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Fastox: A novel, rapid, and short-acting modified botulinum neurotoxin
Abstracts / Toxicon 123 (2016) S2eS90
limb function after stroke. Cochrane Database Syst Rev. 2014;11:CD010820. http://dx.doi.org/10.1002/14651858.CD010820.pub2. Ramachandran VS, Rogers-Ramachandran D, Cobb S. Touching the phantom limb. Nature. 1995;377(6549):489-490. Simpson DM, Gracies JM, Graham HK, et al. Assessment: botulinum neurotoxin for the treatment of spasticity (an evidence-based review): Report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology. Neurology. 2008;70(19):1691-1698. Urban PP, Wolf T, Uebele M, et al. Occurrence and clinical predictors of spasticity after ischemic stroke. Stroke. 2010;41(9):2016-2020. 196. MODIFIED RBONT/A1 MUTANTS WITH ACCELERATED ONSET AND SHORTER DURATION OF EFFECT pez de la Daniel Scheps, Fred Hofmann, Marcel Jurk, Manuela Lo Paz, Jürgen Frevert*. Merz Pharmaceuticals GmbH, Potsdam, Germany * Corresponding author: Merz Pharmaceuticals GmbH Potsdam, Hermannswerder Haus 15, 14473 Potsdam, Germany. E-mail address:[email protected].
Introduction and objectives: Serotypes of botulinum neurotoxins are hallmarked by different onsets and durations of effect. BoNT/E shows a fast onset but a short duration of paralysis, whereas the onset of BoNT/A is delayed in time and the effect lasts markedly longer. The kinetics of the paralysis may be controlled by the amino acid sequence of the respective neurotoxins. Methods: rBoNT/A1 with different mutations in the C-terminus of the light chain (LC) was expressed in Escherichia coli, purified to homogeneity, and subsequently activated with thrombin. The in vivo activity of the mutants was analyzed in the digit abduction score (DAS) assay and in the voluntary mouse running assay (MRA). The wild-type BoNT/A (Xeomin) was analyzed in parallel. Results: Mutations in the C-terminus of BoNT/A-LC resulted in a markedly faster onset and a shorter duration of the paralytic effect in the MRA compared with the wild-type BoNT/A (Xeomin). The effect was dependent on the position of the mutation in the sequence of the C-terminus. A mutant with 4 mutations showed behavior similar to that of BoNT/E in the MRA: the maximum effect was achieved at day 2, and the effect ended at days 5 to 6. Conclusions: The sequence of the C-terminus of BoNT/A-LC at least partly controls onset and duration of effect. It is possible to create a mutated BoNT/A with different pharmacologic properties that might be useful in the therapy of new indications. Keywords: Duration of effect; Light chain; Mouse running assay; Onset of effect; Recombinant BoNT/A 197. PROPERTIES OF A CHIMERIC BONT/A1-A4 Daniel Scheps, Michael Schmidt, Fred Hofmann, Marcel Jurk, Manuela pez de la Paz, Jürgen Frevert*. Lo Merz Pharmaceuticals GmbH, Potsdam, Germany * Corresponding author: Merz Pharmaceuticals GmbH Potsdam, Hermannswerder Haus 15, 14473 Potsdam, Germany. E-mail address:[email protected].
Introduction and objectives: According to reports in the literature, BoNT/ A4 cannot be expressed in Escherichia coli. To prepare this BoNT/A subtype, it was necessary to employ a clostridial expression system. The recombinant BoNT/A4 showed a 1000-fold reduced specific potency compared with BoNT/A1 wild type. To analyze the pharmacologic properties of the light chain (LC) of this subtype, a chimeric BoNT/A1-A4 was designed to be expressed in E coli. Methods: rBoNT/A1-A4 consisting of the LC of A4 and the heavy chain (HC) of BoNT/A1 equipped with a thrombin cleavage site was expressed in E coli. After purification, BoNT/A1-A4 was activated by cleavage with thrombin, whereby the affinity tags were removed as well. The in vivo activity of the chimeric protein was analyzed in the digit abduction score assay (DAS) and in the voluntary mouse running assay (MRA). The wild type BoNT/A (Xeomin) was analyzed in parallel.
S75
Results: The specific potency of the activated BoNTA1-A4 was reduced when compared with BoNT/A1 but markedly higher than the specific potency of BoNT/A4 according to the reported value in the literature. In comparison with the BoNT/A1 wild type (Xeomin), the chimera showed a later onset of effect in the DAS and in the MRA. The recovery from the paralytic effect was also shifted to a slightly later time point when assessed in the MRA. Conclusions: In contrast to BoNT/A4, the chimeric BoNT/A1-A4 could be purified from E coli. Pharmacologic properties of the chimeric protein differed markedly from the parent neurotoxins BoNT/A1 and BoNT/A4. It can be speculated that the low specific potency of BoNT/A4 is based on a low binding affinity of its HC. Keywords: BoNT/A1; BoNT/A4; Duration of effect; Onset of paralysis; Recombinant BoNT/A subtypes 198. TRAFFICKING OF TETANUS AND BOTULINUM NEUROTOXINS IN THE CNS Giampietro Schiavo*, Ione Meyer, Sergey Novoselov, Sunaina Surana, Deniz Tiknaz, Andrew Tosolini. Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, University College London, UK * Corresponding author: Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, University College London, London WC1N 3BG, UK. E-mail address:[email protected].
Introduction and objectives: Tetanus neurotoxin (TeNT) is a major cause of neonatal death in nonvaccinated areas, while botulinum neurotoxins (BoNTs) are responsible for botulism in humans and animals. TeNT and BoNTs block neurotransmitter release by cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, yet their clinical symptoms are very different. BoNTs enter neuromuscular junctions (NMJs) and halt synaptic vesicle fusion mainly at this location, thus inducing a flaccid paralysis. In contrast, TeNT is sorted to the axonal retrograde transport pathway, and it is targeted to the plasma membrane of spinal cord motor neurons. It is then internalized and blocks inhibitory interneurons, causing a persistent spastic paralysis. TeNT targets the NMJ with high affinity, yet understanding of the nature of the TeNT receptor complex at this site is lacking. This work aims to identify the mechanism of internalization of TeNT at the NMJ. Methods: We used a combination of biochemistry of neuronal signaling endosomes, proteomics, imaging, and mouse genetics to identify the NMJ receptor of TeNT. Results: We showed that nidogens are the main determinant for TeNT binding at the NMJ. Inhibition of TeNT-nidogen interaction prevented the binding of TeNT to neurons. Furthermore, a nidogen-derived peptide specifically protected mice from TeNT-induced spastic paralysis. Our findings demonstrated that TeNT is endocytosed via an efficient capture mechanism at specialized NMJ sites, which could concentrate TeNT as well as physiologic ligands for their sorting to axonal transport organelles. The relevance of this mechanism for BoNT uptake and retargeting will be discussed, together with the potential application of these peptides for therapeutic use. Conclusions: Specific components of the basal membrane play a major role in the recognition, uptake, and targeting of TeNT along the axonal retrograde transport route, raising the possibility that this is a general pathway for distribution of ligand-receptor complexes in the CNS. Keywords: Axonal retrograde transport; Basal lamina; Botulinum neurotoxins; Nidogens; Tetanus toxin 199. FASTOX: A NOVEL, RAPID, AND SHORT-ACTING MODIFIED BOTULINUM NEUROTOXIN Michael Schmidt a, Fred Hofmann a, Tim Stoeveken b, Kerstin Hoelscher b, Swen Grein b, Nadine Krause c, Cara Heers c, Christine Janaitis c, Gerd Mander b, Harold V. Taylor b, Klaus Fink b, *, Juergen Frevert a. a Department of Botulinum Toxin Research, Merz Pharmaceuticals, Potsdam, Germany; b Department of Biotechnology, Merz Pharma GmbH & Co KGaA, Frankfurt, Germany; c Department of Non-Clinical Science, Merz Pharmaceuticals, Frankfurt, Germany
limb function after stroke. Cochrane Database Syst Rev. 2014;11:CD010820. http://dx.doi.org/10.1002/14651858.CD010820.pub2. Ramachandran VS, Rogers-Ramachandran D, Cobb S. Touching the phantom limb. Nature. 1995;377(6549):489-490. Simpson DM, Gracies JM, Graham HK, et al. Assessment: botulinum neurotoxin for the treatment of spasticity (an evidence-based review): Report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology. Neurology. 2008;70(19):1691-1698. Urban PP, Wolf T, Uebele M, et al. Occurrence and clinical predictors of spasticity after ischemic stroke. Stroke. 2010;41(9):2016-2020. 196. MODIFIED RBONT/A1 MUTANTS WITH ACCELERATED ONSET AND SHORTER DURATION OF EFFECT pez de la Daniel Scheps, Fred Hofmann, Marcel Jurk, Manuela Lo Paz, Jürgen Frevert*. Merz Pharmaceuticals GmbH, Potsdam, Germany * Corresponding author: Merz Pharmaceuticals GmbH Potsdam, Hermannswerder Haus 15, 14473 Potsdam, Germany. E-mail address:[email protected].
Introduction and objectives: Serotypes of botulinum neurotoxins are hallmarked by different onsets and durations of effect. BoNT/E shows a fast onset but a short duration of paralysis, whereas the onset of BoNT/A is delayed in time and the effect lasts markedly longer. The kinetics of the paralysis may be controlled by the amino acid sequence of the respective neurotoxins. Methods: rBoNT/A1 with different mutations in the C-terminus of the light chain (LC) was expressed in Escherichia coli, purified to homogeneity, and subsequently activated with thrombin. The in vivo activity of the mutants was analyzed in the digit abduction score (DAS) assay and in the voluntary mouse running assay (MRA). The wild-type BoNT/A (Xeomin) was analyzed in parallel. Results: Mutations in the C-terminus of BoNT/A-LC resulted in a markedly faster onset and a shorter duration of the paralytic effect in the MRA compared with the wild-type BoNT/A (Xeomin). The effect was dependent on the position of the mutation in the sequence of the C-terminus. A mutant with 4 mutations showed behavior similar to that of BoNT/E in the MRA: the maximum effect was achieved at day 2, and the effect ended at days 5 to 6. Conclusions: The sequence of the C-terminus of BoNT/A-LC at least partly controls onset and duration of effect. It is possible to create a mutated BoNT/A with different pharmacologic properties that might be useful in the therapy of new indications. Keywords: Duration of effect; Light chain; Mouse running assay; Onset of effect; Recombinant BoNT/A 197. PROPERTIES OF A CHIMERIC BONT/A1-A4 Daniel Scheps, Michael Schmidt, Fred Hofmann, Marcel Jurk, Manuela pez de la Paz, Jürgen Frevert*. Lo Merz Pharmaceuticals GmbH, Potsdam, Germany * Corresponding author: Merz Pharmaceuticals GmbH Potsdam, Hermannswerder Haus 15, 14473 Potsdam, Germany. E-mail address:[email protected].
Introduction and objectives: According to reports in the literature, BoNT/ A4 cannot be expressed in Escherichia coli. To prepare this BoNT/A subtype, it was necessary to employ a clostridial expression system. The recombinant BoNT/A4 showed a 1000-fold reduced specific potency compared with BoNT/A1 wild type. To analyze the pharmacologic properties of the light chain (LC) of this subtype, a chimeric BoNT/A1-A4 was designed to be expressed in E coli. Methods: rBoNT/A1-A4 consisting of the LC of A4 and the heavy chain (HC) of BoNT/A1 equipped with a thrombin cleavage site was expressed in E coli. After purification, BoNT/A1-A4 was activated by cleavage with thrombin, whereby the affinity tags were removed as well. The in vivo activity of the chimeric protein was analyzed in the digit abduction score assay (DAS) and in the voluntary mouse running assay (MRA). The wild type BoNT/A (Xeomin) was analyzed in parallel.
S75
Results: The specific potency of the activated BoNTA1-A4 was reduced when compared with BoNT/A1 but markedly higher than the specific potency of BoNT/A4 according to the reported value in the literature. In comparison with the BoNT/A1 wild type (Xeomin), the chimera showed a later onset of effect in the DAS and in the MRA. The recovery from the paralytic effect was also shifted to a slightly later time point when assessed in the MRA. Conclusions: In contrast to BoNT/A4, the chimeric BoNT/A1-A4 could be purified from E coli. Pharmacologic properties of the chimeric protein differed markedly from the parent neurotoxins BoNT/A1 and BoNT/A4. It can be speculated that the low specific potency of BoNT/A4 is based on a low binding affinity of its HC. Keywords: BoNT/A1; BoNT/A4; Duration of effect; Onset of paralysis; Recombinant BoNT/A subtypes 198. TRAFFICKING OF TETANUS AND BOTULINUM NEUROTOXINS IN THE CNS Giampietro Schiavo*, Ione Meyer, Sergey Novoselov, Sunaina Surana, Deniz Tiknaz, Andrew Tosolini. Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, University College London, UK * Corresponding author: Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, University College London, London WC1N 3BG, UK. E-mail address:[email protected].
Introduction and objectives: Tetanus neurotoxin (TeNT) is a major cause of neonatal death in nonvaccinated areas, while botulinum neurotoxins (BoNTs) are responsible for botulism in humans and animals. TeNT and BoNTs block neurotransmitter release by cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, yet their clinical symptoms are very different. BoNTs enter neuromuscular junctions (NMJs) and halt synaptic vesicle fusion mainly at this location, thus inducing a flaccid paralysis. In contrast, TeNT is sorted to the axonal retrograde transport pathway, and it is targeted to the plasma membrane of spinal cord motor neurons. It is then internalized and blocks inhibitory interneurons, causing a persistent spastic paralysis. TeNT targets the NMJ with high affinity, yet understanding of the nature of the TeNT receptor complex at this site is lacking. This work aims to identify the mechanism of internalization of TeNT at the NMJ. Methods: We used a combination of biochemistry of neuronal signaling endosomes, proteomics, imaging, and mouse genetics to identify the NMJ receptor of TeNT. Results: We showed that nidogens are the main determinant for TeNT binding at the NMJ. Inhibition of TeNT-nidogen interaction prevented the binding of TeNT to neurons. Furthermore, a nidogen-derived peptide specifically protected mice from TeNT-induced spastic paralysis. Our findings demonstrated that TeNT is endocytosed via an efficient capture mechanism at specialized NMJ sites, which could concentrate TeNT as well as physiologic ligands for their sorting to axonal transport organelles. The relevance of this mechanism for BoNT uptake and retargeting will be discussed, together with the potential application of these peptides for therapeutic use. Conclusions: Specific components of the basal membrane play a major role in the recognition, uptake, and targeting of TeNT along the axonal retrograde transport route, raising the possibility that this is a general pathway for distribution of ligand-receptor complexes in the CNS. Keywords: Axonal retrograde transport; Basal lamina; Botulinum neurotoxins; Nidogens; Tetanus toxin 199. FASTOX: A NOVEL, RAPID, AND SHORT-ACTING MODIFIED BOTULINUM NEUROTOXIN Michael Schmidt a, Fred Hofmann a, Tim Stoeveken b, Kerstin Hoelscher b, Swen Grein b, Nadine Krause c, Cara Heers c, Christine Janaitis c, Gerd Mander b, Harold V. Taylor b, Klaus Fink b, *, Juergen Frevert a. a Department of Botulinum Toxin Research, Merz Pharmaceuticals, Potsdam, Germany; b Department of Biotechnology, Merz Pharma GmbH & Co KGaA, Frankfurt, Germany; c Department of Non-Clinical Science, Merz Pharmaceuticals, Frankfurt, Germany