prion proteins. The abnormal folding and aggregation of the soluble cellular prion proteins (PrPC) into scrapie isoform (PrPSc) in the Central nervous system (CNS) resulted in brain damage and other neurological symptoms. Different therapeutic approaches, including stalling PrPC to PrPSc conversion, increasing PrPSc removal, and PrPC stabilization, for which a spectrum of compounds, ranging from organic compounds to antibodies, have been explored. Additionally, a non-PrP targeted drug strategy using serpin inhibitors has been discussed. Despite numerous scaffolds being screened for anti-prion activity in vitro, only a few were effective in vivo and unfortunately, almost none of them proved effective in the clinical studies, most likely due to toxicity and lack of permeability. Recently, encouraging results from a prion-protein monoclonal antibody, PRN100, were presented in the first human trial on CJD patients, which gives a hope for better future for the discovery of other new molecules to treat prion diseases. In this comprehensive review, we have re-visited the history and discussed various classes of anti-prion agents, their structure, mode of action, and toxicity. Understanding pathogenesis would be vital for developing future treatments for prion diseases. Based on the outcomes of existing therapies, new anti-prion agents could be identified/synthesized/designed with reduced toxicity and increased bioavailability, which could probably be effective in treating prion diseases.

Recent advances in cellular models for discovering prion disease therapeutics.

INTRODUCTION: Prion diseases are a group of rare and lethal, rapidly progressive neurodegenerative diseases arising due to conversion of the physiological cellular prion protein into its pathological counterparts, denoted as 'prions.' These agents are resistant to inactivation by standard decontamination procedures and can be transmitted between individuals, consequently driving the irreversible brain damage typical of the diseases. AREAS COVERED: Since its infancy, prion research has mainly depended on animal models for untangling the pathogenesis of the disease as well as for the drug development studies. With the advent of prion-infected cell lines, relevant animal models have been complemented by a variety of cell-based models presenting a much faster, ethically acceptable alternative. EXPERT OPINION: To date, there are still either no effective prophylactic regimens or therapies for human prion diseases. Therefore, there is an urgent need for more relevant cellular models that best approximate in vivo models. Each cellular model presented and discussed in detail in this review has its own benefits and limitations. Once embarking in a drug screening campaign for the identification of molecules that could interfere with prion conversion and replication, one should carefully consider the ideal cellular model.

Cardiac glycoside-mediated turnover of Na, K-ATPases as a rational approach to reducing cell surface levels of the cellular prion protein.

It is widely anticipated that a reduction of brain levels of the cellular prion protein (PrPC) can prolong survival in a group of neurodegenerative diseases known as prion diseases. To date, efforts to decrease steady-state PrPC levels by targeting this protein directly with small molecule drug-like compounds have largely been unsuccessful. Recently, we reported Na,K-ATPases to reside in immediate proximity to PrPC in the brain, unlocking an opportunity for an indirect PrPC targeting approach that capitalizes on the availability of potent cardiac glycosides (CGs). Here, we report that exposure of human co-cultures of neurons and astrocytes to non-toxic nanomolar levels of CGs causes profound reductions in PrPC levels. The mechanism of action underpinning this outcome relies primarily on a subset of CGs engaging the ATP1A1 isoform, one of three α subunits of Na,K-ATPases expressed in brain cells. Upon CG docking to ATP1A1, the ligand receptor complex, and PrPC along with it, is internalized by the cell. Subsequently, PrPC is channeled to the lysosomal compartment where it is digested in a manner that can be rescued by silencing the cysteine protease cathepsin B. These data signify that the repurposing of CGs may be beneficial for the treatment of prion disorders.

Innovative Non-PrP-Targeted Drug Strategy Designed to Enhance Prion Clearance.

Prion diseases are a group of neurodegenerative disorders characterized by the accumulation of misfolded prion protein (called PrPSc). Although conversion of the cellular prion protein (PrPC) to PrPSc is still not completely understood, most of the therapies developed until now are based on blocking this process. Here, we propose a new drug strategy aimed at clearing prions without any direct interaction with neither PrPC nor PrPSc. Starting from the recent discovery of SERPINA3/SerpinA3n upregulation during prion diseases, we have identified a small molecule, named compound 5 (ARN1468), inhibiting the function of these serpins and effectively reducing prion load in chronically infected cells. Although the low bioavailability of this compound does not allow in vivo studies in prion-infected mice, our strategy emerges as a novel and effective approach to the treatment of prion disease.

Pentosan polysulfate induces low-level persistent prion infection keeping measurable seeding activity without PrP-res detection in Fukuoka-1 infected cell cultures.

Each prion strain has its own characteristics and the efficacy of anti-prion drugs varies. Screening of prion disease therapeutics is typically evaluated by measuring amounts of protease-resistant prion protein (PrP-res). However, it remains unclear whether such measurements correlate with seeding activity, which is evaluated by real-time quaking-induced conversion (RT-QuIC). In this study, the effects of anti-prion compounds pentosan polysulfate (PPS), Congo red, and alprenolol were measured in N2a58 cells infected with Fukuoka-1 (FK1) or 22L strain. The compounds abolished PrP-res and seeding activity, except for N2a58/FK1 treated with PPS. Interestingly, the seeding activity of N2a58/FK1, which was reduced in the presence of PPS, was not lost and remained at low levels. However, upon removal of PPS, both were gradually restored to their original levels. These results indicate that low-level persistent prion infection keeping measurable seeding activity is induced by PPS in a strain-dependent manner. Furthermore, for protein misfolding cyclic amplification (PMCA), the anti-prion effect of PPS decreased in FK1 compared to 22L, suggesting that the differences occur at the level of the direct conversion. Our findings demonstrate that the advantages of RT-QuIC and PMCA can be exploited for more accurate assessment of therapeutic drug screening, reflecting strain differences.