Author Identifier
Date of Award
2026
Keywords
dementia, central nervous system, Alzheimer's disease, Aβ, autophagy, proteasome, C. elegans, AI screening, small molecules
Document Type
Thesis
Publisher
Edith Cowan University
Degree Name
Doctor of Philosophy
School
School of Medical and Health Sciences
First Supervisor
Prashant Bharadwaj
Second Supervisor
Ralph Martins
Third Supervisor
Binosha Fernando
Abstract
Alzheimer's disease (AD) is a progressive neurodegenerative disease marked by the aggregation of amyloid-β (Aβ) peptides and hyperphosphorylated tau, disruption of protein homeostasis (proteostasis), synaptic impairment, and cognitive decline. Growing evidence suggests that the impaired clearance of Aβ, rather than their overproduction, is a significant contributor to the pathogenesis of AD.
This thesis examines regulation of intracellular protein clearance mechanisms in AD, specifically the ubiquitin proteasome system (UPS) and autophagy lysosomal pathway (ALP) through small molecule-based interventions. Furthermore, this thesis explores how UPS and ALP pathways are affected by Aβ accumulation using in vitro and in vivo AD models. This study characterizes the proteostasis dysfunction induced by Aβ accumulation using Aβ producing neuronal cell MC65 and Caenorhabditis elegans (C. elegans) AD models. Findings from this study demonstrates that Aβ significantly disrupts the UPS and the ALP, resulting in markedly diminished proteasome activity and dysregulated autophagy. Mechanistic analysis revealed that inhibiting autophagy provided protection, while activating autophagy enhanced Aβ toxicity in both cellular and animal models. Screening a library of small-molecule proteostasis modulators led to the identification of the USP14 inhibitor IU1 as a potent compound that restores proteostasis and alleviates Aβ-associated neurotoxicity. IU1 inhibits USP14 through a steric blockade mechanism by binding to the thumb-palm cleft of its catalytic domain, thereby inhibiting substrate access. Treatment with IU1 markedly diminished Aβ toxicity in both cellular and C. elegans AD models while maintaining protein homeostasis. This first part of the thesis presents the initial evidence that pharmacological inhibition of USP14 using IU1 offers neuroprotective effects against Aβ-induced proteostasis dysfunction and neurodegeneration in AD.
This thesis further employs USP14 structure and IU1 ligand-based virtual screening approaches, in collaboration with Atomwise/AIMS programs, to identify and develop novel IU1 analogues and USP14 inhibitors that alleviate Aβ toxicity. The hit compounds identified via virtual screening were then assessed via deubiquitinating enzyme assays and functional analysis in cellular and C. elegans AD models to prioritize candidates that can restore proteostasis and mitigate neurodegeneration.
This thesis also offers two significant methodological contributions to the discovery of drugs for AD, in addition to the biological findings. A live-cell, high-throughput Amytracker assay was developed and validated, facilitating real-time quantification of intracellular Aβ accumulation and clearance in pertinent neuronal models. This platform offers a scalable and translationally pertinent screening tool that connects biochemical assays with animal studies, enabling swift mechanistic assessment of potential therapeutics. This study established complementary computational discovery pipelines for the identification of novel USP14 inhibitors. Utilizing AI-driven structure-based screening and ligand-based virtual screening of around eight million drug-like compounds, several novel USP14 inhibitor candidates were identified and biochemically validated. These methodologies broadened the chemical landscape beyond established IU1-derived scaffolds and illustrated the viability of integrating computational screening with experimental validation for drug discovery aimed at proteostasis.
While development of disease modifying treatment strategies is of the highest priority for dementia, AD is increasingly recognized as a lifestyle disorder, with non-pharmacological prevention strategies including exercise, a healthy diet, and cognitive engagement. This thesis comprehensively reviewed existing literature on how non-pharmacological factors-such as diet, physical activity, and sleep-affect autophagy and broader proteostasis pathways, our review focused on understanding how lifestyle affects proteostasis pathways and underlying mechanisms, as well as identifying gaps, which is the central theme of this thesis.
This thesis offers novel mechanistic insights into the varying impacts of Aβ on the UPS and autophagy pathways, confirms USP14 as a viable target for drug development in proteostasis regulation, establishes a high-throughput platform for assessing amyloid clearance, and broadens the chemical landscape of USP14 inhibitors via computational drug discovery. These advancements offer novel instruments, therapeutic avenues, and conceptual paradigms that facilitate the formulation of both pharmacological and non-pharmacological approaches for Alzheimer’s disease and associated neurodegenerative disorders.
Access Note
Access to this thesis is embargoed until 12th August 2027
Recommended Citation
Ariyath, A. (2026). Targeting proteostasis dysfunction in Alzheimer’s disease using small molecule modulators. Edith Cowan University. https://doi.org/10.25958/taqt-pf88