Identification of benzothiazoles as potential polyglutamine aggregation inhibitors of Huntington's disease by using an automated filter retardation assay

Abstract

Preventing the formation of insoluble polyglutamine containing protein aggregates in neurons may represent an attractive therapeutic strategy to ameliorate Huntington's disease (HD). Therefore, the ability to screen for small molecules that suppress the self-assembly of huntingtin would have potential clinical and significant research applications. We have developed an automated filter retardation assay for the rapid identification of chemical compounds that prevent HD exon 1 protein aggregation in vitro. Using this method, a total of 25 benzothiazole derivatives that inhibit huntingtin fibrillogenesis in a dose-dependent manner were discovered from a library of Ϸ184,000 small molecules. The results obtained by the filter assay were confirmed by immunoblotting, electron microscopy, and mass spectrometry. Furthermore, cell culture studies revealed that 2-amino-4,7-dimethylbenzothiazol-6-ol, a chemical compound similar to riluzole, significantly inhibits HD exon 1 aggregation in vivo. These findings may provide the basis for a new therapeutic approach to prevent the accumulation of insoluble protein aggregates in Huntington's disease and related glutamine repeat disorders.

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

“…Previous investigations have demonstrated that chemical agents can be used to perturb amyloid formation pathways 22 . Most of these studies focused on small molecules or -sheet breaker peptides that inhibit or slow down spontaneous amyloid polymerization 1,40 .…”

“…1a). Using this method, we screened a focused library of 300 chemical compounds that were previously identified as modulators of polyglutamine-mediated huntingtin exon 1 aggregation 22 . We detected 6 A42 aggregation-promoting compounds, three of which shared a phenoxazine structure.…”

Lithostathine Quadruple-helical Filaments Form Proteinase K-resistant Deposits in Creutzfeldt-Jakob Disease

“…The molar ratio of lithostathine/PGL-034 was 3/20, slightly more than used previously for EM analysis of PGL-034 with hungtingtin (1.5/20; see Ref. 13). The final preparation was then viewed on a Philips CM100 EM (FEI Company, Hillsboro, OR).…”

“…Lithostathine Fibrils Can Be Dissociated by [6,6Ј]Bibenzothiazolyl-2,2Ј-diamine-Studies were also carried out to investigate whether QHF-litho could be dissociated by compounds targeted against amyloid structures such as polyglutamine (13). Tests were carried out with one such compound, the benzothiazole, PGL-034, that has been shown previously to perturb very effectively the aggregation events associated with Huntington's disease.…”

Spontaneous self-assembly of pathogenic huntingtin exon 1 protein into amyloid structures

“…Besides small molecule promoters of functional proteostasis, compounds that directly target the aggregation process have been identified. They include substances such as Congo red, Thioflavine S, PGL-135 or EGCG ((-)-epigallocatechin-3-gallate), which reduce the formation of polyQ-containing HTTex1 aggregates [41][42][43]. To date, the mode of action of EGCG has been studied most extensively (Fig.…”