Acridine and phenothiazine derivatives as pharmacotherapeutics for prion disease

Abstract

Prion diseases in humans and animals are invariably fatal. Prions are composed of a disease-causing isoform (PrP(Sc)) of the normal host prion protein (PrP(C)) and replicate by stimulating the conversion of PrP(C) into nascent PrP(Sc). We report here that tricyclic derivatives of acridine and phenothiazine exhibit half-maximal inhibition of PrP(Sc) formation at effective concentrations (EC(50)) between 0.3 microM and 3 microM in cultured cells chronically infected with prions. The EC(50) for chlorpromazine was 3 microM, whereas quinacrine was 10 times more potent. A variety of 9-substituted, acridine-based analogues of quinacrine were synthesized, which demonstrated variable antiprion potencies similar to those of chlorpromazine and emphasized the importance of the side chain in mediating the inhibition of PrP(Sc) formation. Thus, our studies show that tricyclic compounds with an aliphatic side chain at the middle ring moiety constitute a new class of antiprion reagents. Because quinacrine and chlorpromazine have been used in humans for many years as antimalarial and antipsychotic drugs, respectively, and are known to pass the blood-brain barrier, we suggest that they are immediate candidates for the treatment of Creutzfeldt-Jakob disease and other prion diseases.

Figure 1

PrP^Sc-inhibiting effects of phenothiazine derivatives and other psychopharmacological substances. Anti-PrP immunoblots of protease-digested ScN2a cell lysates treated with the respective substance for 6 days at the concentration indicated. Molecular mass markers at the right of the immunoblot correspond to 37 kDa (upper bands) and 20 kDa (lower bands). The characteristic PrP immunostain depicted corresponds to the amount of PrP^Sc and prion infectivity present in the neuroblastoma cells after treatment with the concentration indicated above. The schematic molecular structure of the compound used is depicted on the left side of each immunoblot.

Figure 2

Dose-response relationship of ScN2a cells treated with chlorpromazine, quinacrine, and methylene blue. Anti-PrP immunoblots of the same ScN2a cell lysates treated with proteinase K (1st row), without proteinase K (2nd row), anti-tubulin immunoblot of the same lysate (3rd row), and average immunoblot densitometry values (IDV) of three independent immunoblots (Bottom; bars represent standard error). ScN2a cells were treated for 6 days with chlorpromazine (a), quinacrine (b), or methylene blue (c). EC₅₀ levels are ≈2 μM for chlorpromazine and ≈250 nM for quinacrine. Methylene blue does not inhibit PrP^Sc formation and is cytotoxic at concentrations >500 nM (see text for discussion).

Figure 3

Quinacrine treatment permanently cures ScN2a cells. Anti-PrP immunoblots of protease-digested ScN2a cell lysates treated with 0.2–0.8 μM quinacrine for 6 days (left) and then left without treatment and weekly splitting for 3 weeks. The lack of reappearance of protease-resistant PrP^Sc after discontinuation of treatment demonstrates that quinacrine permanently cures ScN2a cells. Molecular mass markers at the right of the immunoblot correspond to 37 kDa (upper band) and 20 kDa (lower band).