Repurposed small molecule toxin inhibitors neutralise a diversity of venoms from the Neotropical viperid snake genus Bothrops
- Biology Department, Edge Hill University, United Kingdom
- Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, United Kingdom
- Biomedical & Life Sciences, Lancaster University, United Kingdom
Figures
Figure 1
Variation in the distribution and venom composition of Bothrops species.
(A) Geographical distribution of the Bothrops species relevant to this manuscript. The distribution is based on the ICUN Red List accessed in April 2025 presented using QGIS 3.4. The images of B. alternatus, B. asper, B. atrox, B. jararaca, B. moojeni, and B. neuwiedi are held under all rights reserved copyright and have been published with permission from W. Wüster, while the image of B. lanceolatus is published with permission from J. Florentin. (B) A summary of the published percentage venom compositions for the three key toxins of relevance to this manuscript; snake venom metalloproteinases (SVMPs) in beige, snake venom serine proteases (SVSPs) in orange, and snake venom phospholipase A2 (PLA2) in green. The extracted data (Supplementary file 1) vary from an individual to large pools of specimens, including captive and wild caught. The data is summarised from the following number of publications per species: B. alternatus (n=2), B. asper (n=2), B. atrox (n=6), B. jararaca (n=3), B. lanceolatus (n=2), B. moojeni (n=2), and B. neuwiedi (n=1). The data is displayed as a box and violin plot (interleaved low-high with line at the mean) using Prism software version 11 (GraphPad).
Figure 2
In vitro profiling of venom samples from seven different Bothrops species.
(A) Protein profiles of the venom protein components per species. Whole venom (5 µg per lane) was prepared under denaturing and reducing conditions, then separated by SDS-PAGE using a 4–20% gel. The gel was then stained with Coomassie blue and destained to show all proteinaceous components. M=molecular weight marker, and approximate masses of the markers are given in kDa. (B–D) Enzymatic activity of the seven Bothrops species in toxin specific assays. The results were analysed using area under the curve (AUC) in seconds and plotted using Prism software version 11 (GraphPad). (A) Snake venom metalloproteinase (SVMP) activity using fluorogenic substrate ES010 read at an excitation wavelength of 320 nm and an emission wavelength of 405 nm over 108 min (n=3, ±SE), with 1 µg of venom per reaction. (C) Phospholipase A2 (PLA2) activity using commercially available secretory PLA2 kit (Abcam) read at an absorbance wavelength of 405 nm over 12 min (n=≥2,±range), with 100 ng of venom per reaction. (D) Snake venom serine protease (SVSP) activity using commercially available chromogenic substrate S-2288 read at an absorbance of 405 nm over 35 min (n=≥3 ±SE) with 1 µg of venom per reaction.
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Figure 2—source data 1
Original SDS-PAGE gel displayed in Figure 2A.
- https://cdn.elifesciences.org/articles/110419/elife-110419-fig2-data1-v1.zip
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Figure 2—source data 2
Original SDS-PAGE gel labelled with the 7 Bothrops profiled as displayed in Figure 2A.
- https://cdn.elifesciences.org/articles/110419/elife-110419-fig2-data2-v1.zip
Figure 3
In vitro coagulation profile of seven different Bothrops species and inhibition of procoagulant activity by small molecule drugs.
(A) The coagulopathic profile of the seven Bothrops species at a comparable 100 ng dose in bovine plasma over 25 min at an absorbance of 595 nm at 25°C (n=≥6, ± SD). (B) Coagulation profile in the absence of venom for snake venom metalloproteinase (SVMP) inhibitors (DMPS, a metal chelator, and the matrix metalloproteinase [MMP] inhibitors marimastat and prinomastat), the phospholipase A2 (PLA2) inhibitor varespladib, and the serine protease inhibitor nafamostat. The profiles indicate no direct coagulopathy for any of the inhibitors except for nafamostat which has a strong anticoagulant profile at this dose (n=2, ±range). (C–I) The percentage inhibition in the coagulation assay of the small molecules (excluding nafamostat due to the inherent anticoagulant activity) in each of the seven Bothrops venoms, tested at 5 µM (n=2, ±range) with a 25 min preincubation at 37°C. The adjusted dose for each venom indicated in the graph title was selected to provide comparable profiles for all seven venoms. Lines and p-values indicate the significant differences determined by one-way ANOVA with Dunnett’s multiple comparisons test to the DMSO control (threshold p=<0.05). (J) Dose-response testing was performed for the four inhibitors in the coagulation assays and EC50s (nM) calculated using Prism software version 11 (GraphPad) as presented in this table (n=2, ±95% CI).
Figure 4
In vitro inhibition of seven different Bothrops species by small molecule drugs.
The enzymatic activities of the seven Bothrops species, with relevant inhibitors in the three toxin specific assays, are presented, with the results analysed using area under the curve (AUC) and plotted using Prism software version 11 (GraphPad), with all drugs preincubated with the relevant drug for 25 min at 37°C. (A) Dose-response testing was performed in the snake venom metalloproteinase (SVMP) and phospholipase A2 (PLA2) assays using varespladib for the latter, but a wider panel of matrix metalloproteinase inhibitors (MMPis) and metal chelators with prior evidence of SVMP inhibition in the SVMP assay. This allowed for the calculation of EC50s (with 95% CI shown) using Prism software version 10 (GraphPad) which are displayed in this table (n=≥2, ±95% CI). (B) SVMP activity (1 µg venom per reaction) and inhibition by the MMPi, marimastat at 10 µM (AUC over 178 min, n=4, ±SE). (C) PLA2 activity (100–250 ng venom per reaction, depending on venom) and inhibition by the PLA2 inhibitor, varespladib at 10 µM (AUC over 13 min, n=≥3, ±SE). (D) Snake venom serine protease (SVSP) activity (1 µg venom per reaction) and inhibition by the SP inhibitor, nafamostat at 10 µM (AUC over 35 min, n=4, ±SE). Statistical analysis of the data presented in panels B–D was assessed via two-way ANOVA with Šídák’s multiple comparisons test of each venom control compared to the matched venom treated with inhibitor (GraphPad Prism v11.0); **=p=0.0012, ****=p<0.0001.
Figure 5
In vitro coagulation profile of two coagulopathic Bothrops species and inhibition by combination therapy of two small molecule drugs.
The effect of Bothrops jararaca (A–B, purple) and Bothrops atrox (C–D, green) with and without preincubated small molecule inhibitors is presented alongside the direct effect of these inhibitors (E and F) in the coagulation assay. (A and C) The kinetic curves demonstrate the procoagulant effect of both venoms, compared to normal clotting control (black circles). The snake venom serine protease (SVSP) inhibitor nafamostat (orange triangles) fails to rescue the procoagulant effect, whilst marimastat (5 µM, white triangles) fully inhibits the procoagulant effect of B. atrox and partial rescue of B. jararaca (n=≥6, ± SD). (B and D) Percentage inhibition of coagulopathy by Bothrops species following single treatment with small molecule inhibitors (n=6, ±SE) matched that of the kinetic curves for the top 5 µM dose of marimastat, with subsequent lower doses slightly reducing the percentage inhibition, whereas nafamostat resulted in only minor inhibition at all doses. (E and F) Combination testing of marimastat and nafamostat for both venoms appears to reflect an additive effect; however, in the no venom control (E, kinetic curve; F, % inhibition; n=≥6 ,±SE), nafamostat shows a dose-dependent increase beyond 100% in the absence of venom, demonstrating that it is directly anticoagulant at higher doses. Marimastat has no effect on plasma clotting, indicating that the effects seen in graphs (A–D) are through inhibition of venom. Statistical analysis of the data presented in panels B, D, and F was assessed via one-way ANOVA with Šídák’s multiple comparisons test, *p=0.0163, **p=0.0047, ***p=0.0009, ****p=<0.0001.
Figure 6
Thromboelastography (TEG) profiles of two representative Bothrops species and inhibition by the snake venom metalloproteinase (SVMP) inhibitor marimastat.
(A and B) Thromboelastography profile of respectively B. atrox and B. jararaca using an XY plot of clotting time vs clotting strength. Both venoms (0.6 µg per 300 µL reaction) have strong procoagulant activity (circles) compared to the no venom controls (black diamond) or drug-only control (grey diamond). Marimastat inhibits the procoagulant activity of both venoms (squares) (n>3). (C) The parameters reported by the ROTEM instrument are displayed, including the clotting time (CT; s), maximum clot firmness (MCF; mm), clot formation time (CFT; s), alpha angle (°), and amplitude at 5 min (A5; mm), and each is the mean of the triplicate results ± the related range. Independent statistical analysis of CT and MCF were performed against either the no venom# (pale grey rows) or venom-only controls$ (dark grey rows) using one-way ANOVA with Tukey’s multiple comparisons test. n.s.=not statistically significant.
Figure 7 with 1 supplement
Survival curves of chicken embryos dosed with B. atrox venom with and without representative small molecule drugs.
Groups of 6-day post-fertilisation chicken egg embryos were dosed on to the vitelline vein with either phosphate-buffered saline (PBS) (negative control) or B. atrox venom (20 µg) with or without a subsequent dose of 0.5 µg, 1 µg, and 5 µg of (A) marimastat, (B) DMPS, and (C) nafamostat. All treatment group sizes were n=5 (with the exception of n=4 for the 0.5 µg nafamostat group), the PBS control was n=12, while the venom-only control was n=20. (D) Representative pathology images of chicken embryos 1 hr post-dosing with B. atrox venom with and without 5 µg of the inhibitory drugs, showing varying degrees of protection against the vascular damage shown in the venom-only control (see also Figure 7—figure supplement 1). Log-rank test (Mantel-Cox) with Holm-Šídák’s multiple comparisons test was carried out for all treatments against the relevant venom-only control, and significant differences are indicated by asterisks found to the right-hand side of the relevant legends in each panel; *p=0.0498, **p=0.0306, and ***p=0.0130.
Figure 7—figure supplement 1
Representative pathology images of chicken embryos 1 hr post-dosing with B. atrox venom with and without representative small molecule drugs.
Groups of 6-day post-fertilisation chicken egg embryos were dosed on to the vitelline vein with B. atrox venom (20 µg) with or without a subsequent dose of 0.5 µg, 1 µg, and 5 µg of the inhibitory drugs marimastat, DMPS, and nafamostat. The panel on the right includes representative images of control embryos dosed with venom-only, no venom, or nafamostat-only (5 µg).
Additional files
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Supplementary file 1
Table of Bothrops venom compositions from published proteomics data.
- https://cdn.elifesciences.org/articles/110419/elife-110419-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/110419/elife-110419-mdarchecklist1-v1.docx
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Repurposed small molecule toxin inhibitors neutralise a diversity of venoms from the Neotropical viperid snake genus Bothrops
eLife 15:RP110419.
https://doi.org/10.7554/eLife.110419.3
