Innovative Non-PrP-Targeted Drug Strategy Designed to Enhance Prion Clearance

Abstract

Prion diseases are a group of neurodegenerative disorders characterized by the accumulation of misfolded prion protein (called PrP^Sc). Although conversion of the cellular prion protein (PrP^C) to PrP^Sc is still not completely understood, most of the therapies developed until now are based on blocking this process. Here, we propose a new drug strategy aimed at clearing prions without any direct interaction with neither PrP^C nor PrP^Sc. Starting from the recent discovery of SERPINA3/SerpinA3n upregulation during prion diseases, we have identified a small molecule, named compound 5 (ARN1468), inhibiting the function of these serpins and effectively reducing prion load in chronically infected cells. Although the low bioavailability of this compound does not allow in vivo studies in prion-infected mice, our strategy emerges as a novel and effective approach to the treatment of prion disease.

Figure 1

Chemical structures of compounds A–U.

Figure 2

Chemical structures of compounds 1–8.

Figure 3

Anti-prion effect of anti-SERPINA3 hit compounds in ScGT1 RML cell lines. (A,C) Representative Western blot image of PrP^Sc, total PrP, and β-actin in lysates from ScGT1 RML treated with vehicle (CTRL) or compounds G and H at 40 μM, or compound 5 (ARN1468) at 20 μM. Molecular weight markers are represented on the right (kDa). (B,D) Densitometric analysis of normalized PrP^Sc levels in ScGT1 RML treated with the vehicle or the drug. Experiments have been performed in triples (n = 6). Statistical significance was assessed using Friedman test and Dunn’s multiple comparison compared to control cells (B) or Wilcoxon matched-pair signed rank test (D) *p < 0.05.

Figure 4

Anti-prion effect of compound 5 in ScGT1 22L, ScN2a RML, and ScN2a 22L cell lines. (A–C) Representative Western blot images of PrP^Sc, total PrP, and β-actin in lysates from ScGT1 22L (A), ScN2a RML (B), and ScN2a 22L (C) treated with vehicle (CTRL) or compound 5 at 20 μM. Molecular weight markers are shown on the right (kDa). (D–F) Densitometric analysis of normalized PrP^Sc levels in ScGT1 22L (D), ScN2a RML (E), and ScN2a 22L (F) treated with the vehicle or the drug. Experiments have been performed in six times (n = 6). Statistical significance was assessed by Wilcoxon matched pairs signed rank test, *p < 0.05.

Figure 5

Dose–response curve of compound 5 on RML- and 22L-infected GT1 and N2a cell lines. Densitometric analysis of PrP^Sc level clearance after 3 days treatment with compound 5 in ScGT1 RML (A, EC₅₀ = 8.64), ScGT1 22L (B, EC₅₀ = 19.3), ScN2a RML (C, EC₅₀ = 11.2), and ScN2a 22L (D, EC₅₀ = 6.27) (for all titrations, n = 3).

Figure 6

Chronic treatment of prion-infected GT1 and N2a cell line with compound 5. Western blot analysis of PrP^Sc in lysates from ScGT1 RML (A), ScN2a RML (B), ScGT1 22L (C), and ScN2a 22L (D) treated with vehicle or compound 5 at 20 μM for four passages (up to 1 month). β-actin was used as a loading control. Molecular weight markers are shown on the right of each Western blot (kDa).

Figure 7

Compound 5 and quinacrine treatment of ScN2a RML cells. (A,B) Representative Western blot images of PrP^Sc, total PrP, and β-actin in lysates from ScN2a RML cells treated with (A) vehicles, quinacrine (1 μM), or compound 5 (10 μM) and (B) vehicles or a dose gradient of compound 5 and quinacrine (2–10 μM and 0.2–1 μM, respectively). (C) Pairwise dose–response data (left) and isobologram synergy plot (right) were presented for the combination treatment. Dose–response data are derived from a nonlinear regression of 3 experimental replicates, plotted with mean ± SD. The diagonal line of the isobologram represents the additive line. EC_50,add dot corresponds to the calculated, theoretical paired value, when assuming an additive interaction between the two drugs. EC_50,mix represents the paired value of drug concentrations assessed or synergism. The interaction index (γ) was calculated, and it corresponds to a value of 0.19.

Figure 8

Pharmacokinetic profile of compound 5 in vivo. The pharmacokinetic profiles of compound 5 in plasma following intravenous (I.V.) and oral (P.O.) administration to male C57BL/6 mice (n = 3 per time point).