Natural Compounds and Their Analogues as Potent Antidotes against the Most Poisonous Bacterial Toxin

Abstract

Botulinum neurotoxins (BoNTs), the most poisonous proteins known to humankind, are a family of seven (serotype A to G) immunologically distinct proteins synthesized primarily by different strains of the anaerobic bacterium Clostridium botulinum Being the causative agents of botulism, the toxins block neurotransmitter release by specifically cleaving one of the three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, thereby inducing flaccid paralysis. The development of countermeasures and therapeutics against BoNTs is a high-priority research area for public health because of their extreme toxicity and potential for use as biowarfare agents. Extensive research has focused on designing antagonists that block the catalytic activity of BoNTs. In this study, we screened 300 small natural compounds and their analogues extracted from Indian plants for their activity against BoNT serotype A (BoNT/A) as well as its light chain (LCA) using biochemical and cellular assays. One natural compound, a nitrophenyl psoralen (NPP), was identified to be a specific inhibitor of LCA with an in vitro 50% inhibitory concentration (IC₅₀) value of 4.74 ± 0.03 µM. NPP was able to rescue endogenous synaptosome-associated protein 25 (SNAP-25) from cleavage by BoNT/A in human neuroblastoma cells with an IC₅₀ of 12.2 ± 1.7 µM, as well as to prolong the time to the blocking of neutrally elicited twitch tensions in isolated mouse phrenic nerve-hemidiaphragm preparations.IMPORTANCE The long-lasting endopeptidase activity of BoNT is a critical biological activity inside the nerve cell, as it prompts proteolysis of the SNARE proteins, involved in the exocytosis of the neurotransmitter acetylcholine. Thus, the BoNT endopeptidase activity is an appropriate clinical target for designing new small-molecule antidotes against BoNT with the potential to reverse the paralysis syndrome of botulism. In principle, small-molecule inhibitors (SMIs) can gain entry into BoNT-intoxicated cells if they have a suitable octanol-water partition coefficient (log P) value and other favorable characteristics (P. Leeson, Nature 481:455-456, 2012, https://doi.org/10.1038/481455a). Several efforts have been made in the past to develop SMIs, but inhibitors effective under in vitro conditions have not in general been effective in vivo or in cellular models (L. M. Eubanks, M. S. Hixon, W. Jin, S. Hong, et al., Proc Natl Acad Sci U S A 104:2602-2607, 2007, https://doi.org/10.1073/pnas.0611213104). The difference between the in vitro and cellular efficacy presumably results from difficulties experienced by the compounds in crossing the cell membrane, in conjunction with poor bioavailability and high cytotoxicity. The screened nitrophenyl psoralen (NPP) effectively antagonized BoNT/A in both in vitro and ex vivo assays. Importantly, NPP inhibited the BoNT/A light chain but not other general zinc endopeptidases, such as thermolysin, suggesting high selectivity for its target. Small-molecule (nonpeptidic) inhibitors have better oral bioavailability, better stability, and better tissue and cell permeation than antitoxins or peptide inhibitors.

Keywords: IC50; botulinum neurotoxin type A; cell-based SNAP-25 assay; high-throughput screening; phrenic nerve-hemidiaphragm preparation.

FIG 1

Substrate design for the HTS endopeptidase assays. (A) Schematic of the substrate SNAP-25 (aa 1 to 206) and proteolytic fragments generated following cleavage of SNAP-25 at residues Q197 and R198 by BoNT/A. The residues shown in red were used for FRET peptide substrate development. (B) Sequence of FRET peptide substrate used for HTS of inhibitors. (C) Full-length substrate (GST [26.31 kDa, purple], SNAP25a [23.82 kDa, blue], GFP [26.94 kDa, green], SNAG [77.03 kDa]) for confirmation of inhibition by NPP. SNAG cleaves into 48.73-kDa and 28.33-kDa fragments when incubated with BoNT/A and into 46.78-kDa and 30.27-kDa fragments when incubated with BoNT/E. EGCP, enhanced green fluorescent protein.

FIG 2

Structure of 3-(4-nitrophenyl)-7H-furo[3,2-g]chromen-7-one, which is a nitrophenyl psoralen (NPP).

FIG 3

Concentration-response curves to determine the inhibition of LCA by NPP using a 14-aa SNAP-25 reporter peptide as a substrate. Various concentrations of NPP were incubated with LCA and fluorescent peptide substrate. Percent inhibition was determined by measuring the fluorescence using an excitation wavelength of 490 nm and an emission wavelength of 523 nm. The experiments were performed in 10 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 1.25 mM freshly prepared dithiothreitol (DTT), and 0.05% Tween 20 at 37°C. The symbols represent the mean ± SD for data obtained from three replicate samples. The curve was fit by nonlinear regression analysis and yielded an IC₅₀ of 4.74 ± 0.03 µM. [C], concentration.

FIG 4

Confirmation of inhibitory activity of NPP using the SNAG construct (GST–SNAP-25–GFP). SNAG is labeled as the negative control (NC; lane 1). SNAG incubated with the 2 nM LCA served as the positive control (PC; lane 2). LCA was incubated with serially diluted (2×) inhibitor (80, 40, 20, 10, 5, 2.5, and 1.25 μM; lanes 4 to 10). Percent inhibition was determined using densitometry analysis with Bio-Rad Image Lab (v.5.2.1) software. SNAP-25 cleavage by NPP was reduced in a concentration-dependent manner. The IC₅₀ was determined to be 6.3 ± 2.02 µM (mean ± SD, n =3). The gel is representative of the gels from 3 independent experiments.

FIG 5

NPP inhibition specificity analysis using thermolysin as an enzyme. Two different concentrations of thermolysin (50 nM and 500 nM) were preincubated with 6.5 µM NPP (lane 2 and lane 5, respectively), followed by incubation with SNAG for comparison with the thermolysin reaction in the absence of NPP (lane 3 and lane 6, respectively). SNAG at 1.5 µM (lane 4), thermolysin at 50 nM (lane 1), and thermolysin at 500 nM (lane 7) were used as controls. Lanes 1 and 7 had thermolysin in the absence of a substrate or inhibitor. Lane 8, SDS-PAGE molecular weight standard (the numbers on the right are in kilodaltons).

FIG 6

Inhibition by NPP of BoNT/A-mediated cleavage of SNAP-25 in M17 neuroblastoma cells, determined using Western blot analysis. (A) Lane 1, marker lane; lane 2, control cells without BoNT/A as a negative control; lane 3, cells incubated with 30 nM BoNT/A as a positive control; lanes 4 to 8, 30 nM BoNT/A was mixed with 1 of 5 concentrations of NPP for 30 min, respectively, and the cells were incubated with the toxin-inhibitor mixture for 24 h prior to assay. (B) Percent inhibition was determined by densitometry. The IC₅₀ was calculated using nonlinear regression analysis. Symbols represent the mean ± SD from three independent experiments.

FIG 7

Time course of BoNT/A-mediated muscle inhibition of twitch tension elicited at a frequency of 0.03 Hz in isolated mouse hemidiaphragm muscles. Muscles were exposed to 5 pM BoNT/A at zero time for 30 min at 22°C (first arrowhead). At the end of this incubation period, the muscles were washed to remove unbound BoNT/A to limit entry of the toxin during the recording period and to minimize interactions of BoNT/A and NPP in the extracellular environment. The temperature was increased to 37°C to initiate muscle paralysis. Subsequently, 60 µM NPP dissolved in DMSO or vehicle (0.12% DMSO) was added from concentrated stock solutions to determine the effects of NPP on BoNT/A-mediated paralysis (second arrowhead). Additions were made slowly (30 to 60 s) to minimize the precipitation of NPP and to prevent the generation of muscle fasciculations from transient formation of high concentrations of the solvent prior to its equilibration in the bath. Other than muscle fasciculations (which were observed in one of six experiments), DMSO had no effect on muscle tension at the concentration examined.

FIG 8

The cellular toxicity of NPP was determined using the MTT assay. At 80% confluence, cells were incubated with different concentrations of inhibitors for 24 h. Six wells with 0.5% DMSO without inhibitors were used as a positive control. Six empty wells without any cells were also used as a negative control to remove the background signal. SD is a representation of 6 wells.