A therapeutic combination of two small molecule toxin inhibitors provides broad preclinical efficacy against viper snakebite

Abstract

Snakebite is a medical emergency causing high mortality and morbidity in rural tropical communities that typically experience delayed access to unaffordable therapeutics. Viperid snakes are responsible for the majority of envenomings, but extensive interspecific variation in venom composition dictates that different antivenom treatments are used in different parts of the world, resulting in clinical and financial snakebite management challenges. Here, we show that a number of repurposed Phase 2-approved small molecules are capable of broadly neutralizing distinct viper venom bioactivities in vitro by inhibiting different enzymatic toxin families. Furthermore, using murine in vivo models of envenoming, we demonstrate that a single dose of a rationally-selected dual inhibitor combination consisting of marimastat and varespladib prevents murine lethality caused by venom from the most medically-important vipers of Africa, South Asia and Central America. Our findings support the translation of combinations of repurposed small molecule-based toxin inhibitors as broad-spectrum therapeutics for snakebite.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1. The geographical distributions and varying proteomic venom compositions of the medically important viper species used in this study.

The previously defined venom proteomes of Echis ocellatus (Nigeria), Echis carinatus (India), Bothrops asper (Costa Rica), Bitis arietans (Nigeria) and Daboia russelii (Sri Lanka) are presented in pie charts. Toxin family key: SVMP snake venom metalloproteinase; SVSP snake venom serine protease; PLA2 phospholipase A2; CTL C-type lectin; LAAO l-amino acid oxidase; DIS disintegrin; CRISP cysteine-rich secretory protein; KUN Kunitz-type serine protease inhibitor. Geographical species distributions were drawn using QGIS v3.10 software based on data downloaded from the World Health Organization Venomous Snake Distribution database and the IUCN Red List of Threatened Species database.

Fig. 2. Small molecule toxin inhibitors inhibit the in vitro SVMP activities of several geographically distinct viper venoms.

a SVMP activities of the five viper venoms quantified by fluorogenic assay. The data presented represent mean measurements and SEMs of area under the curves of fluorescent arbitrary units taken from three independent experimental runs. EOC E. ocellatus; ECAR E. carinatus; BAS Bothrops asper; BAR Bitis arietans; DRUS Daboia russelii. b The effectiveness of metal chelators and peptidomimetic hydroxamate inhibitors at inhibiting the SVMP activity of the various viper venoms. Drug concentrations from 150 µM to 150 nM (highest to lowest dose tested) are presented. The data is expressed as percentage of the venom-only sample (100%, dashed red line). The negative control is presented as an interval (dashed black lines) and represents the values recorded in the PBS-only samples (expressed as percentage of venom activity), where the highest and the lowest values in each set of independent experiments are depicted. Inhibitors are color-coded (dimercaprol, red; DMPS, turquoise; marimastat, purple). c Comparison of SVMP inhibition by marimastat (purple) and batimastat (orange) at two concentrations (1.5 µM, left; 0.15 µM, right), expressed as the percentage of the venom-only sample (100%, dashed red line). All data represent triplicate independent repeats with SEMs, where each technical repeat represents the mean of n ≥ 2 technical replicates. Source data provided in Supplementary Data S1.

Fig. 3. SVMP-inhibitors neutralize the in vitro procoagulant activities of several geographically distinct viper venoms.

a The coagulopathic activities of the viper venoms, showing that all, except B. arietans (BAR), exhibit procoagulant effects by increasing the clotting velocity in comparison with the normal plasma control. The data presented represents the maximum clotting velocity, calculated as the maximum of the first derivative of each clotting curve, from triplicate independent repeat experiments with SEMs, where each technical repeat represents the mean of n ≥ 2 technical replicates. EOC E. ocellatus; ECAR E. carinatus; BAS Bothrops asper; BAR, Bitis arietans; DRUS, Daboia russelii. b Neutralization of procoagulant venom activity by four SVMP-inhibitors across four drug concentrations (150 µM to 150 nM). The data is expressed as the maximum clotting velocity at each dose. The negative (PBS) and positive (venom-only) controls are presented as intervals (dashed black and red lines, respectively), with the latter representing the mean maximum clotting velocity in these samples ± SEM. Inhibitors are color-coded (dimercaprol, red; DMPS, turquoise; marimastat, purple; batimastat, blue). The data represent triplicate independent repeats with SEMs, where each technical repeat represents the mean of n ≥ 2 technical replicates. Note the different y-axis scales. Source data provided in Supplementary Data S1.

Fig. 4. Nafamostat inhibits the in vitro serine protease activities of several geographically distinct viper venoms.

a The serine protease (SVSP) activity of five viper venoms expressed as the rate (ΔAbs/time/µg venom) of substrate consumption determined by kinetic chromogenic assay. The data represents triplicate independent repeats with SEMs, where each technical repeat represents the mean of n ≥ 2 technical replicates. EOC E. ocellatus; ECAR E. carinatus; BAS Bothrops asper; BAR Bitis arietans; DRUS Daboia russelii. b Neutralization of SVSP venom activity by the serine protease-inhibitor nafamostat. The data is expressed as rates (ΔAbs/time/µg venom) and represents triplicate independent repeats with SEMs, where each technical repeat represents the mean of n ≥ 2 technical replicates. Venom only activity (venom) is displayed alongside venom incubated with decreasing molarities of nafamostat (150 µM to 150 nM). Note the different y-axis scales. Source data provided in Supplementary Data S1.

Fig. 5. Combinations of small molecule toxin inhibitors broadly protect against venom lethality in an in vivo ‘preincubation’ model of snake envenoming.

Kaplan–Meier survival graphs for experimental animals (n = 5) receiving venom (Ven) preincubated (30 min at 37 °C) with different small molecule inhibitors or inhibitor mixes via the intravenous route and monitored for 6 h. Drug-only controls are presented as black dashed lines at the top of each graph (none of the drugs exhibited any observable toxicity at the given doses). a Survival of mice receiving 45 µg of E. ocellatus venom (2.5 × LD50 dose) with and without 60 µg of marimastat or varespladib or nafamostat. b Survival of mice receiving 45 µg of E. ocellatus venom (2.5 × LD50 dose) with and without a dual combination mixture of marimastat and varespladib (MV, 60 µg each) or a triple combination mixture of marimastat, varespladib and nafamostat (MVN, 60 µg each). c Quantified thrombin-antithrombin (TAT) levels in the envenomed animals from (a) and (b). Where the time of death was the same within experimental groups (e.g., early deaths or complete survival) TAT levels were quantified for n = 3, and where times of death varied, n = 5. The data displayed represents means of the duplicate technical repeats plus SDs. d–g Kaplan–Meier survival graphs for experimental animals (n = 5) receiving inhibitor mixes (MV or MVN) preincubated with 2.5 × LD50 dose of E. carinatus (47.5 µg, d), B. asper (47 µg, e), B. arietans (54 µg, f) or D. russelii (20 µg, g) venom. h Quantified TAT levels in the envenomed animals from (d) to (g), with data presented as described for (c). Source data provided in Supplementary Data S1.

Fig. 6. The inhibitor combination of marimastat and varespladib (MV) provides broad preclinical efficacy against venom lethality in an in vivo ‘challenge then treat’ model of envenoming.

Kaplan–Meier survival graphs for experimental animals (n = 5) receiving venom (Ven), followed by delayed drug treatment (15 min later) with a dual combination of marimastat and varespladib. Both venom and treatment were delivered via the intraperitoneal route, and the end of the experiment was at 24 h. Survival of mice receiving: aE. ocellatus (90 µg, 5× i.v. LD50), bE. carinatus (95 µg, 5× i.v. LD50), cB. asper (303 µg, 3× i.p. LD50), dB. arietans (108 µg, 5× i.v. LD50) and eD. russelii (105 µg, 13× i.v. LD50) venom, with and without the inhibitor mix (120 µg of each drug) 15 min later. The drug-only control is presented as a black dashed line at the top of each graph (no toxicity was observed at the given dose). f Quantified thrombin-antithrombin (TAT) levels in the envenomed animals from (a) to (e). Where the time of death was the same within experimental groups (e.g., early deaths or complete survival) TAT levels were quantified for n = 3, and where times of death varied, n = 5. The data displayed represents means of the duplicate technical repeats plus SDs. Source data provided in Supplementary Data S1.