Neglected tropical diseases (NTDs) are a group of uncurable, viral, and bacterial diseases that affect more than one billion people globally, particularly those countries that are affected by high poverty. The causative agents are eukaryotic organisms that are responsible of different pathological conditions in the human. For the majority of these diseases the currently available treatments are highly inadequate. Poor efficacy, severe side effects and the insurgence of new mechanism of resistance towards the existing therapies result in an urgent need for new treatments. The limited understanding of the diseases’ biology and the discovery of only few validated drug targets, hinders the drug discovery effort in this therapeutic area. With the aim to develop better treatments for neglected tropical diseases, we have been investigating the drug target of enzymes that are involved in the protein synthesis. Our attention has been focused on the aminoacyl-tRNA synthetases (aaRSs), a family of proteins involved in the aminoacylation process. Only few of them have been already validated as promising targets in different diseases, e.g. LysRS as validated drug target in malaria and cryptosporidiosis, but the knowledge around this largely unexplored protein family is still limited. A deeper understanding of the structures of this enzyme family, their sequences and the structural conservation across the whole family is important to explore the possibility of exploiting aaRSs as potential drug targets. Considering the importance of aaRSs biological function, an in-depth analysis evidenced a number of binding sites. From a drug discovery point of view, our attention was focused on the ATP-amino acid binding site (catalytic site) to understand how we could exploit the catalytic site from a drug design/development point of view. Initially, a set of residues that are involved in ATP-amino acid recognition has been identified, then the sequence/structural conservation of the catalytic site across the whole aaRS family has been analysed. The lack of structural information for some of the aaRS members was addressed with the generation of homology models of the missing structures; then, we analysed the interactions between ATP, amino acid and the aaRS enzymes. Using molecular interaction fingerprints and mapping the binding site with chemical probes, other interaction points that could potentially engage a small molecule ligand have been explored. In addition to similarities between the different aaRS members, we wanted also to identify structural/sequence differences that could be exploited to design selective compounds for a given organism and to enhance the selectivity toward the human isoform. As an additional way of achieving selectivity, the possibility of developing covalent inhibitors by targeting cysteine residues was also evaluated across the aaRS family. This could be crucial to optimize the selectivity for a pathogen and to reduce the inhibition of the human isoform, or to avoid resistances. So, a further analysis involved also the 3D-cavity variability other than the sequence one. The results allowed to characterize the differences on aaRS catalytic sites and to identify which ones present the highest 3D similarity. In addition, the chemical space associated to this target family has been investigated. AaRS active compounds were retrieved from several chemical databases and analysed in terms of molecular properties, structural similarities and privileged chemical scaffolds. This work has allowed to gain a deeper insight of the aaRS target family and to evaluate its potentiality as drug target. These results could be useful to understand the targets’ variability; they provide also the possibility of deploying the chemistry known for a target towards other ones, by considering the similarity information. This could suggest new hypothesis of potential drug targets that might be used to expand the common therapies.
Le malattie tropicali neglette sono un gruppo di malattie non curabili che colpiscono più di un miliardo di persone. Sono causate da organismi eucariotici che generano diverse condizioni patologiche nell'uomo e caratterizzate da trattamenti inadeguati. La scarsa efficacia, gli effetti collaterali e l'insorgenza di meccanismi di resistenza verso le terapie esistenti determinano la necessità di nuovi trattamenti. Nel tentativo di sviluppare soluzioni per queste malattie, abbiamo studiato le potenzialità come bersagli farmacologici di enzimi coinvolti nella sintesi proteica. La nostra attenzione è focalizzata sulle aminoacil-tRNA sintetasi (aaRSs). Le aaRSs sono una famiglia di proteine coinvolte nel processo di amminoacilazione. Alcune di esse sono convalidate come promettenti bersagli in diverse malattie, ad esempio LysRS come bersaglio farmacologico convalidato nella malaria e nella criptosporidiosi, ma la conoscenza di questa inesplorata famiglia di proteine è ancora limitata. Una comprensione delle strutture di questa famiglia di enzimi, delle sequenze e della conservazione strutturale è fondamentale. Ciò ha il fine di sfruttare le aaRSs come bersagli farmacologici. Considerando l’importanza della funzione delle aaRSs, un’analisi approfondita ha evidenziato una serie di siti di legame. Da un punto di vista della scoperta di farmaci, l’attenzione si è concentrata sul sito di legame dell'ATP e dell’amminoacido (sito catalitico), per caratterizzarlo da un punto di vista di progettazione di farmaci. Inizialmente, è stato identificato un insieme di residui coinvolti nel riconoscimento dell'ATP e dell’amminoacido; quindi, è stata analizzata la conservazione strutturale e sequenziale del sito catalitico nell'intera famiglia aaRSs. La mancanza di informazioni strutturali per alcune aaRSs è stata affrontata con la generazione di modelli di omologia delle strutture mancanti; quindi, abbiamo analizzato le interazioni tra ATP, amminoacidi e gli enzimi aaRSs. Utilizzando impronte di interazione molecolare e mappando il sito di legame con sonde chimiche, sono stati esplorati altri punti di interazione che potrebbero coinvolgere una nuova small molecule. Oltre alle somiglianze tra i diversi membri aaRSs, volevamo identificare differenze utilizzabili per progettare composti selettivi per un organismo o per migliorare la selettività rispetto all’isoforma umana. Nel tentativo di ottenere selettività, è stato valutato lo sviluppo di inibitori covalenti valutando i residui di cisteina in tutta la famiglia aaRSs. Ciò potrebbe essere cruciale per ottimizzare la selettività nei confronti di un patogeno e per ridurre l'inibizione dell'isoforma umana, o per evitare resistenze al trattamento. Un’ulteriore analisi ha coinvolto la variabilità della cavità 3D. I risultati caratterizzano le differenze tra i siti catalitici nella famiglia aaRSs, identificando quelli che presentano la maggiore somiglianza 3D. Inoltre, è stato studiato lo spazio chimico associato a questa famiglia target. I composti attivi come inibitori aaRSs sono stati recuperati da diversi database chimici e analizzati in termini di proprietà molecolari, somiglianze strutturali e scaffolds chimici privilegiati. Questo lavoro ha prodotto una visione approfondita della famiglia target delle aaRSs e una valutazione del potenziale come bersagli farmacologici. Questi risultati potrebbero essere utili per comprendere la variabilità dei targets. Essi suggeriscono la possibilità di distribuire la chimica nota per un target verso altri, grazie alle informazioni di similarità. Ciò potrebbe aiutare nel generare nuove ipotesi di potenziali bersagli farmacologici, utilizzabili per espandere le terapie comuni.
AMINOACYL-tRNA SYNTHETASES AS DRUG TARGET FAMILY.
GENTILE, ROCCO
2019/2020
Abstract
Neglected tropical diseases (NTDs) are a group of uncurable, viral, and bacterial diseases that affect more than one billion people globally, particularly those countries that are affected by high poverty. The causative agents are eukaryotic organisms that are responsible of different pathological conditions in the human. For the majority of these diseases the currently available treatments are highly inadequate. Poor efficacy, severe side effects and the insurgence of new mechanism of resistance towards the existing therapies result in an urgent need for new treatments. The limited understanding of the diseases’ biology and the discovery of only few validated drug targets, hinders the drug discovery effort in this therapeutic area. With the aim to develop better treatments for neglected tropical diseases, we have been investigating the drug target of enzymes that are involved in the protein synthesis. Our attention has been focused on the aminoacyl-tRNA synthetases (aaRSs), a family of proteins involved in the aminoacylation process. Only few of them have been already validated as promising targets in different diseases, e.g. LysRS as validated drug target in malaria and cryptosporidiosis, but the knowledge around this largely unexplored protein family is still limited. A deeper understanding of the structures of this enzyme family, their sequences and the structural conservation across the whole family is important to explore the possibility of exploiting aaRSs as potential drug targets. Considering the importance of aaRSs biological function, an in-depth analysis evidenced a number of binding sites. From a drug discovery point of view, our attention was focused on the ATP-amino acid binding site (catalytic site) to understand how we could exploit the catalytic site from a drug design/development point of view. Initially, a set of residues that are involved in ATP-amino acid recognition has been identified, then the sequence/structural conservation of the catalytic site across the whole aaRS family has been analysed. The lack of structural information for some of the aaRS members was addressed with the generation of homology models of the missing structures; then, we analysed the interactions between ATP, amino acid and the aaRS enzymes. Using molecular interaction fingerprints and mapping the binding site with chemical probes, other interaction points that could potentially engage a small molecule ligand have been explored. In addition to similarities between the different aaRS members, we wanted also to identify structural/sequence differences that could be exploited to design selective compounds for a given organism and to enhance the selectivity toward the human isoform. As an additional way of achieving selectivity, the possibility of developing covalent inhibitors by targeting cysteine residues was also evaluated across the aaRS family. This could be crucial to optimize the selectivity for a pathogen and to reduce the inhibition of the human isoform, or to avoid resistances. So, a further analysis involved also the 3D-cavity variability other than the sequence one. The results allowed to characterize the differences on aaRS catalytic sites and to identify which ones present the highest 3D similarity. In addition, the chemical space associated to this target family has been investigated. AaRS active compounds were retrieved from several chemical databases and analysed in terms of molecular properties, structural similarities and privileged chemical scaffolds. This work has allowed to gain a deeper insight of the aaRS target family and to evaluate its potentiality as drug target. These results could be useful to understand the targets’ variability; they provide also the possibility of deploying the chemistry known for a target towards other ones, by considering the similarity information. This could suggest new hypothesis of potential drug targets that might be used to expand the common therapies.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/20.500.14239/12086