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 (EC50) between 0.3 M and 3 M in cultured cells chronically infected with prions. The EC 50 for chlorpromazine was 3 M, 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. P rion diseases are uniquely manifest as spontaneous, inherited, and infectious maladies. These diseases include Gerstmann-Sträussler-Scheinker (GSS) disease, fatal insomnia, and Creutzfeldt-Jakob disease (CJD). Most cases of CJD are sporadic with 10-15% being inherited (1). Although the infectious human prion diseases are most notorious, they account for less than 1% of all prion disorders (2). Concern about these infectious disorders has been heightened by the identification of more than 100 young adults and teenagers who have developed new variant CJD (nvCJD) in Europe after exposure to bovine prions from cattle with bovine spongiform encephalopathy (BSE; refs. 3 and 4). Other infectious prion diseases include kuru, which is found among New Guinea natives and is caused by ritualistic cannibalism, and iatrogenic CJD, which is caused by prioncontaminated cadaveric growth hormone and dura mater grafts (2,5,6).A wealth of experimental data indicates that prions are composed solely of a misfolded prion protein (PrP) isoform (PrP Sc ) of a glycolipid-anchored host protein (PrP C ; refs. 7 and 8). Unlike all other infectious agents, prions are devoid of nucleic acid (9, 10). For years, the existence of prion strains caused many investigators to argue that a small nucleic acid encodes prion diversity (11). Eventually, convincing data accumulated arguing that prion diversity is enciphered in the conformation of PrP Sc (3,12,13).Patients with CJD and other prion diseases develop progressive neurologic dysfunction. Prion diseases are invariably fatal, and death frequently occurs in less than 1 year after the first symptoms appear (2). No effective therapy exists for prion diseases in humans or animals (14).Many compounds have been identified that inhibit prion propagation when adm...

Many amyloid inhibitors resemble molecules that form chemical aggregates, which are known to inhibit many proteins. Eight known chemical aggregators inhibited amyloid formation of the yeast and mouse prion proteins Sup35 and recMoPrP in a manner characteristic of colloidal inhibition. Similarly, three known anti-amyloid molecules inhibited β-lactamase in a detergent-dependent manner, which suggests that they too form colloidal aggregates. The colloids localized to preformed fibers and prevented new fiber formation in electron micrographs. They also blocked infection of yeast cells with Sup35 prions, which suggests that colloidal inhibition may be relevant in more biological milieus.The aggregation of proteins into amyloid fibers is associated with a growing list of diseases, including diabetes, Alzheimer's, Parkinson's, Huntington's and the prion diseases. In these disorders, proteins aggregate into long, unbranched fibers after misfolding into a β-sheet-rich conformation 1 . Though there are no approved therapies targeting amyloid formation directly, many organic molecules inhibit fibrillization in vitro [2][3][4][5][6][7] . Some, such as the chelator clioquinol (1), even have activity in vivo 4 . These results have inspired the hope of therapeutic applications for some molecules 3-5 . Curiously, many fibrillization inhibitors resemble molecules known to form promiscuous chemical aggregates. These colloidal particles are composed of small organic molecules and range in size from 50 to over 600 nm 8 . Once formed, they physically sequester proteins and inhibit enzymes nonspecifically 8,9 . Like many inhibitors of amyloid polymerization, these colloidal inhibitors are typically highly conjugated, hydrophobic and dye-like (Supplementary Table 1 online) 8,9 . A good example is the amyloid inhibitor Congo red (2), a dye that was one of the first molecules observed to exhibit colloidal inhibition 8 . The flavonoid baicalein (3), an inhibitor of α-synuclein polymerization 6 , resembles the known chemical aggregator quercetin (4), and 4,5-dianilinophthalimide (DAPH, 5), an inhibitor of Alzheimer's amyloid formation 2 , resembles the aggregator bisindoylmaleimide (6 ; Supplementary Fig. 1 online).Given that chemical aggregates function through enzyme sequestration, we wondered whether they might also sequester protein molecules from each other, thereby preventing amyloid polymerization. Here, we investigate this hypothesis in two classic amyloid-forming proteins: the yeast prion protein Sup35 (ref. 10 ) and the recombinant mouse prion protein recMoPrP 89-230 (ref. 11 ). We ask whether known chemical aggregators can inhibit amyloid fiber formation, whether known fibrillization inhibitors form colloidal aggregates and whether amyloid inhibition by these molecules is in fact mediated via colloidal aggregation.Eight known chemical aggregators and two known nonaggregators 8,9 were tested for inhibition of Sup35 fibrillization in a thioflavin T (ThT, 7) fluorescence assay. All eight inhibited Sup35 fibrillization b...

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