Development of advanced analytical methodologies for Alzheimer’s disease drug discovery

Davani, Lara (2022) Development of advanced analytical methodologies for Alzheimer’s disease drug discovery, [Dissertation thesis], Alma Mater Studiorum Università di Bologna. Dottorato di ricerca in Scienza e cultura del benessere e degli stili di vita, 34 Ciclo. DOI 10.48676/unibo/amsdottorato/10295.
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Advanced analytical methodologies were developed to characterize new potential active MTDLs on isolated targets involved in the first stages of Alzheimer’s disease (AD). In addition, the methods investigated drug-protein bindings and evaluated protein-protein interactions involved in the neurodegeneration. A high-throughput luminescent assay allowed the study of the first in class GSK-3β/ HDAC dual inhibitors towards the enzyme GSK-3β. The method was able to identify an innovative disease-modifying agent with an activity in the micromolar range both on GSK-3β, HDAC1 and HDAC6. Then, the same assay reliably and quickly selected true positive hit compounds among natural Amaryllidaceae alkaloids tested against GSK-3β. Hence, given the central role of the amyloid pathway in the multifactorial nature of AD, a multi-methodological approach based on mass spectrometry (MS), circular dichroism spectroscopy (CD) and ThT assay was applied to characterize the potential interaction of CO releasing molecules (CORMs) with Aβ1-42 peptide. The comprehensive method provided reliable information on the different steps of the fibrillation process and regarding CORMs mechanism of action. Therefore, the optimal CORM-3/Aβ1−42 ratio in terms of inhibitory effect was identified by mass spectrometry. CD analysis confirmed the stabilizing effect of CORM-3 on the Aβ1−42 peptide soluble form and the ThT Fluorescent Analysis ensured that the entire fibrillation process was delayed. Then the amyloid aggregation process was studied in view of a possible correlation with AD lipid brain alterations. Therefore, SH-SY5Y cells were treated with increasing concentration of Aß1-42 at different times and the samples were analysed by a RP-UHPLC system coupled with a high-resolution quadrupole TOF mass spectrometer in comprehensive data-independent SWATH acquisition mode. Each lipid class profiling in SH-SY5Y cells treated with Aß1-42 was compared to the one obtained from the untreated. The approach underlined some peculiar lipid alterations, suitable as biomarkers, that might be correlated to Aß1-42 different aggregation species.

Tipologia del documento
Tesi di dottorato
Davani, Lara
Dottorato di ricerca
Settore disciplinare
Settore concorsuale
Parole chiave
Alzheimer’s Disease; Drug Discovery; MTLDs; Advanced Analytical Methods; High-throughput Luminescent Assay, Liquid Chromatography, Mass Spectrometry, Circular Dichroism, Lipidomics, GSK-3β, HDAC, Amyloid Aggregation, Biomarkers
Data di discussione
15 Giugno 2022

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