Novel Anti Prion Compounds Identified by Targeting a Functional Activity of PrP C

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1 Novel Anti Prion Compounds Identified by Targeting a Functional Activity of PrP C Emiliano Biasini Department of Biochemistry Boston University School of Medicine

2 PrP C & PrP Sc Non infectious Infectious Soluble/monomeric Insoluble/aggregated Protease sensitive Protease resistant (PrP 27 30) PrP C 43% α helix, 3% β sheet PrP Sc 30% α helix, 43% β sheet PrP Sc PrP C Infectivity and Pathology PK + PrP27-30kDa

3 Uncoupling prion toxicity from infectivity Prion infectivity and toxicity are temporally uncoupled Prion propagation and toxicity in vivo occur in two distinct mechanistic phases Sandberg MK. et al. Nature Infectivity in absence of toxicity PrP C is necessary to develop disease Normal host prion protein necessary for scrapie induced neurotoxicity. Brandner S. et al. Nature Silencing PrP C specifically in neurons of infected mice prevents disease, even if PrP Sc replicates in astrocytes. Mallucci G. et al. Science 2003

4 PrP C and PrP Sc : partners in crime? PrP C Infectivity Pathology PrP C could not only be a substrate for infectivity, but also a receptor for toxicity!

5 PrP C may transduce toxicity of other aggregates A normal, physiological activity of PrP C could be subverted by PrP Sc or other protein aggregates to generate toxicity. What is this activity?

6 What is the normal function of PrP C?

7 An artificial PrP mutant deleted for the central region ( CR) is highly neurotoxic in absence of infectivity PrP WT PrP CR WT PrP suppresses mutant PrP toxicity! CR is not infectious and non convertible into PrP Sc Toxicity in absence of infectivity ΔCR is relevant to study the physiological function of PrP The deletion doesn t change protein conformation Is soluble like PrP C Is localized like PrP C Its toxicity is rescued by expression of wild type PrP

8 The Drug Based Cell Assay (DBCA) CR PrP induces hypersensitivity to several antibiotics, including Zeocin, G418 and Hygromicin PrP WT PrP CR % Cell Viability Time window of ~10 days Days

9 DBCA based High Throughput Screening DBCA miniaturized (384 well plates) High Throughput Drug Screening of 70,000 compounds

10 Screening results 68 compounds potently inhibited ΔCR PrP in DBCA (DBCA performed with two antibiotics with different mode of action) 61 molecules available for purchase and tested in secondary screenings: PrP C expression 0/61 decrease PrP C expression PrP C localization 0/61 change PrP C localization Direct binding to PrP C 0/61 bind to PrP C Citotoxicity 61/61 have LD50 > 50μM PrP Sc propagation in cells 24/61 inhibit prion replication

11 DBCA based screening identifies several new anti prion compounds

12 Testing the curing activity in cells Number of passages

13 Some of the new anti prion compounds eradicate PrP Sc from cells LD49 PK res levels (% DMSO) Number of passages

14 CONCLUSIONS The DBCA based drug screening is a completely novel approach, and allowed screening of the largest compound library ever tested in prion diseases We identified 61 compounds that block ΔCR toxicity 24 compounds also block prion replication in cell cultures IMPLICATIONS Some of the newly identified compounds may be effective in blocking the toxicity of prions in mice Defining the target of these molecules may have a tremendous impact on our understanding of both prion replication and toxicity

15 David Harris (Head of the lab) Tania Massignan (Cell assays) Brian Fluharty (SPR studies) Marco Gobbi Matteo Stravalaci (SPR studies) Isaac Solomon (Channel activity) Natasha Khatry (Toxicity assay) Jessie Turnbaugh (PMCA) Rick Bowman (Tissues and mice) Marcie Glicksman Kathleen Seib Greg Cuny (In vitro screening)