The great majority of therapeutic strategies tested so far for prion diseases, fatal transmissible neurodegenerative disorders, tackled PrPSc, the infectious isoform of the cellular prion protein (PrPC), with largely unsuccessful results. Conversely, targeting PrPC is a poorly explored strategy. In this thesis, I exploited the concepts of altering PrPC cell surface localization and tackling PrPC-mediated cytotoxicity to design two different screening paradigms and study the effect of novel anti-prion compounds. We recently shed light on the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. In the first part of my thesis, I employed cells expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically re-localizing PrPC from the plasma membrane as well as inhibiting prion replication and toxicity in cell cultures. I found four candidate hits inducing a significant reduction in cell surface PrPC, one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. In a previous publication, an artificial mutant of PrPC (ΔCR), sensitizing cells to several cationic antibiotics as Zeocin, was used to screen a library of compounds rescuing Zeocin-induced cytotoxicity. However, the main hit of the screening, named LD24, had low efficiency and high toxicity. In the second part of my thesis, I coupled cycles of chemical rearrangement and screening steps using ΔCR cells, to test a small library of derivatives of LD24 and validated the selected compounds with a panel of cellular assays. I found that one molecule, SM231 and its derivative SM884, counteracted PrPC-mediated toxicity in cellular and ex vivo models of prion disease and Alzheimer's disease. Collectively, these studies define new screening methods and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface and blocking its cytotoxicity could represent viable therapeutic strategies for prion diseases and other neurodegenerative conditions.
Tackling Prion Replication and Toxicity by Targeting the Cellular Prion Protein with Different Pharmacological Strategies / Biggi, Silvia. - (2019 Dec 19), pp. 1-199.
|Titolo:||Tackling Prion Replication and Toxicity by Targeting the Cellular Prion Protein with Different Pharmacological Strategies|
|Anno di pubblicazione:||2019-12-19|
|Struttura:||Dipartimento di Biologia Cellulare, Computazionale e Integrata - CIBIO|
|Corso di dottorato:||Biomolecular Sciences|
|Supervisori e coordinatori:||Massignan, Tania|
|Tesi in cotutela:||no|
|Digital Object Identifier (DOI):||10.15168/11572_246789|
|Appare nelle tipologie:||08.1 Tesi di dottorato (Doctoral Thesis)|
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|Thesis Biggi S. 9 Dec 19.pdf||Tesi di dottorato (Doctoral Thesis)||Tutti i diritti riservati (All rights reserved)||Embargo: 10/12/2021|