Using budding yeast to screen for anti-prion drugs

Prions are misfolded proteins capable of propagating their altered conformation which are commonly considered as the causative agent of transmissible spongiform encephalopathies, a class of fatal neurodegenerative diseases. Currently, no treatment for prion-based diseases is available. Recently we have developed a rapid, yeast-based, two-step assay to screen for anti-prion drugs [1]. This new method allowed us to identify several compounds that are effective in vivo against budding yeast [PSI+] and [URE3] prions but also able to promote mammalian prion clearance in three different cell culture-based assays. Taken together, these results validate our method as an economic and efficient high-throughput screening approach to identify novel prion inhibitors or to carry on comprehensive structure-activity studies for already isolated anti-mammalian prion drugs. These results suggest furthermore that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to human and thus amenable to pharmacological and genetic analysis. Finally, it would be very interesting to test active drugs isolated using the yeast-based assay in models for other diseases (neurodegenerative or not) involving amyloid fibers like Huntington's, Parkinson's or Alzheimer's diseases.     

A yeast-based assay to isolate drugs active against mammalian prions

Recently, we have developed a yeast-based (Saccharomyces cerevisiae) assay to isolate drugs active against mammalian prions. The initial assumption was that mechanisms controlling prion appearance and/or propagation could be conserved from yeast to human, as it is the case for most of the major cell biology regulatory mechanisms. Indeed, the vast majority of drugs we isolated as active against both [PSI(+)] and [URE3] budding yeast prions turned out to be also active against mammalian prion in three different mammalian cell-based assays. These results strongly argue in favor of common prion controlling mechanisms conserved in eukaryotes, thus validating our yeast-based assay and also the use of budding yeast to identify antiprion compounds and to study the prion world.     

The Toll-Like Receptor Agonist Imiquimod Is Active against Prions

Using a yeast-based assay, a previously unsuspected antiprion activity was found for imiquimod (IQ), a potent Toll-like receptor 7 (TLR7) agonist already used for clinical applications. The antiprion activity of IQ was first detected against yeast prions [PSI ⁺] and [URE3], and then against mammalian prion both ex vivo in a cell-based assay and in vivo in a transgenic mouse model for prion diseases. In order to facilitate structure-activity relationship studies, we conducted a new synthetic pathway which provides a more efficient means of producing new IQ chemical derivatives, the activity of which was tested against both yeast and mammalian prions. The comparable antiprion activity of IQ and its chemical derivatives in the above life forms further emphasizes the conservation of prion controlling mechanisms throughout evolution. Interestingly, this study also demonstrated that the antiprion activity of IQ and IQ-derived compounds is independent from their ability to stimulate TLRs. Furthermore, we found that IQ and its active chemical derivatives inhibit the protein folding activity of the ribosome (PFAR) in vitro.     

Yeast-based screening of natural product extracts results in the identification of prion inhibitors from a marine sponge

ABSTRACT

One of the major medical challenges of the twenty-first century is the treatment of incurable and fatal neurodegenerative disorders caused by misfolded prion proteins. Since the discovery of these diseases a number of studies have been conducted to identify small molecules for their treatment, however to date no curative treatment is available. These studies can be highly expensive and time consuming, but more recent experimental approaches indicate a significant application for yeast prions in these studies. We therefore used yeast prions to optimize previous high-throughput methods for the cheaper, easier and more rapid screening of natural extracts. Through this approach we aimed to identify natural yeast-prion inhibitors that could be useful in the development of novel treatment strategies for neurodegenerative disorders. We screened 500 marine invertebrate extracts from temperate waters in Australia allowing the identification of yeast-prion inhibiting extracts. Through the bioassay-driven chemical investigation of an active Suberites sponge extract, a group of bromotyrosine derivatives were identified as potent yeast-prion inhibitors. This study outlines the importance of natural products and yeast prions as a first-stage screen for the identification of new chemically diverse and bioactive compounds.     

A Cell-Free Fluorometric High-Throughput Screen for Inhibitors of Rtt109-Catalyzed Histone Acetylation

The lysine acetyltransferase (KAT) Rtt109 forms a complex with Vps75 and catalyzes the acetylation of histone H3 lysine 56 (H3K56ac) in the Asf1-H3-H4 complex. Rtt109 and H3K56ac are vital for replication-coupled nucleosome assembly and genotoxic resistance in yeast and pathogenic fungal species such as Candida albicans. Remarkably, sequence homologs of Rtt109 are absent in humans. Therefore, inhibitors of Rtt109 are hypothesized as potential and minimally toxic antifungal agents. Herein, we report the development and optimization of a cell-free fluorometric high-throughput screen (HTS) for small-molecule inhibitors of Rtt109-catalyzed histone acetylation. The KAT component of the assay consists of the yeast Rtt109-Vps75 complex, while the histone substrate complex consists of full-length Drosophila histone H3-H4 bound to yeast Asf1. Duplicated assay runs of the LOPAC demonstrated day-to-day and plate-to-plate reproducibility. Approximately 225,000 compounds were assayed in a 384-well plate format with an average Z'' factor of 0.71. Based on a 3σ cut-off criterion, 1,587 actives (0.7%) were identified in the primary screen. The assay method is capable of identifying previously reported KAT inhibitors such as garcinol. We also observed several prominent active classes of pan-assay interference compounds such as Mannich bases, catechols and p-hydroxyarylsulfonamides. The majority of the primary active compounds showed assay signal interference, though most assay artifacts can be efficiently removed by a series of straightforward counter-screens and orthogonal assays. Post-HTS triage demonstrated a comparatively small number of confirmed actives with IC₅₀ values in the low micromolar range. This assay, which utilizes five label-free proteins involved in H3K56 acetylation in vivo, can in principle identify compounds that inhibit Rtt109-catalyzed H3K56 acetylation via different mechanisms. Compounds discovered via this assay or adaptations thereof could serve as chemical probes or leads for a new class of antifungals targeting an epigenetic enzyme.     

Selective inhibitors of bacterial DNA adenine methyltransferases

The authors describe the discovery and characterization of several structural classes of small-molecule inhibitors of bacterial DNA adenine methyltransferases. These enzymes are essential for bacterial virulence (DNA adenine methyltransferase [DAM]) and cell viability (cell cycle-regulated methyltransferase [CcrM]). Using a novel high-throughput fluorescence-based assay and recombinant DAM and CcrM, the authors screened a diverse chemical library. They identified 5 major structural classes of inhibitors composed of more than 350 compounds: cyclopentaquinolines, phenyl vinyl furans, pyrimidine-diones, thiazolidine-4-ones, and phenyl-pyrroles. DNA binding assays were used to identify compounds that interact directly with DNA. Potent compounds selective for the bacterial target were identified, whereas other compounds showed greater selectivity for the mammalian DNA cytosine methyltransferase, Dnmt1. Enzyme inhibition analysis identified mechanistically distinct compounds that interfered with DNA or cofactor binding. Selected compounds demonstrated cell-based efficacy. These small-molecule DNA methyltransferase inhibitors provide useful reagents to probe the role of DNA methylation and may form the basis of developing novel antibiotics.     

Small-molecule inhibitors of the Rce1p CaaX protease

The Rce1p protease is required for the maturation of the Ras GTPase and certain other isoprenylated proteins and is considered a chemotherapeutic target. To identify new small-molecule inhibitors of Rce1p, the authors screened the National Cancer Institute Diversity Set compound library using in vitro assays to monitor the proteolytic processing of peptides derived from Ras and the yeast a-factor mating pheromone. Of 46 inhibitors initially identified with a Ras-based assay, only 9 were effective in the pheromone-based assay. The IC(50) values of these 9 compounds were in the low micromolar range for both yeast (6-35 microM) and human Rce1p (0.4-46 microM). Four compounds were somewhat Rce1p selective in that they partially inhibited the Ste24p protease and did not inhibit Ste14p isoprenylcysteine carboxyl methyltransferase, 2 enzymes also involved in the maturation of isoprenylated proteins. The remaining 5 compounds inhibited all 3 enzymes. The 2 most Rce1p-selective agents were ineffective trypsin inhibitors, further supporting the specificity of these agents for Rce1p. The 5 least specific compounds formed colloidal aggregates, a proposed common feature of promiscuous inhibitors. Interestingly, the most specific Rce1p inhibitor also formed a colloidal aggregate. In vivo studies revealed that treatment of wild-type yeast with 1 compound induced a Ras2p delocalization phenotype that mimics observed effects in rce1 ste24 null yeast. The 9 compounds identified in this study represent new tools for understanding the enzymology of postisoprenylation-modifying enzymes and provide new insight for the future development of Rce1p inhibitors.     

New aspects of research upon the yeast Saccharomyces cerevisiae [PSI+] prion

One of the key feature of prions is the ability to be stable in two alternative conformations. Besides the intensively studied mammalian prions, there are also prion proteins present in the yeast Saccharomyces cerevisiae. Research in this field has lead to opposite hypotheses that explain the sense of presence of [PSI+] prion in yeast cells. Some authors postulate e of role of the prions in the evolution of S. cerevisiae, whereas other investigators point out the negative influence of these particles upon the yeast physiology. In recent years, yeast prions are used for anti-prion drug screening, because of common features with mammalian prions. This work presents the most intensively studied fields of the research carried out on [PSI+] prion in yeast.     

Expanding the yeast prion world

Mammalian and fungal prion proteins form self-perpetuating β-sheet-rich fibrillar aggregates called amyloid. Prion inheritance is based on propagation of the regularly oriented amyloid structures of the prion proteins. All yeast prion proteins identified thus far contain aggregation-prone glutamine/asparagine (Gln/Asn)-rich domains, although the mammalian prion protein and fungal prion protein HET-s do not contain such sequences. In order to fill this gap, we searched for novel yeast prion proteins lacking Gln/Asn-rich domains via a genome-wide screen based on cross-seeding between two heterologous proteins and identified Mod5, a yeast tRNA isopentenyltransferase, as a novel non-Gln/Asn-rich yeast prion protein. Mod5 formed self-propagating amyloid fibers in vitro and the introduction of Mod5 amyloids into non-prion yeast induced dominantly and cytoplasmically heritable prion state [MOD⁺], which harbors aggregates of endogenous Mod5. [MOD⁺] yeast showed an increased level of membrane lipid ergosterol and acquired resistance to antifungal agents. Importantly, enhanced de novo formation of [MOD⁺] was observed when non-prion yeast was grown under selective pressures from antifungal drugs. Our findings expand the family of yeast prions to non-Gln/Asn-rich proteins and reveal the acquisition of a fitness advantage for cell survival through active prion conversion.     

Cross-talk between RNA and prions

As concepts evolve in mammalian and yeast prion biology, rather preliminary research investigating the interplay between prion and RNA processes are gaining momentum. The yeast prion [PSI+] represents an aggregated state of the translation termination factor Sup35 resulting in the tendency of ribosomes to readthrough stop codons. This "nonsense suppression" activity is investigated for its possible physiological role to engender on Saccharomyces cerevisiae the ability to respond to stress or variable growth conditions and thereby act as a capacitor to evolve. The interaction between prion and RNA is a two way street--the cell may have adopted RNA processes in translation to govern the presence of prions and the [PSI+] prion's nonsense suppressor phenotype may exhibit different growth phenotypes by its control of translation termination. RNA processes in the mammalian cell also effect and are affected by prions.     

Human cellular prion protein interacts directly with clusterin protein

Prion protein is a glycosyl-phosphatidyl-inositol anchored glycoprotein localized on the surface and within a variety of cells. Its conformation change is thought to be essential for the proliferation of prion neurodegenerative diseases. Using the yeast two-hybrid assay we identified an interaction between prion protein and clusterin, a chaperone glycoprotein. This interaction was confirmed in a mammalian system by in vivo co-immunoprecipitation and in vitro by circular dichroism analysis. Through deletion mapping analysis we demonstrated that the alpha subunit, but not the beta subunit, of clusterin binds to prion and that the C-terminal 62 amino acid segment of the putative alpha helix region of clusterin is essential for the binding interaction. The full prion protein as well as the N-terminal section (aa 23-95) and C-terminal (aa 96-231) were shown to interact with clusterin. These findings provide new insights into the molecular mechanisms of interaction between prion and clusterin protein and contribute to the understanding of prion protein's physiological function.     

Probing the role of PrP repeats in conformational conversion and amyloid assembly of chimeric yeast prions

Oligopeptide repeats appear in many proteins that undergo conformational conversions to form amyloid, including the mammalian prion protein PrP and the yeast prion protein Sup35. Whereas the repeats in PrP have been studied more exhaustively, interpretation of these studies is confounded by the fact that many details of the PrP prion conformational conversion are not well understood. On the other hand, there is now a relatively good understanding of the factors that guide the conformational conversion of the Sup35 prion protein. To provide a general model for studying the role of oligopeptide repeats in prion conformational conversion and amyloid formation, we have substituted various numbers of the PrP octarepeats for the endogenous Sup35 repeats. The resulting chimeric proteins can adopt the [PSI+] prion state in yeast, and the stability of the prion state depends on the number of repeats. In vitro, these chimeric proteins form amyloid fibers, with more repeats leading to shorter lag phases and faster assembly rates. Both pH and the presence of metal ions modulate assembly kinetics of the chimeric proteins, and the extent of modulation is highly sensitive to the number of PrP repeats. This work offers new insight into the properties of the PrP octarepeats in amyloid assembly and prion formation. It also reveals new features of the yeast prion protein, and provides a level of control over yeast prion assembly that will be useful for future structural studies and for creating amyloid-based biomaterials.     

Specificity of prion assembly in vivo. [PSI+] and [PIN+] form separate structures in yeast

The yeast prions [PSI+] and [PIN+] are self-propagating amyloid aggregates of the Gln/Asn-rich proteins Sup35p and Rnq1p, respectively. Like the mammalian PrP prion "strains," [PSI+] and [PIN+] exist in different conformations called variants. Here, [PSI+] and [PIN+] variants were used to model in vivo interactions between co-existing heterologous amyloid aggregates. Two levels of structural organization, like those previously described for [PSI+], were demonstrated for [PIN+]. In cells with both [PSI+] and [PIN+] the two prions formed separate structures at both levels. Also, the destabilization of [PSI+] by certain [PIN+] variants was shown not to involve alterations in the [PSI+] prion size. Finally, when two variants of the same prion that have aggregates with distinct biochemical characteristics were combined in a single cell, only one aggregate type was propagated. These studies demonstrate the intracellular organization of yeast prions and provide insight into the principles of in vivo amyloid assembly.     

Yeast as a model for studying the prion and amyloid occurrence

Recent data on the use of yeast as a model for studying the molecular basis of prion infection are summarized. In contrast to mammalian prions, which are related to incurable neurodegenerative diseases, yeast prions determine the appearance of non-chromosomally inherited phenotypic traits. Prions of yeast are structurally similar to amyloids of mammals and their replication involves not only growth, but also fragmentation of prion amyloid-like fibrils. In mammals the fragmentation should lead to an increase in infectious titer. The use of yeast for study of the mechanisms of human amyloidoses, development of new anti-prion drugs and search for new proteins with prion properties is described.     

Supporting the structural basis of prion strains: induction and identification of [PSI] variants

The [PSI] genetic element, which enhances the nonsense suppression efficiency in the yeast Saccharomyces cerevisiae, is thought to be amyloid-like aggregates of the Sup35 protein, and to self-propagate by a prion-like mechanism. Analogous to strains of the mammalian prion, variants of [PSI], with different nonsense suppression efficiencies and mitotic stabilities, can be isolated from the same yeast genetic background. In the framework of the "protein-only" hypothesis, variants of prion are assumed to be distinct conformers of the same prion polypeptide. This study aims to provide further support for the structural basis of [PSI] variation. Three variants of [PSI] were induced and distinguished by a panel of 11 single point mutations of the Sup35 protein. The variant phenotypes are intrinsically associated with [PSI] elements, presumably structurally different amyloids, rather than produced from variations in the genetic background. Differential incorporation to [PSI] variants of a Sup35 point mutation as well as N and C-terminally truncated Sup35 fragments is further demonstrated in vivo, suggesting that distinct patches of amino acid residues are involved in the assembly of [PSI] variants. These results establish a method for [PSI] variant-typing and indicate that heritable variations of amyloid structures can be derived from the same polypeptide.     

The [PSI+] prion of yeast: a problem of inheritance

The [PSI(+)] prion of the yeast Saccharomyces cerevisiae was first identified by Brian Cox some 40 years ago as a non-Mendelian genetic element that modulated the efficiency of nonsense suppression. Following the suggestion by Reed Wickner in 1994 that such elements might be accounted for by invoking a prion-based model, it was subsequently established that the [PSI(+)] determinant was the prion form of the Sup35p protein. In this article, we review how a combination of classical genetic approaches and modern molecular and biochemical methods has provided conclusive evidence of the prion basis of the [PSI(+)] determinant. In so doing we have tried to provide a historical context, but also describe the results of more recent experiments aimed at elucidating the mechanism by which the [PSI(+)] (and other yeast prions) are efficiently propagated in dividing cells. While understanding of the [PSI(+)] prion and its mode of propagation has, and will continue to have, an impact on mammalian prion biology nevertheless the very existence of a protein-based mechanism that can have a beneficial impact on a cell's fitness provides equally sound justification to fully explore yeast prions.     

A high-throughput screen to identify novel calcineurin inhibitors

Calcineurin is a eukaryotic protein phosphatase important for many signalling and developmental processes in cells. Inhibitors of this enzyme are used clinically and there is interest in identifying novel inhibitors for therapeutic applications. This report describes a high-throughput assay that can be used to screen natural or chemical libraries of compounds to identify new calcineurin inhibitors. The microtitre plate assay is based on a yeast reporter strain and was validated with known inhibitors and tested in a pilot screen of bacterial extracts.     

A non-Q/N-rich prion domain of a foreign prion, [Het-s], can propagate as a prion in yeast

Prions are self-propagating, infectious aggregates of misfolded proteins. The mammalian prion, PrP(Sc), causes fatal neurodegenerative disorders. Fungi also have prions. While yeast prions depend upon glutamine/asparagine (Q/N)-rich regions, the Podospora anserina HET-s and PrP prion proteins lack such sequences. Nonetheless, we show that the HET-s prion domain fused to GFP propagates as a prion in yeast. Analogously to native yeast prions, transient overexpression of the HET-s fusion induces ring-like aggregates that propagate in daughter cells as cytoplasmically inherited, detergent-resistant dot aggregates. Efficient dot propagation, but not ring formation, is dependent upon the Hsp104 chaperone. The yeast prion [PIN(+)] enhances HET-s ring formation, suggesting that prions with and without Q/N-rich regions interact. Finally, HET-s aggregates propagated in yeast are infectious when introduced into Podospora. Taken together, these results demonstrate prion propagation in a truly foreign host. Since yeast can host non-Q/N-rich prions, such native yeast prions may exist.     

Molecular basis of a yeast prion species barrier

The yeast [PSI+] factor is inherited by a prion mechanism involving self-propagating Sup35p aggregates. We find that Sup35p prion function is conserved among distantly related yeasts. As with mammalian prions, a species barrier inhibits prion induction between Sup35p from different yeast species. This barrier is faithfully reproduced in vitro where, remarkably, ongoing polymerization of one Sup35p species does not affect conversion of another. Chimeric analysis identifies a short domain sufficient to allow foreign Sup35p to cross this barrier. These observations argue that the species barrier results from specificity in the growing aggregate, mediated by a well-defined epitope on the amyloid surface and, together with our identification of a novel yeast prion domain, show that multiple prion-based heritable states can propagate independently within one cell.     

Inhibitors of human indoleamine 2,3-dioxygenase identified with a target-based screen in yeast

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan degradation enzyme that is emerging as an important drug target. IDO is expressed by many human tumors to help them escape immune detection, and it has been implicated in depression and in the formation of senile nuclear cataracts. There is a need for potent and selective IDO inhibitors for use in research and as lead compounds for drug development. We show that expression of human IDO in a Saccharomyces cerevisiae tryptophan auxotroph restricts yeast growth in the presence of low tryptophan concentrations and that inhibition of IDO activity can restore growth. We use this assay to screen for IDO inhibitors in collections of pure chemicals and crude natural extracts. We identify NSC 401366 (imidodicarbonimidic diamide, N-methyl-N'-9-phenanthrenyl-, monohydrochloride) as a potent nonindolic IDO inhibitor (Ki=1.5 +/- 0.2 microM) that is competitive with respect to tryptophan. We also use this assay to identify the active compound caulerpin from a crude algal extract. The yeast growth restoration assay is simple and inexpensive. It combines desirable attributes of cell- and target-based screens and is an attractive tool for chemical biology and drug screening.