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dc.contributor.authorFerraro, Giusy
dc.date.accessioned2024-07-15T08:46:32Z
dc.date.available2024-07-15T08:46:32Z
dc.date.issued2023-04-24
dc.identifier.urihttp://elea.unisa.it/xmlui/handle/10556/7258
dc.description2021 - 2022it_IT
dc.description.abstractThe identification of natural products and synthetic compounds target proteins is pivotal to understand their mechanism of action for the development of molecular probes and/or potential drugs. Functional proteomics is a mass spectrometry-based discipline focused on the analysis of the interactome of small molecules and their targets discovery. Functional proteomics has become an invaluable tool in targets identification of small molecules since Fishing for Partners strategy, also named affinity purification mass spectrometry coupled approach (AP-MS), successfully disclosed a multitude of bioactive compounds interacting proteins in the past 15 years (Rix and Superti-Furga, 2009). Unfortunately, this strategy is not universally applicable being limited by the need of a covalent modification of the molecular probe that should contain at least one reactive chemical group and, most importantly, the compound modification should not influence its original bioactivity. Thus, an alternative functional proteomics platform, based on a combination of untargeted Drug Affinity Responsive Target Stability (DARTS) with targeted Limited Proteolysis coupled to Multiple Reaction Monitoring (t-LiP-MRM), has been exploited during my PhD project to disclose and characterize the interacting proteins of bioactive compounds (Lomenick et al., 2009; Feng et al., 2014). At first, DARTS has been exploited to identify small molecules most reliable cellular partners, then t-LiP-MRM has been carried out to investigate the molecules/target proteins interaction features. Moreover, the proteomics results were validated by Western Blotting to confirm small molecules interaction with their DARTS-identified targets and by in silico molecular docking to corroborate t- LiP-MRM information about the target region(s) involved in the binding. For an in-deep analysis of the binding between the investigated compounds and their protein counterparts and for moving through the activity of such compounds on their putative targets, proper in vitro and/or in cell biological assays were also employed. In particular, during this PhD project, MS-based proteomics approaches have been exploited to profile the interactomes of two natural compounds (Artemetin and Tatridin A), abundant in the extracts from Achillea millefolium (De Souza et al., 2011) and from Anthemis melanolepis (Saroglou et al., 2010), respectively, and a synthetic benzodiazepine derivative, called 1g (Parenti et.al., 2016). The interactomes of Artemetin, 1g and Tatridin A in HeLa, U87MG and THP-1 cells proteome have been examined unveiling, respectively, the Filamin A and Filamin B (crucial role in the organization of the cytoskeleton interacting with F-actin) (Zhou et al., 2021; Xu et al., 2017), the Brain Glycogen Phosphorylase (crucial role in the degradation of the glycogen clusters in the brain and in the regulation of the cellular glucose concentrations) (Mathieu et al., 2017) and the Phosphoglycerate Kinase 1 (crucial role in glycolysis) (Zieker et al., 2010) as their principal cellular interactors. [edited by Author]it_IT
dc.language.isoenit_IT
dc.publisherUniversita degli studi di Salernoit_IT
dc.subjectMass spectrometryit_IT
dc.subjectProteomicsit_IT
dc.subjectSmall moleculesit_IT
dc.titleTargeted and untargeted proteomic approaches to disclose small molecules interactomeit_IT
dc.typeDoctoral Thesisit_IT
dc.subject.miurCHIM/06 CHIMICA ORGANICAit_IT
dc.contributor.coordinatoreSbardella, Gianlucait_IT
dc.description.cicloXXXVit_IT
dc.contributor.tutorMonti, Maria Chiarait_IT
dc.identifier.DipartimentoFarmaciait_IT
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