Fred Hofmann

Design of modified botulinum neurotoxin A1 variants with a shorter persistence of paralysis and duration of action

ABSTRACT Botulinum neurotoxins (BoNTs) are classified by their antigenic properties into seven serotypes (A‐G) and in addition by their corresponding subtypes. They are further characterized by divergent onset and duration of effect. Injections of low doses of botulinum neurotoxins cause localized muscle paralysis that is beneficial for the treatment of several medical disorders and aesthetic indications. Optimizing the therapeutic properties could offer new treatment opportunities. This report describes a rational design approach to modify the pharmacological properties by mutations in the C‐terminus of BoNT/A1 light chain (LC). Toxins with C‐terminal modified LCs displayed an altered onset and duration of the paralytic effect in vivo. The level of effect was dependent on the kind of the mutation in the sequence of the C‐terminus. A mutant with three mutations (T420E F423M Y426F) revealed a faster onset and a shorter duration than BoNT/A1 wild type (WT). It could be shown that the C‐terminus of BoNT/A1‐Lc controls both onset and duration of effect. Thus, it is possible to create a mutated BoNT/A1 with different pharmacological properties which might be useful in the therapy of new indications. This strategy opens the way to design BoNT variants with novel and useful properties. Highlights6 Mutants of Botulinum neurotoxin type A with mutation in the C‐Terminus of the light chain were investigated.Two mutants with mutations in positions 420, 426 and 429 resulted in a faster onset and a shorter duration of effect.The potency of these two mutants in vivo was only slightly reduced in contrast to other mutants.The mutants are possible candidates for therapeutics for indications requiring only a short paralysis of injected muscleso.

Rational design of botulinum neurotoxin A1 mutants with improved oxidative stability

&NA; Botulinum neurotoxins (BoNTs) are the most potent toxic proteins to mankind known but applied in low doses trigger a localized muscle paralysis that is beneficial for the therapy of several neurological disorders and aesthetic treatment. The paralytic effect is generated by the enzymatic activity of the light chain (LC) that cleaves specifically one of the SNARE proteins responsible for neurotransmitter exocytosis. The activity of the LC in a BoNT‐containing therapeutic can be compromised by denaturing agents present during manufacturing and/or in the cell. Stabilization of the LC by reducing vulnerability towards denaturants would thus be advantageous for the development of BoNT‐based therapeutics. In this work, we focused on increasing the stability of LC of BoNT/A1 (LC/A1) towards oxidative stress. We tackled this task by rational design of mutations at cysteine and methionine LC/A1 sites. Designed mutants showed improved oxidative stability in vitro and equipotency to wildtype toxin in vivo. Our results suggest that suitable modification of the catalytic domain can lead to more stable BoNTs without impairing their therapeutic efficacy. HighlightsStabilization of the light chain of botulinum neurotoxin A1 (LC/A1) towards oxidation was approached by rational design.All methionine and cysteine residues of LC/A1 were considered for the in silico design of mutants.Designed mutants were selected based on predicted hydropathy and stability changes respect to wildtype (WT) LC/A1.The mutant M106L, M253L, M411Q was more resistant against oxidation in vitro than WT toxin without loss of potency in vivo.