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

Date Published: January 17, 2017

Publisher: Public Library of Science

Author(s): Jae Wook Hyeon, Su Yeon Kim, Sol Moe Lee, Jeongmin Lee, Seong Soo A. An, Myung Koo Lee, Yeong Seon Lee, Ina Maja Vorberg.


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.

Partial Text

Prion diseases are fatal neurodegenerative diseases that trigger the accumulation of pathogenic prion protein (PrPSc) and neuronal death in humans and animals [1]. The process of prion propagation involves the structural alteration of host-encoded cellular prion protein (PrPC) to PrPSc and the autocatalytic amplification of pathogenic protein [2]. PrPSc is largely protease-resistant, insoluble, β-sheet rich, and capable of aggregation as a hallmark of prion disease [3]. Therefore, inhibiting the conversion of PrPC to PrPSc and/or facilitating the degradation of PrPSc are primary strategies for anti-prion pharmaceutical development.

A majority of anti-prion compounds have been confirmed in vitro and to some extent in vivo [6, 8]. However, compounds can affect a variety of factors and components in cells, including DNA, protein, and metal/non-metal ions. Therefore, some compounds may not directly inhibit the conversion of PrPC or reduce the amplification of PrPres, but may have indirect effects on other cellular factors. Alternatively, the RT-QuIC assay has a relatively simple reaction environment. In the present study, possible environmental influences (especially ThT quenching by compounds) were in part excluded by performing follow-up western blotting of RT-QuIC reaction products and MDS. However, it is still unclear whether acridine inhibited PrPres and quenched ThT at the same time. It has been reported that, in dye-dependent seeded aggregation assays, low molecular weight substances can interfere with the fluorescence of dye compounds bound to amyloid fibrils [27]. Indeed, some compounds have been posited to compete with ThT for the same binding site on fibrils rather than directly inhibit the fibrilization process. Other studies have similarly described the interference of small molecules with ThT fluorescence during fibril formation [28, 29]. To address this issue, we performed a dye-independent conversion assay in order to determine whether the test compounds indeed inhibited fibrilization. We concluded that the compounds in this study were unlikely to produce ThT quenching.




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