A simple in vitro assay for assessing the efficacy, mechanisms and kinetics of anti-prion fibril compounds

ABSTRACT Prion diseases are caused by the conversion of normal cellular prion proteins (PrP) into lethal prion aggregates. These prion aggregates are composed of proteinase K (PK) resistant fibrils and comparatively PK-sensitive oligomers. Currently there are no anti-prion pharmaceuticals available to treat or prevent prion disease. Methods of discovering anti-prion molecules rely primarily on relatively complex cell-based, tissue slice or animal-model assays that measure the effects of small molecules on the formation of PK-resistant prion fibrils. These assays are difficult to perform and do not detect the compounds that directly inhibit oligomer formation or alter prion conversion kinetics. We have developed a simple cell-free method to characterize the impact of anti-prion fibril compounds on both the oligomer and fibril formation. In particular, this assay uses shaking-induced conversion (ShIC) of recombinant PrP in a 96-well format and resolution enhanced native acidic gel electrophoresis (RENAGE) to generate, assess and detect PrP fibrils in a high throughput fashion. The end-point PrP fibrils from this assay can be further characterized by PK analysis and negative stain transmission electron microscopy (TEM). This cell-free, gel-based assay generates metrics to assess anti-prion fibril efficacy and kinetics. To demonstrate its utility, we characterized the action of seven well-known anti-prion molecules: Congo red, curcumin, GN8, quinacrine, chloropromazine, tetracycline, and TUDCA (taurourspdeoxycholic acid), as well as four suspected anti-prion compounds: trans-resveratrol, rosmarinic acid, myricetin and ferulic acid. These findings suggest that this in vitro assay could be useful in identifying and comprehensively assessing novel anti-prion fibril compounds. Abbreviations: PrP, prion protein; PK, proteinase K; ShIC, shaking-induced conversion; RENAGE, resolution enhanced native acidic gel electrophoresis; TEM, transmission electron microscopy; TUDCA, taurourspdeoxycholic acid; BSE, bovine spongiform encephalopathy; CWD, chronic wasting disease; CJD, Creutzfeldt Jakob disease; GSS, Gerstmann–Sträussler–Scheinker syndrome; FFI, fatal familial insomnia; PrPc, cellular prion protein; recPrPC, recombinant monomeric prion protein; PrPSc, infectious particle of misfolded prion protein; RT-QuIC, real-time quaking-induced conversion; PMCA, Protein Misfolding Cyclic Amplification; LPS, lipopolysaccharide; EGCG, epigallocatechin gallate; GN8, 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide; DMSO, dimethyl sulfoxide; ScN2A, scrapie infected neuroblastoma cells; IC50, inhibitory concentration for 50% reduction; recMoPrP 23−231, recombinant full-length mouse prion protein residues 23-231; EDTA; PICUP, photo-induced cross-linking of unmodified protein; BSA, bovine serum albumin;; PMSF, phenylmethanesulfonyl fluoride.

[1]  S. Stephenson,et al.  Two new species of dictyostelid cellular slime molds in high-elevation habitats on the Qinghai-Tibet Plateau, China , 2019, Scientific Reports.

[2]  A. M. Troncoso,et al.  In Vitro Effects of Serotonin, Melatonin, and Other Related Indole Compounds on Amyloid‐&bgr; Kinetics and Neuroprotection , 2018, Molecular nutrition & food research.

[3]  P. Halbur,et al.  Integrated Organotypic Slice Cultures and RT-QuIC (OSCAR) Assay: Implications for Translational Discovery in Protein Misfolding Diseases , 2017, Scientific Reports.

[4]  S. Y. Kim,et al.  Anti-Prion Screening for Acridine, Dextran, and Tannic Acid using Real Time–Quaking Induced Conversion: A Comparison with PrPSc-Infected Cell Screening , 2017, PloS one.

[5]  K. Doh-ura,et al.  A Single Subcutaneous Injection of Cellulose Ethers Administered Long before Infection Confers Sustained Protection against Prion Diseases in Rodents , 2016, PLoS pathogens.

[6]  D. Wishart,et al.  Role of polysaccharide and lipid in lipopolysaccharide induced prion protein conversion , 2016, Prion.

[7]  Aaron B. Beeler,et al.  Identification of Anti-prion Compounds using a Novel Cellular Assay* , 2016, The Journal of Biological Chemistry.

[8]  S. Guan,et al.  Optimization of Aryl Amides that Extend Survival in Prion-Infected Mice , 2016, The Journal of Pharmacology and Experimental Therapeutics.

[9]  B. Caughey,et al.  Factors That Improve RT-QuIC Detection of Prion Seeding Activity , 2016, Viruses.

[10]  N. Makarava,et al.  Strain-dependent profile of misfolded prion protein aggregates , 2016, Scientific Reports.

[11]  J. Collinge,et al.  A systematic investigation of production of synthetic prions from recombinant prion protein , 2015, Open Biology.

[12]  Jong Young Joung,et al.  Discovery of Novel Anti-prion Compounds Using In Silico and In Vitro Approaches , 2015, Scientific Reports.

[13]  V. Sim,et al.  Bile Acids Reduce Prion Conversion, Reduce Neuronal Loss, and Prolong Male Survival in Models of Prion Disease , 2015, Journal of Virology.

[14]  S. C. Nayaka,et al.  Rosmarinic acid mediated neuroprotective effects against H2O2-induced neuronal cell damage in N2A cells. , 2014, Life sciences.

[15]  D. Wishart,et al.  Shaking Alone Induces De Novo Conversion of Recombinant Prion Proteins to β-Sheet Rich Oligomers and Fibrils , 2014, PLoS ONE.

[16]  S. Prusiner,et al.  Mouse Models for Studying the Formation and Propagation of Prions* , 2014, The Journal of Biological Chemistry.

[17]  S. Ghaemmaghami,et al.  Successes and Challenges in Phenotype-Based Lead Discovery for Prion Diseases , 2014, Journal of medicinal chemistry.

[18]  D. Wishart,et al.  Lipopolysaccharide induced conversion of recombinant prion protein , 2014, Prion.

[19]  A. Aguzzi,et al.  Structural basis of prion inhibition by phenothiazine compounds. , 2014, Structure.

[20]  M. Gobbi,et al.  Doxycycline in Creutzfeldt-Jakob disease: a phase 2, randomised, double-blind, placebo-controlled trial , 2014, The Lancet Neurology.

[21]  P. Pascutti,et al.  Anti-Prion Activity of a Panel of Aromatic Chemical Compounds: In Vitro and In Silico Approaches , 2014, PloS one.

[22]  S. Prusiner,et al.  Quinacrine treatment trial for sporadic Creutzfeldt-Jakob disease , 2013, Neurology.

[23]  V. Sim,et al.  Early Increase and Late Decrease of Purkinje Cell Dendritic Spine Density in Prion-Infected Organotypic Mouse Cerebellar Cultures , 2013, PloS one.

[24]  Zhe Li,et al.  Biaryl Amides and Hydrazones as Therapeutics for Prion Disease in Transgenic Mice , 2013, The Journal of Pharmacology and Experimental Therapeutics.

[25]  P. Tavan,et al.  Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson’s disease , 2013, Acta Neuropathologica.

[26]  Wei Chen,et al.  Small Protease Sensitive Oligomers of PrPSc in Distinct Human Prions Determine Conversion Rate of PrPC , 2012, PLoS pathogens.

[27]  D. Wishart,et al.  Resolution-enhanced native acidic gel electrophoresis: a method for resolving, sizing, and quantifying prion protein oligomers. , 2012, Analytical biochemistry.

[28]  H. Nishijo,et al.  Phenolic compounds prevent beta-amyloid-protein oligomerization and synaptic dysfunction by site-specific binding , 2012, Alzheimer's & Dementia.

[29]  P. Tavan,et al.  From High‐Throughput Cell Culture Screening to Mouse Model: Identification of New Inhibitor Classes against Prion Disease , 2011, ChemMedChem.

[30]  S. Prusiner,et al.  A Survey of Antiprion Compounds Reveals the Prevalence of Non-PrP Molecular Targets* , 2011, The Journal of Biological Chemistry.

[31]  Alaina S. DeToma,et al.  Myricetin: A Naturally Occurring Regulator of Metal‐Induced Amyloid‐β Aggregation and Neurotoxicity , 2011, Chembiochem : a European journal of chemical biology.

[32]  Constance A. Sobsey,et al.  Detailed biophysical characterization of the acid-induced PrP(c) to PrP(β) conversion process. , 2011, Biochemistry.

[33]  O. Andréoletti,et al.  Recovery of Small Infectious PrPres Aggregates from Prion-infected Cultured Cells* , 2011, The Journal of Biological Chemistry.

[34]  J. Dordick,et al.  Resveratrol Selectively Remodels Soluble Oligomers and Fibrils of Amyloid Aβ into Off-pathway Conformers* , 2010, The Journal of Biological Chemistry.

[35]  S. Prusiner,et al.  Discovery of 2-Aminothiazoles as Potent Antiprion Compounds , 2009, Journal of Virology.

[36]  A. Murase,et al.  Phenolic compounds prevent Alzheimer's pathology through different effects on the amyloid-beta aggregation pathway. , 2009, The American journal of pathology.

[37]  David W. Colby,et al.  Design and construction of diverse mammalian prion strains , 2009, Proceedings of the National Academy of Sciences.

[38]  M. Lazzarino,et al.  Structural insights into alternate aggregated prion protein forms. , 2009, Journal of molecular biology.

[39]  K. Doh-ura,et al.  Continuous intraventricular infusion of pentosan polysulfate: Clinical trial against prion diseases , 2009, Neuropathology : official journal of the Japanese Society of Neuropathology.

[40]  M. Rossor,et al.  Safety and efficacy of quinacrine in human prion disease (PRION-1 study): a patient-preference trial , 2009, The Lancet Neurology.

[41]  M. Beal,et al.  Dietary supplementation with resveratrol reduces plaque pathology in a transgenic model of Alzheimer's disease , 2009, Neurochemistry International.

[42]  S. Hornemann,et al.  De novo generation of a transmissible spongiform encephalopathy by mouse transgenesis , 2009, Proceedings of the National Academy of Sciences.

[43]  Hironori K. Nakamura,et al.  Variety of Antiprion Compounds Discovered through an In Silico Screen Based on Cellular-Form Prion Protein Structure: Correlation between Antiprion Activity and Binding Affinity , 2008, Antimicrobial Agents and Chemotherapy.

[44]  I. Bone,et al.  Intraventricular pentosan polysulphate in human prion diseases: an observational study in the UK , 2008, European journal of neurology.

[45]  Jonathan Weissman,et al.  Small-molecule aggregates inhibit amyloid polymerization. , 2008, Nature chemical biology.

[46]  Iva Hafner-Bratkovič,et al.  Curcumin binds to the α‐helical intermediate and to the amyloid form of prion protein – a new mechanism for the inhibition of PrPSc accumulation , 2008 .

[47]  S. Mok,et al.  Evaluation of drugs for treatment of prion infections of the central nervous system. , 2008, The Journal of general virology.

[48]  John Collinge,et al.  A General Model of Prion Strains and Their Pathogenicity , 2007, Science.

[49]  K. Doh-ura,et al.  Orally Administered Amyloidophilic Compound Is Effective in Prolonging the Incubation Periods of Animals Cerebrally Infected with Prion Diseases in a Prion Strain-Dependent Manner , 2007, Journal of Virology.

[50]  B. Caughey,et al.  Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein , 2007, Nature Methods.

[51]  Kazuo Kuwata,et al.  Hot spots in prion protein for pathogenic conversion , 2007, Proceedings of the National Academy of Sciences.

[52]  Weimin Guo,et al.  Chemical structure of flavonols in relation to modulation of angiogenesis and immune-endothelial cell adhesion. , 2006, The Journal of nutritional biochemistry.

[53]  K. Ono,et al.  Ferulic acid destabilizes preformed β-amyloid fibrils in vitro , 2005 .

[54]  J. Benito‐León Compassionate use of quinacrine in Creutzfeldt–Jakob disease fails to show significant effects , 2005, Neurology.

[55]  O. Bocharova,et al.  Semiautomated cell-free conversion of prion protein: applications for high-throughput screening of potential antiprion drugs. , 2005, Analytical biochemistry.

[56]  J. Hauw,et al.  Compassionate use of quinacrine in Creutzfeldt–Jakob disease fails to show significant effects , 2004, Neurology.

[57]  T. Iwaki,et al.  Amyloid imaging probes are useful for detection of prion plaques and treatment of transmissible spongiform encephalopathies. , 2004, The Journal of general virology.

[58]  E. Irle,et al.  Efficacy of flupirtine on cognitive function in patients with CJD , 2004, Neurology.

[59]  B. Caughey,et al.  New Inhibitors of Scrapie-Associated Prion Protein Formation in a Library of 2,000 Drugs and Natural Products , 2003, Journal of Virology.

[60]  G. Forloni,et al.  Evaluation of Quinacrine Treatment for Prion Diseases , 2003, Journal of Virology.

[61]  G. J. Raymond,et al.  Inhibition of Protease-Resistant Prion Protein Accumulation In Vitro by Curcumin , 2003, Journal of Virology.

[62]  C. Masters,et al.  Quinacrine does not prolong survival in a murine Creutzfeldt‐Jakob disease model , 2002, Annals of neurology.

[63]  G. Forloni,et al.  Tetracyclines affect prion infectivity , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[64]  F. Cohen,et al.  Pathway Complexity of Prion Protein Assembly into Amyloid* , 2002, The Journal of Biological Chemistry.

[65]  B. Caughey,et al.  Lysosomotropic Agents and Cysteine Protease Inhibitors Inhibit Scrapie-Associated Prion Protein Accumulation , 2000, Journal of virology.

[66]  M. Pocchiari,et al.  Congo red prolongs the incubation period in scrapie-infected hamsters , 1995, Journal of virology.

[67]  B. Caughey,et al.  Potent Inhibition of Scrapie‐Associated PrP Accumulation by Congo Red , 1992, Journal of neurochemistry.

[68]  Iva Hafner-Bratkovič,et al.  Curcumin binds to the alpha-helical intermediate and to the amyloid form of prion protein - a new mechanism for the inhibition of PrP(Sc) accumulation. , 2008, Journal of neurochemistry.

[69]  K. Ono,et al.  Ferulic acid destabilizes preformed beta-amyloid fibrils in vitro. , 2005, Biochemical and biophysical research communications.

[70]  J. Collinge Prion diseases of humans and animals: their causes and molecular basis. , 2001, Annual review of neuroscience.

[71]  V. Neuhoff,et al.  Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: A systematic analysis , 1985 .