Cancer constitutes a major cause of mortality on a global scale, manifesting itself in a wide range of diseases that can affect any organ or tissue in the body. According to the World Health Organization, there were approximately 10 million deaths attributable to cancer during 2020. The deadliest forms of cancer include lung cancer, with about 2 million deaths, followed by those involving colorectum, stomach and breast. One of the defining characteristics of cancer cells is their propensity for immortalization, a process by which, due to the constant activation of the mutated telomerase enzyme, cell proliferation becomes almost unlimited resulting in the impairment of the normal cellular life cycle. Apoptosis thus plays a fundamental role in cancer research, as it is possible to study and counteract all those mechanisms involved in cellular immortality. One of the avenues taken in scientific research of anticancer therapies is the development of drugs (small organic molecules or biotechnological drugs) aimed at controlling, usually inhibitory, proteins overexpressed in cancer cells. One example of an important possible drug target is the B-cell lymphoma 2 (BCL-2) family of proteins. BCL-2 is a family of proteins with regulatory activity that controls cell death by inhibiting or inducing apoptosis. In a physiological context, BCL-2 proteins are present on the outer membrane of mitochondria, where they play a key role on the balance between cell growth and cell death. Specifically, BCL-2 proteins, act on the physiology of the mitochondrion by promoting or inhibiting permeabilization and release of ROS, precursors of the apoptotic cascade, and regulating the dynamics of mitochondrial fusion and fission . Possible drugs that act on the function of BCL-2 proteins have been the subject of intense study and research over the past 30 years, but to date only a few molecules with antitumor activity have been clinically tested. One protein in the BCL-2 family that has been relatively little studied is Bfl-1, a protein with anti-apoptotic action whose mechanism is still not well identified. While in a physiological context Bfl-1 prevents apoptosis, overexpression of it contributes to tumor growth and drug resistance. The research laboratory of Professor Mattia Sturlese (Department of Pharmaceutical Sciences, University of Padua) has recently identified compounds (hit compounds) with affinity for Bfl-1. The identification of these compounds was made possible thanks to the Fragment Based Drug Discovery approach, an innovative technique that allows, through the use of Nuclear Magnetic Resonance Imaging, to analyze libraries of fragments (small-sized, low-molecular-weight organic molecules) and determine which ones have higher binding affinity to the drug target. The fragments that yielded the best affinity results in this screening were two: a fluoroindoline affinity to an allosteric site of Bfl-1 (near the binding site), and a substituted imidazo-pyridine, which showed affinity to the binding site of Bfl-1, and the ability to irreversibly bind the protein at this site. This thesis work, carried out at the research laboratory of Prof Andrea Mattarei (Department of Pharmaceutical Sciences, University of Padua), involved the synthesis of some organic compounds, consisting of the union of the two fragments with higher affinity for the protein. Molecular targets for this project were designed based on the hypothesized binding site for the individual fragments in order to optimize the affinity and specificity of the resulting compounds for Bfl-1. Synthetic strategies were then designed and put into practice through the synthesis and characterization of the desired compounds and the synthetic intermediates required to obtain them.
Il cancro costituisce una delle principali cause di mortalità su scala mondiale, manifestandosi in una vasta gamma di patologie che possono colpire qualsiasi organo o tessuto nell’organismo. Secondo l'Organizzazione Mondiale della Sanità, nel corso del 2020 si sono registrati approssimativamente 10 milioni di decessi attribuibili al cancro. Tra le forme tumorali più letali figurano quelle polmonari, con circa 2 milioni di morti, seguite da quelle che coinvolgono colon-retto, stomaco e seno. Una delle caratteristiche peculiari delle cellule cancerogene è la loro propensione all’immortalizzazione, processo attraverso cui, grazie alla costante attivazione dell’enzima telomerasi mutato, la proliferazione cellulare diventa pressoché illimitata con la conseguente compromissione del normale ciclo di vita cellulare. L’apoptosi gioca quindi un ruolo fondamentale nella ricerca contro il cancro, poiché è possibile studiare e contrastare tutti quei meccanismi implicati nell’immortalità cellulare.Una delle vie intraprese nella ricerca scientifica di terapie antitumorali è lo sviluppo di farmaci (piccole molecole organiche o farmaci biotecnologici) mirati al controllo, solitamente inibitorio, di proteine iper-espresse nelle cellule tumorali. Un esempio di importante possibile target farmacologico è la famiglia delle proteine BCL-2 (B-cell lymphoma 2). In particolare, le proteine BCL-2, agiscono sulla fisiologia del mitocondrio promuovendo o inibendo la permeabilizzazione e il rilascio di ROS, precursori della cascata apoptotica e regolando le dinamiche di fusione e fissione mitocondriali . Possibili farmaci che agiscano sul funzionamento delle proteine BCL-2 sono stati oggetto di intenso studio e ricerca negli ultimi 30 anni, ma ad oggi solo poche molecole ad attività antitumorale sono state clinicamente testate. Una proteina della famiglia BCL-2 relativamente poco studiata è Bfl-1, proteina ad azione anti-apoptotica il cui meccanismo risulta non ancora ben identificato. Mentre in un contesto fisiologico Bfl-1 previene l’apoptosi, una sua sovraespressione contribuisce alla crescita tumorale e alla resistenza farmacologica. Il laboratorio di ricerca del Professor Mattia Sturlese ha recentemente individuato dei composti (hit compounds) con affinità per Bfl-1. L’identificazione di questi composti è stata resa possibile grazie all’approccio Fragment Based Drug Discovery, una tecnica innovativa che permette, grazie all’utilizzo della Risonanza Magnetica Nucleare, di analizzare librerie di frammenti (molecole organiche di piccola dimensione, a basso peso molecolare) e di determinare quali hanno maggiore affinità di legame con il bersaglio farmacologico.I frammenti che hanno dato i migliori risultati di affinità in questo screening sono risultati due: una fluoroindolina affine ad un sito allosterico di Bfl-1 (in prossimità del sito di binding), e una imidazo-piridina sostituita, che ha mostrato affinità al sito di binding di Bfl-1, e la capacità di legare irreversibilmente la proteina in questo sito.Questo lavoro di tesi, svolto presso il laboratorio di ricerca del Prof Andrea Mattarei, ha previsto la sintesi di alcuni composti organici, costituiti dall’unione dei due frammenti a maggiore affinità per la proteina. I target molecolari per questo progetto sono stati disegnati sulla base del sito di legame ipotizzato per i singoli frammenti, allo scopo di ottimizzare l’affinità e la specificità dei composti risultanti per Bfl-1. Sono state quindi progettate delle strategie sintetiche, che sono state messe in pratica attraverso la sintesi e la caratterizzazione deicomposti desiderati e degli intermedi sintetici necessari al loro ottenimento.
Sviluppo Farmaceutico "Fragment-to-Lead": Progettazione e Sintesi di inibitori irreversibili di Bfl-1
BALDIN, MARIAVITTORIA
2023/2024
Abstract
Cancer constitutes a major cause of mortality on a global scale, manifesting itself in a wide range of diseases that can affect any organ or tissue in the body. According to the World Health Organization, there were approximately 10 million deaths attributable to cancer during 2020. The deadliest forms of cancer include lung cancer, with about 2 million deaths, followed by those involving colorectum, stomach and breast. One of the defining characteristics of cancer cells is their propensity for immortalization, a process by which, due to the constant activation of the mutated telomerase enzyme, cell proliferation becomes almost unlimited resulting in the impairment of the normal cellular life cycle. Apoptosis thus plays a fundamental role in cancer research, as it is possible to study and counteract all those mechanisms involved in cellular immortality. One of the avenues taken in scientific research of anticancer therapies is the development of drugs (small organic molecules or biotechnological drugs) aimed at controlling, usually inhibitory, proteins overexpressed in cancer cells. One example of an important possible drug target is the B-cell lymphoma 2 (BCL-2) family of proteins. BCL-2 is a family of proteins with regulatory activity that controls cell death by inhibiting or inducing apoptosis. In a physiological context, BCL-2 proteins are present on the outer membrane of mitochondria, where they play a key role on the balance between cell growth and cell death. Specifically, BCL-2 proteins, act on the physiology of the mitochondrion by promoting or inhibiting permeabilization and release of ROS, precursors of the apoptotic cascade, and regulating the dynamics of mitochondrial fusion and fission . Possible drugs that act on the function of BCL-2 proteins have been the subject of intense study and research over the past 30 years, but to date only a few molecules with antitumor activity have been clinically tested. One protein in the BCL-2 family that has been relatively little studied is Bfl-1, a protein with anti-apoptotic action whose mechanism is still not well identified. While in a physiological context Bfl-1 prevents apoptosis, overexpression of it contributes to tumor growth and drug resistance. The research laboratory of Professor Mattia Sturlese (Department of Pharmaceutical Sciences, University of Padua) has recently identified compounds (hit compounds) with affinity for Bfl-1. The identification of these compounds was made possible thanks to the Fragment Based Drug Discovery approach, an innovative technique that allows, through the use of Nuclear Magnetic Resonance Imaging, to analyze libraries of fragments (small-sized, low-molecular-weight organic molecules) and determine which ones have higher binding affinity to the drug target. The fragments that yielded the best affinity results in this screening were two: a fluoroindoline affinity to an allosteric site of Bfl-1 (near the binding site), and a substituted imidazo-pyridine, which showed affinity to the binding site of Bfl-1, and the ability to irreversibly bind the protein at this site. This thesis work, carried out at the research laboratory of Prof Andrea Mattarei (Department of Pharmaceutical Sciences, University of Padua), involved the synthesis of some organic compounds, consisting of the union of the two fragments with higher affinity for the protein. Molecular targets for this project were designed based on the hypothesized binding site for the individual fragments in order to optimize the affinity and specificity of the resulting compounds for Bfl-1. Synthetic strategies were then designed and put into practice through the synthesis and characterization of the desired compounds and the synthetic intermediates required to obtain them.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/62624