Abstract
Neurological diseases constitute a quarter of global disease burden and are expected to rise worldwide with the ageing of human populations. There is an increasing need to develop new molecular systems which can deliver drugs specifically into neurons, non-dividing cells meant to last a human lifetime. Neuronal drug delivery must rely on agents which can recognise neurons with high specificity and affinity. Here we used a recently introduced ‘stapling’ system to prepare macromolecules carrying duplicated binding domains from the clostridial family of neurotoxins. We engineered individual parts of clostridial neurotoxins separately and combined them using a strong alpha-helical bundle. We show that combining two identical binding domains of tetanus and botulinum type D neurotoxins, in a sterically defined way by protein stapling, allows enhanced intracellular delivery of molecules into neurons. We also engineered a botulinum neurotoxin type C variant with a duplicated binding domain which increased enzymatic delivery compared to the native type C toxin. We conclude that duplication of the binding parts of tetanus or botulinum neurotoxins will allow production of high avidity agents which could deliver imaging reagents and large therapeutic enzymes into neurons with superior efficiency.
Highlights
The intractable nature of many neurological disorders demands new approaches to deliver medications into neurons with high precision
Identification of the sensitive factor Attachment Protein REceptors (SNAREs) proteins in the early 1990s led to a breakthrough in our understanding of the actions of the most potent toxins known to man – clostridial neu rotoxins, tetanus toxin and botulinum neurotoxins (BoNTs), all of which cause long-lasting and yet reversible paralysis (Rossetto et al, 2014; Hayashi et al, 1994)
We investigated whether duplication of the tetanus binding domain can enhance neuronal delivery of the botulinum type A protease (LCHn/A)
Summary
The intractable nature of many neurological disorders demands new approaches to deliver medications into neurons with high precision. These neurotoxins are precisely engineered proteins composed of three domains with their specific autonomous functions (Montal, 2010) Both tetanus toxin and BoNTs are 150-kDa proteins consisting of a 50-kDa enzymatic SNARE-cleaving light chain (LC) linked by a disulphide bond to a 100-kDa heavy chain, which contains two major structural domains – binding and translocation do mains (Montal, 2010). The lasting persistence of cleaved SNAP-25 in BoNT/A-intoxicated neurons allowed a design of a unique antibody which recognises the BoNT/A-generated cleaved end of SNAP-25 (Yadirgi et al, 2017) This neoepitope antibody proved to be useful to observe the location of BoNT/A action both in vitro and in vivo and we exploited this antibody to study the effects of delivering botulinum enzyme using duplicated clostridial binding domains. We observed an increase in the speed of action of novel botulinum con structs, which could be exploited for potential therapeutic benefits
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