Proteins, as dynamic and versatile molecules, engage in intricate interaction networks with various partners, including other proteins, nucleic acids, lipids, and small molecules, playing central roles in numerous biological processes. Understanding these protein-ligand interactions is essential not only for deciphering the molecular basis of cellular and extracellular functions but also for developing targeted therapeutic interventions. This thesis, while exploring two distinct biological systems, bridges them through the application of different biophysical techniques developed to analyse protein-ligand and protein-protein interactions. The first case study investigates the zinc-binding properties of aaAG5-3, a mosquito salivary protein, using both computational predictions and biochemical assays. The second case study focuses on identifying small molecule fragments that interact with the SARS-CoV-2 Spike protein, previously identified in a computational screening by Frasnetti (2022), using biophysical methods such as Microscale Thermophoresis. By adopting these methodological approaches, I demonstrate the versatility of these techniques in elucidating critical protein-ligand interactions across different biological contexts. The first case study examines aaAG5-3, a protein belonging to the CAP (Cysteine-Rich Secretory Protein, Antigen 5, and Pathogenesis-Related 1) superfamily, which is found in the saliva of Aedes albopictus, the Asian tiger mosquito. The investigation focuses on the potential zinc-binding properties of aaAG5-3, a feature hypothesized to play a role in the biological function of CAP proteins. Through computational predictions and biochemical assays, including the development of a novel protocol for a colorimetric assay, potential zinc-binding sites were identified and the interaction between zinc ions and aaAG5-3 was evaluated. The analysis provided insights into the zinc-binding ability of and the role of the ion in determining the protein conformation and function. The findings contribute to a broader understanding of the molecular mechanisms underlying blood-feeding and pathogen transmission in mosquitoes. The second case study involves the screening of a small library of molecule fragments for their ability to bind to the Spike glycoprotein of SARS-CoV-2, the virus responsible for COVID-19. The Spike protein is crucial for viral entry into host cells, making it a prime target for therapeutic intervention. Using MST the study identified promising fragment candidates that interact with the receptor-binding domain (RBD) of the Spike protein. These fragments serve as potential starting points for the development of novel therapeutic agents aimed at disrupting the virus's ability to infect host cells. The results highlight the potential of fragment-based drug discovery (FBDD) coupled with the use of Microscale Thermophoresis as a strategy for identifying new antiviral compounds. Overall, this thesis underscores the importance of understanding protein-ligand interactions in both fundamental biology and drug discovery. Through the integration of experimental and computational approaches, the research presented here offers contributions to vector-borne disease biology and antiviral drug development. This thesis prompts further structural characterization of aaAG5-3 in complex with zinc through advanced biophysical techniques, as well as the optimization of the identified Spike-binding fragments, providing a foundation for the development of novel interventions targeting key biological processes in disease contexts.
Le proteine, in quanto molecole dinamiche e versatili, sono coinvolte in complessi network di interazione con vari partner, tra cui altre proteine, acidi nucleici, lipidi e piccole molecole, svolgendo ruoli centrali in numerosi processi biologici. Comprendere queste interazioni proteina-ligando è essenziale non solo per decifrare le basi molecolari delle funzioni cellulari ed extracellulari, ma anche per sviluppare interventi terapeutici mirati. Questa tesi, pur esplorando due distinti sistemi biologici, li collega attraverso l'applicazione di diverse tecniche biofisiche sviluppate per analizzare le interazioni proteina-ligando e proteina-proteina. Il primo caso di studio indaga le proprietà di legame allo zinco di aaAG5-3, una proteina salivare della zanzara, utilizzando sia predizioni computazionali che saggi biochimici. Il secondo caso di studio si concentra sull'identificazione di frammenti molecolari che interagiscono con la proteina Spike di SARS-CoV-2, identificati in precedenza da Frasnetti (2022) attraverso uno screening computazionale, sfruttando metodi biofisici come la Miscroscale Thermophoresis (termoforesi su scala microscopica, MST). Adottando questi approcci metodologici, dimostro la versatilità di queste tecniche nell'elucidare le interazioni proteina-ligando in diversi contesti biologici. Il primo caso di studio esamina aaAG5-3, una proteina appartenente alla superfamiglia CAP (Cysteine-Rich Secretory Protein, Antigen 5, and Pathogenesis-Related 1), che si trova nella saliva di Aedes albopictus, la zanzara tigre asiatica. L'indagine si concentra sulle potenziali proprietà di legame allo zinco di aaAG5-3, una caratteristica ipotizzata come rilevante per la funzione biologica delle proteine CAP. Attraverso previsioni computazionali e saggi biochimici, inclusa la messa a punto di un nuovo protocollo per un saggio colorimetrico, sono stati identificati potenziali siti di legame per lo zinco e valutata l'interazione tra ioni zinco e aaAG5-3. L'analisi ha fornito spunti sulle capacità di legame allo zinco e sul ruolo dello ione nel determinare la conformazione e la funzione della proteina. I risultati contribuiscono a una comprensione più ampia dei meccanismi molecolari che sottostanno all’ematofagia e alla trasmissione di patogeni nelle zanzare. Il secondo caso di studio riguarda lo screening di una piccola libreria di frammenti molecolari per la loro capacità di legarsi alla glicoproteina Spike di SARS-CoV-2, il virus responsabile di COVID-19. La proteina Spike è cruciale per l'ingresso nelle cellule ospiti, rendendola un obiettivo primario per l'intervento terapeutico. Utilizzando la tecnica MST, lo studio ha identificato candidati promettenti tra i frammenti che interagiscono con il dominio di legame al recettore (RBD) della proteina Spike. Questi frammenti servono come potenziali punti di partenza per lo sviluppo di nuovi agenti terapeutici mirati a interrompere la capacità del virus di infettare le cellule ospiti. I risultati evidenziano il potenziale del Fragment-based Drug Design (FBDD) associata all'uso della termoforesi a scala microscopica come strategia per l'identificazione di nuovi composti antivirali. Nel complesso, questa tesi sottolinea l'importanza di comprendere le interazioni proteina-ligando sia nella biologia fondamentale che nella scoperta di farmaci. Attraverso l'integrazione di approcci sperimentali e computazionali, la ricerca qui presentata offre contributi alla biologia delle malattie trasmesse da vettori e allo sviluppo di farmaci antivirali. Questa tesi promuove ulteriori caratterizzazioni strutturali di aaAG5-3 in complesso con lo zinco mediante tecniche biofisiche avanzate, nonché l'ottimizzazione dei frammenti che legano Spike identificati, fornendo una base per lo sviluppo di nuovi interventi mirati ai principali processi biologici in contesti patologici.
Indagine sulle interazioni proteina-ligando nei sistemi biologici: prospettive dalla saliva di zanzara e dalle proteine virali
GENNARI, NICOLÒ
2023/2024
Abstract
Proteins, as dynamic and versatile molecules, engage in intricate interaction networks with various partners, including other proteins, nucleic acids, lipids, and small molecules, playing central roles in numerous biological processes. Understanding these protein-ligand interactions is essential not only for deciphering the molecular basis of cellular and extracellular functions but also for developing targeted therapeutic interventions. This thesis, while exploring two distinct biological systems, bridges them through the application of different biophysical techniques developed to analyse protein-ligand and protein-protein interactions. The first case study investigates the zinc-binding properties of aaAG5-3, a mosquito salivary protein, using both computational predictions and biochemical assays. The second case study focuses on identifying small molecule fragments that interact with the SARS-CoV-2 Spike protein, previously identified in a computational screening by Frasnetti (2022), using biophysical methods such as Microscale Thermophoresis. By adopting these methodological approaches, I demonstrate the versatility of these techniques in elucidating critical protein-ligand interactions across different biological contexts. The first case study examines aaAG5-3, a protein belonging to the CAP (Cysteine-Rich Secretory Protein, Antigen 5, and Pathogenesis-Related 1) superfamily, which is found in the saliva of Aedes albopictus, the Asian tiger mosquito. The investigation focuses on the potential zinc-binding properties of aaAG5-3, a feature hypothesized to play a role in the biological function of CAP proteins. Through computational predictions and biochemical assays, including the development of a novel protocol for a colorimetric assay, potential zinc-binding sites were identified and the interaction between zinc ions and aaAG5-3 was evaluated. The analysis provided insights into the zinc-binding ability of and the role of the ion in determining the protein conformation and function. The findings contribute to a broader understanding of the molecular mechanisms underlying blood-feeding and pathogen transmission in mosquitoes. The second case study involves the screening of a small library of molecule fragments for their ability to bind to the Spike glycoprotein of SARS-CoV-2, the virus responsible for COVID-19. The Spike protein is crucial for viral entry into host cells, making it a prime target for therapeutic intervention. Using MST the study identified promising fragment candidates that interact with the receptor-binding domain (RBD) of the Spike protein. These fragments serve as potential starting points for the development of novel therapeutic agents aimed at disrupting the virus's ability to infect host cells. The results highlight the potential of fragment-based drug discovery (FBDD) coupled with the use of Microscale Thermophoresis as a strategy for identifying new antiviral compounds. Overall, this thesis underscores the importance of understanding protein-ligand interactions in both fundamental biology and drug discovery. Through the integration of experimental and computational approaches, the research presented here offers contributions to vector-borne disease biology and antiviral drug development. This thesis prompts further structural characterization of aaAG5-3 in complex with zinc through advanced biophysical techniques, as well as the optimization of the identified Spike-binding fragments, providing a foundation for the development of novel interventions targeting key biological processes in disease contexts.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/28451