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.

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.

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.