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.

Botulinum Neurotoxins (BoNTs) are Proteins produced by the anaerobic bacterium clostridium botulinum. Thees proteins are very potent neuro toxins which prevents muscle contraction. The bacteria occur in rotten flesh and cause the disease botulism. This occurs especially in nutztieren but can also occur in humans and in infants. Left untreated, the disease can lead to respiratory paralysis and death. There are several serotypes A-H of the BoNTs. They all have the same basic structure. The toxins consist of a heavy chain and a light chain which are connectet with a high conserved disulfied bond and noncovalent interactions. Both chains have special tasks that make the toxins so special and provied the extreme toxicity. The HC is responsible for the specific binding to the presynaptic membrane, ensuing of the whole Protein and translocation of the LC into the neuronal cytosol. The LC is a metalloproteas, cleaving specific Snare Proteins in the neuron. As a result, no neurotrasmitters are released and the muscles no longer contract. mechanism of the Toxin The first step is the specific binding to the presynaptic neuronal membrane through the HC c-terminus. This leads to endoocytosis of the Botulinum toxin in to the neuron. Next, the translocation of the LC from the endosome into the cytosol takes place through the N-terminal part of the HC. There, the Zn2 + protease specifically cuts Snap-25 and Synthaxin. Both proteins play a major role in the release of neurotransmitters. The LC cuts these proteins so that no release of neurotransmitters takes place and so no muscle contraction. Scheme of uptake of botulinum toxin The mechanism of action has two very interesting features that we want to take advantage of: 1) high specificity of neurons (a spdc cell type) 2) Infiltration of the cell / mechanism to overcome the cell membrane We want to use these properties to establish a shuttle mechanism to specifically target proteins and other molecules to neurons. To achieve this, however, the toxicity of botulinum toxin must be neutralized while preserving the transport mechanism. By 3 published point mutations in the protein sequence of the LC of the BoNT C, the activity of the metalloprotease is extremely lowered and thereby also the toxicity by a factor of about 10 6.