Antiprion Drugs as Chemical Tools to Uncover Mechanisms of Prion Propagation

Abstract

A number of drugs active against prions either in vitro, in cellular systems or in vivo in animal models have been isolated in various screening assays. In this minireview, we would like to suggest, that in addition to their direct interest as potential therapeutic agents, these molecules could be used as original research tools to understand prion propagation. The use of antiprion compounds as tool to understand fundamentals of prion propagation relies on reverse screening approaches. These global genetic and/or biochemical approaches aim to identify the intracellular target(s) and mechanism of action of the drugs. Once those are known, the biological activity of the compounds can be optimized on a rational basis, their potential side effects understood and minimized. In vitro enzyme-based screening assays can then be designed to allow discovery of new, more potent and selective molecules. Here we describe the main comprehensive biochemical and genetical approaches to realize reverse screening approaches based on antiprion drugs. We will finish by discussing the interest of using drug inactivation of specific targets as a substitute to genetic inactivation.

A number of drugs have been isolated as active against mammalian prion (reviewed in ref. 1). For most of these molecules, the mode of action and targets remain largely unknown. In principle, two main modes of action for antiprion drugs can be envisioned: either in cis directly on PrP^C/PrP^Sc, or in trans by interfering with the activity of cellular factors required for prion propagation. Some compounds are thought to bind directly to PrP^C or PrP^Sc (action in cis). Among these compounds are Congo Red (CR), Pentosan Polysulfate (PPS) or Glycosaminoglycans (GAGs). Other compounds are thought to act in trans by affecting PrP^C or PrP^Sc indirectly. Among these molecules are various lysosomotropic factors including the antimalarial drugs Quinacrine (QC) and Chloroquine. Indeed, the lysosome is a potential site of conversion of PrP^C to PrP^Sc.2 In addition, a recent report,3 proposes that QC's antiprion activity is related to its ability to redistribute cholesterol from the plasma membrane to intracellular compartments, thereby destabilizing membrane domains. This conclusion was drawn from correlation experiments indicating that drugs known to display cholesterol-redistributing activity (but structurally unrelated to QC) also have antiprion potency. Finally, QC was also shown to interact directly with PrP.4,5 The example of QC, with these conflicting results, thus illustrates the need for unbiaised and global approaches not driven by a preconcieved hypothesis on the drug mode of action. To our knowledge, no such approaches were applied for any of the known antiprion drugs, with the noticeable exception of chlorpromazine for which a haploinsufficiency profiling screen (HIP, see in the later section) has been published.6 In this minireview, we discuss the various advantages of defining extensively intracellular targets of antiprion drugs. We then would like to present some global approaches that can be applied to uncover, in an unbiaised manner, cellular mechanism(s) of action of compounds active against prions. To finish we propose that, once intracellular targets have been clearly identified, the drugs can be used to do “chemical genetics” to inactivate cellular target(s) which could be especially useful in situations where classical inactivation by mutagenesis is unpractical (for instance if redundant multigenic families are involved).

The Benefits of the Use of Global Approaches to Determine the Mode of Action of Antiprion Drugs

Similar to classical genetic screens, global approaches allow to address the question of the drug mode of action in an unbiaised manner and without any preconceived idea. As such, these approaches can lead to discovery of new prion controlling mechanisms, especially mechanisms in trans which remain largely unknown. Once the target and drug mechanism are known, drug efficiency can be improved on a rational basis. In addition, these global approaches allow identifying and understanding potential side effects of the molecules. This point is of special importance for compounds evaluated in preclinical and clinical trials. Together, these approaches can also help to define new screening targets for the development of highly selective mechanism-based, cell-free screening assays.

Antiprion Drugs as Baits in Affinity Chromatography on Immobilized Compounds

Protein purification using affinity chromatography based on immobilized substrates or cofactors has been used for several decades.7–10 In contrast, the use of affinity chromatography with immobilized drugs to comprehensively purify and identify cellular factors that interact with these compounds is relatively recent and undoubtedly linked to the improvements of proteomics and mass spectrometry (MS). Affinity chromatography experiments on immobilized drugs has been already successfully applied to protein kinase inhibitors like flavopiridol,11 purvalanol,12 (R)-roscovitine (CYC202, Seliciclib, at present in phase IIb of clinical trials)13 or Gefitinib (ZD1839, Iressa, already in clinic for the treatment of non small cells lung cancers)14 or to Hsp90 chaperone inhibitor geldanamycin.15 It should be noted that the affinity chromatography approach on immobilized drugs to investigate the actual antiprion mechanism of a compound of interest should be considered only as a first step of the analysis, as it only shows a specific interaction between the drug and a cellular component. Whether or not this interaction affects the biological activity of this component (inhibition or activation) needs to be determined in a second step. In a third step, the link between this biological effect and the antiprion activity of the drug also needs to be established. Finally, it should be kept in mind that affinity chromatography allows purification of protein (or nucleic acid) targets but potentially misses other types of targets (lipids for example).

The first important technical point when starting the affinity chromatography approach is the preparation of the drug-linked affinity matrix. This point is of the uppermost importance. Indeed, to avoid possible steric hindrance between target(s) and the matrix (generally Sepharose beads), it is necessary to introduce a spacer between the antiprion drug of interest and the Sepharose beads. A good linker should be long enough to avoid steric hindrance and should not be too hydrophobic to avoid auto-aggregation or unspecific hydrophobic interactions with macromolecules from the cell lysate (Fig. 1A). Different linkers have been described. Among them (Fig. 1B) are Polyethylene glycol (PEG) linkers,13,16–18 divalent epoxide linkers (ethyleneglycol diglycidylether, EGDE)19 and amino-caproylaminopentyloxy (ACAP) linkers (Gug F and Galons H, in preparation). We believe the ACAP linker is an interesting choice because, in addition to its optimal length, its internal peptidic bond lowers its hydrophobicity. Furthermore, the amide function increases the rigidity of the linker. An important point is also to check that introduction of the linker does not interfere with the antiprion activity of the considered drug (or at least that some of its activity is retained). Several positions should be tested in order to determine the most appropriate.

These matrices can then be used for affinity chromatography experiments to purify biological targets of the considered antiprion drug (Fig. 1C). Crude extracts of various tissues or cell types, prion-infected or not, should then be incubated with the immobilized drug, the matrix extensively washed and the bound proteins eluted with electrophoresis sample buffer, and analyzed by SDS-PAGE.20 Several specificity controls should be included (Fig. 1C, right panel). In a first control, the Sepharose beads without the drug are used. In second control, competitions with an excess of free antiprion drug are performed. Proteins interacting specifically with the antiprion compound should be displaced by the free drug. A last important control is to realize the same type of competition with an inactive, close chemical derivative of the antiprion drug, if available. The macromolecules specifically bound to the antiprion drug are thus those which bind the drug matrix, but not the matrix alone and which are competed away by the active antiprion drug but not by its inactive derivative. However, it should be kept in mind, as a limit of this biochemical method, that some other potential biological targets (in particular those not related to the antiprion activity but rather to undesired side effects) might be missed due to the position chosen for the linker. For this reason global genetic approaches can be considered as complement to the biochemical approach with immobilized antiprion compounds.

Antiprion Drugs as Baits in Yeast-based Genetic Approaches

A number of global approaches based on the use of the budding yeast Saccharomyces cerevisiae have been developed to uncover drug targets.21 The main advantage of these methods compared to the biochemical approach described above is the absence of chemical linker added onto the tested compound. Among these cell-based, high-throughput screening methods are: (i) haploinsufficiency profiling (HIP), (ii) synthetic lethality screens (SLS) and (iii) genome wide overexpression screens (OES).

In the HIP approach, a library of diploid yeast strains with heterozygous deletion of each single gene is screened for drug sensitivity either in a single culture with a competitive growth assay or by screening the 6000+ strains separately in parallel. This assay is based on the observation that reducing the copy number of a gene encoding a drug target from two copies (in diploid strain) to one copy (in a diploid strain heterozygote for deletion of this gene) often results in a strain sensitized to the drug of interest. In one of these HIP assays, Chlorpromazine, a tricyclic antidepressant shown to promote PrP^Sc clearance in N2a cell system22 exhibited genetic interaction with integral membrane ATPase activities.6 It should be noted, however, that in this screen only the effect of chlorpromazine on toxicity was analyzed and not its effect on prion propagation.

In the SLS approach, the drug is screened at a concentration that is normally subefficient against a library of haploid yeast strains with individual gene deletions. Genes whose deletion results in increased drug sensitivity might be one of several direct drug targets or genes that are involved in the same cellular pathways as the drug target.23

In the OES method (Fig. 2A), the rationale is to identify genes or cDNA whose overexpression confers resistance to a drug in yeast. This screen is based on the idea that cells overexpressing a target or a component related to the target, should tolerate higher levels of the active compound. The main interest of this approach is that overexpressed genes or cDNAs can be of any origin (from yeast but also from mouse or human). In addition, because this method can be based on yeast prions, convenient red/white colony color assays can be used in conjunction with GAL-inducible promoters (highly active when yeast cells are grown in galactose medium and inactive when cells are grown in glucose medium). The utility of this approach in the analysis of small molecules targets has been demonstrated.24 Naturally, typical false positive in such approaches might correspond to proteins involved in drug import/export and stability. In addition, genes whose overexpression is toxic in yeast cells are counterslected.

Finally, another yeast-based method can be used: the yeast three-hybrid system (Y3H, Fig. 2B). This method is derived from the yeast two-hybrid system (Y2H) which has proved to be a powerful tool for detecting protein-protein interactions.25 In the Y2H system, protein-protein interaction lead to reconstitution of a transcriptional activator by close positioning in space of its DNA binding (DBD) and activation domain (AD) expressed separately as fusion proteins with the two potentially interacting proteins. Activity of the reconstituted transcriptional activator is evaluated using convenient reporter genes like LacZ or HIS3. In 1996, Licitra and Liu extended this method to small molecule target identification by dimerization of two receptor proteins via heterodimeric ligand.26 As described in Figure 2B, one ligand/receptor pair (for example methotrexate/DHFR in fusion with DBD) serves as bait. The ligand (e.g., methotrexate) is covalently attached to the antiprion molecule of interest (6AP in the example given) via a linker. DHFR-DBD fusion protein is expressed in cells treated with the dimer methotrexate-6AP. In the same cells, a library of protein (here again of any origin) in fusion with the AD is expressed. If some of these fusion proteins have affinity with 6AP, an active transcriptional activator can be reconstituted, as in the case of Y2H experiments. Y3H can thus be considered as another way of scanning the proteome (of any species) to identify the targets of antiprion drugs. However, one of the limit of this method is the same than the one highlighted for affinity chromatography on immobilized drug. Because a linker is attached to the antiprion drug it is necessary to verify that the modified drug retains activity. Also, the linker can potentially prevent binding of cellular factors—not related to the antiprion mechanism—that can be important for the understanding of undesired side effects.

Screening for Drugs Instead of Mutants, Antiprion Drugs as Tools in Genetic Experiments

In this last section, we would like to present how antiprion drugs could be used as fundamental tools in model systems like yeast as substitutes to genetic inactivation.

A classical random mutagenesis screen aimed at the isolation of mutations destabilizing or exacerbating a prion phenotype can theoretically only lead to identification of non-redundant genes. In contrast, a multigenic family involved in prion propagation and/or stability would not be identified in such classical genetic screen. Conversely, all members of a family could be targeted by a single drug. Therefore, a drug screen could potentially uncover involvement of a redundant gene family in prion propagation while a classical genetic screen cannot.

In addition, drugs can be used as alternative approaches to genetic inactivation in fundamental studies. There are several advantages of using a drug rather than mutations to observe an effect on a prion phenotype. First, it is much more rapid and simple; second, it is reversible; and third, the effect can be adjusted (dose-dependent effect), which is especially important in the case of essential genes. Thus, drug induced inactivation can be considered as analogous to conditional mutations with the additional and important advantage to be able to affect multigenic families. This aspect is perfectly illustrated by the widespread use of GuHCl, as inhibitor of Hsp104 in yeast prion studies. In budding yeast, Guanidine hydrochoride (GuHCl) is known to efficiently cure all known prions. The GuHCl is a chemical inhibitor of Hsp104p,27–31 a protein chaperone essential for propagation of prions in budding yeast but not conserved in animals. GuHCl is probably the only known antiprion molecule for which the critical biological target is clearly identified and numerous studies made use of GuHCl as a convenient tool for rapid and transient Hsp104-inactivation. It should be noted, however, that some published data tend to indicate that, in addition to Hsp104p, GuHCl might affect additional targets32–34 involved in prion propagation.

To finish, drugs can be used in more sophisticated genetic screens like those leading to synthetic phenotype. In such screens, classically, geneticists look for a second site mutation able to either exacerbate or suppress the effect of a first mutation. If the target of an antiprion drug is known, this compound can be used to mimic the effect of inactivation of the corresponding gene. By looking among libraries of mutants (for example Euroscarf collection of dele- tion of every non essential budding yeast single gene) for those hypersensitive or resistant to the effect of the drug, it is possible to easily identify second site mutation able to exacerbate or suppress effect of inactivation of the drug target. For instance, by looking for mutations that suppress or exacerbate, the curing effect of GuHCl, when cells are treated by subefficient dose of this chemical compound, new cellular factors involved in yeast prions propagation might be identified.

Closing Remarks

Recently, we have developed a rapid two-step yeast-based assay to screen for antiprion drugs.35,36 The first step of the screening is based on the [PSI⁺] phenotype and then, active molecules are tested against [URE3] phenotype. Given the structural and functional divergences between the proteins responsible for these prion phenotypes (respectively Sup35p for the [PSI⁺] phenotype and Ure2p for the [URE3] phenotype), compounds active against both yeast prions can be considered as active against all yeast prions. Our 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 regu- latory mechanisms. Indeed, the vast majority of drugs we isolated as active against both [PSI⁺] and [URE3] prions turned out to be also active against mammalian prion in three different mammalian cell-based assays35,37 but also in vivo in animal models (Tribouillard et al in preparation). These results strongly argue in favor of common prion controlling mechanisms conserved in eukaryotes, from yeast to human, thus validating the yeast-based assay to isolate antiprion compounds but also the yeast model to study prions in general.

In this way, in the prion field, in addition to be an appropriate model system, yeast is also a nice tool not only for drug screening but also for reverse screening approaches aiming at understanding the mode of action of active compounds and thus potential new prion controlling mechanisms.

Figures and Tables

Figure 1 Antiprion drugs as baits in affinity chromatography on immobilized compounds. (A) The structure of the linker connecting the drug to the matrix (Sepharose bead) is crucial. A short spacer can prevent interaction with cellular targets because of steric hindrance (upper scheme). If the spacer is too hydrophobic (depicted in red in the middle panel), it can lead to its auto aggregation thus also preventing target binding. In addition, a hydrophobic linker can generate unspecific hydrophobic interactions with macromolecules from cell lysates. An optimal spacer (lower panel) should be only mildly hydrophobic (hydrophilic part depicted in blue) and long enough to avoid steric hindrance. In this case, specific interactions can occur, as long as the covalent link of the linker to the drug does not affect its antiprion activity too strongly. (B) Possible linker chemical structures are depicted. Polyethylene glycol (PEG) (upper panel) and amino caproylaminopentyloxy linker (lower panel). (C) Affinity chromatography purification on immobilized 6AP. In the example given, the antiprion drug 6AP was covalently liked to Sepharose beads via an amino caproylaminopentyloxy linker. Cell extracts were then incubated with this matrix. After extensive washing, the affinity matrix bound cellular components were analyzed by SDS PAGE followed by silver staining. The interacting proteins were identified by mass spectrometry.

Figure 2 Antiprion drugs as baits in genetic approaches. (A) Genome wide overexpression screen (OES). A cDNA library (of any origin) expressed from the GAL-inducible promoter in a yeast plasmid was transformed in a [PRION⁺] yeast strain (in the example given [PSI⁺] forming white colonies) and cells were streaked on a Galactose medium (in condition where strong expression of cDNA was induced) and containing an antiprion drug. The curing effect produces red [psi^-] colonies. Colonies which remain white despite presence of the antiprion compound might express a cDNA whose overexpression prevent the curing effect of the drug. These colonies are restreaked on the same Galactose medium containing the antiprion drug to check for resistance to the antiprion drug and also on Glucose medium containing antiprion drug to check that the isolated clone is sensitive to the drug in this condition where expression of the cDNA is largely repressed. In the example given, one clone appears to be a false positive for which resistance is not caused by expression of the cDNA (it remains white on Glucose medium). (B) Three-hybrid assay (Y3H). This method is based on the use of a heterodimeric ligand constituted of a ligand of the receptor used as bait covalently linked to the antiprion drug (6AP in the example given). The heterodimeric ligand can be used to screen against a library of cDNAs (from any origin) in fusion with the activation domain (AD) of a transcriptional activator (here Gal4p). The HIS3 reporter gene will only be activated in cells expressing a cDNA encoding a protein to which the antiprion drug (6AP here) binds, and thus only these cells will be able to grow in a medium lacking histidine.

Acknowledgements

Most of our work in the prion field was carried out in the laboratory of Laurent Meijer (CNRS Roscoff ) who is warmly acknowledged for his continuing support, friendship and helpful discussions. Thanks are also due to Elodie Couplan for critical proof-reading of the manuscript and for helpful suggestions. This work was supported by the following grants: GIS “Infections à prion”, ACI “Jeune Chercheur” from the french government, “CRITT Santé Bretagne”, “Ministère de la Recherche” and FRM grants (to Déborah Tribouillard), “Ingénieur de valorisation” fellowship from the CNRS and ANR “blanche” from the french government.