- Email: [email protected]
New anti-prion drugs make yeast blush
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2 Caceres, A. et al. (1995) Antigonorrhoeal activity of plants used in Guatemala for the treatment of sexually transmitted diseases. J. Ethnopharmacol. 48, 85 – 88 3 Otero, R. et al. (2000) Snakebites and ethnobotany in the northwest region of Colombia. Part III: neutralization of the haemorrhagic effect of Bothrops atrox venom. J. Ethnopharmacol. 73, 233– 241 4 Nish, W.A. et al. (1991) Anaphylaxis to annatto dye: a case report. Ann. Allergy 66, 129 – 131 5 Hallagan, J.B. et al. (1995) The safety and regulatory status of food, drug and cosmetics colour additives exempt from certification. Food Chem. Toxicol. 33, 515 – 528 6 Mercadante, A.Z. et al. (1996) Isolation of methyl 90 Z-apo-60 -lycopenoate from Bixa orellana. Phytochemistry 41, 1201 – 1203 7 Mercadante, A.Z. et al. (1997) Isolation and identification of new apocarotenoids from annatto (Bixa orellana) seeds. J. Agric. Food Chem. 45, 1050– 1054 8 Mercadante, A.Z. et al. (1997) Isolation and structure elucidation of minor carotenoids from annatto (Bixa orellana L.) seeds. Phytochemistry 46, 1379– 1383 9 Bouvier, F. et al. (2003) Biosynthesis of the food and cosmetic plant pigment bixin (annatto). Science 300, 2089 – 2091 10 Giuliano, G. et al. (2003) Carotenoid oxygenases: cleave it or leave it. Trends Plant Sci. 8, 145– 149 11 Bouvier, F. et al. (2003) Oxidative remodeling of chromoplast carotenoids: identification of the carotenoid dioxygenase CsCCD and CsZCD genes involved in Crocus secondary metabolite biogenesis. Plant Cell 15, 47 – 62
Vol.21 No.12 December 2003
12 Skibbe, D.S. et al. (2002) Characterization of the aldehyde dehydrogenase gene families of Zea mays and Arabidopsis. Plant Mol. Biol. 48, 751– 764 13 Cui, X.Q. et al. (1996) The rf2 nuclear restorer gene of male-sterile T-cytoplasm maize. Science 272, 1334 – 1336 14 Ross, J.R. et al. (1999) S-Adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, an enzyme involved in floral scent production and plant defense, represents a new class of plant methyltransferases. Arch. Biochem. Biophys. 367, 9 – 16 15 Zubieta, C. et al. (2003) Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family. Plant Cell 15, 1704– 1716 16 Rathinasabapathi, B. et al. (1994) Metabolic engineering of glycine betaine synthesis: plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance. Planta 193, 155 – 162 17 Trossat, C. et al. (1996) Evidence that the pathway of dimethylsulfoniopropionate biosynthesis begins in the cytosol and ends in the chloroplast. Plant Physiol. 111, 965– 973 18 Thompson, J.D. et al. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment throught sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673– 4680
0167-7799/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2003.10.001
New anti-prion drugs make yeast blush Sven J. Saupe Laboratoire de Ge´ne´tique Mole´culaire des Champignons, Institut de Biochimie et de Ge´ne´tique Cellulaire, UMR 5095, CNRS Universite´ de Bordeaux II, Bordeaux, France
Prions are misfolded proteins capable of propagating their altered conformational state. They have been identified as the causative agents of a class of neurodegenerative diseases termed spongiform encephalopathies. No treatment for prion diseases is currently available. In a recent paper, Bach et al. describe a yeastbased approach for the development of anti-prion drugs. This approach could prove convenient for the identification and improvement of anti-prion pharmacologicals. From a fundamental point of view this study underscores the mechanistic similarities between mammalian and yeast prions. Rarely in modern biology has a protein received as much attention as the membrane bound PrP prion protein. Since the outbreak of the mad cow disease crisis, prions have been brought to general public attention and have stimulated scientific debate. Stanley Prusiner’s hypothesis that a protein on its own (without the informational support of a nucleic acid) can become a devastating infectious element [1] was received with fierce scepticism. Scientific evidence gathered over several decades now leaves little room for an alternate explanation. It is now generally accepted that transmissible spongiform encephalopathies are caused by Corresponding author: Sven J. Saupe ([email protected]). http://tibtec.trends.com
an abnormal form of the PrP protein, a protein expressed at the surface of numerous cell types, including neurons. During the course of the disease, an abnormal proteaseresistant form of PrP (termed PrPSc for scrapie) accumulates and is capable of converting the normal form, PrPC, to this altered conformation [2]. In 1994, the extensively debated ‘protein-only’ hypothesis received unexpected support from the field of yeast genetics [3]. In a brilliant intuition, Reed Wickner realized that a long-known bizarre genetic element of yeast is in fact a prion protein, and provided genetic evidence to demonstrate this [3]. The concept of a nucleic acid-free transmission of genetic information was thus widened and PrPSc no longer stood as the isolated daredevil defying the central dogma of molecular biology. The most intensively studied yeast prions [URE3] and [PSI þ ] correspond to the prion forms of the Ure2p and Sup35p proteins, respectively (for a review see [4]). Ure2p is a protein involved in the regulation of the use of nitrogen sources, and Sup35p is a translation termination factor (it is the ortholog of the mammalian eRF3 polypeptide chain release factor). In yeast, prions are detected by the loss of function of the corresponding proteins. For example, the [PSI þ ] prion is detected by the partial loss of function of the Sup35p protein. In its prion state, Sup35p becomes insoluble and is thus inactivated [5,6]. Because Sup35p is
Update
TRENDS in Biotechnology
part of the translation-termination complex, transition to the prion state causes an increase in translational readthrough of stop codons. Beyond their conceptual identities, how similar are yeast and mammalian prions? There are several differences between these two types of prion. First, yeast prions do not kill cells; they do not cause ‘disease’ [7]. Prioninfected yeast strains grow nearly as well as wild-type and even better in certain growth conditions [8]. Thus it has even been suggested that in yeast, prions might be adaptative. In addition, unlike PrP, fungal prion proteins are cytosolic proteins. PrP and the fungal prion proteins share no primary sequence homology but have a similar domain organization with a poorly structured domain appended to a well-folded globular domain. Fungal prion propagation is intimately connected to amyloid aggregation. PrP and the fungal prion proteins both have the ability to polymerize into amyloid aggregates. Whether these amyloid aggregates represent the replicative form of prions or terminal aggregation by-products remains to be confirmed. Several avenues for the development of prion disease therapies are currently being explored, in particular immunological and pharmacological approaches [9 – 11]. Various classes of molecules were found to promote PrPSc clearance in cell culture assays, including branched polyamines, cysteine protease inhibitors, tetrapyrrole compounds and tricyclic derivatives [12]. Among these tricyclic derivatives are the antimalarial drug quinacrine and the antipsychotic chlorpromazine [13]. Similarly, various chemical treatments are known to cure yeast prions. In particular, it has been known for some time that mM concentrations of guanidine chloride (GuHCl) lead to loss of both [URE3] and [PSI þ ]. This effect most certainly results from inactivation of the Hsp104p chaperone protein, which resolubilizes protein aggregates, notably after heat denaturation [14,15]. However, targeting of Hsp104p is not an option for inhibiting prion replication in mammals because although it is conserved in bacteria, fungi and plants, animals appear to lack a Hsp104p homolog. Identification of new anti-prion drugs Bach and co-workers recently published a report that allowed the identification of new antiprion drugs, using a remarkably simple approach [16]. They screened a chemical library for molecules active against yeast prions and made use of a clever colorimetric assay which allows easy detection of prion loss [17] (see Box 1). In yeast, ade1 mutants affected in the adenine biosynthesis pathway accumulate a red pigment when grown on complete medium. In the ade1 – 14 mutant, the ADE1 gene is interrupted by a premature stop codon. In normal cells, translation is terminated at that codon. Because of accumulation of metabolic by-products, ade1 – 14 strains plated on complete medium thus form red colonies. In a [PSI þ ] background Sup35p protein is in its prion conformation and therefore inactivated, translation read through occurs and the ade1 – 14 mutation is suppressed. [PSI þ ] prion-infected ade1 – 14 strains thus form white colonies. Using this genetic trick, inhibition of prion http://tibtec.trends.com
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replication is easily detected because prion-infected strains will turn from white to red upon prion loss. Bach and co-workers spotted 2500 compounds from a chemical library onto lawns of prion-infected [PSI þ ] white colonies. Compounds active against [PSI þ ] are detected as the ones leading to formation of a halo of red colonies indicative of prion loss (Figure 1). To increase the sensitivity of the assay, the yeast strain was engineered by deletion of the ERG6 gene, which increased permeability to various chemical compounds [18]. The authors also included sub-effective amounts of GuHCl in the culture media, which was also found to dramatically increase the sensitivity of the assay. In this screen, six active molecules were identified – all are tricyclic derivatives. Five form a new class of molecules termed kastellpaolitines, the sixth is phenanthridine (Figure 1). In a structure – activity approach 6-amino derivatives of phenanthridine with increased activity were generated. These compounds were not only active against the [PSI þ ] prion but also efficiently cured the [URE3] prion, suggesting that they could have a broad effect on various prion proteins. All these molecules act synergistically with GuHCl, which could mean that they target a protein distinct from Hsp104. Pharmacology identifies similarities in the mechanisms of prion propagation in yeast and mammals Remarkably, two molecules identified as being able to clear PrPSc in mammalian cell culture models, namely quinacrine and chlorpromazine, could also clear yeast prions in the assay developed by Bach et al. They were also found to act in synergy with GuHCl. This indicates that quinacrine and chlorpromazine could have been identified as anti-prion drugs using this yeast-based assay. Moreover, the new molecules identified in this study also inhibited generation of PrPSc in a cell-culture assay. These findings have important practical and fundamental implications. First, it appears that this yeast-based assay can be a convenient screen for identifing new anti-prion molecules. It could also improve the potency of previously identified compounds by allowing rapid testing of various chemical derivatives. It would be interesting to perform a comprehensive comparative analysis of anti-prion activity of various compounds (tricyclic derivatives and others) in the yeast-based and the cellculture assay to determine how much overlap exists between the assays. Clearly, efficient inhibition of prion replication in either assay does not mean that the identified molecules will be efficient in infected animals. To date, the drugs identified using the in vitro cell-culture assay, including quinacrine, failed to prevent development of prion disease in live animals or when administered as compassionate treatment in rare clinical trials [19]. However, because it is inexpensive, fast and simple, this yeast-based screening assay might prove useful as an initial high-throughput screen used upstream of secondary screens using mammalian cell cultures, and ultimately, infected animals. Because yeast prion propagation is intimately linked to amyloid aggregation, the assay developed by Bach et al. could also be instrumental in the identification of molecules involved in other protein misfolding disorders with great effects on human health, such as Parkinson’s and
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Box 1. Yeast prions can be detected using colorimetric reporter systems The colorimetric plate assay used to discriminate [PSIþ] (prioninfected) from [psi-] (prion-free) cells was developed by Brian Cox [17]. Yeast ade1 and ade2 mutants are blocked in the adenine biosynthesis pathway and accumulate an intermediate (termed AIR) when grown on complete medium (Figure I). This compound is then metabolized into a red pigment (R). In the ade1 –14 mutant, the ADE1 gene is interrupted by a premature opale UGA stop codon. In [psi-] cells carrying Sup35p in its normal soluble form, translation termination proceeds normally and translation of ADE1 is terminated at the premature opale codon. Because of the accumulation of AIR, [psi-] ade1 –14 strains plated on complete medium form red colonies. In a [PSIþ ] background (i.e. when the Sup35p protein is in its prion conformation and thus aggregated and inactivated), termination is
[psi-] Sup35p in normal state
impaired and read through of the UGA codon occurs. The ade1 –14 mutation is therefore suppressed and [PSIþ] prion-infected ade1 –14 strains form white colonies. A similar colorimetric reporter system has also been set up to monitor for presence of the [URE3] prion [23] (Figure II). In this assay, the yeast strain expressing ADE2 ORF is under the control of the DAL5 promoter (in a chromosomal ade2 mutant background). In normal prion-free [ure3 –0] cells, Ure2p represses expression of DAL5 by sequestering the Gln3p GATA transcription factor in the cytoplasm. Expression of the DAL5 promoter-driven ADE2 allele is therefore repressed and thus [ure3– 0] colonies are red. In [URE3] cells, Ure2p is inactivated, Gln3p can enter the nucleus and the DAL5 promoter-driven ADE2 allele is expressed, so [URE3] colonies are white.
[PSI+] Sup35p in prion state
UGA
[psi-]
[PSI+]
No Ade1p AIR Adenine
Soluble Sup35p
Ade1p AIR
Aggregated Sup35p
Adenine
R TRENDS in Biotechnology
Figure I. Reporter system for colorimetric detection of the [PSIþ ] prion
[ure3-0] Ure2p in normal state
[URE3] Ure2p in prion state Gln3p
Gln3p
PDAL5
Soluble Ure2p
ADE2
PDAL5
No Ade2p AIR Adenine R
ADE2
[URE3]
Ade2p Aggregated Ure2p
AIR
[ure3-0] Adenine
TRENDS in Biotechnology
Figure II. Reporter system for colorimetric detection of the [URE3] prion
Alzheimer’s diseases. This system might be superior to other microbial protein aggregation assays [20] because it employs a eukaryotic cellular environment, which is more relevant to the study of protein-folding disorders in humans than a bacterial cell environment. Apart from these practical aspects, the finding that molecules curing yeast prions inhibit PrPSc generation has important fundamental implications. If one makes the reasonable assumption that these drugs target homologous proteins in both systems, then replication of yeast and mammalian prions seem to rely on common co-factor proteins. This is unexpected considering that Sup35p, Ure2p and PrP are confined to distinct cellular compartments. The yeast prion proteins are cytosolic whereas PrP http://tibtec.trends.com
travels through the endoplasmic reticulum (ER) and the Golgi to the cell surface. The recent suggestion that a mistargeted cytosolic form of PrP plays an important role in prion conversion and neurotoxicity might help to explain this apparent paradox [21,22]. Because yeast allows the use of powerful genetic approaches, it certainly should not be long before the molecular target(s) of these tricyclic antiprion drugs have been identified, at least in yeast. This will allow a better understanding of the fundamental mechanisms governing prion replication and could lead to the generation of highly specific structurebased inhibitors. Even if no one has questioned the fundamental importance of the discovery of fungal prion models for demonstrating
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model system might represent an interesting short cut for the identification of a curative approach to prion diseases.
(a)
Note added in proofs In a recent paper Kocisko and co-workers described a highthroughput screen for PrPSc inhibitors using cell cultures and identified new inhibitors from a library of 2000 molecules [24]. References (b)
Anti-prion molecule
Potency against yeast prions
Kastellpaolitine 1 (Kp1)
(+++)
Phenanthridine
(++)
6AP
(++++)
6A-8CP
(+++++)
Quinacrine
(+)
Chlorpromazine
(+)
NH2 N
S Cl N
NH2 N
NH2 N
Cl
CH3
CH3 N
HN
OCH3
Cl
CH3
N
N
N
Cl
S
Figure 1. Yeast-based screening method for identifying anti-prion drugs. (a) Compounds of a chemical library are spotted onto filter paper deposited on lawns of [PSI þ ] (prion-infected) yeast colonies. A colorimetric reporter system allows detection of the [PSI þ ] prion (see Box 1). Original [PSI þ ] prion-infected cells form white colonies whereas cured [psi-] prion-free cells form red colonies. Active molecules capable of curing cells of the [PSI þ ] prion lead to the formation of a red halo around the filter paper. Note that toxicity of certain compounds prevents colony formation in the direct vicinity of the filter paper. (b) The chemical structure of several newly identified anti-prion molecules and the structure of quinacrine and chlorpromazine, two tricyclic compounds known to inhibit mammalian prion replication in mammalian cell-culture assays. Abbreviations: 6AP, 6-amino phenanthridine; 6A-8CP, 6-amino 8-chloro phenanthridine.
and generalizing the bold concept of protein-based genetic inheritance, it seemed at first unrealistic to believe that these models might provide direct benefits for the development of cures for prion diseases. The recent report by Bach et al. suggests that as in many other cases, exploration of a http://tibtec.trends.com
1 Prusiner, S.B. (1982) Novel proteinaceous infectious particles cause scrapie. Science 216, 136– 144 2 Prusiner, S.B. (1998) Prions. Proc. Natl. Acad. Sci. U. S. A. 95, 13363 – 13383 3 Wickner, R.B. (1994) [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. Science 264, 566 – 569 4 Uptain, S.M. and Lindquist, S. (2002) Prions as protein-based genetic elements. Annu. Rev. Microbiol. 56, 703 – 741 5 Paushkin, S.V. et al. (1996) Propagation of the yeast prion-like [psi þ ] determinant is mediated by oligomerization of the SUP35-encoded polypeptide chain release factor. EMBO J. 15, 3127 – 3134 6 Patino, M.M. et al. (1996) Support for the prion hypothesis for inheritance of a phenotypic trait in yeast. Science 273, 622– 626 7 Couzin, J. (2002) Molecular biology. In yeast, prions’ killer image doesn’t apply. Science 297, 758 – 761 8 True, H.L. and Lindquist, S.L. (2000) Ayeast prion provides a mechanism for genetic variation and phenotypic diversity. Nature 407, 477–483 9 Aguzzi, A. et al. (2001) Interventional strategies against prion diseases. Nat. Rev. Neurosci. 2, 745 – 749 10 White, A.R. et al. (2003) Monoclonal antibodies inhibit prion replication and delay the development of prion disease. Nature 422, 80–83 11 Meier, P. et al. (2003) Soluble dimeric prion protein binds PrP(Sc) in vivo and antagonizes prion disease. Cell 113, 49– 60 12 Supattapone, S. et al. (2002) Pharmacological approaches to prion research. Biochem. Pharmacol. 63, 1383– 1388 13 Korth, C. et al. (2001) Acridine and phenothiazine derivatives as pharmacotherapeutics for prion disease. Proc. Natl. Acad. Sci. U. S. A. 98, 9836–9841 14 Ferreira, P.C. et al. (2001) The elimination of the yeast [PSI þ ] prion by guanidine hydrochloride is the result of Hsp104 inactivation. Mol. Microbiol. 40, 1357– 1369 15 Jung, G. and Masison, D.C. (2001) Guanidine hydrochloride inhibits Hsp104 activity in vivo: a possible explanation for its effect in curing yeast prions. Curr. Microbiol. 43, 7 – 10 16 Bach, S. et al. (2003) Isolation of drugs active against mammalian prions using a yeast-based screening assay. Nat. Biotechnol. 21, 1075 – 1081 17 Cox, B.S. (1965) PSI, a cytoplasmic suppressor of super-suppressor in yeast. Heredity 20, 505– 521 18 Gaber, R.F. et al. (1989) The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cellcycle-sparking sterol. Mol. Cell. Biol. 9, 3447 – 3456 19 Follette, P. (2003) New perspectives for prion therapeutics meeting. Prion disease treatment’s early promise unravels. Science 299, 191– 192 20 Wigley, W.C. et al. (2001) Protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein. Nat. Biotechnol. 19, 131 – 136 21 Ma, J. and Lindquist, S. (2002) Conversion of PrP to a self-perpetuating PrPSc-like conformation in the cytosol. Science 298, 1785–1788 22 Ma, J. et al. (2002) Neurotoxicity and neurodegeneration when PrP accumulates in the cytosol. Science 298, 1781– 1785 23 Schlumpberger, M. et al. (2001) Induction of distinct [URE3] yeast prion strains. Mol. Cell. Biol. 21, 7035– 7046 24 Kocisko, D.A. et al. (2003) New inhibitors of Scrapie-associated prion protein formation in a library of 2000 drugs and natural products. J. Virol 77, 10288 – 10294 0167-7799/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2003.10.004
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2 Caceres, A. et al. (1995) Antigonorrhoeal activity of plants used in Guatemala for the treatment of sexually transmitted diseases. J. Ethnopharmacol. 48, 85 – 88 3 Otero, R. et al. (2000) Snakebites and ethnobotany in the northwest region of Colombia. Part III: neutralization of the haemorrhagic effect of Bothrops atrox venom. J. Ethnopharmacol. 73, 233– 241 4 Nish, W.A. et al. (1991) Anaphylaxis to annatto dye: a case report. Ann. Allergy 66, 129 – 131 5 Hallagan, J.B. et al. (1995) The safety and regulatory status of food, drug and cosmetics colour additives exempt from certification. Food Chem. Toxicol. 33, 515 – 528 6 Mercadante, A.Z. et al. (1996) Isolation of methyl 90 Z-apo-60 -lycopenoate from Bixa orellana. Phytochemistry 41, 1201 – 1203 7 Mercadante, A.Z. et al. (1997) Isolation and identification of new apocarotenoids from annatto (Bixa orellana) seeds. J. Agric. Food Chem. 45, 1050– 1054 8 Mercadante, A.Z. et al. (1997) Isolation and structure elucidation of minor carotenoids from annatto (Bixa orellana L.) seeds. Phytochemistry 46, 1379– 1383 9 Bouvier, F. et al. (2003) Biosynthesis of the food and cosmetic plant pigment bixin (annatto). Science 300, 2089 – 2091 10 Giuliano, G. et al. (2003) Carotenoid oxygenases: cleave it or leave it. Trends Plant Sci. 8, 145– 149 11 Bouvier, F. et al. (2003) Oxidative remodeling of chromoplast carotenoids: identification of the carotenoid dioxygenase CsCCD and CsZCD genes involved in Crocus secondary metabolite biogenesis. Plant Cell 15, 47 – 62
Vol.21 No.12 December 2003
12 Skibbe, D.S. et al. (2002) Characterization of the aldehyde dehydrogenase gene families of Zea mays and Arabidopsis. Plant Mol. Biol. 48, 751– 764 13 Cui, X.Q. et al. (1996) The rf2 nuclear restorer gene of male-sterile T-cytoplasm maize. Science 272, 1334 – 1336 14 Ross, J.R. et al. (1999) S-Adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, an enzyme involved in floral scent production and plant defense, represents a new class of plant methyltransferases. Arch. Biochem. Biophys. 367, 9 – 16 15 Zubieta, C. et al. (2003) Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family. Plant Cell 15, 1704– 1716 16 Rathinasabapathi, B. et al. (1994) Metabolic engineering of glycine betaine synthesis: plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance. Planta 193, 155 – 162 17 Trossat, C. et al. (1996) Evidence that the pathway of dimethylsulfoniopropionate biosynthesis begins in the cytosol and ends in the chloroplast. Plant Physiol. 111, 965– 973 18 Thompson, J.D. et al. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment throught sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673– 4680
0167-7799/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2003.10.001
New anti-prion drugs make yeast blush Sven J. Saupe Laboratoire de Ge´ne´tique Mole´culaire des Champignons, Institut de Biochimie et de Ge´ne´tique Cellulaire, UMR 5095, CNRS Universite´ de Bordeaux II, Bordeaux, France
Prions are misfolded proteins capable of propagating their altered conformational state. They have been identified as the causative agents of a class of neurodegenerative diseases termed spongiform encephalopathies. No treatment for prion diseases is currently available. In a recent paper, Bach et al. describe a yeastbased approach for the development of anti-prion drugs. This approach could prove convenient for the identification and improvement of anti-prion pharmacologicals. From a fundamental point of view this study underscores the mechanistic similarities between mammalian and yeast prions. Rarely in modern biology has a protein received as much attention as the membrane bound PrP prion protein. Since the outbreak of the mad cow disease crisis, prions have been brought to general public attention and have stimulated scientific debate. Stanley Prusiner’s hypothesis that a protein on its own (without the informational support of a nucleic acid) can become a devastating infectious element [1] was received with fierce scepticism. Scientific evidence gathered over several decades now leaves little room for an alternate explanation. It is now generally accepted that transmissible spongiform encephalopathies are caused by Corresponding author: Sven J. Saupe ([email protected]). http://tibtec.trends.com
an abnormal form of the PrP protein, a protein expressed at the surface of numerous cell types, including neurons. During the course of the disease, an abnormal proteaseresistant form of PrP (termed PrPSc for scrapie) accumulates and is capable of converting the normal form, PrPC, to this altered conformation [2]. In 1994, the extensively debated ‘protein-only’ hypothesis received unexpected support from the field of yeast genetics [3]. In a brilliant intuition, Reed Wickner realized that a long-known bizarre genetic element of yeast is in fact a prion protein, and provided genetic evidence to demonstrate this [3]. The concept of a nucleic acid-free transmission of genetic information was thus widened and PrPSc no longer stood as the isolated daredevil defying the central dogma of molecular biology. The most intensively studied yeast prions [URE3] and [PSI þ ] correspond to the prion forms of the Ure2p and Sup35p proteins, respectively (for a review see [4]). Ure2p is a protein involved in the regulation of the use of nitrogen sources, and Sup35p is a translation termination factor (it is the ortholog of the mammalian eRF3 polypeptide chain release factor). In yeast, prions are detected by the loss of function of the corresponding proteins. For example, the [PSI þ ] prion is detected by the partial loss of function of the Sup35p protein. In its prion state, Sup35p becomes insoluble and is thus inactivated [5,6]. Because Sup35p is
Update
TRENDS in Biotechnology
part of the translation-termination complex, transition to the prion state causes an increase in translational readthrough of stop codons. Beyond their conceptual identities, how similar are yeast and mammalian prions? There are several differences between these two types of prion. First, yeast prions do not kill cells; they do not cause ‘disease’ [7]. Prioninfected yeast strains grow nearly as well as wild-type and even better in certain growth conditions [8]. Thus it has even been suggested that in yeast, prions might be adaptative. In addition, unlike PrP, fungal prion proteins are cytosolic proteins. PrP and the fungal prion proteins share no primary sequence homology but have a similar domain organization with a poorly structured domain appended to a well-folded globular domain. Fungal prion propagation is intimately connected to amyloid aggregation. PrP and the fungal prion proteins both have the ability to polymerize into amyloid aggregates. Whether these amyloid aggregates represent the replicative form of prions or terminal aggregation by-products remains to be confirmed. Several avenues for the development of prion disease therapies are currently being explored, in particular immunological and pharmacological approaches [9 – 11]. Various classes of molecules were found to promote PrPSc clearance in cell culture assays, including branched polyamines, cysteine protease inhibitors, tetrapyrrole compounds and tricyclic derivatives [12]. Among these tricyclic derivatives are the antimalarial drug quinacrine and the antipsychotic chlorpromazine [13]. Similarly, various chemical treatments are known to cure yeast prions. In particular, it has been known for some time that mM concentrations of guanidine chloride (GuHCl) lead to loss of both [URE3] and [PSI þ ]. This effect most certainly results from inactivation of the Hsp104p chaperone protein, which resolubilizes protein aggregates, notably after heat denaturation [14,15]. However, targeting of Hsp104p is not an option for inhibiting prion replication in mammals because although it is conserved in bacteria, fungi and plants, animals appear to lack a Hsp104p homolog. Identification of new anti-prion drugs Bach and co-workers recently published a report that allowed the identification of new antiprion drugs, using a remarkably simple approach [16]. They screened a chemical library for molecules active against yeast prions and made use of a clever colorimetric assay which allows easy detection of prion loss [17] (see Box 1). In yeast, ade1 mutants affected in the adenine biosynthesis pathway accumulate a red pigment when grown on complete medium. In the ade1 – 14 mutant, the ADE1 gene is interrupted by a premature stop codon. In normal cells, translation is terminated at that codon. Because of accumulation of metabolic by-products, ade1 – 14 strains plated on complete medium thus form red colonies. In a [PSI þ ] background Sup35p protein is in its prion conformation and therefore inactivated, translation read through occurs and the ade1 – 14 mutation is suppressed. [PSI þ ] prion-infected ade1 – 14 strains thus form white colonies. Using this genetic trick, inhibition of prion http://tibtec.trends.com
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replication is easily detected because prion-infected strains will turn from white to red upon prion loss. Bach and co-workers spotted 2500 compounds from a chemical library onto lawns of prion-infected [PSI þ ] white colonies. Compounds active against [PSI þ ] are detected as the ones leading to formation of a halo of red colonies indicative of prion loss (Figure 1). To increase the sensitivity of the assay, the yeast strain was engineered by deletion of the ERG6 gene, which increased permeability to various chemical compounds [18]. The authors also included sub-effective amounts of GuHCl in the culture media, which was also found to dramatically increase the sensitivity of the assay. In this screen, six active molecules were identified – all are tricyclic derivatives. Five form a new class of molecules termed kastellpaolitines, the sixth is phenanthridine (Figure 1). In a structure – activity approach 6-amino derivatives of phenanthridine with increased activity were generated. These compounds were not only active against the [PSI þ ] prion but also efficiently cured the [URE3] prion, suggesting that they could have a broad effect on various prion proteins. All these molecules act synergistically with GuHCl, which could mean that they target a protein distinct from Hsp104. Pharmacology identifies similarities in the mechanisms of prion propagation in yeast and mammals Remarkably, two molecules identified as being able to clear PrPSc in mammalian cell culture models, namely quinacrine and chlorpromazine, could also clear yeast prions in the assay developed by Bach et al. They were also found to act in synergy with GuHCl. This indicates that quinacrine and chlorpromazine could have been identified as anti-prion drugs using this yeast-based assay. Moreover, the new molecules identified in this study also inhibited generation of PrPSc in a cell-culture assay. These findings have important practical and fundamental implications. First, it appears that this yeast-based assay can be a convenient screen for identifing new anti-prion molecules. It could also improve the potency of previously identified compounds by allowing rapid testing of various chemical derivatives. It would be interesting to perform a comprehensive comparative analysis of anti-prion activity of various compounds (tricyclic derivatives and others) in the yeast-based and the cellculture assay to determine how much overlap exists between the assays. Clearly, efficient inhibition of prion replication in either assay does not mean that the identified molecules will be efficient in infected animals. To date, the drugs identified using the in vitro cell-culture assay, including quinacrine, failed to prevent development of prion disease in live animals or when administered as compassionate treatment in rare clinical trials [19]. However, because it is inexpensive, fast and simple, this yeast-based screening assay might prove useful as an initial high-throughput screen used upstream of secondary screens using mammalian cell cultures, and ultimately, infected animals. Because yeast prion propagation is intimately linked to amyloid aggregation, the assay developed by Bach et al. could also be instrumental in the identification of molecules involved in other protein misfolding disorders with great effects on human health, such as Parkinson’s and
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Box 1. Yeast prions can be detected using colorimetric reporter systems The colorimetric plate assay used to discriminate [PSIþ] (prioninfected) from [psi-] (prion-free) cells was developed by Brian Cox [17]. Yeast ade1 and ade2 mutants are blocked in the adenine biosynthesis pathway and accumulate an intermediate (termed AIR) when grown on complete medium (Figure I). This compound is then metabolized into a red pigment (R). In the ade1 –14 mutant, the ADE1 gene is interrupted by a premature opale UGA stop codon. In [psi-] cells carrying Sup35p in its normal soluble form, translation termination proceeds normally and translation of ADE1 is terminated at the premature opale codon. Because of the accumulation of AIR, [psi-] ade1 –14 strains plated on complete medium form red colonies. In a [PSIþ ] background (i.e. when the Sup35p protein is in its prion conformation and thus aggregated and inactivated), termination is
[psi-] Sup35p in normal state
impaired and read through of the UGA codon occurs. The ade1 –14 mutation is therefore suppressed and [PSIþ] prion-infected ade1 –14 strains form white colonies. A similar colorimetric reporter system has also been set up to monitor for presence of the [URE3] prion [23] (Figure II). In this assay, the yeast strain expressing ADE2 ORF is under the control of the DAL5 promoter (in a chromosomal ade2 mutant background). In normal prion-free [ure3 –0] cells, Ure2p represses expression of DAL5 by sequestering the Gln3p GATA transcription factor in the cytoplasm. Expression of the DAL5 promoter-driven ADE2 allele is therefore repressed and thus [ure3– 0] colonies are red. In [URE3] cells, Ure2p is inactivated, Gln3p can enter the nucleus and the DAL5 promoter-driven ADE2 allele is expressed, so [URE3] colonies are white.
[PSI+] Sup35p in prion state
UGA
[psi-]
[PSI+]
No Ade1p AIR Adenine
Soluble Sup35p
Ade1p AIR
Aggregated Sup35p
Adenine
R TRENDS in Biotechnology
Figure I. Reporter system for colorimetric detection of the [PSIþ ] prion
[ure3-0] Ure2p in normal state
[URE3] Ure2p in prion state Gln3p
Gln3p
PDAL5
Soluble Ure2p
ADE2
PDAL5
No Ade2p AIR Adenine R
ADE2
[URE3]
Ade2p Aggregated Ure2p
AIR
[ure3-0] Adenine
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Figure II. Reporter system for colorimetric detection of the [URE3] prion
Alzheimer’s diseases. This system might be superior to other microbial protein aggregation assays [20] because it employs a eukaryotic cellular environment, which is more relevant to the study of protein-folding disorders in humans than a bacterial cell environment. Apart from these practical aspects, the finding that molecules curing yeast prions inhibit PrPSc generation has important fundamental implications. If one makes the reasonable assumption that these drugs target homologous proteins in both systems, then replication of yeast and mammalian prions seem to rely on common co-factor proteins. This is unexpected considering that Sup35p, Ure2p and PrP are confined to distinct cellular compartments. The yeast prion proteins are cytosolic whereas PrP http://tibtec.trends.com
travels through the endoplasmic reticulum (ER) and the Golgi to the cell surface. The recent suggestion that a mistargeted cytosolic form of PrP plays an important role in prion conversion and neurotoxicity might help to explain this apparent paradox [21,22]. Because yeast allows the use of powerful genetic approaches, it certainly should not be long before the molecular target(s) of these tricyclic antiprion drugs have been identified, at least in yeast. This will allow a better understanding of the fundamental mechanisms governing prion replication and could lead to the generation of highly specific structurebased inhibitors. Even if no one has questioned the fundamental importance of the discovery of fungal prion models for demonstrating
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model system might represent an interesting short cut for the identification of a curative approach to prion diseases.
(a)
Note added in proofs In a recent paper Kocisko and co-workers described a highthroughput screen for PrPSc inhibitors using cell cultures and identified new inhibitors from a library of 2000 molecules [24]. References (b)
Anti-prion molecule
Potency against yeast prions
Kastellpaolitine 1 (Kp1)
(+++)
Phenanthridine
(++)
6AP
(++++)
6A-8CP
(+++++)
Quinacrine
(+)
Chlorpromazine
(+)
NH2 N
S Cl N
NH2 N
NH2 N
Cl
CH3
CH3 N
HN
OCH3
Cl
CH3
N
N
N
Cl
S
Figure 1. Yeast-based screening method for identifying anti-prion drugs. (a) Compounds of a chemical library are spotted onto filter paper deposited on lawns of [PSI þ ] (prion-infected) yeast colonies. A colorimetric reporter system allows detection of the [PSI þ ] prion (see Box 1). Original [PSI þ ] prion-infected cells form white colonies whereas cured [psi-] prion-free cells form red colonies. Active molecules capable of curing cells of the [PSI þ ] prion lead to the formation of a red halo around the filter paper. Note that toxicity of certain compounds prevents colony formation in the direct vicinity of the filter paper. (b) The chemical structure of several newly identified anti-prion molecules and the structure of quinacrine and chlorpromazine, two tricyclic compounds known to inhibit mammalian prion replication in mammalian cell-culture assays. Abbreviations: 6AP, 6-amino phenanthridine; 6A-8CP, 6-amino 8-chloro phenanthridine.
and generalizing the bold concept of protein-based genetic inheritance, it seemed at first unrealistic to believe that these models might provide direct benefits for the development of cures for prion diseases. The recent report by Bach et al. suggests that as in many other cases, exploration of a http://tibtec.trends.com
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