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Zanetti, Giulia (2018) Study of the mechanism of action of botulinum neurotoxins to develop inhibitors and to improve their pharmacological application. [Ph.D. thesis]

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Abstract (english)

Seven antigenically different botulinum neurotoxin types (BoNT/A through /G) and many subtypes (BoNT/A1, BoNT/A2, etc.) constitute a growing family of bacterial exotoxins that specifically paralyze the cholinergic peripheral nerve terminals of vertebrates. Most notably, BoNTs intoxicate the neuromuscular junction, thereby causing a severe neuro-muscular paralysis known as botulism.
Despite the heterogeneity of their primary sequence, BoNTs are structurally and functionally conserved and composed of a 50 kDa light chain (L) and a 100 kDa heavy chain (H), linked via a unique, and fundamental, disulphide bridge. The C-terminal (HC, 50 kDa) and the N-terminal halves (HN, 50 kDa) of H constitute a sophisticated nanomolecular machine that mediates both the neurospecific binding of the molecule to peripheral nerve endings and the delivery of L into the neuronal cytosol. L is a zinc-dependent protease that specifically cleaves SNARE proteins (SNAP-25 (synaptosomal-associated protein of 25 kDa), VAMP (vesicle-associated membrane protein) and Stx (syntaxin)), the three proteins that form the SNARE complex which is the core of the nanomachine that mediates the fusion of synaptic vesicles (SV) with the presynaptic membrane, thus allowing neurotransmitter release. Cleavage causes impairment of SNARE complex assembly/function and thus a blockade of neuroexocytosis, which results in the flaccid paralysis typical of botulism. Patients can die for respiratory failure but, if vital functions are maintained by intensive care, they fully recover as the botulism neuroparalysis is completely reversible.
The mechanism of action of BoNTs can be conveniently divided into five fundamental steps: 1) binding to nerve terminals, 2) internalization by SV recycling, 3) pH-dependent translocation of L into the cytoplasm, 4) reduction of the interchain disulphide bond and 5) hydrolysis of SNARE proteins. BoNT/A and /E cleave SNAP-25, BoNT/B, /D, /F and /G cleave VAMP. BoNT/C is unique because it cleaves two substrates, SNAP-25 and syntaxins.
BoNTs are the most poisonous substances known to vertebrates, and are classified as potential biological weapons. Currently, the only treatment available consists in passive immunisation with antisera raised against the seven main toxin types. Unfortunately, antisera are variably reactive against subtypes and, moreover, no licensed vaccine are available for human use. This situation has promoted an intense research to develop new antitoxins.
At the same time, neurospecificity and reversibility of action make BoNTs the therapeutic of choice for the treatment of a heterogeneous number of human diseases characterized by the hyperactivity of peripheral nerve terminals.
Given this dichotomy of BoNTs, the aim of my PhD has been double: i) to develop pan-inhibitors that would prevent/treat botulism and ii) to better understand the toxin functioning in vivo to improve its use in human therapy.
i) BoNT’s intoxication strictly depends on the reduction of the interchain disulphide bond. Without it, L remains attached to H and cannot exert its catalytic activity. By using a pharmacological approach, I found that the Thioredoxin-Thioredoxin Reductase (Trx-TrxR) system is responsible for the reduction of all BoNT serotypes and that inhibitors of Trx-TrxR strongly reduce their neurotoxicity in vitro and in vivo in a model that recapitulates clinical botulism. These results are remarkable because they show for the first time that the different BoNTs can be inhibited by a single drug (a pan-inhibitor) by impacting on their common mechanism of action.
Following the same concept, the trafficking of BoNTs within the synaptic terminal represents another rational target to develop pan-inhibitors. Recently, it was reported that the chemical compound EGA inhibits pathogens or toxins that enter cells via acidic endosomes. Since also BoNTs have a similar requirement to trigger the translocation of L into the cytosol, I tested the activity of EGA and I found that it significantly inhibits the neurotoxic activity of BoNT/A, BoNT/B and BoNT/D in vitro and in vivo, tested because are serotypes frequently involved in human and animal botulism, respectively. Interestingly, none of the main steps underlying toxin’s cellular mechanism is directly affected by the drug. Rather, I provided indirect evidence that EGA interferes with the sorting of BoNTs inside nerve terminals, hampering their trafficking toward acidic compartments essential for L translocation.
Together, these studies show that BoNT’s activity can be significantly mitigated independently from their intertypic differences by using drugs targeting common steps of their mechanism of action. These inhibitors represent lead compounds for the development of new drugs against botulism.
ii) BoNTs are successful human therapeutic agents. Despite their use is almost invariably restricted to BoNT/A and BoNT/B, recent data on human volunteers suggest that BoNT/C can be used to treat non-responder individuals with similarly effective pharmacological outcomes. However, little is known about the mechanism by which BoNT/C paralyzes peripheral nerve terminals in vivo. In fact, at variance from all the other BoNTs, BoNT/C cleaves two substrates, SNAP-25 and syntaxin-1A/1B. Therefore, I undertook a study to evaluate the individual contribution of SNAP-25 and syntaxin cleavage to BoNT/C activity in vivo. I took advantage from a recent publication where two triple-mutated BoNT/C L, L200W/M221W/I226W (BoNT/C α-3W) and S51T/R52N/N53P (BoNT/C α-51), were reported to cleave selectively syntaxins.
Thanks to a collaboration with Dr. T. Binz, I received the full-length BoNT/C mutants produced by recombinant methods and I tested their biochemical and toxicological properties. I found that both mutants cleave syntaxin with similar efficiency with respect to wild type BoNT/C (BoNT/C-wt), but unexpectedly, they maintain a residual activity on SNAP-25 which is higher for BoNT/C α-3W than for BoNT/C α-51. Interestingly, this different activity on SNAP-25 strictly correlates with the lethality of mutant toxins in vivo. At the same time, the proteolysis of syntaxin provides a substantial and prolonged neuromuscular impairment without the complete blockage of neurotransmission. These results suggest that SNAP-25 cleavage is the main determinant of BoNT/C neuroparalyzing activity and that BoNT/C derivatives with selective activity for syntaxins represent an appealing strategy to develop BoNTs endowed with long lasting activity and a wide safety margin.

Abstract (italian)

Le neurotossine botuliniche (BoNTs) sono esotossine batteriche, agenti eziologici del botulismo. In base alla diversa antigenicità si possono dividere in sette sierotipi principali (BoNT/A-/G) che comprendono ulteriori sottotipi (BoNT/A1, BoNT/A2 ecc.). Presentano tutte tropismo specifico per la giunzione neuromuscolare dove esercitano un’azione neuroparalizzante.
Nonostante la sequenza amminoacidica eterogenea, dal punto di vista strutturale e funzionale le BoNTs appaiono altamente conservate: sono composte da due catene polipeptidiche, una pesante di 100 kDa (H) e una leggera di 50 kDa (L), unite da un unico, ma fondamentale, ponte disolfuro. Il C-terminale (HC, 50 kDa) e l’N-terminale (HN, 50 kDa) di H costituiscono una sofisticata macchina molecolare che media sia il legame neurospecifico della molecola ai terminali nervosi periferici che la traslocazione di L nel citoplasma dei motoneuroni. L è una proteasi zinco-dipendente che idrolizza in modo specifico le proteine SNARE (SNAP-25 (synaptosomal-associated protein of 25 kDa), VAMP (vesicle-associated membrane protein) and Stx (syntaxin)), tre proteine che costituiscono il nucleo della macchina molecolare, il cosiddetto “SNARE complex”, che media la fusione delle vescicole sinaptiche (SV) con la membrana presinaptica permettendo il rilascio di neurotrasmettitore. Il taglio di una di queste importanti proteine provoca una riduzione nella funzionalità del complesso e quindi un blocco della neuroesocitosi: ciò determina la paralisi flaccida tipica del botulismo. I pazienti possono morire di insufficienza respiratoria ma, se le funzioni vitali vengono sostenute, essi recuperano completamente la mobilità in quanto la neuroparalisi è reversibile.
Il meccanismo d’azione delle BoNTs può essere riassunto in cinque fasi fondamentali: 1) riconoscimento specifico e legame al terminale sinaptico, 2) internalizzazione tramite il riciclo delle SV, 3) traslocazione pH-dipendente di L nel citoplasma 4) riduzione del ponte disolfuro intercatena e 5) idrolisi delle proteine SNARE.
In particolare, la BoNT/A e BoNT/E agiscono sulla SNAP-25, mentre BoNT/B, /D, /F e /G idrolizzano la VAMP. La BoNT/C è unica perché taglia sia SNAP-25 che la proteina sintaxina.
Le BoNTs sono le sostanze più velenose note, classificate dal Centro per il Controllo e la Prevenzione delle Malattie (CDC) come agenti in categoria A, cioè tossine potenzialmente utilizzabili come armi biologiche. Attualmente non esiste nessun vaccino approvato per l’uso umano e l'unico trattamento disponibile consiste nell'immunizzazione passiva con antisieri prodotti per contrastare i sette sierotipi principali e di conseguenza variabilmente reattivi nei confronti dei sottotipi. Questa situazione ha promosso un'intensa ricerca per sviluppare nuove antitossine.
Tuttavia, allo stesso tempo, la loro neurospecificità e la reversibilità della paralisi rendono le BoNTs degli agenti terapeutici di prima scelta per il trattamento di un’ampia varietà di malattie umane caratterizzate da iperattività dei terminali nervosi periferici.
Considerando questa dicotomia, il mio progetto di dottorato si può dividere in due parti principali aventi lo scopo di: i) sviluppare pan-inibitori in grado di prevenire/trattare il botulismo e ii) capire meglio il funzionamento delle BoNTs in vivo per migliorare e ampliare l'uso di queste molecole in terapia.
i) Considerando il meccanismo d’intossicazione delle BoNTs, uno step fondamentale è la riduzione del ponte disolfuro intercatena, senza la quale L rimane attaccata a H e non può esercitare la sua attività catalitica. Utilizzando un approccio farmacologico ho dimostrato che il sistema Tioredossina-Tioredossina Reduttasi (Trx-TrxR) è il principale responsabile della riduzione per tutti i sierotipi di tossina botulinica e che inibitori di questa coppia redox producono una sostanziale protezione dall’intossicazione sia in vitro che in vivo in un modello che ricapitola il botulismo. Questo risultato è importante perché è il primo che mostra che sierotipi diversi di BoNTs possono essere inibiti da un'unica molecola (pan-inibitore) che agisce sul meccanismo d’azione comune.
Un altro aspetto importante nella tossicità di queste neurotossine è il loro traffico all'interno del terminale nervoso. Questo potrebbe essere un altro target razionale.
Recentemente, è stato riportato che il composto chimico EGA inibisce agenti patogeni o tossine che necessitano del passaggio attraverso compartimenti acidi (endosomi) per poter penetrare nel citoplasma di cellule bersaglio. Dal momento che anche le BoNTs necessitano di condizioni simili perché avvenga la traslocazione di L nel citoplasma, ho saggiato l’effetto di questa molecola sull’azione delle BoNTs. Consistentemente, ho trovato che EGA inibisce significativamente l’attività neurotossica della BoNT/A, BoNT/B e BoNT/D in vitro e in vivo. Sono stati scelti questi tre sierotipi perché sono quelli più comunemente associati al botulismo umano (BoNT/A e /B) ed animale (BoNT/D). È interessante notare come nessuno degli steps del meccanismo d’azione sia direttamente inibito dalla molecola: ciò è compatibile con la possibilità che EGA interferisca con il traffico delle BoNTs all’interno del terminale nervoso ostacolando il raggiungimento del compartimento acido essenziale per la traslocazione di L.
Complessivamente, questi studi mostrano come l’attività delle BoNTs possa essere significativamente inibita, indipendentemente dalle loro differenze antigeniche, usando molecole che agiscano su steps comuni del meccanismo d’azione. Questi inibitori rappresentano lead compounds per lo sviluppo di farmaci capaci di prevenire il botulismo.
ii) Le BoNTs sono agenti terapeutici di successo. Nonostante il loro impiego sia quasi esclusivamente limitato alle BoNT/A e BoNT/B, dati recenti ottenuti su volontari umani suggeriscono che la BoNT/C possa essere utilizzata per trattare individui non rispondenti a BoNT/A e BoNT/B con risultati farmacologici altrettanto efficaci.
Tuttavia, al momento ci sono poche conoscenze riguardo al meccanismo con il quale la BoNT/C paralizza i terminali nervosi periferici in vivo: infatti, a differenza di tutti gli altri sierotipi, la BoNT/C è l'unica che idrolizza due substrati, SNAP-25 e sintaxina-1A/1B. Per questo motivo, ho intrapreso uno studio per valutare il contributo individuale del taglio di SNAP-25 e di quello della sintaxina nell’attività della BoNT/C in vivo.
Per fare questo mi sono basata su una recente pubblicazione in cui è stato riportato che due BoNT/C triple-mutanti, L200W/M221W/I226W (BoNT/C α-3W) e S51T/R52N/N53P (BoNT/C α-51), sono risultate selettive per la sintaxina.
Grazie alla collaborazione col gruppo del Dr. T. Binz, ho ottenuto queste due BoNT/C mutanti, prodotte con metodi ricombinanti, e ne ho testato le loro proprietà biochimiche e tossicologiche. Quello che ho scoperto è che entrambe le triple-mutanti idrolizzano la sintaxina con un’efficienza simile alla tossina WT (BoNT/C-wt) ma, inaspettatamente, entrambe mantengono anche un'attività residua nei confronti della SNAP-25 (più elevata per la BoNT/C α-3W rispetto alla BoNT/C α-51). È interessante notare come questa attività sulla SNAP-25 sia strettamente correlata alla letalità delle tossine mutanti in vivo. D’altra parte, la proteolisi della sintaxina fornisce una sostanziale e prolungata compromissione neuromuscolare senza provocare il completo blocco della neurotrasmissione. Questi risultati suggeriscono che il taglio di SNAP-25 sia il principale determinante dell'attività neuroparalizzante e che derivati della BoNT/C, aventi attività selettiva per le sintaxina, potrebbero rappresentare una buona strategia per lo sviluppo di BoNTs dotate di attività prolungata e con un ampio margine di sicurezza.

EPrint type:Ph.D. thesis
Tutor:Montecucco, Cesare
Ph.D. course:Ciclo 30 > Corsi 30 > SCIENZE BIOMEDICHE SPERIMENTALI
Data di deposito della tesi:12 January 2018
Anno di Pubblicazione:12 January 2018
Key Words:Neurotossine botuliniche; Sistema della tioredossina; Neuroparalisi periferica; Tossine mutanti Botulinum neurotoxins; Thioredoxin system; Peripheral neuroparalysis; Mutant toxins
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/04 Patologia generale
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Biomediche
Codice ID:10717
Depositato il:25 Oct 2018 15:41
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