Bimodal modulation of the botulinum neurotoxin protein-conducting channel

Abstract

Clostridium botulinum neurotoxin (BoNT) is the causative agent of botulism, a neuroparalytic disease. We describe here a semisynthetic strategy to identify inhibitors based on toosendanin, a traditional Chinese medicine reported to protect from BoNT intoxication. Using a single molecule assay of BoNT serotypes A and E light chain (LC) translocation through the heavy chain (HC) channel in neurons, we discovered that toosendanin and its tetrahydrofuran analog selectively arrest the LC translocation step of intoxication with subnanomolar potency, and increase the unoccluded HC channel propensity to open with micromolar efficacy. The inhibitory profile on LC translocation is accurately recapitulated in 2 different BoNT intoxication assays, namely the mouse protection and the primary rat spinal cord cell assays. Toosendanin has an unprecedented dual mode of action on the protein-conducting channel acting as a cargo-dependent inhibitor of translocation and as cargo-free channel activator. These results imply that the bimodal modulation by toosendanin depends on the dynamic interactions between channel and cargo, highlighting their tight interplay during the progression of LC transit across endosomes.natural product ͉ protein translocation ͉ small molecule modulator

The Case of Botulinum Toxin in Milk: Experimental Data

“…After crossing the intestinal mucosa, BoNT is circulated through the blood, ultimately reaching the neuromuscular nerve endings (22,29). Specific binding of the heavy chain to receptors on the nerve cell surface triggers the translocation of BoNT into the lumen of the cell (3,14,34). Once BoNT reaches the cytosol of the nerve cell, the release of acetylcholine is inhibited by the endopeptidase activity of the BoNT light chain, leading to symmetric descending, flaccid paralysis (38).…”

The Anti-Botulism Triterpenoid Toosendanin Elicits Calcium Increase and Exocytosis in Rat Sensory Neurons

“…Botulinum neurotoxin is known to block synaptic vesicles exocytosis in a 4-step process: binding, internalization, translocation of the toxin, and cleavage of its substrates (synaptobrevin, SNAP-25, and syntaxin) (Hanson et al 1997;Zhou et al 2003). Toosendanin blockade of Clostridium botulinum neurotoxin (BoTNT) light chain translocation through the heavy chain channel (Fischer et al 2009;Li and Shi 2006), and activation of unoccupied BoTNT heavy chain channels itself (Fischer et al 2009;Nakai et al 2009) may partially explain the anti-botulismic effect of toosendanin. The direct modulation by toosendanin of neuronal exocytosis in both motor neurons and sensory neurons will need to be further investigated in the future.…”

“…Toosendanin may block BoTNT binding to its substrates, by inhibiting heavy chain pore-forming activity, but does not inhibit light chain endopeptidase activity (Li and Shi 2006;Shi and Wang 2004;Zhou et al 2003). Toosendanin inhibits light chain translocation at nanomolar but stimulates heavy chain channel opening at micromolar concentrations (Fischer et al 2009;Nakai et al 2010).…”

Exploiting endocytic pathways to prevent bacterial toxin infection

“…Other inhibitors prevented endosomal acidification, impairing the pore-forming ability of the toxins. Toosendanin, which blocks translocation of the BoNT/A enzyme, also prevented anthrax toxicity [109]; however, it did so through a different mechanism [8]. The steps that were inhibited and the cellular targets of the drug were not well defined and require further investigation.…”

“…The endosomal pore formed by anthrax, α-hemolysin, C2 and Iota toxin are inhibited by β-cyclodextrin derivatives in cells, and a number of studies have also been carried out in mice [110,[113][114][115][116][117][118][119][120]. The compound toosendanin prevents translocation of the enzymatic light chain of BoNT/A and is partially effective in preventing botulism in mice [109]. Novel classes of inhibitors based on preventing the cytoplasmic release of the BoNT light chain are currently being investigated [121].…”