Investigation of Salicylanilides as Botulinum Toxin Antagonists

Abstract

Botulinum neurotoxin serotype A (BoNT/A) is recognized by the Centers for Disease Control and Prevention (CDC) as the most potent toxin and as a Tier 1 biowarfare agent. The severity and longevity of botulism stemming from BoNT/A is of significant therapeutic concern, and early administration of antitoxin-antibody therapy is the only approved pharmaceutical treatment for botulism. Small molecule therapeutic strategies have targeted both the heavy chain (HC) and the light chain (LC) catalytic active site and α-/β-exosites. The LC translocation mechanism has also been studied, but an effective, nontoxic inhibitor remains underexplored. In this work, we screened a library of salicylanilides as potential translocation inhibitors. Potential leads following a primary screen were further scrutinized to identify sal30, which has a cellular minimal concentration of a drug that is required for 50% inhibition (IC₅₀) value of 141 nM. The inquiry of salicylanilide sal30's mechanism of action was explored through a self-quenched fluorogenic substrate conjugated to bovine serum albumin (DQ-BSA) fluorescence, confocal microscopy, and vacuolar H⁺-ATPase (V-ATPase) inhibition assays. The summation of these findings imply that endolysosomal proton translocation through the protonophore mechanism of sal30 causes endosome pH to increase, which in turn prevents LC translocation into cytosol, a process that requires an acidic pH. Thus, the inhibition of BoNT/A activity by salicylanilides likely occurs through disruption of pH-dependent endosomal LC translocation. We further probed BoNT inhibition by sal30 using additivity analysis studies with bafilomycin A1, a known BoNT/A LC translocation inhibitor, which indicated the absence of synergy between the two ionophores.

Keywords: botulinum neurotoxin; protonophore; salicylanilide; small molecule antagonists.

Figure 1

General structure of salicylanilide and library of halogenated salicylanilides used for screening

Figure 2

A) Salicylanilides selected for IC50 assessment based on initial and secondary cell activity assay results. Cellular IC50 values of salicylanilides sal17, sal30, sal31, sal36, sal50, and sal51 determined by monitoring cleaved SNAP-25 over a range of concentrations. Cellular IC50 values were established through Western blot analysis and graphs were prepared in PRISM using nonlinear regression and a variable four parameter fit . B)Time dependent inhibition of BoNT/A by sal30. The graph on the right depicts densitometry data.

Figure 3

A) Protection of SNAP-25 cleavage in BoNT/A treated hiPSC-derived GABA neurons by sal30, BafA1, and the two in combination. Data was fit in Graphpad Prism using Nonlinear fit-inhibitor vs response (three parameter least squares ordinary fit). B) Loewe additivity analysis using Synergyfinder web application.38

Figure 4

A) Neuro-2A cells seeded in 96 well plates and grown overnight were treated with DMSO or inhibitor at indicated concentrations for 6 hours, followed by 1 hour treatment with DQ-BSA (10 μg/mL). After washing, fluorescence at 515 nM (ex = 495 nm) was recorded. Values and error bars indicate mean ± SD, n=3. B and C) Lysosomes in HeLa cells were prelabelled by internalization of DQ-BSA (Green) for 1 hour. Subsequently, cells were treated with DMSO or inhibitor for 6 hours and nuclei were stained with Hoechst (Blue) and observed through sequential scanning with a LSM 880 Confocal microscope using an oil immersion objective. Pixel intensity was normalized to cell count (150 cells). Values and error bars represent mean ± SD D) V-ATPase assay measuring ATPase activity through lysosomal Dx-OG fluorescence, after treatment with inhibitors, n=2.