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 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...

Comparison of Three Amyloid Assembly Inhibitors: The Sugar scyllo-Inositol, the Polyphenol Epigallocatechin Gallate, and the Molecular Tweezer CLR01

“…The importance of understanding the mechanism of inhibition has been highlighted 36, 37 following evidence suggesting that many small molecule inhibitors of fibrillogenesis might act nonspecifically, likely making them unsuitable for treating amyloid-related disorders. 38 Inhibition of fibril formation or dissociation of existing fibrils actually may yield toxic oligomers under certain circumstances. 39 Importantly, understanding the details of target−drug interaction is essential not only for subsequent drug development but also for preventing potential side effects.…”

Pharmacological chaperone for the structured domain of human prion protein

“…We have measured and characterized this interaction by several independent biophysical techniques. The binding site, monomeric structure, and positive charge of Fe(III)-TMPyP make it entirely different from polyanions and colloidal inhibitors, such as anionic tetrapyrroles, which appear to be nonspecific inhibitors of amyloid formation (29). As such this cationic tetrapyrrole represents a distinct class of human PrP-binding molecule with proven activity in prion-infected cells (32), animal models (33), and now in PMCA.…”

“…In contrast, a number of molecules have been demonstrated to bind to the unstructured region of PrP including pentosan polysulfate (25), Congo red (26), and a number of anionic tetrapyrroles (27,28). Congo red and anionic tetrapyrroles can stack in solution, and are believed to behave like polyanions and bind with low specificity to the N-terminal domain (29)(30)(31). Indeed, the ability of anionic tetrapyrroles to stack in solution correlated well with their antiprion activity (31).…”

Small-molecule conversion of toxic oligomers to nontoxic β-sheet–rich amyloid fibrils

“…Thus, our studies demonstrate that O4 specifically targets hydrophobic regions critical for efficient amyloid polymerization 33 in A40 and A42 peptides. Previous studies indicate that effects of small molecules on amyloidogenesis might be caused by compound aggregates 34 . We therefore investigated whether O4 forms aggregates in aqueous solution.…”