The voltage gated anion channel (VDAC), also known as mitochondrial porin, is the most abundant protein in the outer mitochondrial membrane (OMM) of all eukaryotes, covering up to 80% of the membrane surface. The isoform 1 acts as a gatekeeper for the entry and exit of metabolites through OMM. VDAC1 exhibits a β-barrel structure formed by 19 β-strands. It also has an N-terminal α-helix, folded towards the inside of the barrel. The N-terminal helix is highly dynamic and can move out of the pore as the voltage gradient varies, playing the role of binding site for various proteins including the enzyme hexokinase (HK). VDAC1, therefore, appears to be a convergence point for a variety of survival and death cell signals mediated by its association with various ligands and proteins. Studies have shown that, in cancer cells, HK is over-expressed by interacting with and inhibiting VDAC1, modulating mitochondrial functionality and increasing glycolysis rate. In cancer cells, elevated levels of mitochondrialbound HK play a key role in promoting cell growth, in the survival of highly glycolytic tumours, and in protecting against mitochondria-mediated cell death. Targeting the VDAC1-HK interaction has been proposed as a promising target for anticancer therapy since the detachment of HK from VDAC1 would lead to the activation of apoptosis. In this thesis we have studied the interaction between a pool of ligands preselected and VDAC1. We compared the unfolding temperatures of VDAC1 in the presence of the ligands and screened among all the ligands only those capable of leading to a considerable increase in the inflection temperature correlated to the stability of VDAC1. The selected ligands have been visualized by docking studies inside the VDAC1 in a pocket formed by the N-terminal. The general goal of the project is to find small molecules able to block the movement of the N-terminal of VDAC1 without precluding its role as a channel. By doing so, the HK binding site with VDAC1 will be missing and therefore it will not be able to activate the metabolic pathway typical of neoplastic cells.
Il canale anionico voltaggio dipendente (VDAC), noto anche come porina mitocondriale, è la proteina più abbondante nella outer mitochondrial membrane (OMM) di tutti gli eucarioti, andando a coprire fino all'80% della superficie della membrana. L’isoforma umana di tipo 1 funge da gatekeeper per l'entrata e l'uscita dei metaboliti attraverso OMM. VDAC1 presenta una struttura a β-barile. Presenta anche una α-elica N-terminale altamente dinamica e può traslocare fuori dal poro al variare del gradiente di tensione, svolgendo il ruolo di sito di legame per diverse proteine tra cui l’enzima esochinasi (HK). VDAC1, quindi, sembra essere un punto di convergenza per una varietà di segnali di sopravvivenza cellulare e segnali di morte mediati dalla sua associazione con vari ligandi e proteine. Studi hanno dimostrato che, nelle cellule tumorali, HK viene iper-espressa andando ad interagire, e ad inibire, il VDAC1, modulando la funzionalità mitocondriale e incrementando il tasso della glicolisi. Nelle cellule tumorali elevati livelli di HK legato ai mitocondri svolgono un ruolo chiave nella promozione della crescita cellulare, nella sopravvivenza dei tumori altamente glicolitici e nella protezione contro la morte cellulare mediata dai mitocondri. Il targeting dell'interazione VDAC1-HK è stato proposto come un bersaglio promettente per la terapia antitumorale poiché il distacco di HK da VDAC1 porterebbe all’attivazione dell’apoptosi. In questa tesi abbiamo studiato l’interazione tra un pool di ligandi preselezionati e il VDAC1. Abbiamo confrontato le temperature di unfolding del VDAC1 in presenza dei ligandi e selezionato tra tutti i ligandi solo quelli in grado di portare ad un considerevole aumento della inflection temperature correlata alla stabilità del VDAC1. I ligandi selezionati sono stati visualizzati tramite studi di docking all’interno del VDAC1 in una tasca formata dal N-terminale. L’obiettivo generale del progetto è trovare piccole molecole in grado di bloccare il movimento dell’N-terminale del VDAC1 senza precluderne il ruolo di canale. Così facendo verrà a mancare il sito di legame dell’HK con il VDAC1 e quindi non potrà attivare il pathway metabolico tipico delle cellule neoplastiche.
Il canale mitocondriale VDAC1 come bersaglio farmacologico per una nuova terapia antitumorale
MINAKOVA, ANNA
2020/2021
Abstract
The voltage gated anion channel (VDAC), also known as mitochondrial porin, is the most abundant protein in the outer mitochondrial membrane (OMM) of all eukaryotes, covering up to 80% of the membrane surface. The isoform 1 acts as a gatekeeper for the entry and exit of metabolites through OMM. VDAC1 exhibits a β-barrel structure formed by 19 β-strands. It also has an N-terminal α-helix, folded towards the inside of the barrel. The N-terminal helix is highly dynamic and can move out of the pore as the voltage gradient varies, playing the role of binding site for various proteins including the enzyme hexokinase (HK). VDAC1, therefore, appears to be a convergence point for a variety of survival and death cell signals mediated by its association with various ligands and proteins. Studies have shown that, in cancer cells, HK is over-expressed by interacting with and inhibiting VDAC1, modulating mitochondrial functionality and increasing glycolysis rate. In cancer cells, elevated levels of mitochondrialbound HK play a key role in promoting cell growth, in the survival of highly glycolytic tumours, and in protecting against mitochondria-mediated cell death. Targeting the VDAC1-HK interaction has been proposed as a promising target for anticancer therapy since the detachment of HK from VDAC1 would lead to the activation of apoptosis. In this thesis we have studied the interaction between a pool of ligands preselected and VDAC1. We compared the unfolding temperatures of VDAC1 in the presence of the ligands and screened among all the ligands only those capable of leading to a considerable increase in the inflection temperature correlated to the stability of VDAC1. The selected ligands have been visualized by docking studies inside the VDAC1 in a pocket formed by the N-terminal. The general goal of the project is to find small molecules able to block the movement of the N-terminal of VDAC1 without precluding its role as a channel. By doing so, the HK binding site with VDAC1 will be missing and therefore it will not be able to activate the metabolic pathway typical of neoplastic cells.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/20.500.14239/13897