Small-molecule aggregates inhibit amyloid polymerization

Abstract

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 beta-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.

Figure 1

Chemical aggregators inhibit amyloid formation in biochemical assays. (a) The known chemical aggregators TIPT and clotrimazole inhibit seeded Sup35 polymerization in a dose-dependent manner. Inhibition of Sup35 polymerization was measured by ThT fluorescence and plotted as a function of time. Percent inhibition was calculated as the ratio of the initial rate of the inhibited reaction compared with control. Error bars represent the s.d. of triplicate measurements. (b) Transmission electron micrographs of control Sup35 prion fibers grown in the absence of compound. (c) Preformed Sup35 fibers mixed with chemical aggregates of TIPT (concentration of compound 100 μM). Scale bar, 100 nm.

Figure 2

Chemical aggregators inhibit prion infection in yeast culture. (a) Representative cultures of infected and noninfected yeast cells. The infected phenotype, PSI⁺, appears as white or pink colonies, whereas the uninfected PSI⁻ colonies are red. Rottlerin, a known chemical aggregator, inhibits infection. (+) and (−) correspond to positive and negative controls for infection. (b) The known chemical aggregate-forming molecules nicardipine, DAPH and baicalein block infection to varying degrees. (c) Infection is blocked in a dose-dependent manner by TIPT. *Indicates that slightly fewer than 56 viable colonies were observed due to mild cell toxicity. Error bars represent the s.d. in colonies counted.