Dataset Information

Anti-Prion Screening for Acridine, Dextran, and Tannic Acid using Real Time-Quaking Induced Conversion: A Comparison with PrPSc-Infected Cell Screening.

ABSTRACT: Prion propagation is mediated by the structural alteration of normal prion protein (PrPC) to generate pathogenic prion protein (PrPSc). To date, compounds for the inhibition of prion propagation have mainly been screened using PrPSc-infected cells. Real time-quaking-induced conversion (RT-QuIC) is one alternative screening method. In this study, we assessed the propagation inhibition effects of known anti-prion compounds using RT-QuIC and compared the results with those from a PrPSc-infected cell assay. Compounds were applied to RT-QuIC reactions at 0 h or 22 h after prion propagation to determine whether they inhibited propagation or reduced amplified aggregates. RT-QuIC reactions in presence of acridine, dextran sulfate sodium (DSS), and tannic acid inhibited seeded aggregation with sporadic Creutzfeldt-Jakob disease at 0 h. After treatment at 22 h, amplified fluorescence was decreased in wells treated with either acridine or tannic acid. Compound activities were verified by western blot of RT-QuIC products and in a dye-independent conversion assay, the Multimer Detection System. Protease K-resistant PrPSc fragments (PrPres) were reduced by DSS and tannic acid in the PrPSc-infected cell assay. Importantly, these inhibitory effects were similar despite different treatment times (0 h versus 3 days). Consequentially, RT-QuIC enabled the more specific classification of compounds according to action (i.e., inhibition of prion propagation versus reduction of amplified aggregates). RT-QuIC addresses the limitations of cell-based screening methods and can be used to further aid our understanding of the mechanisms of action of anti-prion compounds.

SUBMITTER: Hyeon JW

PROVIDER: S-EPMC5240994 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Anti-Prion Screening for Acridine, Dextran, and Tannic Acid using Real Time-Quaking Induced Conversion: A Comparison with PrPSc-Infected Cell Screening.

Hyeon Jae Wook JW Kim Su Yeon SY Lee Sol Moe SM Lee Jeongmin J An Seong Soo A SS Lee Myung Koo MK Lee Yeong Seon YS

PloS one 20170117 1

Prion propagation is mediated by the structural alteration of normal prion protein (PrPC) to generate pathogenic prion protein (PrPSc). To date, compounds for the inhibition of prion propagation have mainly been screened using PrPSc-infected cells. Real time-quaking-induced conversion (RT-QuIC) is one alternative screening method. In this study, we assessed the propagation inhibition effects of known anti-prion compounds using RT-QuIC and compared the results with those from a PrPSc-infected cel ...[more]

PMID: 28095474

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Project description:UNLABELLED:A key challenge in managing transmissible spongiform encephalopathies (TSEs) or prion diseases in medicine, agriculture, and wildlife biology is the development of practical tests for prions that are at or below infectious levels. Of particular interest are tests capable of detecting prions in blood components such as plasma, but blood typically has extremely low prion concentrations and contains inhibitors of the most sensitive prion tests. One of the latter tests is quaking-induced conversion (QuIC), which can be as sensitive as in vivo bioassays, but much more rapid, higher throughput, and less expensive. Now we have integrated antibody 15B3-based immunoprecipitation with QuIC reactions to increase sensitivity and isolate prions from inhibitors such as those in plasma samples. Coupling of immunoprecipitation and an improved real-time QuIC reaction dramatically enhanced detection of variant Creutzfeldt-Jakob disease (vCJD) brain tissue diluted into human plasma. Dilutions of 10(14)-fold, containing ~2 attogram (ag) per ml of proteinase K-resistant prion protein, were readily detected, indicating ~10,000-fold greater sensitivity for vCJD brain than has previously been reported. We also discriminated between plasma and serum samples from scrapie-infected and uninfected hamsters, even in early preclinical stages. This combined assay, which we call "enhanced QuIC" (eQuIC), markedly improves prospects for routine detection of low levels of prions in tissues, fluids, or environmental samples. IMPORTANCE:Transmissible spongiform encephalopathies (TSEs) are largely untreatable and are difficult to diagnose definitively prior to irreversible clinical decline or death. The transmissibility of TSEs within and between species highlights the need for practical tests for even the smallest amounts of infectivity. A few sufficiently sensitive in vitro methods have been reported, but most have major limitations that would preclude their use in routine diagnostic or screening applications. Our new assay improves the outlook for such critical applications. We focused initially on blood plasma because a practical blood test for prions would be especially valuable for TSE diagnostics and risk reduction. Variant Creutzfeldt-Jakob disease (vCJD) in particular has been transmitted between humans via blood transfusions. Enhanced real-time quaking-induced conversion (eRTQ) provides by far the most sensitive detection of vCJD to date. The 15B3 antibody binds prions of multiple species, suggesting that our assay may be useful for clinical and fundamental studies of a variety of TSEs of humans and animals.

Dataset Information

Anti-Prion Screening for Acridine, Dextran, and Tannic Acid using Real Time-Quaking Induced Conversion: A Comparison with PrPSc-Infected Cell Screening.

Publications

Anti-Prion Screening for Acridine, Dextran, and Tannic Acid using Real Time-Quaking Induced Conversion: A Comparison with PrPSc-Infected Cell Screening.

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