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
Safety