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.

BMD42-2910, a Novel Benzoxazole Derivative, Shows a Potent Anti-prion Activity and Prolongs the Mean Survival in an Animal Model of Prion Disease

“…As a result, ThT-compound interference is not observed in optical (data not shown). This result was consistent with that of our previous experiment [30].…”

“…Human recombinant PrP (rPrP, residues 23-231; accession no. K02234) was produced, and the RT-QuIC reaction was performed as described in our previous report, as a modified method of Wilham et al and Cramm et al [28][29][30]. Briefly, Escherichia coli transfected with vector with human PrP sequence was grown in Luria broth medium with kanamycin and chloramphenicol.…”

Cellulose ether treatment in vivo generates chronic wasting disease prions with reduced protease resistance and delayed disease progression

“…In order to determine whether CEs exert anti-prion effects against CWD prions, we first tested CEs in prion conversion assays in vitro. RT-QuIC has been used to assess the antiprion effects of numerous drugs, whether as a screening tool to discriminate various compounds or as an assay to analyze the specific effect of a given compound (Ferreira et al 2014;Vieira et al 2014;Schmitz et al 2016;Hyeon et al 2017;Ferreira et al 2018). Thus, we used RT-QuIC to assess the inhibitory effect of CEs on CWD prion seeding activity ( Fig.…”

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

“…Also, the effect of TUDCA on RT-QuIC converted prions is now available [37]. Previous attempts to perform time-dependent analysis of quinacrine activity on prion conversion failed because the required concentrations of quinacrine interfere with Thioflavin T fluorescence detection [50]. Notably, previous cell-free full-length PrP conversion methods for identifying and characterizing anti-prion molecules have relied on denaturants or detergents that often disrupt ligand to protein interactions [22,24,51,52].…”