Enrico Ferrari †‡, Chunjing Gu †, Dhevahi Niranjan†, Laura Restani §, Christine Rasetti-Escargueil 
,Ilona Obara 
#, Sandrine M. Geranton , Jason Arsenault †, Tom A. Goetze ¶, Callista B. Harper □,Tam H. Nguyen □, Elizabeth Maywood †, John O’Brien †, Giampietro Schiavo ●, Daniel W. Wheeler
, Frederic A. Meunier □, Michael Hastings †, J. Michael Edwardson ¶, Dorothea Sesardic , Matteo Caleo §, Stephen P. Hunt , and Bazbek Davletov *†
,Ilona Obara #, Sandrine M. Geranton , Jason Arsenault †, Tom A. Goetze ¶, Callista B. Harper □,Tam H. Nguyen □, Elizabeth Maywood †, John O’Brien †, Giampietro Schiavo ●, Daniel W. Wheeler, Frederic A. Meunier □, Michael Hastings †, J. Michael Edwardson ¶, Dorothea Sesardic , Matteo Caleo §, Stephen P. Hunt , and Bazbek Davletov *†
† MRC Laboratory of Molecular Biology, Cambridge,United Kingdom
‡ School of Life Sciences, University of Lincoln, Lincoln, United Kingdom
§ Istituto di Neuroscienze, Consiglio Nazionale delle Ricerche, Pisa, Italy
National Institute for Biological Standard and Control, Health Protection Agency, Potters Bar, United Kingdom Department of Cell and Developmental Biology,University College London, London, United Kingdom
# School of Medicine, Pharmacy and Health, University of Durham, Stockton on Tees, United Kingdom
¶ Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom
□ The Queensland Brain Institute, University of Queensland, St Lucia, Australia
● London Research Institute, Cancer Research U.K., London, United Kingdom
Department of Anaesthesia, Addenbrooke’s Hospital, Cambridge, United Kingdom
Bioconjugate Chem., 2013, 24 (10), pp 1750–1759
DOI: 10.1021/bc4003103
Publication Date (Web): September 6, 2013
Copyright © 2013 American Chemical Society
*E-Mail: b.davletov@sheffield.ac.uk; Tel.: +44-114-2225111.
ABSTRACTClostridial neurotoxins reversibly block neuronal communication for weeks and months. While these proteolytic neurotoxins hold great promise for clinical applications and the investigation of brain function, their paralytic activity at neuromuscular junctions is a stumbling block. To redirect the clostridial activity to neuronal populations other than motor neurons, we used a new self-assembling method to combine the botulinum type A protease with the tetanus binding domain, which natively targets central neurons. The two parts were produced separately and then assembled in a site-specific way using a newly introduced ‘protein stapling’ technology. Atomic force microscopy imaging revealed dumbbell shaped particles which measure 23 nm. The stapled chimera inhibited mechanical hypersensitivity in a rat model of inflammatory pain without causing either flaccid or spastic paralysis. Moreover, the synthetic clostridial molecule was able to block neuronal activity in a defined area of visual cortex. Overall, we provide the first evidence that the protein stapling technology allows assembly of distinct proteins yielding new biomedical properties.
PRESS RELEASE FROM THE UNIVERSITY OF LINCOLN, October 2013
Scientists have manufactured a new bio-therapeutic molecule that could be used to treat neurological disorders such as chronic pain and epilepsy.
A team of 22 scientists from 11 research institutes, including Dr Enrico Ferrari from the University of Lincoln, UK, created and characterised a new molecule that was able to alleviate hypersensitivity to inflammatory pain.
The work is featured on the cover of the October 2013 issue of the scientific journal Bioconjugate Chemistry.
Dr Ferrari joined the School of Life Sciences in October last year from the Medical Research Council’s Laboratory of Molecular Biology in Cambridge, where he took part in the development of a new way of joining and rebuilding molecules in the research group of Professor Bazbek Davletov - now at the University of Sheffield.
Now, by separating elements of clostridium botulinum and clostridium tetani neurotoxins, commonly known as Botox and tetanus toxin respectively, the scientists were able to develop a model to re-join the molecule proteins yielding new biomedical properties, without unwanted toxic effects.
While the Botox element is able to block neuronal communication – and therefore pain signals - for months, the tetanus component targets the engineered toxin to the central nervous system, rather than stopping at exterior neurons that are the normal target of Botox. The combination of the two effects is of great interest for neuroscience and can be applied to the treatment of several neurological disorders, particularly chronic pain conditions.
Botox and tetanus neurotoxins hold great promise for clinical applications, but since they are the most lethal proteins known to man, their paralytic activity was a stumbling block until now.
Dr Ferrari, who is one of the lead authors of the study, said: “The toxins were split into parts so they were unable to function. Then later they were reassembled using a ‘zipping’ system so they can operate in a safe way. The re-engineered chimera toxin has very similar characteristics to Botox and is still able to block neurotransmission release, but the paralytic effect is a lot less. We then added a tetanus molecule which targets the chimera to where the pain signals travel towards the central nervous system.”
Preliminary data on animal models has now been collated at University College London and future clinical trials are expected to fully characterise the new bio-therapeutic.
Dr Ferrari added: “Many painkillers relieve the pain temporarily and have various side effects. The selling point of this molecule is that the pain relief could last up to seven months, in a similar way that Botox injections for removing wrinkles last for several months. Engineering this kind of toxin has many uses and would be a major improvement in the quality of life for those people who suffer from chronic pain. It is very exciting to know that a protein you made could be one of the future drug treatments.”
The crux of Dr Ferrari’s research is now aimed at creating a method where more than two protein elements can be combined together and their exact order dictated, which will open up further avenues to explore possible medical uses in the future.
Synthetic Self-Assembling Clostridial Chimera for Modulation of Sensory Functions in Bioconjugate Chemistry, DOI: 10.1021/bc4003103
© 2013 L. Ryan
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