Team:Tuebingen/Description
Project
A creative project is a moving target. You never end up where you start.- Evangeline Lilly
Introduction
BoNT C - Licence to enter
In modern medicine treatment options involve many substances modified from natural sources, occasionally even toxins.
We modify botulinum toxin in a way that leads to its detoxification. Thus, it can be coupled with a variety of other substances while not losing its specific shuttle mechanism for neuronal cells. In detail, we develop a library of different detoxified botulinum toxin derivatives which can accommodate other proteins, small molecules, and fluorochromes by specific linkers.
To investigate the influence of the point mutations leading to detoxification in the active site, we conduct MD simulations. Since our shuttle mechanism could potentially be used in patients, we remove the most prevalent immune epitopes by a theoretical bioinformatics approach.
Ultimately, our system is supposed to be utilized for therapy strategies and specific neuronal targeting in basic research.
With our project we want to encourage future teams to think outside the box while keeping safety in mind.
Background
Botulinum Neurotoxins (BoNTs) are proteins produced by the bacterium Clostridium botulinum. These proteins are very potent neuro-toxins which prevent muscle contraction via inhibition of motor neurons. The bacteria themselves can be mostly found in forest soil and start to proliferate under anaerobic conditions. These conditions are met after uptake by animals in their intestine. After release of the toxin, infection of motor neurons and finally the animal’s death, bacteria proliferation even increases in the rotting process of the carcass. After uptake of the rotten flesh by scavengers the cycle begins anew. For humans, botulism - the disease caused by BoNT - becomes mainly a threat when tinned food is insufficiently conserved which provides an anaerobic environment for the clostridia. Left untreated, the disease can lead to respiratory paralysis and death.
The BoNTs belong to the family of AB toxins and can be divided into the serotypes A-H. As an AB-toxin, BoNT consists of two protein chains, the heavy chain (HC) and light chain (LC), which are connected by a highly conserved disulfide bond and multiple noncovalent interactions.
The mechanism underlying the cell specific infiltration by BoNT is partly understood, yet many questions remain: For cell recognition, BoNT uses gangliosides in the presynaptic membrane to select for neuronal cells (Benson et al 2011). Besides the gangliosides themselves, which are enriched in neuronal cells, binding of BoNT requires a linkage between gangliosides and N-acetylneuraminic acid (Sia5) (Strotmeier et al 2011). The subsequent infiltration process is performed by the HC binding to the membrane and further unknown receptors. HC and LC then undergo endocytosis until only the LC is released into the cytosol. Lastly, the LC, being a zinc metalloprotease, cleaves Snap-25 and other proteins of the SNARE complex depending on the toxin’s serotype. This leads to prevention of neurotransmitter release into the synaptic cleft and therefore muscle contraction at the neuromuscular end plate.
Since BoNT is so highly specific for neuronal cells, its’ potency and therefore toxicity is among the highest for peptide toxins in the world. For BoNT serotype C the lethal concentration (LD50) in humans is approximately 1ng/kg (parenteral application) and 1µg/kg (oral application). The high toxicity would therefore prevent from any usage of the specific shuttle mechanism. In 2017, three missense mutations in the active site of the LC were reported that conserve the overall structure and binding affinity of BoNTC, however reduce the activity and overall toxicity by the factor 10^6. (Vazquez-Cintron et al 2017)
This option of an efficient detoxification provides us with a very powerful tool that might be used to transport a variety of molecules into neuronal cells with a high specitivity: Synthetic drugs, therapeutic peptides and labeling proteins for fundamental research imported this way might revolutionize neuronal targeting strategies to increase our overall knowledge about the complexity of the brain and the chances to treat severe neuronal diseases.
Project Development
Chemistry
For our alkynyl-botulinus toxin we planned to use a fluorescent molecule as a proof of principle for bioorthogonal conjugation. We already tried to create a drug-conjugate useful for further investigations by using the Na+-channel blocker Eslicarbazepine.
Fluorescent molecule
Since there are already commercially available azide-linked fluorophores we saw no need to functionalise them further.
We obtained Azide-Fluor488 (figure 1) as a kind sponsorship from Sigma-Aldrich.
To see if a site-specific modification is indeed beneficial, we also employed a BODIPY-fluorophore as NHS-ester to nonspecifically modify solvent-exposed lysines of our botulinus toxin.
This NHS-ester is also commercially available and was obtained as a sponsorship from Lumiprobe. (See figure 2)
Hier sollen die Bilder hin
Hier sollen die Bilder hin
Eslicarbazepine
Having an actual drug as a conjugate is more complicated than the use of a fluorophore by several orders of magnitude.
For instance a 50 kDa peptide covalently linked to a 250 Da small molecule would likely inhibit it’s function to act on the target proteins. Therefore we chose to investigate a cleavable linker. As already applied by the group of Peter G. Schulz in 2015 we chose a disulfide linker that would be cleaved by the intracellular glutathione.
Therefore, the general strategy from Eslicarbazepine was: R-OH to R-SH to R-SS-R’-NH3 to R-SS-R’-N3 to create an azide group for bioorthogonal chemistry linked to our drug via a disulfide linker.
Hier ganz toll das Bild vom flow einbinden
A method for a one-pot synthesis of sulfhydryls from hydroxy compounds has already been published. Therefore we planned to follow their strategy to employ Lawesson's reagent in Dimethoxyethane.
We then wished to use a disulfide exchange to create a mixed disulfide using cysteamine. A similar reaction has been used to create a disulfide linker with antibody drug conjugates.
An alternative option was found with S-tosyl mercaptoethanolamine. A similar reaction has been used in the total synthesis of ajoene in 2018.
For the final step we would have to change the amine group to an azide group to enable this compound for azide-alkyne click reactions. These transformations usually involve rather dangerous substances, such as triflyl azide or imidazole-1-sulfonyl azide which is known to be explosive. The latter of which, however, can be prepared as a hydrogen sulfate as a stable and safe reagent. As this compound is to our knowledge not commercially available for a reasonable price, we decided to synthesise it ourselves from imidazole, sulfuryl chloride and sodium azide. (See figure 4)
Hier ganz toll das Bild vom Azid-D einbinden
Safety
F. Silva, S. S. Khokhar, D. M. Williams, R. Saunders, G. J. S. Evans, M. Graz, T. Wirth, Angew. Chem. 2018, 130, 12470.
An Efficient, Inexpensive, and Shelf-Stable Diazotransfer Reagent: Imidazole-1-sulfonyl Azide Hydrochloride Ethan D. Goddard-Borger and and Robert V. Stick Organic Letters 2007 9 (19), 3797-3800 DOI: 10.1021/ol701581g
Sensitivities of Some Imidazole-1-sulfonyl Azide Salts Niko Fischer, Ethan D. Goddard-Borger, Robert Greiner, Thomas M. Klapötke, Brian W. Skelton, and Jörg Stierstorfer The Journal of Organic Chemistry 2012 77 (4), 1760-1764 DOI: 10.1021/jo202264r