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Azarnia Tehran, Domenico (2016) Novel insights into botulinum neurotoxins mechanism of action and the discovery of prophylactic inhibitors against botulism. [Ph.D. thesis]

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Abstract (english)

Botulinum neurotoxins (BoNTs), the most poisonous substances identified so far, are protein toxins that cause botulism, a severe neuroparalytic disease. They are produced by different species of neurotoxigenic Clostridia and can be grouped into seven serotypes (BoNT/A to /G). Using genomic and proteomic approach, many novel BoNTs have been recently identified and are classified as subtypes, though they cannot be completely neutralized by currently available immunological methods. However, all BoNTs have a similar molecular architecture which reflects a conserved mechanism of action. Therefore this situation can be tackled by developing inhibitors targeting the BoNT intracellular intoxication process.
The BoNTs consist of two main chains linked by a unique inter-chain disulfide bond: the heavy chain (H, 100 kDa) and the catalytic light chain (L, 50 kDa). The C-terminal part of H (HC) is responsible for the neurospecific binding and the internalization within an endocytic compartment, whilst the N-terminal part (HN) is involved in the translocation of L across the endosome membrane. L is a Zn2+ dependent metalloprotease that targets specifically the SNARE proteins, the three proteins constituting the core of neuroexocytosis. This cleavage results in a prolonged inactivation of neurotransmitter release and causes the flaccid paralysis typical of botulism. To penetrate into neurons, BoNTs exploit synaptic vesicles (SV) recycling and their lumen acidification induces the HN-mediated membrane translocation of L. It has been demonstrated that, once on the cytosolic side, the L metalloprotease remains connected to H via the interchain disulphide bridge and the reduction of this bond is necessary to release the protease in the cytosol and enable their catalytic activity.
Using a series of well characterized inhibitors of Thioredoxin Reductase (TrxR)/Thioredoxin (Trx) system, we found that this redox system is involved in the cytosolic reduction of the interchain disulphide bond of BoNTs. In neuronal cultures, these molecules prevent the metalloproteolytic activity of all toxin serotypes without significantly affecting cell viability. Moreover, such compounds are very effective in vivo, lowering the severity and the duration of paralysis caused by a local BoNT injection. More importantly, one of these drugs elicits a remarkable protection in mice systemically injected with lethal doses of different serotypes. These results entail that the reduction of the interchain disulphide bond is a strict prerequisite for the activity of BoNTs and that this class of inhibitors can prevent the neurotoxicity regardless of their different immunogenicity. Intriguingly, we also found that the TrxR/Trx system is bound to the cytosolic side of the SV membrane and that it is enriched in those SV that are docked to active zones. We speculated that this redox system may play a role in maintaining SV protein function by controlling the redox state of the different SV protein disulfides.
Another step in BoNTs mechanism of action that might offer a good template for drug design is their trafficking. Recently, an inhibitor of different pathogens that require a passage through acidic endosomes to invade cells has been identified and dubbed EGA. We tested the effect of this molecule in neurons treated with BoNTs as also their neurotoxicity is strictly dependent on the passage through an intracellular acidic compartment. We focused our investigation on BoNT/A and BoNT/B, the two serotypes mainly associated with human botulism and used in therapy, and BoNT/D, that scarcely affects humans, but frequently causes botulism in animals. We found that EGA inhibits BoNTs activity on neuronal cultures, without interfering with any of the main steps characterising their cellular mechanism of intoxication. We speculated that, rather than having a direct effect on BoNTs, this compound impinges on an intracellular target which is responsible for their trafficking. Importantly, we found that EGA is not toxic per se in vivo, and is particularly efficacious in preventing botulism induced by BoNT/B and BoNT/D. Instead, in the case of BoNT/A the lethality was not reduced, but botulism symptoms developed later. We argued that the trafficking of the different BoNT types might be differently impacted by EGA and this compound may be used as a new tool for studying different intracellular routes exploited by BoNTs.
On the basis of the present knowledge about BoNTs mechanism of action, it is clear that once the LC has been released in the cytosol, the inhibitors tested here are no longer effective. Therefore, these drugs are to be considered as prophylactics. However, if given soon after diagnosis, these compounds could reduce symptoms severity by preventing the entry into neurons of circulating BoNTs, thus reducing the severity of poisoning and shortening the period of hospitalization that is related to the high costs of intensive care. Moreover, these molecules may be administered without knowing the BoNT serotype and subtype, therefore saving the time needed for toxin characterization.

Abstract (italian)

Le neurotossine botuliniche (BoNTs) sono le esotossine più potenti attualmente conosciute nonché gli agenti eziologici di una grave malattia neuroparalitica, il botulismo. Storicamente classificate in 7 sierotipi (A-B-C-D-E-F-G), perché antigenicamente differenti, il loro numero risulta in rapida crescita poiché ogni sierotipo esiste in più sottotipi, la cui presenza sta progressivamente palesandosi grazie all’introduzione delle moderne tecniche di next generation sequencing (NGS). Sebbene il botulismo rappresenti un problema sanitario minore, la scoperta di nuovi inibitori contro le BoNTs è di assoluto rilievo dal punto di vista socio-economico visti i limitati trattamenti ad oggi disponibili e il possibile utilizzo delle tossine botuliniche come potenziali agenti di bioterrorismo.
Dal punto di vista strutturale, tutti i diversi sierotipi sono costituiti da due catene polipeptidiche unite covalentemente da un unico ponte disolfuro: una catena pesante (H, 100 kDa) e una leggera (L, 50 kDa). Dal punto di vista funzionale, la stessa struttura può essere invece suddivisa in tre principali domini con un determinato ruolo nel processo di intossicazione: 1) HC, definito anche dominio di legame, media l’adsorbimento specifico della tossina alla membrana plasmatica del motoneurone, 2) HN, denominato anche dominio di traslocazione, costituisce un canale di permeazione attraverso cui 3) L, riconosciuto essere il dominio catalitico, viene traslocato nel citoplasma. Qui, la catena leggera (L) viene liberata attraverso la riduzione del legame disolfuro intercatena ed è quindi pronta ad esercitare la sua funzione enzimatica. In dettaglio, le BoNTs sono metalloproteasi zinco-dipendenti capaci di idrolizzare in maniera specifica le proteine SNARE. Ogni sierotipo presenta un preciso bersaglio molecolare: BoNT/A e /E idrolizzano un diverso legame peptidico della proteina SNAP-25, BoNT/B, /D, /F e /G proteolizzano la proteina VAMP2, mentre, BoNT/C è in grado di idrolizzare due substrati differenti, sintaxina e SNAP-25. Le proteine SNARE costituisco il cuore del macchinario biochimico che permette il riconoscimento e la fusione delle vescicole sinaptiche con la membrana presinaptica del terminale nervoso a livello della giunzione neuromuscolare, pertanto, la loro idrolisi porta al blocco del rilascio di acetilcolina, inducendo neuroparalisi di tipo flaccido, conseguenza tipica del botulismo.
In una prima parte del lavoro, utilizzando un approccio farmacologico, si è dimostrato come il sistema ossido-riduttivo Tioredossina (Trx)-Tioredossina Reduttasi (TrxR) dell’ospite abbia un ruolo chiave nella riduzione citosolica del ponte disolfuro intercatena di tutti i sierotipi di BoNTs. In dettaglio, utilizzando colture neuronali primarie, si è potuto dimostrare come tali molecole siano in grado di proteggere la coltura modello dall'intossicazione. Inoltre, i dati ottenuti in vitro sono stati confermati in vivo: la somministrazione dei differenti inibitori, in un modello murino, porta ad una diminuzione della severità e della durata della paralisi flaccida nonché ad una sostanziale protezione in topi trattati con dosi letali di tossina. Infine, si è riusciti ad identificare il sistema della TrxR/Trx a livello delle vescicole sinaptiche. In particolare, si è compreso come entrambi le proteine si arricchiscano a livello delle vescicole sinaptiche docked, ossia quelle legate alla membrana presinaptica pronte a rilasciare il neurotrasmettitore in esse contenuto. Questa evidenza è di particolare importanza se si prende in considerazione un possibile ruolo di tali proteine nel processo di neuroesocitosi.
In un successivo lavoro, si è dimostrato come l'endocitosi all'interno del terminale nervoso, può essere considerato un altro passaggio chiave nel meccanismo d’azione delle tossine botuliniche, da prendere in considerazione nello sviluppo di nuovi inibitori. In dettaglio, un gruppo americano nel 2014 ha dimostrato come una piccola molecola, chiamata EGA, sia capace di bloccare l’azione di diverse tossine batteriche e virus che utilizzano gli endosomi come “cavallo di Troia” per il loro ingresso nelle cellule. Sebbene il target intracellulare risulta ancora non noto, si è deciso di sintetizzare e testare tale inibitore per capire se anche nel caso delle tossine botuliniche sia in grado di inibire il loro ingresso in vitro e in vivo. I risultati ottenuti evidenziano come EGA sia capace di inibire in vitro l’azione di molteplici sierotipi di BoNTs: A e B, comunemente associati a casi di botulismo umano e utilizzati in terapia, e D, coinvolto in casi di botulismo animale. Inoltre, questa molecola risulta efficace nel prevenire la paralisi in vivo dovuta ai sierotipi B e D e ritarda quella dovuta al sierotipo A. Di conseguenza, i nostri risultati suggeriscono come questa molecola possa essere presa in considerazione come lead farmacologico per lo sviluppo di nuovi antidoti.
L’identificazione di questi inibitori potrà avere importanti implicazioni applicative volte a compensare il gap attualmente presente nel campo della prevenzione/terapia del botulismo. Infatti, il grande numero di sottotipi (>70) e la potenziale (probabile) esistenza di varianti non ancora identificate, è notevolmente limitante al controllo della loro azione patogena con il solo utilizzo di strumenti immunologici, quali antisieri e vaccinazione. In questa tesi verrà discusso come il nostro approccio risulta essere, invece, indipendente dal sierotipo di BoNTs coinvolta nell'intossicazione, dunque indipendente dall'antigenicità delle diverse tossine.

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EPrint type:Ph.D. thesis
Tutor:Montecucco, Cesare
Data di deposito della tesi:29 January 2016
Anno di Pubblicazione:29 January 2016
Key Words:neurotoxins/paralysis/trafficking/endosome/neuromuscular junction/inhibitors/thioredoxin/botulinum
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/04 Patologia generale
Struttura di riferimento:Dipartimenti > Dipartimento di Biologia
Codice ID:9384
Depositato il:21 Oct 2016 10:35
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