One of the fundamental aspects of life is cellular communication. To properly carry out biochemical tasks and regulate the processes needed for survival, replication and motion, cells need to communicate with other cells and to elaborate signals that originate from the extracellular environment. Proteins are the class of biomolecules that are in charge for most of these processes. A set of protein-protein interactions that is particularly critical for the correct maintenance of cell processes is represented by the interactions that involve molecular chaperones. Molecular chaperones are fundamental proteins whose main role is the supervision of the folding, activation, aggregation suppression and regulation of degradation of other proteins, named client proteins. Chaperones can be arranged in classes according to sequence homology; a main group is that of stress proteins or heat shock proteins (HSPs). Inside the cell compartments are hosted different paralogs, which supervise diverse and specific interaction networks. In humans, the principal paralogs are HSP90 in the cytoplasm, Grp94 in the endoplasmatic reticulum and Trap1 in the mitochondria. The many biological roles of this class of molecular chaperones all rely on finely tuned structural dynamics, which are dependent on enzymatic ATP hydrolysis; dysregulation of these functions has been related to a wide range of diseases, ranging from inflammatory conditions to cancerous states and neurodegeneration. The interest in targeting HSPs is based on the consideration that, in pathological states, the cellular machinery is subjected to stress; this leads to the involvement of various type of HSPs, including HSP90 and its mitochondrial paralog Trap1, in these kinds of conditions. Allostery is one of the prime mechanisms of regulation of protein energy landscape and consequently of cell life in general and has recently attracted increasing interest from medicinal chemists and drug discovery partnership in the search for new types of targets and strategies for drug development. In this framework, it became clear that the development of Trap1 allosteric inhibitors which can interfere with its functional processes can be exploited for perspective therapeutic application. In particular, compounds that target Trap1, without targeting HSP90, would provide exciting new chemical tools to disentangle the complicacies of the roles of specific chaperones in the regulation of normal and transformed cells. By exploiting the intrinsic structural asymmetry of the chaperone, a set of allosteric compounds have recently been designed by the Colombo Lab based on the relationship between the kind of NTD-bound nucleotide (ATP/ATP, ATP/ADP, ADP/ADP) and the structural dynamics of the middle domain, where the client-binding region lies. A first set of molecules was designed on the basis of the stereoelectronic properties of the putative allosteric pocket, and since these compounds need to be transferred to the mitochondria to fully express their potential on Trap1 inhibition, in this thesis we set out to modify them with mitochondrial targeting functionalities, i.e. triphenylphosphonium (TPP). To make progress along this route, we first set out to design efficient synthetic pathways and then synthesize TPP-conjugated allosteric modulators. The final molecules will be transferred to the laboratory of Professor Andrea Rasola, at the University of Padua, to carry out a series of biochemical and cell tests to evaluate their (improved over cognates) perturbation of the Trap1 system.
Uno degli aspetti fondamentali della vita è la comunicazione cellulare. Per regolare i processi necessari per la sopravvivenza, la replicazione e il movimento, le cellule devono comunicare fra loro ed elaborare i segnali provenienti dall'ambiente extracellulare. Le proteine sono la classe di biomolecole addette al controllo della maggior parte di questi processi. Un insieme di interazioni proteina-proteina particolarmente critico per il corretto funzionamento dei processi cellulari è rappresentato dalle interazioni che coinvolgono gli chaperone molecolari. Gli chaperon molecolari sono proteine fondamentali il cui ruolo principale è la supervisione del ripiegamento, attivazione, soppressione dell'aggregazione e regolazione della degradazione di altre proteine, chiamate proteine client. Gli chaperone possono essere organizzati in classi in base all'omologia della sequenza; uno dei gruppi principali è quello delle proteine dello stress o delle proteine dello shock termico (HSP). All'interno dei compartimenti cellulari sono ospitati diversi paraloghi che supervisionano reti di interazione diverse e specifiche. Nell'uomo, i principali paraloghi sono HSP90 nel citoplasma, Grp94 nel reticolo endoplasmatico e Trap1 nei mitocondri. I numerosi ruoli biologici di questa classe di chaperone molecolari si basano su dinamiche strutturali dipendenti dall'idrolisi enzimatica dell'ATP; la mancata regolazione di queste funzioni è stata correlata a una vasta gamma di patologie, come condizioni infiammatorie, stati cancerosi e neurodegenerazioni. L'interesse nel target delle HSP si basa sulla considerazione che, negli stati patologici, il sistema cellulare è sottoposto a stress; questo porta al coinvolgimento di vari tipi di HSP, tra cui HSP90 e il suo paralogo mitocondriale Trap1, in questo tipo di condizioni. L'allosteria è uno dei principali meccanismi di regolazione dellenergy landscape proteico e, di conseguenza, della vita cellulare in generale; ha recentemente attirato un crescente interesse nellambito della chimica farmaceutica nella ricerca di nuovi tipi di target e nuove strategie per lo sviluppo di farmaci. In questo contesto, è diventato chiaro come lo sviluppo di inibitori allosterici per Trap1 che possano interferire con i suoi processi funzionali possa essere sfruttato per possibili applicazioni terapeutiche. In particolare, i composti che modulano Trap1 senza interferire con HSP90, fornirebbero nuovi strumenti chimici per comprendere al meglio il ruolo di specifici chaperone nella regolazione delle cellule normali e trasformate. Sfruttando l'intrinseca asimmetria strutturale dello chaperone, un gruppo di composti allosterici è stato recentemente progettato dal Colombo Lab in base alla relazione tra il tipo di nucleotide legato al NTD (ATP / ATP, ATP / ADP, ADP / ADP) e la dinamica strutturale del Middle Domain, in cui si trova la regione deputata al legame con le proteine client. Una prima serie di molecole è stata progettata sulla base delle proprietà stereoelettroniche dellipotetica tasca allosterica; poiché questi composti devono essere trasferiti ai mitocondri per esprimere pienamente il loro potenziale nell'inibizione di Trap1, in questo progetto di tesi si è deciso di modificarli con funzioni adatte al targeting mitocondriale, cioè il gruppo trifenilfosfonio (TPP). Per questo motivo sono stati prima progettati opportuni percorsi sintetici e quindi sintetizzati i modulatori allosterici coniugati con il TPP. Le molecole finali verranno trasferite nel laboratorio del professor Andrea Rasola, presso l'Università di Padova, per effettuare una serie di test biochimici e cellulari per valutare la loro perturbazione del sistema Trap1.
Sintesi di modulatori allosterici per chaperone molecolari
DALMONTE, IRENE
2018/2019
Abstract
One of the fundamental aspects of life is cellular communication. To properly carry out biochemical tasks and regulate the processes needed for survival, replication and motion, cells need to communicate with other cells and to elaborate signals that originate from the extracellular environment. Proteins are the class of biomolecules that are in charge for most of these processes. A set of protein-protein interactions that is particularly critical for the correct maintenance of cell processes is represented by the interactions that involve molecular chaperones. Molecular chaperones are fundamental proteins whose main role is the supervision of the folding, activation, aggregation suppression and regulation of degradation of other proteins, named client proteins. Chaperones can be arranged in classes according to sequence homology; a main group is that of stress proteins or heat shock proteins (HSPs). Inside the cell compartments are hosted different paralogs, which supervise diverse and specific interaction networks. In humans, the principal paralogs are HSP90 in the cytoplasm, Grp94 in the endoplasmatic reticulum and Trap1 in the mitochondria. The many biological roles of this class of molecular chaperones all rely on finely tuned structural dynamics, which are dependent on enzymatic ATP hydrolysis; dysregulation of these functions has been related to a wide range of diseases, ranging from inflammatory conditions to cancerous states and neurodegeneration. The interest in targeting HSPs is based on the consideration that, in pathological states, the cellular machinery is subjected to stress; this leads to the involvement of various type of HSPs, including HSP90 and its mitochondrial paralog Trap1, in these kinds of conditions. Allostery is one of the prime mechanisms of regulation of protein energy landscape and consequently of cell life in general and has recently attracted increasing interest from medicinal chemists and drug discovery partnership in the search for new types of targets and strategies for drug development. In this framework, it became clear that the development of Trap1 allosteric inhibitors which can interfere with its functional processes can be exploited for perspective therapeutic application. In particular, compounds that target Trap1, without targeting HSP90, would provide exciting new chemical tools to disentangle the complicacies of the roles of specific chaperones in the regulation of normal and transformed cells. By exploiting the intrinsic structural asymmetry of the chaperone, a set of allosteric compounds have recently been designed by the Colombo Lab based on the relationship between the kind of NTD-bound nucleotide (ATP/ATP, ATP/ADP, ADP/ADP) and the structural dynamics of the middle domain, where the client-binding region lies. A first set of molecules was designed on the basis of the stereoelectronic properties of the putative allosteric pocket, and since these compounds need to be transferred to the mitochondria to fully express their potential on Trap1 inhibition, in this thesis we set out to modify them with mitochondrial targeting functionalities, i.e. triphenylphosphonium (TPP). To make progress along this route, we first set out to design efficient synthetic pathways and then synthesize TPP-conjugated allosteric modulators. The final molecules will be transferred to the laboratory of Professor Andrea Rasola, at the University of Padua, to carry out a series of biochemical and cell tests to evaluate their (improved over cognates) perturbation of the Trap1 system.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/21602