Post-Intoxication Inhibition of Botulinum Neurotoxin Serotype A within Neurons by Small-Molecule, Non-Peptidic Inhibitors

Abstract

Botulinum neurotoxins (BoNTs) comprise seven distinct serotypes that inhibit the release of neurotransmitter across neuromuscular junctions, resulting in potentially fatal flaccid paralysis. BoNT serotype A (BoNT/A), which targets synaptosomal-associated protein of 25kDa (SNAP-25), is particularly long-lived within neurons and requires a longer time for recovery of neuromuscular function. There are currently no treatments available to counteract BoNT/A after it has entered the neuronal cytosol. In this study, we examined the ability of small molecule non-peptidic inhibitors (SMNPIs) to prevent SNAP-25 cleavage post-intoxication of neurons. The progressive cleavage of SNAP-25 observed over 5 h following 1 h BoNT/A intoxication was prevented by addition of SMNPIs. In contrast, anti-BoNT/A neutralizing antibodies that strongly inhibited SNAP-25 cleavage when added during intoxication were completely ineffective when added post-intoxication. Although Bafilomycin A1, which blocks entry of BoNT/A into the cytosol by preventing endosomal acidification, inhibited SNAP-25 cleavage post-intoxication, the degree of inhibition was significantly reduced versus addition both during and after intoxication. Post-intoxication application of SMNPIs, on the other hand, was nearly as effective as application both during and after intoxication. Taken together, the results indicate that competitive SMNPIs of BoNT/A light chain can be effective within neurons post-intoxication.

Small-Molecule Quinolinol Inhibitor Identified Provides Protection against BoNT/A in Mice

“…The majority of such molecules reportedly demonstrated to be effective in enzymatic assays [21], [23], [27], [28], [32], [33] and a few small-molecules have been tested in cell-based assays [34], [35], [36], [37]. But the information shows that small-molecules can significantly protect mammals against BoNT/A is scanty [31], [36].…”

“…Though the above approaches have resulted in the identification of a number of small-molecules as BoNT/A inhibitors, no compound has yet advanced to pre-clinical development [24] , [29] , [30] , [31] . The majority of such molecules reportedly demonstrated to be effective in enzymatic assays [21] , [23] , [27] , [28] , [32] , [33] and a few small-molecules have been tested in cell-based assays [34] , [35] , [36] , [37] . But the information shows that small-molecules can significantly protect mammals against BoNT/A is scanty [31] , [36] .…”

Targeting Botulinum A Cellular Toxicity: A Prodrug Approach

“…Next, the cells were incubated for an additional 8 h in the presence of compound before quantifying the amount of cleaved and uncleaved SNAP-25 by Western blot analysis. It is important to note that this type of post-intoxication scenario is most relevant because it relies solely on the intracellular inhibition of LC/A, 23, 43 in contrast to assays where toxin and compound are preincubated prior to their addition to cells or where toxin and compound are simultaneously added to the cells. Unfortunately, dioxazole 5 did not provide any protection against SNAP-25 cleavage; however, benzyl carbamate 6a exhibited ~50% inhibition (Figure 3).…”

Identification of Small Molecules against Botulinum Neurotoxin B Binding to Neuronal Cells at Ganglioside GT1b Binding Site with Low to Moderate Affinity

“…They were successful in finding three lead candidate hits among a library of 1990 compounds from 39 potential inhibitor candidates; using unique pharmacophores they screened compound repositories such as those at http://dtp.nci.nih.gov. For BoNT studies, the application of this approach has led to the development of small molecule non-peptidic inhibitors of the light chain proteases of botulinum neurotoxin serotype A (Tang et al, 2007;Capkova et al, 2009;Burnett et al, 2003;Burnett et al, 2007a;Burnett et al, 2007b;Ruthel et al, 2011;Roxas-Duncan et al, 2009). Roxas-Duncan et al (2009) discovered small molecule inhibitors against BoNT/A with 92 to 97% inhibition.…”