Advertisement

Identification of Anti-prion Compounds using a Novel Cellular Assay*

  • Thibaut Imberdis
    Affiliations
    From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118,
    Search for articles by this author
  • James T. Heeres
    Affiliations
    From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118,
    Search for articles by this author
  • Han Yueh
    Affiliations
    the Department of Chemistry, Boston University, Boston, Massachusetts 02115, and
    Search for articles by this author
  • Cheng Fang
    Affiliations
    From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118,
    Search for articles by this author
  • Jessie Zhen
    Affiliations
    the Department of Chemistry, Boston University, Boston, Massachusetts 02115, and
    Search for articles by this author
  • Celeste B. Rich
    Affiliations
    From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118,
    Search for articles by this author
  • Author Footnotes
    1 Present address: ORIG3N, Inc., 27 Drydock Ave., 6th Floor, Boston, MA 02210.
    Marcie Glicksman
    Footnotes
    1 Present address: ORIG3N, Inc., 27 Drydock Ave., 6th Floor, Boston, MA 02210.
    Affiliations
    the Laboratory for Drug Discovery in Neurodegeneration, Brigham and Women's Hospital, Harvard Medical School, Cambridge, Massachusetts 02139
    Search for articles by this author
  • Aaron B. Beeler
    Affiliations
    the Department of Chemistry, Boston University, Boston, Massachusetts 02115, and
    Search for articles by this author
  • David A. Harris
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118,
    Search for articles by this author
  • Author Footnotes
    * This work was supported by National Institutes of Health Grants R01 NS065244 (to D. A. H.) and U24 NS049339 (to M. G.) and by the Harvard NeuroDiscovery Center (to M. G.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the National Science Foundation.
    This article contains supplemental Figs. S1–S3.
    1 Present address: ORIG3N, Inc., 27 Drydock Ave., 6th Floor, Boston, MA 02210.
Open AccessPublished:November 01, 2016DOI:https://doi.org/10.1074/jbc.M116.745612

      Abstract

      Prion diseases are devastating neurodegenerative disorders with no known cure. One strategy for developing therapies for these diseases is to identify compounds that block conversion of the cellular form of the prion protein (PrPC) into the infectious isoform (PrPSc). Most previous efforts to discover such molecules by high-throughput screening methods have utilized, as a read-out, a single kind of cellular assay system: neuroblastoma cells that are persistently infected with scrapie prions. Here, we describe the use of an alternative cellular assay based on suppressing the spontaneous cytotoxicity of a mutant form of PrP (Δ105–125). Using this assay, we screened 75,000 compounds, and identified a group of phenethyl piperidines (exemplified by LD7), which reduces the accumulation of PrPSc in infected neuroblastoma cells by >90% at low micromolar doses, and inhibits PrPSc-induced synaptotoxicity in hippocampal neurons. By analyzing the structure-activity relationships of 35 chemical derivatives, we defined the pharmacophore of LD7, and identified a more potent derivative. Active compounds do not alter total or cell-surface levels of PrPC, and do not bind to recombinant PrP in surface plasmon resonance experiments, although at high concentrations they inhibit PrPSc-seeded conversion of recombinant PrP to a misfolded state in an in vitro reaction (RT-QuIC). This class of small molecules may provide valuable therapeutic leads, as well as chemical biological tools to identify cellular pathways underlying PrPSc metabolism and PrPC function.

      Introduction

      Prion diseases are fatal neurodegenerative disorders that are due to the conversion of a normal, neuronal glycoprotein (PrPC)
      The abbreviations used are: PrPC, cellular isoform of the prion protein, PrP, prion protein, ΔCR, Δ105–125 deletion mutant of the prion protein, DBCA, drug-based cellular assay, DMSO, dimethyl sulfoxide, LD50, lethal dose 50%, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, PK, proteinase K, PPS, pentosan polysulfate, PrPSc, scrapie isoform of the prion protein, RML, Rocky Mountain Laboratory, RT-QuIC, real-time quaking-induced conversion, ScN2a, N2 cells persistently infected with scrapie prions, SPR, surface plasmon resonance, TMPyP, Fe(III) meso-tetra(N-methyl-4-pyridyl)porphine pentachloride, ThT, thioflavin T, LDDN, Laboratory for Drug Discovery in Neurodegeneration.
      The abbreviations used are: PrPC, cellular isoform of the prion protein, PrP, prion protein, ΔCR, Δ105–125 deletion mutant of the prion protein, DBCA, drug-based cellular assay, DMSO, dimethyl sulfoxide, LD50, lethal dose 50%, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, PK, proteinase K, PPS, pentosan polysulfate, PrPSc, scrapie isoform of the prion protein, RML, Rocky Mountain Laboratory, RT-QuIC, real-time quaking-induced conversion, ScN2a, N2 cells persistently infected with scrapie prions, SPR, surface plasmon resonance, TMPyP, Fe(III) meso-tetra(N-methyl-4-pyridyl)porphine pentachloride, ThT, thioflavin T, LDDN, Laboratory for Drug Discovery in Neurodegeneration.
      into an infectious isoform (PrPSc) that propagates itself by an autocatalytic templating process (
      • Prusiner S.B.
      Prions.
      ,
      • Aguzzi A.
      • Polymenidou M.
      Mammalian prion biology: one century of evolving concepts.
      ). In addition to their intrinsic interest to biologists, prion diseases are of enormous medical and public health concern. A global epidemic of bovine spongiform encephalopathy, a prion disease of cattle, emerged in the 1980s and 1990s, resulting in contamination of food supplies and transmission of the disease to a small number of human beings (
      • Bruce M.E.
      • Will R.G.
      • Ironside J.W.
      • McConnell I.
      • Drummond D.
      • Suttie A.
      • McCardle L.
      • Chree A.
      • Hope J.
      • Birkett C.
      • Cousens S.
      • Fraser H.
      • Bostock C.J.
      Transmissions to mice indicate that “new variant” CJD is caused by the BSE agent.
      ,
      • Wells G.A.
      • Wilesmith J.W.
      The neuropathology and epidemiology of bovine spongiform encephalopathy.
      ). Prion contamination has also increased the risk of blood transfusions, and organ transplants (
      • Belay E.D.
      • Schonberger L.B.
      The public health impact of prion diseases.
      ). Most recently, a prion-like process has been found to play a role in the CNS dissemination of misfolded proteins in more common neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and tauopathies, and there is even evidence that these diseases can be spread between individuals by iatrogenic means (
      • Jucker M.
      • Walker L.C.
      Self-propagation of pathogenic protein aggregates in neurodegenerative diseases.
      ,
      • Jaunmuktane Z.
      • Mead S.
      • Ellis M.
      • Wadsworth J.D.
      • Nicoll A.J.
      • Kenny J.
      • Launchbury F.
      • Linehan J.
      • Richard-Loendt A.
      • Walker A.S.
      • Rudge P.
      • Collinge J.
      • Brandner S.
      Evidence for human transmission of amyloid-β pathology and cerebral amyloid angiopathy.
      ).
      There are currently no cures for prion diseases. A great deal of effort has been invested over the past 25 years in identifying compounds that block the conversion of PrPC into PrPSc as a therapeutic strategy. Most of these efforts have used as a read-out a single kind of cellular assay system: N2a neuroblastoma cells that are chronically infected with scrapie prions (designated ScN2a cells). N2a cells are one of the few cell lines capable of propagating prions, and they can maintain a chronic state of infection with no outward signs of cytotoxicity (
      • Race R.E.
      • Caughey B.
      • Graham K.
      • Ernst D.
      • Chesebro B.
      Analyses of frequency of infection, specific infectivity, and prion protein biosynthesis in scrapie-infected neuroblastoma cell clones.
      ,
      • Butler D.A.
      • Scott M.R.
      • Bockman J.M.
      • Borchelt D.R.
      • Taraboulos A.
      • Hsiao K.K.
      • Kingsbury D.T.
      • Prusiner S.B.
      Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins.
      ). A number of inhibitory compounds have been discovered with this assay, either by educated guessing, or by systematic screens of compound libraries consisting of up to several thousand entries (
      • Trevitt C.R.
      • Collinge J.
      A systematic review of prion therapeutics in experimental models.
      ,
      • Sim V.L.
      Prion disease: chemotherapeutic strategies.
      ). Although some of these compounds are able to prolong the incubation time between prion exposure and the onset of symptoms in mice, none of them prevents the eventual occurrence of disease, and none has proven effective in limited human trials (
      • Collinge J.
      • Gorham M.
      • Hudson F.
      • Kennedy A.
      • Keogh G.
      • Pal S.
      • Rossor M.
      • Rudge P.
      • Siddique D.
      • Spyer M.
      • Thomas D.
      • Walker S.
      • Webb T.
      • Wroe S.
      • Darbyshire J.
      Safety and efficacy of quinacrine in human prion disease (PRION-1 study): a patient-preference trial.
      ,
      • Haïk S.
      • Marcon G.
      • Mallet A.
      • Tettamanti M.
      • Welaratne A.
      • Giaccone G.
      • Azimi S.
      • Pietrini V.
      • Fabreguettes J.R.
      • Imperiale D.
      • Cesaro P.
      • Buffa C.
      • Aucan C.
      • Lucca U.
      • Peckeu L.
      • et al.
      Doxycycline in Creutzfeldt-Jakob disease: a phase 2, randomised, double-blind, placebo-controlled trial.
      ). On a mechanistic level, there is evidence that many anti-prion compounds discovered in high-throughput screens of ScN2a cells do not interact with either PrPC or PrPSc, and presumably target non-PrP molecules (
      • Poncet-Montange G.
      • St. Martin S.J.
      • Bogatova O.V.
      • Prusiner S.B.
      • Shoichet B.K.
      • Ghaemmaghami S.
      A survey of antiprion compounds reveals the prevalence of non-PrP molecular targets.
      ). However, in virtually every case, the identity of these targets remains unknown, limiting the extension of target-based, drug discovery approaches beyond the PrP molecule itself.
      In addition to blocking conversion of PrPC to PrPSc, another possible strategy for treating prion diseases would be to inhibit downstream neurotoxic signaling pathways. However, there is very little known about how the interaction of PrPSc with cell-surface PrPC results in pathogenic effects such as synapse loss (
      • Biasini E.
      • Turnbaugh J.A.
      • Unterberger U.
      • Harris D.A.
      Prion protein at the crossroads of physiology and disease.
      ,
      • Mallucci G.
      • Dickinson A.
      • Linehan J.
      • Klöhn P.C.
      • Brandner S.
      • Collinge J.
      Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis.
      • Mallucci G.R.
      Prion neurodegeneration: starts and stops at the synapse.
      ). We and others have found that expression in transgenic mice of PrP molecules harboring deletions spanning the central domain of the protein (residues 105–125) cause spontaneous neurodegenerative phenotypes that can be suppressed by co-expression of wild-type PrP (
      • Li A.
      • Christensen H.M.
      • Stewart L.R.
      • Roth K.A.
      • Chiesa R.
      • Harris D.A.
      Neonatal lethality in transgenic mice expressing prion protein with a deletion of residues 105–125.
      ,
      • Baumann F.
      • Tolnay M.
      • Brabeck C.
      • Pahnke J.
      • Kloz U.
      • Niemann H.H.
      • Heikenwalder M.
      • Rülicke T.
      • Bürkle A.
      • Aguzzi A.
      Lethal recessive myelin toxicity of prion protein lacking its central domain.
      • Shmerling D.
      • Hegyi I.
      • Fischer M.
      • Blättler T.
      • Brandner S.
      • Götz J.
      • Rülicke T.
      • Flechsig E.
      • Cozzio A.
      • von Mering C.
      • Hangartner C.
      • Aguzzi A.
      • Weissmann C.
      Expression of amino-terminally truncated PrP in the mouse leading to ataxia and specific cerebellar lesions.
      ). These mice have been the object of considerable interest, because they are likely to provide insights into PrP-mediated neurotoxic pathways. Our laboratory created mice with the most toxic (and paradoxically the shortest) of these PrP deletions, Δ105–125 (referred to as ΔCR) (for deletion of the central region) (
      • Li A.
      • Christensen H.M.
      • Stewart L.R.
      • Roth K.A.
      • Chiesa R.
      • Harris D.A.
      Neonatal lethality in transgenic mice expressing prion protein with a deletion of residues 105–125.
      ). In the course of investigating what makes ΔCR and the other deletion mutants so neurotoxic, we discovered that they induce spontaneous ionic currents, recordable by patch-clamping techniques, when expressed in cultured cell lines and neurons (
      • Solomon I.H.
      • Huettner J.E.
      • Harris D.A.
      Neurotoxic mutants of the prion protein induce spontaneous ionic currents in cultured cells.
      ,
      • Solomon I.H.
      • Khatri N.
      • Biasini E.
      • Massignan T.
      • Huettner J.E.
      • Harris D.A.
      An N-terminal polybasic domain and cell surface localization are required for mutant prion protein toxicity.
      ). Coincidentally, we also observed that these same mutant PrP molecules sensitize cells to the cytotoxic effects of certain antibiotics, including G418 and Zeocin, a phenomenon that may be related to enhanced uptake of these cationic antibiotics via the formation of membrane channels or pores (
      • Massignan T.
      • Biasini E.
      • Harris D.A.
      A drug-based cellular assay (DBCA) for studying cytotoxic and cytoprotective activities of the prion protein: a practical guide.
      ,
      • Massignan T.
      • Stewart R.S.
      • Biasini E.
      • Solomon I.H.
      • Bonetto V.
      • Chiesa R.
      • Harris D.A.
      A novel, drug-based, cellular assay for the activity of neurotoxic mutants of the prion protein.
      ).
      In the present study, we have used suppression of this antibiotic hypersensitivity phenotype as a cellular read-out in a high throughput screen of a diverse, small-molecule library. Using this approach, we have discovered a new class of lead compounds, phenethyl piperidines, which display a dual activity: they suppress the toxicity of the deleted forms of PrP, and they also inhibit PrPSc formation in ScN2a cells. The use of this novel cellular screening assay has allowed us to identify compounds that may have escaped detection in previous screens, and that may have value as therapeutic leads as well as chemical biological tools.

      Discussion

      In this study, we have used a novel assay (DBCA) (
      • Massignan T.
      • Biasini E.
      • Harris D.A.
      A drug-based cellular assay (DBCA) for studying cytotoxic and cytoprotective activities of the prion protein: a practical guide.
      ,
      • Massignan T.
      • Stewart R.S.
      • Biasini E.
      • Solomon I.H.
      • Bonetto V.
      • Chiesa R.
      • Harris D.A.
      A novel, drug-based, cellular assay for the activity of neurotoxic mutants of the prion protein.
      ), based on the cellular toxicity of a mutant form of PrP (Δ105–125, designated ΔCR), to perform a high-throughput screen of 75,000 compounds to identify those that significantly improved cell viability in the presence of two cationic antibiotics (G418 and Zeocin) normally toxic to ΔCR PrP-expressing cells. Surprisingly, several of the compounds identified in this screen also proved to be efficacious in reducing PrPSc levels in scrapie-infected N2a cells. Structure-activity relationships were established for one of these compounds, a phenethyl piperidine (LD7), based on analysis of 34 chemical derivatives, resulting in creation of a molecule, JZ107, that was more potent at reducing PrPSc levels. We showed that the mechanism of action of JZ107 does not involve decreasing total or cell-surface levels of the precursor molecule, PrPC. Our results using an in vitro PrPC-PrPSc seeding assay (RT-QuIC) raise the possibility that, although the compound does not bind with high affinity to recombinant PrP in SPR experiments, it might interact with PrPC in a cellular context, perhaps in conjunction with other cell-surface receptors. Altogether, our results establish a new assay, orthogonal to the standard ScN2a test, for discovering anti-prion compounds. Such molecules may have therapeutic potential in both prion and Alzheimer's disease, and may prove useful as tool compounds to further investigate PrP-mediated neurotoxic pathways.
      Over the past 20 years, a number of different approaches to anti-prion therapy have been pursued. Perhaps the most effective has been to reduce the amount of PrPC substrate available for conversion into PrPSc. The validity of this approach is demonstrated by the key observation that mice in which the PrP gene has been knocked out are completely resistant to prion infection (
      • Büeler H.
      • Aguzzi A.
      • Sailer A.
      • Greiner R.A.
      • Autenried P.
      • Aguet M.
      • Weissmann C.
      Mice devoid of PrP are resistant to scrapie.
      ). A second major strategy has been the use of compounds that block the conversion of PrPC into PrPSc. By far, the most widely employed system for discovering and testing such compounds relies on ScN2a cells, which are chronically infected with scrapie prions. A number of inhibitory compounds have been identified using this assay (
      • Trevitt C.R.
      • Collinge J.
      A systematic review of prion therapeutics in experimental models.
      ,
      • Sim V.L.
      Prion disease: chemotherapeutic strategies.
      ). Some of these compounds bind to PrPC, and may act by sterically occluding critical interactions, or by stabilizing the structure of the folded, C-terminal domain (e.g. glycosaminoglycans, Congo Red, and other sulfonated dyes, nucleic acids, cationic tetrapyrroles, anti-PrP antibodies). Other compounds appear to act by binding to PrPSc, destabilizing or otherwise altering its structure, and possibly enhancing its clearance (e.g. polyamine and phosphorus-containing dendrimers, polyoxometalates, and polythiophenes). Importantly, there is evidence that many anti-prion compounds discovered in high-throughput screens of ScN2a cells do not interact with either PrPC or PrPSc, and presumably target non-PrP molecules (
      • Poncet-Montange G.
      • St. Martin S.J.
      • Bogatova O.V.
      • Prusiner S.B.
      • Shoichet B.K.
      • Ghaemmaghami S.
      A survey of antiprion compounds reveals the prevalence of non-PrP molecular targets.
      ). However, in virtually every case, the identity of these targets remains unknown.
      We predicted that the DBCA might represent an orthogonal assay that would be useful as a screening tool to identify novel anti-prion compounds, based on our observation that several molecules (PPS, Cong Red, and TMPyP) previously shown to reduce PrPSc levels in ScN2a cells were also active in the DBCA. For these compounds, there may be a mechanistic connection between their effects in the two assays. For example, both PPS and Congo Red bind to the flexible, N-terminal domain of PrPC, in particular the polybasic region encompassing residues 23–31 (
      • Pan T.
      • Wong B.S.
      • Liu T.
      • Li R.
      • Petersen R.B.
      • Sy M.S.
      Cell-surface prion protein interacts with glycosaminoglycans.
      ,
      • Warner R.G.
      • Hundt C.
      • Weiss S.
      • Turnbull J.E.
      Identification of the heparan sulfate binding sites in the cellular prion protein.
      ,
      • Caughey B.
      • Brown K.
      • Raymond G.J.
      • Katzenstein G.E.
      • Thresher W.
      Binding of the protease-sensitive form of PrP (prion protein) to sulfated glycosaminoglycan and Congo Red.
      ). This region is known to be essential both for the toxic activity of ΔCR PrP in the DBCA (
      • Solomon I.H.
      • Khatri N.
      • Biasini E.
      • Massignan T.
      • Huettner J.E.
      • Harris D.A.
      An N-terminal polybasic domain and cell surface localization are required for mutant prion protein toxicity.
      ,
      • Westergard L.
      • Turnbaugh J.A.
      • Harris D.A.
      A nine amino acid domain is essential for mutant prion protein toxicity.
      ), and for productive interaction between PrPC and PrPSc during the prion conversion process (
      • Turnbaugh J.A.
      • Unterberger U.
      • Saá P.
      • Massignan T.
      • Fluharty B.R.
      • Bowman F.P.
      • Miller M.B.
      • Supattapone S.
      • Biasini E.
      • Harris D.A.
      The N-terminal, polybasic region of PrPC dictates the efficiency of prion propagation by binding to PrPSc.
      ,
      • Miller M.B.
      • Geoghegan J.C.
      • Supattapone S.
      Dissociation of infectivity from seeding ability in prions with alternate docking mechanism.
      ). We found that three of the 68 confirmed hits from the DBCA screen, representing 3 of 9 distinct chemical scaffolds, possessed anti-prion activity when tested in ScN2a cells. It remains to be determined whether the parallel activities of these compounds in the two assays are mechanistically related. When we tested 10 compounds selected from the LD7 structure-activity series, we did not find a close correlation between their activities in the DBCA and ScN2a assays (Fig. 4, D and E). Regardless of the underlying mechanisms, however, we have shown that the DBCA does have the ability to identify new anti-scrapie compounds, and may therefore represent a useful preliminary filter in drug screening efforts.
      What is the mechanism of action of the LD7/JZ107 class of compounds? JZ107 does not alter the levels of total or cell-surface PrPC, suggesting that it does not act by altering the amount or distribution of this precursor, as is the case for some other anti-prion drugs (
      • Silber B.M.
      • Gever J.R.
      • Rao S.
      • Li Z.
      • Renslo A.R.
      • Widjaja K.
      • Wong C.
      • Giles K.
      • Freyman Y.
      • Elepano M.
      • Irwin J.J.
      • Jacobson M.P.
      • Prusiner S.B.
      Novel compounds lowering the cellular isoform of the human prion protein in cultured human cells.
      ). LD7 and JZ107 do not bind to immobilized, recombinant PrP in SPR experiments, arguing that PrPC itself may not be a high-affinity target for these compounds. Interestingly, however, we found that JZ107 and other active derivatives suppressed PrPSc-seeded conversion of recombinant PrPC to a misfolded state in the RT-QuIC reaction. In contrast, derivatives that had no activity in the ScN2a assay had no effect on the RT-QuIC reaction. Although we cannot be certain whether the compounds are interacting with the PrPC substrate, the PrPSc seeds, or both, the fact that the inhibitory effect in the RT-QuIC reaction was partially dependent upon preincubation of the compounds with PrPC suggests the first possibility. Notably, the concentrations of the compounds required to observe an effect in the RT-QuIC reaction were quite high (100–200 μm), well above the EC50 of these molecules in the ScN2a assay (<10 μm). This discrepancy may be due to the high concentration of recombinant PrP (∼6 μm) present in the RT-QuIC reaction, compared with what would be present in a cellular context, or to modifications such as glycosylation and GPI membrane anchoring, which could enhance the affinity of cellular PrPC for these molecules. A third possibility is that PrPC functions as part of a complex with other membrane proteins that together constitute high-affinity binding targets that mediate the anti-prion activity of the compounds. Ongoing efforts are now directed toward definitively identifying the molecular target(s) for the LD7/JZ107 class of compounds using forward and reverse chemical genetic approaches.
      There are several important implications of the work presented here. First, we have used a novel, cellular assay (the DBCA), based on a toxic activity of mutant PrP, to discover a new class of anti-prion compounds using high-throughput screening methods. These molecules may serve as a starting point for therapeutic applications in animals and humans, in conjunction with further optimization of their pharmacodynamic and pharmacokinetic properties. Second, these molecules are potentially valuable tool compounds, which may provide insights into pathways controlling PrPSc formation and degradation in cells. Finally, the compounds may provide clues to how PrPC mediates the toxic effects of PrPSc (
      • Biasini E.
      • Turnbaugh J.A.
      • Unterberger U.
      • Harris D.A.
      Prion protein at the crossroads of physiology and disease.
      ). The fact that these compounds were discovered using the DBCA, which we think is related in some way to alterations in a physiological activity of PrPC, makes it more likely that the compounds will affect processes beyond PrPSc metabolism. The combination of the DBCA, the ScN2a assay, and the PrPSc dendritic toxicity assay (
      • Fang C.
      • Imberdis T.
      • Garza M.C.
      • Wille H.
      • Harris D.A.
      A neuronal culture system to detect prion synaptotoxicity.
      ) should allow us to discover additional therapeutic leads that act via novel mechanisms.

      Author Contributions

      D. A. H. conceived and coordinated the study. D. A. H. and T. I. wrote the paper. J. T. H. designed, performed, and analyzed the experiments and results shown in Table 1 and supplemental Fig. S1. J. T. H. and T. I. designed, performed, and analyzed the experiments shown in Fig. 1. T. I. designed, performed, and analyzed the experiments shown in FIGURE 2., FIGURE 3., FIGURE 4., FIGURE 5. and 7, and supplemental S2. C. F. designed, performed, and analyzed the experiments shown in Fig. 6. A. B. B., H. Y., and J. Z. designed, synthesized, and analyzed the compounds shown in supplemental Figs. S2 and S3. C. B. R. provided technical assistance. M. G. coordinated the screening of compound libraries at the LDDN. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We acknowledge Tania Massignan and Emiliano Biasini (University of Trento, Italy) for participation in the initial phases of the project while in the Harris Laboratory. We thank Byron Caughey for providing bacteria expressing the recombinant hamster PrP(90–231) used in the RT-QuIC assay. NMR (CHE-0619339) and MS (CHE-0443618) facilities at Boston University are supported by the National Science Foundation.

      References

        • Prusiner S.B.
        Prions.
        Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13363-13383
        • Aguzzi A.
        • Polymenidou M.
        Mammalian prion biology: one century of evolving concepts.
        Cell. 2004; 116: 313-327
        • Bruce M.E.
        • Will R.G.
        • Ironside J.W.
        • McConnell I.
        • Drummond D.
        • Suttie A.
        • McCardle L.
        • Chree A.
        • Hope J.
        • Birkett C.
        • Cousens S.
        • Fraser H.
        • Bostock C.J.
        Transmissions to mice indicate that “new variant” CJD is caused by the BSE agent.
        Nature. 1997; 389: 498-501
        • Wells G.A.
        • Wilesmith J.W.
        The neuropathology and epidemiology of bovine spongiform encephalopathy.
        Brain Pathol. 1995; 5: 91-103
        • Belay E.D.
        • Schonberger L.B.
        The public health impact of prion diseases.
        Annu. Rev. Public Health. 2005; 26: 191-212
        • Jucker M.
        • Walker L.C.
        Self-propagation of pathogenic protein aggregates in neurodegenerative diseases.
        Nature. 2013; 501: 45-51
        • Jaunmuktane Z.
        • Mead S.
        • Ellis M.
        • Wadsworth J.D.
        • Nicoll A.J.
        • Kenny J.
        • Launchbury F.
        • Linehan J.
        • Richard-Loendt A.
        • Walker A.S.
        • Rudge P.
        • Collinge J.
        • Brandner S.
        Evidence for human transmission of amyloid-β pathology and cerebral amyloid angiopathy.
        Nature. 2015; 525: 247-250
        • Race R.E.
        • Caughey B.
        • Graham K.
        • Ernst D.
        • Chesebro B.
        Analyses of frequency of infection, specific infectivity, and prion protein biosynthesis in scrapie-infected neuroblastoma cell clones.
        J. Virol. 1988; 62: 2845-2849
        • Butler D.A.
        • Scott M.R.
        • Bockman J.M.
        • Borchelt D.R.
        • Taraboulos A.
        • Hsiao K.K.
        • Kingsbury D.T.
        • Prusiner S.B.
        Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins.
        J. Virol. 1988; 62: 1558-1564
        • Trevitt C.R.
        • Collinge J.
        A systematic review of prion therapeutics in experimental models.
        Brain. 2006; 129: 2241-2265
        • Sim V.L.
        Prion disease: chemotherapeutic strategies.
        Infect. Disord. Drug Targets. 2012; 12: 144-160
        • Collinge J.
        • Gorham M.
        • Hudson F.
        • Kennedy A.
        • Keogh G.
        • Pal S.
        • Rossor M.
        • Rudge P.
        • Siddique D.
        • Spyer M.
        • Thomas D.
        • Walker S.
        • Webb T.
        • Wroe S.
        • Darbyshire J.
        Safety and efficacy of quinacrine in human prion disease (PRION-1 study): a patient-preference trial.
        Lancet Neurol. 2009; 8: 334-344
        • Haïk S.
        • Marcon G.
        • Mallet A.
        • Tettamanti M.
        • Welaratne A.
        • Giaccone G.
        • Azimi S.
        • Pietrini V.
        • Fabreguettes J.R.
        • Imperiale D.
        • Cesaro P.
        • Buffa C.
        • Aucan C.
        • Lucca U.
        • Peckeu L.
        • et al.
        Doxycycline in Creutzfeldt-Jakob disease: a phase 2, randomised, double-blind, placebo-controlled trial.
        Lancet Neurol. 2014; 13: 150-158
        • Poncet-Montange G.
        • St. Martin S.J.
        • Bogatova O.V.
        • Prusiner S.B.
        • Shoichet B.K.
        • Ghaemmaghami S.
        A survey of antiprion compounds reveals the prevalence of non-PrP molecular targets.
        J. Biol. Chem. 2011; 286: 27718-27728
        • Biasini E.
        • Turnbaugh J.A.
        • Unterberger U.
        • Harris D.A.
        Prion protein at the crossroads of physiology and disease.
        Trends Neurosci. 2012; 35: 92-103
        • Mallucci G.
        • Dickinson A.
        • Linehan J.
        • Klöhn P.C.
        • Brandner S.
        • Collinge J.
        Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis.
        Science. 2003; 302: 871-874
        • Mallucci G.R.
        Prion neurodegeneration: starts and stops at the synapse.
        Prion. 2009; 3: 195-201
        • Li A.
        • Christensen H.M.
        • Stewart L.R.
        • Roth K.A.
        • Chiesa R.
        • Harris D.A.
        Neonatal lethality in transgenic mice expressing prion protein with a deletion of residues 105–125.
        EMBO J. 2007; 26: 548-558
        • Baumann F.
        • Tolnay M.
        • Brabeck C.
        • Pahnke J.
        • Kloz U.
        • Niemann H.H.
        • Heikenwalder M.
        • Rülicke T.
        • Bürkle A.
        • Aguzzi A.
        Lethal recessive myelin toxicity of prion protein lacking its central domain.
        EMBO J. 2007; 26: 538-547
        • Shmerling D.
        • Hegyi I.
        • Fischer M.
        • Blättler T.
        • Brandner S.
        • Götz J.
        • Rülicke T.
        • Flechsig E.
        • Cozzio A.
        • von Mering C.
        • Hangartner C.
        • Aguzzi A.
        • Weissmann C.
        Expression of amino-terminally truncated PrP in the mouse leading to ataxia and specific cerebellar lesions.
        Cell. 1998; 93: 203-214
        • Solomon I.H.
        • Huettner J.E.
        • Harris D.A.
        Neurotoxic mutants of the prion protein induce spontaneous ionic currents in cultured cells.
        J. Biol. Chem. 2010; 285: 26719-26726
        • Solomon I.H.
        • Khatri N.
        • Biasini E.
        • Massignan T.
        • Huettner J.E.
        • Harris D.A.
        An N-terminal polybasic domain and cell surface localization are required for mutant prion protein toxicity.
        J. Biol. Chem. 2011; 286: 14724-14736
        • Massignan T.
        • Biasini E.
        • Harris D.A.
        A drug-based cellular assay (DBCA) for studying cytotoxic and cytoprotective activities of the prion protein: a practical guide.
        Methods. 2011; 53: 214-219
        • Massignan T.
        • Stewart R.S.
        • Biasini E.
        • Solomon I.H.
        • Bonetto V.
        • Chiesa R.
        • Harris D.A.
        A novel, drug-based, cellular assay for the activity of neurotoxic mutants of the prion protein.
        J. Biol. Chem. 2010; 285: 7752-7765
        • Biasini E.
        • Turnbaugh J.A.
        • Massignan T.
        • Veglianese P.
        • Forloni G.
        • Bonetto V.
        • Chiesa R.
        • Harris D.A.
        The toxicity of a mutant prion protein is cell-autonomous, and can be suppressed by wild-type prion protein on adjacent cells.
        PLoS ONE. 2012; 7: e33472
        • Biasini E.
        • Unterberger U.
        • Solomon I.H.
        • Massignan T.
        • Senatore A.
        • Bian H.
        • Voigtlaender T.
        • Bowman F.P.
        • Bonetto V.
        • Chiesa R.
        • Luebke J.
        • Toselli P.
        • Harris D.A.
        A mutant prion protein sensitizes neurons to glutamate-induced excitotoxicity.
        J. Neurosci. 2013; 33: 2408-2418
        • Westergard L.
        • Turnbaugh J.A.
        • Harris D.A.
        A nine amino acid domain is essential for mutant prion protein toxicity.
        J. Neurosci. 2011; 31: 14005-14017
        • Pan T.
        • Wong B.S.
        • Liu T.
        • Li R.
        • Petersen R.B.
        • Sy M.S.
        Cell-surface prion protein interacts with glycosaminoglycans.
        Biochem. J. 2002; 368: 81-90
        • Warner R.G.
        • Hundt C.
        • Weiss S.
        • Turnbull J.E.
        Identification of the heparan sulfate binding sites in the cellular prion protein.
        J. Biol. Chem. 2002; 277: 18421-18430
        • Caughey B.
        • Raymond G.J.
        Sulfated polyanion inhibition of scrapie-associated PrP accumulation in cultured cells.
        J. Virol. 1993; 67: 643-650
        • Caughey B.
        • Ernst D.
        • Race R.E.
        Congo red inhibition of scrapie agent replication.
        J. Virol. 1993; 67: 6270-6272
        • Caughey W.S.
        • Raymond L.D.
        • Horiuchi M.
        • Caughey B.
        Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines.
        Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 12117-12122
        • Priola S.A.
        • Raines A.
        • Caughey W.S.
        Porphyrin and phthalocyanine antiscrapie compounds.
        Science. 2000; 287: 1503-1506
        • Nicoll A.J.
        • Trevitt C.R.
        • Tattum M.H.
        • Risse E.
        • Quarterman E.
        • Ibarra A.A.
        • Wright C.
        • Jackson G.S.
        • Sessions R.B.
        • Farrow M.
        • Waltho J.P.
        • Clarke A.R.
        • Collinge J.
        Pharmacological chaperone for the structured domain of human prion protein.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 17610-17615
        • Massignan T.
        • Cimini S.
        • Stincardini C.
        • Cerovic M.
        • Vanni I.
        • Elezgarai S.R.
        • Moreno J.
        • Stravalaci M.
        • Negro A.
        • Sangiovanni V.
        • Restelli E.
        • Riccardi G.
        • Gobbi M.
        • Castilla J.
        • Borsello T.
        • Nonno R.
        • Biasini E.
        A cationic tetrapyrrole inhibits toxic activities of the cellular prion protein.
        Sci. Rep. 2016; 6: 23180
        • Büeler H.
        • Aguzzi A.
        • Sailer A.
        • Greiner R.A.
        • Autenried P.
        • Aguet M.
        • Weissmann C.
        Mice devoid of PrP are resistant to scrapie.
        Cell. 1993; 73: 1339-1347
        • Shyng S.-L.
        • Lehmann S.
        • Moulder K.L.
        • Harris D.A.
        Sulfated glycans stimulate endocytosis of the cellular isoform of the prion protein, PrPC, in cultured cells.
        J. Biol. Chem. 1995; 270: 30221-30229
        • Fang C.
        • Imberdis T.
        • Garza M.C.
        • Wille H.
        • Harris D.A.
        A neuronal culture system to detect prion synaptotoxicity.
        PLoS Pathog. 2016; 12: e1005623
        • Wilham J.M.
        • Orrú C.D.
        • Bessen R.A.
        • Atarashi R.
        • Sano K.
        • Race B.
        • Meade-White K.D.
        • Taubner L.M.
        • Timmes A.
        • Caughey B.
        Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays.
        PLoS Pathog. 2010; 6: e1001217
        • Caughey B.
        • Brown K.
        • Raymond G.J.
        • Katzenstein G.E.
        • Thresher W.
        Binding of the protease-sensitive form of PrP (prion protein) to sulfated glycosaminoglycan and Congo Red.
        J. Virol. 1994; 68: 2135-2141
        • Turnbaugh J.A.
        • Unterberger U.
        • Saá P.
        • Massignan T.
        • Fluharty B.R.
        • Bowman F.P.
        • Miller M.B.
        • Supattapone S.
        • Biasini E.
        • Harris D.A.
        The N-terminal, polybasic region of PrPC dictates the efficiency of prion propagation by binding to PrPSc.
        J. Neurosci. 2012; 32: 8817-8830
        • Miller M.B.
        • Geoghegan J.C.
        • Supattapone S.
        Dissociation of infectivity from seeding ability in prions with alternate docking mechanism.
        PLoS Pathog. 2011; 7: e1002128
        • Silber B.M.
        • Gever J.R.
        • Rao S.
        • Li Z.
        • Renslo A.R.
        • Widjaja K.
        • Wong C.
        • Giles K.
        • Freyman Y.
        • Elepano M.
        • Irwin J.J.
        • Jacobson M.P.
        • Prusiner S.B.
        Novel compounds lowering the cellular isoform of the human prion protein in cultured human cells.
        Bioorg. Med. Chem. 2014; 22: 1960-1972
        • Carr R.A.
        • Congreve M.
        • Murray C.W.
        • Rees D.C.
        Fragment-based lead discovery: leads by design.
        Drug Discov. Today. 2005; 10: 987-992
        • Safar J.G.
        • Scott M.
        • Monaghan J.
        • Deering C.
        • Didorenko S.
        • Vergara J.
        • Ball H.
        • Legname G.
        • Leclerc E.
        • Solforosi L.
        • Serban H.
        • Groth D.
        • Burton D.R.
        • Prusiner S.B.
        • Williamson R.A.
        Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice.
        Nat. Biotechnol. 2002; 20: 1147-1150
        • Fluharty B.R.
        • Biasini E.
        • Stravalaci M.
        • Sclip A.
        • Diomede L.
        • Balducci C.
        • La Vitola P.
        • Messa M.
        • Colombo L.
        • Forloni G.
        • Borsello T.
        • Gobbi M.
        • Harris D.A.
        An N-terminal fragment of the prion protein binds to amyloid-β oligomers and inhibits their neurotoxicity in vivo.
        J. Biol. Chem. 2013; 288: 7857-7866