Countering amyloid polymorphism and drug resistance with minimal drug cocktails

Abstract

Several fatal, progressive neurodegenerative diseases, including various prion and prion-like disorders, are connected with the misfolding of specific proteins. These proteins misfold into toxic oligomeric species and a spectrum of distinct self-templating amyloid structures, termed strains. Hence, small molecules that prevent or reverse these protein-misfolding events might have therapeutic utility. Yet it is unclear whether a single small molecule can antagonize the complete repertoire of misfolded forms encompassing diverse amyloid polymorphs and soluble oligomers. We have begun to investigate this issue using the yeast prion protein, Sup35, as an experimental paradigm. We have discovered that a polyphenol, (-)epigallocatechin-3-gallate (EGCG), effectively inhibited the formation of infectious amyloid forms (prions) of Sup35 and even remodeled preassembled prions. Surprisingly, EGCG selectively modulated specific prion strains and even selected for EGCG-resistant prion strains with novel structural and biological characteristics. Thus, treatment with a single small molecule antagonist of amyloidogenesis can select for novel, drug-resistant amyloid polymorphs. Importantly, combining EGCG with another small molecule, 4,5-bis-(4-methoxyanilino)phthalimide, synergistically antagonized and remodeled a wide array of Sup35 prion strains without producing any drug-resistant prions. We suggest that minimal drug cocktails, small collections of drugs that collectively antagonize all amyloid polymorphs, should be identified to besiege various neurodegenerative disorders.

Acknowledgements

M.L.D. is supported by a research grant from the American Federation for Aging Research, from the William Wood Foundation, and the Hereditary Disease Foundation. J.S. is supported by an NIH Director's New Innovator Award (1DP2OD002177-01), an Ellison Medical Foundation New Scholar in Aging Award, an NINDS grant (1R21NS067354-0110), a Bill and Melinda Gates Foundation Grand Challenges Explorations Award and a University of Pennsylvania Diabetes and Endocrinology Research Center Pilot and Feasibility grant.

Figures and Tables

Figure 1 Sup35 prion strains and small-molecule antagonists. (A) Sup35 is a modular protein comprised of a C-terminal GTPase domain (C, amino acids 254–685, black), a highly charged middle domain (M, amino acids 124–253, dark grey) and an N-terminal domain (N, amino acids 1–123, light grey) enriched in glutamine, asparagine, tyrosine and glycine residues. Together N and M (NM) confer all the properties needed to form a stable prion in yeast. NM is termed the prion domain.83 Within N, prion recognition elements termed the “head” (red) and “tail” (green), which flank a “central core” (blue), play important roles in prion formation.33,90 (B) Sup35 prions adopt a polymeric cross-beta structure. In one proposed model (left), this amyloid structure is composed of the head (red), central core (blue) and tail (green) regions of N. The M and C domains are located on the exterior of this structure and are not depicted for clarity. If we zoom in on three adjacent monomers in the Sup35 prion polymer, we find that the prion is proposed to be maintained by an alternating sequence of head-to-head (red) and tail-to-tail (green) intermolecular contacts. The central core is sequestered in intramolecular contacts (blue). Different Sup35 prion strains assemble under different environmental conditions. Thus, NM25 assembles at 25°C or when NM is chemically crosslinked with BMB in the tail region.33,61 NM4 assembles at 4°C or when NM is chemically crosslinked with BMB in the head region.33,61 NM4E assembles in the presence of EGCG at 4°C.61 These prion strains have subtle differences in the precise residues that comprise the head, tail and central core (right). The residues that comprise the head, tail and central core are shown to the right of each central protomer. NM25, NM4 and NM4E are distinguished by their different tail-to-tail contacts and central core region.61 Moreover, NM4E has a distinct head-to-head contact.61 Infection of [psi⁻] [pin⁻] cells with NM25 yields mostly weak [PSI⁺] variants, whereas NM4 yields mostly strong [PSI⁺] variants.61 By contrast, NM4E generates purely strong [PSI⁺] variants61 (pie charts in lower portion). (C) Chemical structure of EGCG. (D) Chemical structure of DAPH-12.