Botulinum neurotoxin (
BoNT), a Category A biodefense agent, delivers a
protease to motor neuron cytosol that cleaves one or more
soluble NSF attachment protein receptors (
SNARE) proteins involved in neurotransmission to cause a flaccid
paralysis. No antidotes exist to reverse symptoms of BoNT intoxication so severely affected patients require
artificial respiration with prolonged
intensive care. Time to recovery depends on toxin serotype because the intraneuronal persistence of the seven known BoNT serotypes varies widely from days to many months. Our therapeutic
antidote strategy is to develop 'targeted F-box' (TFB) agents that target the different intraneuronal BoNT
proteases for accelerated degradation by the
ubiquitin proteasome system (UPS), thus promoting rapid recovery from all serotypes. These agents consist of a camelid heavy chain-only V(H) (VHH) domain specific for a BoNT
protease fused to an F-box domain recognized by an intraneuronal E3-ligase. A fusion
protein containing the 14 kDa anti-
BoNT/A protease VHH, ALcB8, joined to a 15 kDa F-box domain region of TrCP (D5) was sufficient to cause increased ubiquitination and accelerate turnover of the targeted
BoNT/A protease within neurons. Neuronal cells expressing this TFB, called D5-B8, were also substantially resistant to
BoNT/A intoxication and recovered from intoxication at least 2.5 fold quicker than control neurons. Fusion of D5 to a VHH specific for BoNT/B
protease (BLcB10) led to accelerated turnover of the targeted
protease within neurons, thus demonstrating the modular nature of these therapeutic agents and suggesting that development of similar therapeutic agents specific to all botulinum serotypes should be readily achievable.