Snakebite venom inhibition -
We've commissioned the first comprehensive look at funding for snakebite envenoming research globally between We hope this research helps those working in the snakebite field to see the gaps and possibilities for new solutions and collaborations.
Download the report. PLOS Neglected Tropical Diseases Intrageneric cross-reactivity of monospecific rabbit antisera against venoms of the medically most important Bitis spp. and Echis spp. African snakes. BMJ Global Health Situation of snakebite, antivenom market and access to antivenoms in ASEAN countries.
If you have any questions about our programme or the research we are supporting, please contact us via email. snakebites wellcome. Senior Research Manager - Snakebite. Wellcome is formally endorsing the Kigali Declaration at the Malaria and Neglected Tropical Diseases NTDs Summit, hosted by the Government of Rwanda on 23 June Today, Wellcome announces an ambitious new £80 million programme to transform the way snakebite treatments are researched and delivered, to make them better, safer and more accessible for all.
National Geographic — This man gets bitten by deadly snakes in the name of science. The Telegraph — Why camel antibodies could be the key ingredient in a universal snakebite antivenom.
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Share with Facebook Share with X Share with LinkedIn Share with Email. What we want to achieve. Areas of focus. Credit: Ryan Chapman for Wellcome. Case study: Development of Target Product Profiles for Snake Antivenom Products Dr Bernadette Abela-Ridder, World Health Organization, Switzerland WHO has identified a need to develop the first public-interest Target Product Profiles TPPs to support optimisation of current and emerging products to treat snakebite.
Case study: A rapid and substantial increase in supplies of an effective, safe and affordable antivenom to sub-Saharan Africa Professor John Landon, MicroPharm, Wales While exciting progress is being made in the development of the next generation of antivenoms and treatments, this will take time and interim solutions are urgently required to address snakebite.
Ambition 2: Develop the next generation of treatments Antivenom treatments can be expensive to manufacture, have risky side effects, and are not always effective at neutralising snake venom.
Case study: Small molecule antidote to snakebite Dr Matthew Lewin, Ophirex, Inc. a Public Benefit Corp. Ambition 3: Build and sustain snakebite as a global health priority Until now, snakebite envenoming has never been regarded as a public health priority or as an issue of urgent concern.
Case study: Antivenom Market study: Burden of Snakebite and its Situation Analysis in ASEAN countries Professor Iekhsan Othman, Monash University, Malaysia The Association of South East Asia Nations ASEAN is recognised as having one of the highest regional burdens of snakebite.
Case study: Small Grants for snakebite Tamar Ghosh, Royal Society of Tropical Medicine and Hygiene, UK We believe an essential part of building and maintaining snakebite on the global agenda is investment in the next generation of snakebite researchers.
Nicholas Cammack Head of Snakebite Wellcome. Our progress so far. Research location and projects. We will continue helping to build and sustain global funding for snakebite research. Programme outputs. To determine PLA 2 activity under each of these conditions, the slope of each reaction was calculated by dividing the difference in absorbance at nm between the first two time points in the linear range t n —t 0 by time in min where t n was 2 min 41 s in this case.
These values were then inputted into the following equation:. where A represents venom activity, 0. This activity is then converted into specific activity by dividing the overall activity to the amount of venom in the reaction in micrograms. To assess the quality of the assay, we calculated Z prime values across the plate by using the following formula:.
where σ and µ represent the standard deviations and means of the positive varespladib containing wells and negative controls DMSO only containing wells , respectively. Plate means and coefficients of variation for the positive and negative controls were also calculated.
Following assay validation in well format, a bespoke repurposed drug library of 3, compounds was purchased from MedChemExpress. This library a combination of the HY-L and HY-L libraries with overlaps removed was curated by MedChemExpress to contain a diverse chemical panel of post-phase I and approved drugs that have completed preclinical and clinical studies for a wide range of diseases.
All drugs have well-characterised bioactivity, safety, and bioavailability properties, making them suitable for drug repurposing. Once in house this library was formatted as 11 × well stock plates containing drugs at a concentration of 1 mM in DMSO. russelii Sri Lanka snake venom, as determined earlier.
In addition to the blinded compounds on the plates, control compounds were added to confirm that the assay had performed appropriately. Assay-ready plates ARPs were made by transferring 0. These latter wells containing control drugs and assay buffer served as the blank.
The plates were incubated for 25 min at 37°C, then left to reach room temperature for 5 min. The data were analysed as above by calculating the slopes of all reactions on the plate.
To determine percentage inhibition relative to the positive and negative controls we applied the following formula:. where A represents specific activity as determined for each sample, and µ pos and µ neg represent the means of the specific activity in the positive and negative controls, respectively.
These compounds were thus selected and transferred onto one plate for retesting together against D. Drugs were made into 1 mM working stocks in DMSO, after which they were tested at a final concentration of 10 µM against a variety of PLA 2 -rich venoms D. russelli , N. nigricollis , N. naja , B. arietans , C.
atrox and C. durissus terrificus. To further explore the capabilities of the assay, EC 50 curves were generated for drug hits that displayed the best broad-spectrum venom inhibition gossypol, punicalagin and varespladib against three venoms: D. russelii , N.
nigricollis and C. Drugs were plated as before, starting at 10 µM followed by 2-fold dilutions down to 5 pM, with each dilution series plated in quadruplicate. The data were analysed and expressed as described above and the percentage PLA 2 inhibition values were used to generate EC 50 s in Graphpad Prism 9 using a nonlinear regression to fit the dose-response inhibition curve.
The HTS library was screened for common Pan-Assay INterference compoundS PAINS substructures Baell and Holloway, using the in-built PAINS filter in the RDKIT cheminformatics package version Tree Manifold Approximation Projection TMAP visualisation was performed using the TMAP python module version 1.
The DataWarrior open source program was used to calculate the Ligand efficiency metrics Sander et al. To best utilise commercial drug libraries as a source for novel PLA 2 inhibitors, we first needed to identify and optimise a screening method that would allow us to fully explore the chemical space.
To this end, we took advantage of the commercially available Abcam secretory PLA 2 kit ab , which we miniaturised and adapted for use in well format using robotics see Methods, Figure 1A. This resulted in a 5-fold scale-down of the assay volume and decreased substrate concentrations to allow increased sample throughput while reducing the cost.
Using the kit-supplied bee venom as a positive control, we determined working concentration ranges for a representative viper D. russelii and elapid N. nigricollis snake venom, with fold dilutions ranging from pg to 2 µg of venom tested.
Our findings demonstrated that 20 ng of D. russelli and 5 ng of N. nigricollis venom were sufficient to display at least equivalent activity to the positive bee venom control, while still falling within the linear range of the assay Supplementary Figure S1. We next demonstrated that 10 µM of the gold standard PLA 2 inhibitor, varespladib, inhibited venom activity to baseline readings for both viper and elapid venoms Figure 1B.
FIGURE 1. Assay setup and miniaturisation. A Assay reaction principle. B Examples of venoms optimised for work under HTS conditions. Various venom concentrations were explored to ensure they were within the linear range of the assay 20, 10 or 5 ng shown here , and then inhibition of PLA 2 activity by 10 µM varespladib var was tested.
Data shown represents the reaction rate, with bars showing the mean of duplicate readings and error bars representing standard error. To further test the applicability of our adapted assay for large scale screening we next investigated assay reproducibility across the well plate and replicability between different batches of well plates.
To determine intra-plate and inter-plate variability, we ran three identical interleaved plates containing D. russelii venom on different days. Marimastat, a peptidomimetic inhibitor of a different class of venom toxins the snake venom metalloproteinases , and assay buffer, both of which are not expected to neutralise PLA 2 activity, were also included in the panel as additional controls marimastat at a concentration of 10 µM.
No differences were noted between the average signal values on each of the three plates, suggesting good reproducibility of the method Figure 2B. Similarly, when variability was assessed on a column or row basis, we noted no drift across columns 1—24 and only a minimal change in activity from the top to bottom rows — a reflection of the time it takes the instrument to read the plate in real time Figures 2C, D.
Our data suggest that under these conditions we can obtain a reliable assay window between our positive and negative controls in which to measure PLA 2 inhibition by various drugs. The full plate by plate data is provided in Supplementary Figure S2. We calculated resulting Z-primes, which represents a measure of assay quality Zhang et al.
FIGURE 2. Assay optimisation for high throughput screening. A Plate map showing interleaved controls across the well plate. D - DMSO dark blue , B - buffer red , V - varespladib light blue , M - marimastat magenta. No drift is observed from column 1 to D The signal variability for each row containing the same control DMSO, buffer, 10 µM varespladib or 10 µM marimastat.
A minimal decrease in activity is noted from A to M, but this still allows for a significant window of detection between the positive and negative controls.
Following method validation, we next wanted to test the ability of our assay to identify novel PLA 2 inhibitors by screening a commercial drug library. To this end, we purchased a bespoke library sourced from MedChemExpress, which we screened against the medically important and PLA 2 -rich venom of D.
russelii Faisal et al. The library was curated to contain diverse chemistry of 3, approved or post-phase I clinically trialled drugs i. The drugs were plated at a final assay concentration of 10 µM in well format with controls on either side of the plate. FIGURE 3.
Screening of a commercial library and assessing hit reproducibility for strong and mediocre hits. B Percentage PLA 2 inhibition for strong hits. C Percentage PLA 2 inhibition for mediocre hits with SDs, with heat map below showing the original screen value versus the average value in the rescreen bottom.
Drug names are presented underneath each heat map. Of the strong hits recovered, gossypol, a phenolic compound derived from the cotton plant, appeared three times as an independent hit mean inhibition between screen and rescreen of Other plant compounds we recovered were punicalagin mean inhibition of Additionally, prasugrel hydrochloride, a platelet aggregation inhibitor, was also among our top hits mean inhibition of Tannic acid was excluded from this selection as it is a likely false positive since it is known to react with the exposed -SH group present in the substrate and prevent the reaction between the latter and DTNB Chen et al.
All compounds were virtually screened for substructures common to Pan-Assay INterference compoundS PAINS Baell and Holloway, In our approach we screened the structures of all compounds for known PAINS substructures and flagged compounds containing these structures as being more likely to represent a false positive result.
Out of all the strong and mediocre hits identified in the primary HTS, we identified 15 PAINS flagged structures Supplementary Figure S3 of which four were strong hits. Gossypol, punicalagin, tannic acid and salvianolic acid were all flagged as PAINS positive due to containing catechol moieties—a moiety known to be have promiscuous reactivity, redox activity and to chelate metal ions Matlock et al.
As previously mentioned, tannic acid was excluded due to documented reactivity with free thiols Chen et al. Gossypol was also flagged due to containing the catechol moiety, but also for containing reactive aldehyde groups which have been documented to form Schiff bases with primary amines Kovacic, This is supported by the identification of gossypol as a promiscuous compound using data-mining and deep-learning approaches Yu et al.
Despite this limitation, owing to its documented inactivation of PLA 2 in prior biochemical assays, we carried gossypol forward for further inhibitory profiling B. Yu et al. While lacking any reports of promiscuous behaviour in HTS, punicalagin, like tannic acid, is a naturally occurring tannin and contains many catechol moieties which may lead to false positives in our assay.
Nevertheless, punicalagin was further shown via rescreening to inhibit PLA 2 activity and was taken forward for further characterisation.
To visualise the HTS chemical space and to identify trends among closely related scaffolds identified as PLA 2 inhibitory molecules we employed Tree Manifold Approximation Projection TMAP Probst and Reymond, TMAP visually represents chemical space as a tree-like manifold, grouping similar compounds into branches based on their proximity, revealing meaningful patterns and relationships within chemical space.
Visualisation of our dataset Figure 4 shows that mediocre and strong hits are well distributed across the manifold with most mediocre hits isolated from other compounds.
Most strong hits, including those flagged for containing PAINS related substructures, are similarly isolated on sub-branches, with the exception of varespladib and prasugrel, which share their sub-branches with mediocre hits fiboflapon and vicagrel, respectively.
Fiboflapon is a potent inhibitor of 5-lipoxygenase-activating protein which shares a common N -aryl-2,3-disubstituted indole core with varespladib. Similarly, vicagrel, a closely related analogue of prasugrel, is an antiplatelet prodrug based on clopidogrel and which is converted sequentially by esterases and cytochrome P enzymes into an active metabolite common to both prasugrel and clopidogrel.
The close proximity of these two strong hits to closely related moderate hits is suggestive of common inhibitory scaffolds. FIGURE 4. TMAP representation of the chemical space covered by our HTS library. Non-hits are shown as pink crosses, mediocre hits are shown as green squares, and strong hits are shown as blue triangles.
Strong hits containing PAINS fragments are shown as yellow circles. As our original screen only assessed PLA 2 inhibition for D.
russelii venom, we wanted to understand whether our remaining hits were able to inhibit the PLA 2 activity of a variety of PLA 2 -rich venoms at a top dose of 10 µM. As such, we chose venoms spanning a wide spectrum of geographical locations from Asia, Africa, South and North America, including both vipers Crotalus atrox and Crotalus durissus terrificus from the Crotalinae subfamily and Bitis arietans from Viperinae and elapids Naja naja and N.
nigricollis Figure 5A. This is important, because while both vipers and elapids have PLA 2 toxins in their venoms, these evolved independently via the duplication of different genes encoding different PLA 2 subclasses IIA and IB, respectively Lynch, ; Fry et al.
Following approved industry practices, we commercially sourced re-synthesised versions of our top hits to ensure their inhibitory activity was maintained independent of the material present in the commercial drug screening library.
In addition to our selection of remaining strong hits, we also chose to test quercetin dihydrate, a compound which was previously shown to inhibit enzymatic PLA 2 s from C.
durissus terrificus venom Cotrim et al. russelii venom in the rescreen Figure 3C. FIGURE 5. The PLA 2 inhibitory capability of selected strong hits against different snake venoms.
A Geographical distribution of a selection of PLA 2 -rich venoms spanning both viper and elapid snakes. Images either belong in the public domain or are displayed under a creative commons license; photographers Gary Stolz C.
atrox , Leandro Avelar C. durissus terrificus , Kelly Abram B. arietans , Lucy Keith-Diagne N. nigricollis , Dr. Raju Kasambe N. naja and Tushar Mone D. B Heat map displaying PLA 2 inhibition values in percentages of selected drugs against those six venoms.
Drugs were assayed at a top dose of 10 µM against an optimised venom amount which elicited a strong signal in the assay: D. russelii 30 ng , N. naja 40 ng , B arietans 40 ng , C. atrox 30 ng , N. nigricollis 5 ng and C. durissus terrificus russelii venom, and also partly inhibited the PLA 2 activity of C.
Despite being a strong hit in both the primary and rescreen of the library, DL-borneol did not re-test as a hit against any of our six venoms, perhaps indicating issues with compound resynthesis. Contrastingly, the known PLA 2 inhibitor varespladib also our positive control displayed the best cross-species effectiveness, with potent PLA 2 inhibitory activity observed against all six diverse snake venoms, reducing readings to control levels.
These findings highlight that this previously described drug remains the standout inhibitory molecule for inhibiting PLA 2 venom toxins, despite the increased chemical space explored in this commercial drug library screen.
To better explore the potency of our broad-spectrum hits, we next decided to generate EC 50 curves for our three top performing drugs varespladib, gossypol, and punicalagin against three venoms. We chose the venoms of D. russelii , C. durissus terrificus and N.
nigricollis because, with one exception gossypol against N. Our range of drug serial dilutions covered a concentration interval from 5 pM to 10 µM Figure 6A. Varespladib was consistently the most potent PLA 2 inhibitor with EC 50 s ranging from pM against D. russelii venom to 5. Gossypol was slightly more effective than punicalagin for the two viper venoms tested Figure 6B with EC 50 s in the low nanomolar range russelii and C.
durissus terrificus respectively , but provided only partial inhibition for N. nigricollis venom at the top dose tested 10 µM. Similarly, punicalagin was most effective against the venom of C. durissus terrificus EC 50 of nigricollis venoms respectively. Overall, these data demonstrate that the validated screening assay enables the identification of novel inhibitors that display strong to moderate inhibition against snake venom PLA 2 s and that our methodology is appropriate for broad-spectrum efficacy and EC 50 testing.
FIGURE 6. Testing the PLA 2 inhibitory potency of the top three drug hits against diverse snake venoms. A EC 50 curves of venom PLA 2 inhibition for R - - -gossypol acetic acid, punicalagin and varespladib against three snake venoms D.
EC 50 values for the drugs against the three venoms. Here we present the miniaturisation of a colorimetric assay which allowed us to assess PLA 2 activity in snake venoms in a high throughput manner.
Using the commercially sourced MedChemExpress repurposed drug library we demonstrated the applicability of the assay for snakebite drug screening, and identified novel compounds capable of neutralising venom PLA 2 activities. Our screening activities further contextualised varespladib as a potent and broad inhibitor of snake venom phospholipase toxins.
One important consideration for identifying hits was the stringency of our self-imposed cut-offs, with more stringent cut-offs ultimately required to ensure reproducibility given the relatively narrow assay window. One exception was quercetin, a compound retained because it was previously shown to inhibit the enzymatic PLA 2 activity of C.
Although we confirmed this activity against this same venom in our assay Figure 5 , this compound was not effective against other venoms, and thus appears not be a potent broad spectrum PLA 2 inhibitor.
Among the nine strong hits identified from the primary screen, we noted an abundance of plant-derived compounds such as gossypol isolated from cottonseed , punicalagin a compound found in pomegranates , salvianolic acid A from Salvia miltiorrhiza which is used in traditional Chinese medicine and tannic acid a polyphenol found in many plant species.
Tannic acid likely represents a false positive as it is known to react with the exposed -SH group present in the assay substrate, thereby preventing its reaction with DTNB Chen et al.
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The Protein Journal. Gutiérrez JM, Calvete JJ, Habib AG, et al. Snakebite envenoming. Nature Reviews. Disease Primers. Written By Narumi Aoki-Shioi and Cassandra M. Continue reading from the same book View All. Chapter 4 The Effects of Snake Venom Bitis arietans on Emb By Charlotte Peters, Vladimir Petrilla, Lenka Luptako Chapter 5 Toxicosis of Snake, Scorpion, Honeybee, Spider, an By Saganuwan Alhaji Saganuwan downloads.
Chapter 6 Toxicosis of Snake, Scorpion, Honeybee, Spider, an Geographical regions. Africa and the Middle East. Atractaspis andersonii. Dendroaspis viridis, Dendroaspis angusticeps, Dendroaspis jamesoni, Dendroaspis polylepis; Naja anchietaea, Naja annulifera, Naja asheia, Naja arabica, Naja haje, Naja katiensis, Naja melanoleuca, Naja mossambica, Naja nigricollis, Naja nigricincta, Naja nivea, Naja oxiana, Naja senegalensis.
Bitis arietans, Bitis gabonicaa, Bitis nasicornis, Bitis rhinocerosa; Cerastes cerastes, Cerastes gasperettii; Daboia mauritanicaa, Daboia palaestinaea; Echis borkini, Echis carinatus, Echis coloratus, Echis jogeri, Echis leucogaster, Echis ocellatus, Echis omanensisa, Echis leucogaster, Echis pyramidum; Macrovipera lebetina, Montivipera xanthina1; Pseudocerastes persicus,.
Asia and Australasia. Acanthophis laevisa; Bungarus caeruleus, Bungarus candidus, Bungarus niger, Bungarus magnimaculatus, Bungarus multicinctus, Bungarus sindanus, Bungarus walli; Naja atra, Naja kaouthia, Naja naja, Naja mandalayensis, Naja philippinensis, Naja samarensis, Naja siamensis, Naja sumatrana, Naja sputatrix, Naja oxiana; Notechis scutatus; Oxyuranus scutellatus; Pseudonaja affinis, Pseudechis australisb, Pseudonaja mengdeni, Pseudonaja nuchalis, Pseudonaja textilis.
Cryptelytrops albolabrisa, Cryptelytrops erythrurusa, Cryptelytrops insularisa, Calloselasma rhodostoma; Deinagkistrodon acutus, Daboia russeliia, Daboia siamensisa; Deinagkistrodon acutu; Echis carinatus; Gloydius blomhoffii, Gloydius brevicaudus, Gloydius halys; Hypnale hypnale; Macrovipera lebetina, Protobothrops flavoviridis, Protobothrops mucrosquamatus; Viridovipera stejnegeria,.
Vipera ammodytes, Vipera berus, Vipera aspis,. the Americas. Agkistrodon bilineatus, Agkistrodon contortrix, Agkistrodon piscivorus, Agkistrodon tayloria; Bothrops asper, Bothrops atrox, Bothrops cf. atrox Trinidad , Bothrops bilineatus, Bothrops alternatus, Bothrops brazili, Bothrops caribbaeus St Lucia , Bothrops lanceolatus Martinique , Bothrops diporusa, Bothrops jararaca, Bothrops jararacussu, Bothrops leucurus, Bothrops mattogrossensisa, Bothrops moojeni, Bothrops pictus, Bothrops venezuelensis; Crotalus adamanteus, Crotalus atrox, Crotalus durissus, Crotalus durissus Aruba , Crotalus horridus, Crotalus oreganusa, Crotalus simus, Crotalus scutulatus, Crotalus totonacusa, Crotalus viridis, Lachesis muta.
Venom gland transcriptomes. Venomous snake genomes. Naja kaouthia,. Notechis scutatus. Dendroaspis angusticeps,.
Pseudonaja textilis. Dendroaspis jamesoni,. Dendroaspis polylepis,. Dendroaspis viridis. Pseudonaja nuchalis. Agkistrodon piscivorus. Deinagkistrodon acutus. Protobothrops mucrosquamatus. Bitis gabonica. Protobothrops flavoviridis. Bothrops alternatus. Vipera berus.
Bothrops asper. Crotalus viridis. Bothrops atrox. Crotalus horridus. Bothrops jararaca,. Bothrops jararacussu.
When two inhibirion children arrived at Anxiety relief supplements local Tanzanian hospital with snakebites, Andreas Laustsen, then a Snkaebite student, figured Structured meal frequency they Snakebite venom inhibition Low-carb nutrition given a Snakebiet of antivenom, recover in the Snakebie for a inhibktion days, Snakfbite then return home to their families. Doctors amputated one child at the elbow, and the other around the knee. Snakes make their homes throughout the warm, tropical and sub-tropical regions of Africa, Asia, Latin America, and Oceania. Typically shy creatures, snakes are not interested in biting humans unless threatened or provoked. But in rural areas and in developing countries where many people work outside, accidental human-snake interactions are common. Venomous snakes bite approximately 2. Most snakebite victims are young, typically 10 to 40 years of age, and work in agricultural professions.Video
First Aid for Snake BiteSnakebite venom inhibition -
Edited by Pınar Erkekoglu. Open access peer-reviewed chapter Snakebite Therapeutics Based on Endogenous Inhibitors from Vipers Written By Narumi Aoki-Shioi and Cassandra M.
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Impact of this chapter. Abstract Venomous snakebite is a major human health issue in many countries and has been categorized as a neglected tropical disease by the World Health Organization.
Keywords venomous snake snake venom metalloprotease hemorrhagic nonhemorrhagic toxin resistance natural inhibitor endogenous inhibitor. atrox Trinidad , Bothrops bilineatus, Bothrops alternatus, Bothrops brazili, Bothrops caribbaeus St Lucia , Bothrops lanceolatus Martinique , Bothrops diporusa, Bothrops jararaca, Bothrops jararacussu, Bothrops leucurus, Bothrops mattogrossensisa, Bothrops moojeni, Bothrops pictus, Bothrops venezuelensis; Crotalus adamanteus, Crotalus atrox, Crotalus durissus, Crotalus durissus Aruba , Crotalus horridus, Crotalus oreganusa, Crotalus simus, Crotalus scutulatus, Crotalus totonacusa, Crotalus viridis, Lachesis muta Table 1.
Table 2. Cystatin super family Fetuin family [ 87 , ] AB BJ46a Bothrops Jararaca 5. jararaca SVMP jararhagin P-III from B. jararaca atrolysin C P-I from Crotalus atrox [ 88 ] AF D 48 Crude venom Gloydius blomhoffi brevicaudus SVMP from Gloydius blomhoffi brevicaudus brevilysin H2, brevilysin H3, brevilysin H4, brevilysin H6 P-III SVMP from Protobothrops flavoviridis HR2a weak [ 89 ] AB cMSF Chainese MSF Gloydius blomhoffi brevicaudus 5.
D 48 Crude venom Gloydius blomhoffi brevicaudus SVMP from Gloydius blomhoffi brevicaudus brevilysin H3, brevilysin H4, brevilysin H6 P-III SVMP from Protobothrops flavoviridis HR1A, HR1B [ 89 ] Q5KQS4.
D XX Agkistrodon contortrix mokasen N. mokassen Agkistrodon piscivorus conanti N. D XX Crotalus atrox N. D 65—80 Crude venom Crotales atrox SVMP hemorrhagic toxin-e from Crotales atrox N.
D XX Vipera palaestinae N. D TMI Protobothrops mucrosquamatus N. D 47 Crude venom P. mucrosquamatus P. flavoviridis P. stejnegeri SVMP from P.
mucrosquamatus TM-1, TM-2, TM-3 N. D venomous snake 2 neutralized toxin from other species BaltMPI Bothrops alternatus N.
atrox BjussuMP-I P-III MP from B. jararacussu N. D Non-venomous NtAH serum Natrix tessellata N. D , 70, , Crude venom Bothrops asper SVMP from Bothrops asper BaH1 N. D XX Dinodon semicarinatu N. D 59 and 52 Crude venom Trimeremrus flavovlridis N.
D XXX Drymarchon couperi N. D Whole serum Crude venom Agkistrodon contortrix N. Table 3. Antihemorrhagic proteins from snake sera or plasma. References 1. WHO Technical Report Series; No. ISSN: , ISBN: 92 4 6 2.
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Bioscience Reports. The drugs were plated at a final assay concentration of 10 µM in well format with controls on either side of the plate. FIGURE 3. Screening of a commercial library and assessing hit reproducibility for strong and mediocre hits.
B Percentage PLA 2 inhibition for strong hits. C Percentage PLA 2 inhibition for mediocre hits with SDs, with heat map below showing the original screen value versus the average value in the rescreen bottom. Drug names are presented underneath each heat map. Of the strong hits recovered, gossypol, a phenolic compound derived from the cotton plant, appeared three times as an independent hit mean inhibition between screen and rescreen of Other plant compounds we recovered were punicalagin mean inhibition of Additionally, prasugrel hydrochloride, a platelet aggregation inhibitor, was also among our top hits mean inhibition of Tannic acid was excluded from this selection as it is a likely false positive since it is known to react with the exposed -SH group present in the substrate and prevent the reaction between the latter and DTNB Chen et al.
All compounds were virtually screened for substructures common to Pan-Assay INterference compoundS PAINS Baell and Holloway, In our approach we screened the structures of all compounds for known PAINS substructures and flagged compounds containing these structures as being more likely to represent a false positive result.
Out of all the strong and mediocre hits identified in the primary HTS, we identified 15 PAINS flagged structures Supplementary Figure S3 of which four were strong hits.
Gossypol, punicalagin, tannic acid and salvianolic acid were all flagged as PAINS positive due to containing catechol moieties—a moiety known to be have promiscuous reactivity, redox activity and to chelate metal ions Matlock et al. As previously mentioned, tannic acid was excluded due to documented reactivity with free thiols Chen et al.
Gossypol was also flagged due to containing the catechol moiety, but also for containing reactive aldehyde groups which have been documented to form Schiff bases with primary amines Kovacic, This is supported by the identification of gossypol as a promiscuous compound using data-mining and deep-learning approaches Yu et al.
Despite this limitation, owing to its documented inactivation of PLA 2 in prior biochemical assays, we carried gossypol forward for further inhibitory profiling B.
Yu et al. While lacking any reports of promiscuous behaviour in HTS, punicalagin, like tannic acid, is a naturally occurring tannin and contains many catechol moieties which may lead to false positives in our assay.
Nevertheless, punicalagin was further shown via rescreening to inhibit PLA 2 activity and was taken forward for further characterisation. To visualise the HTS chemical space and to identify trends among closely related scaffolds identified as PLA 2 inhibitory molecules we employed Tree Manifold Approximation Projection TMAP Probst and Reymond, TMAP visually represents chemical space as a tree-like manifold, grouping similar compounds into branches based on their proximity, revealing meaningful patterns and relationships within chemical space.
Visualisation of our dataset Figure 4 shows that mediocre and strong hits are well distributed across the manifold with most mediocre hits isolated from other compounds. Most strong hits, including those flagged for containing PAINS related substructures, are similarly isolated on sub-branches, with the exception of varespladib and prasugrel, which share their sub-branches with mediocre hits fiboflapon and vicagrel, respectively.
Fiboflapon is a potent inhibitor of 5-lipoxygenase-activating protein which shares a common N -aryl-2,3-disubstituted indole core with varespladib.
Similarly, vicagrel, a closely related analogue of prasugrel, is an antiplatelet prodrug based on clopidogrel and which is converted sequentially by esterases and cytochrome P enzymes into an active metabolite common to both prasugrel and clopidogrel.
The close proximity of these two strong hits to closely related moderate hits is suggestive of common inhibitory scaffolds. FIGURE 4. TMAP representation of the chemical space covered by our HTS library.
Non-hits are shown as pink crosses, mediocre hits are shown as green squares, and strong hits are shown as blue triangles. Strong hits containing PAINS fragments are shown as yellow circles. As our original screen only assessed PLA 2 inhibition for D.
russelii venom, we wanted to understand whether our remaining hits were able to inhibit the PLA 2 activity of a variety of PLA 2 -rich venoms at a top dose of 10 µM. As such, we chose venoms spanning a wide spectrum of geographical locations from Asia, Africa, South and North America, including both vipers Crotalus atrox and Crotalus durissus terrificus from the Crotalinae subfamily and Bitis arietans from Viperinae and elapids Naja naja and N.
nigricollis Figure 5A. This is important, because while both vipers and elapids have PLA 2 toxins in their venoms, these evolved independently via the duplication of different genes encoding different PLA 2 subclasses IIA and IB, respectively Lynch, ; Fry et al.
Following approved industry practices, we commercially sourced re-synthesised versions of our top hits to ensure their inhibitory activity was maintained independent of the material present in the commercial drug screening library. In addition to our selection of remaining strong hits, we also chose to test quercetin dihydrate, a compound which was previously shown to inhibit enzymatic PLA 2 s from C.
durissus terrificus venom Cotrim et al. russelii venom in the rescreen Figure 3C. FIGURE 5. The PLA 2 inhibitory capability of selected strong hits against different snake venoms.
A Geographical distribution of a selection of PLA 2 -rich venoms spanning both viper and elapid snakes. Images either belong in the public domain or are displayed under a creative commons license; photographers Gary Stolz C.
atrox , Leandro Avelar C. durissus terrificus , Kelly Abram B. arietans , Lucy Keith-Diagne N. nigricollis , Dr. Raju Kasambe N. naja and Tushar Mone D. B Heat map displaying PLA 2 inhibition values in percentages of selected drugs against those six venoms. Drugs were assayed at a top dose of 10 µM against an optimised venom amount which elicited a strong signal in the assay: D.
russelii 30 ng , N. naja 40 ng , B arietans 40 ng , C. atrox 30 ng , N. nigricollis 5 ng and C. durissus terrificus russelii venom, and also partly inhibited the PLA 2 activity of C. Despite being a strong hit in both the primary and rescreen of the library, DL-borneol did not re-test as a hit against any of our six venoms, perhaps indicating issues with compound resynthesis.
Contrastingly, the known PLA 2 inhibitor varespladib also our positive control displayed the best cross-species effectiveness, with potent PLA 2 inhibitory activity observed against all six diverse snake venoms, reducing readings to control levels.
These findings highlight that this previously described drug remains the standout inhibitory molecule for inhibiting PLA 2 venom toxins, despite the increased chemical space explored in this commercial drug library screen.
To better explore the potency of our broad-spectrum hits, we next decided to generate EC 50 curves for our three top performing drugs varespladib, gossypol, and punicalagin against three venoms. We chose the venoms of D. russelii , C. durissus terrificus and N.
nigricollis because, with one exception gossypol against N. Our range of drug serial dilutions covered a concentration interval from 5 pM to 10 µM Figure 6A. Varespladib was consistently the most potent PLA 2 inhibitor with EC 50 s ranging from pM against D. russelii venom to 5. Gossypol was slightly more effective than punicalagin for the two viper venoms tested Figure 6B with EC 50 s in the low nanomolar range russelii and C.
durissus terrificus respectively , but provided only partial inhibition for N. nigricollis venom at the top dose tested 10 µM. Similarly, punicalagin was most effective against the venom of C. durissus terrificus EC 50 of nigricollis venoms respectively.
Overall, these data demonstrate that the validated screening assay enables the identification of novel inhibitors that display strong to moderate inhibition against snake venom PLA 2 s and that our methodology is appropriate for broad-spectrum efficacy and EC 50 testing.
FIGURE 6. Testing the PLA 2 inhibitory potency of the top three drug hits against diverse snake venoms. A EC 50 curves of venom PLA 2 inhibition for R - - -gossypol acetic acid, punicalagin and varespladib against three snake venoms D. EC 50 values for the drugs against the three venoms. Here we present the miniaturisation of a colorimetric assay which allowed us to assess PLA 2 activity in snake venoms in a high throughput manner.
Using the commercially sourced MedChemExpress repurposed drug library we demonstrated the applicability of the assay for snakebite drug screening, and identified novel compounds capable of neutralising venom PLA 2 activities.
Our screening activities further contextualised varespladib as a potent and broad inhibitor of snake venom phospholipase toxins. One important consideration for identifying hits was the stringency of our self-imposed cut-offs, with more stringent cut-offs ultimately required to ensure reproducibility given the relatively narrow assay window.
One exception was quercetin, a compound retained because it was previously shown to inhibit the enzymatic PLA 2 activity of C. Although we confirmed this activity against this same venom in our assay Figure 5 , this compound was not effective against other venoms, and thus appears not be a potent broad spectrum PLA 2 inhibitor.
Among the nine strong hits identified from the primary screen, we noted an abundance of plant-derived compounds such as gossypol isolated from cottonseed , punicalagin a compound found in pomegranates , salvianolic acid A from Salvia miltiorrhiza which is used in traditional Chinese medicine and tannic acid a polyphenol found in many plant species.
Tannic acid likely represents a false positive as it is known to react with the exposed -SH group present in the assay substrate, thereby preventing its reaction with DTNB Chen et al. Further, while salvianolic acid A failed to reproducibly inhibit D. russelii venom Figure 3B.
In addition, gossypol inhibited proliferation in non-small cell lung carcinoma cells Wang et al. While gossypol is flagged for PAINS associated substructures, and is documented as being a promiscuously reactive compound Bisson et al.
This was also the case based on calculated EC 50 s, which were in the nanomolar range for the two viper venoms C. durissus terrificus , russelii at nigricollis due to a lack of complete inhibition at the highest dose tested. Punicalagin was shown to possess antioxidant, anticancer and anti-inflammatory activity Seeram et al.
Punicalagin was also flagged as containing PAINS related substructures in our virtual screen, however promiscuous biological activity has not been reported.
While showing a more reduced spectrum of inhibition against the different snake venoms when compared to gossypol, punicalagin did display higher nanomolar EC 50 s against both viper and elapid venoms, with a value of durissus terrificus and nigricollis venom, though reduced potency was observed against D.
russelii venom While prasugrel was found to possess poor broad-spectrum activity, visualisation of HTS chemical space brought the closely related platelet inhibitor vicagrel as a mediocre hit—this suggests that their common scaffold may have inherent, albeit weak, PLA 2 inhibitory activities.
Despite the application of the optimised PLA 2 inhibition assay for the discovery of novel venom toxin inhibitors via a high throughput screening approach, none of the hits identified exhibited equivalent or superior PLA 2 inhibitory potencies to that of varespladib potent inhibition against the six diverse venoms at 10 µM and EC 50 s in the 0.
Moreover, in addition to the limitations associated with reduced potency and inhibitory breadth, the phenolic compounds identified in our screen are relatively large molecules possessing several structural characteristics Supplementary Figure S3 ; Supplementary Table S1 , which render them suboptimal for progression for drug development purposes.
To be considered for lead development, a compound requires suitable physicochemical properties in addition to specific target affinity. Beyond the rule of five Lipinski, , ligand efficiency LE and ligand lipophilicity efficiency LLE are two metrics in modern early drug discovery that predict the affinity and quality of screened hits against a target Hevener et al.
The LE metric calculates ligand affinity using activity values percentage inhibition values in this case and the number of heavy atoms Hopkins et al. On the other hand, LLE accounts for the lipophilic properties of the molecules LogP.
Candidate drugs possessing higher LE and LLE metrics should be preferred during target-based drug discovery programs when compared to active ligands against that target Leeson and Springthorpe, In addition, Gleeson Gleeson, , suggested that along with good activity profiles, lower molecular weight and LogP values must be emphasized in hit compounds for improved ADMET properties.
When compared to varespladib, gossypol and punicalagin both displayed decreased LE metrics Supplementary Table S1 due to their larger heavy atom count. In addition, while salvianolic acid and punicalagin presented similar LLE metrics to varespladib accounted by their lower logP approximations , they had some of the lowest LE metrics among our strong hits.
Together, these characteristics make gossypol and punicalagin suboptimal molecules for further progression. Although varespladib retains exciting promise pending forthcoming snakebite clinical trial outcomes Lewin et al. To that end, we are currently utilising the methodology described herein to explore the wider chemical space covered by several other drug libraries.
We believe this approach will be useful for identifying novel repurposed drugs in the field of snakebite envenoming, as well as allowing for the initiation of medicinal chemistry campaigns to rationally optimise hits by improving DMPK characteristics amenable to snakebite indication.
Despite our success in miniaturising this assay and markedly reducing cost, we are aware that research kits for determining PLA 2 activity remain scarce and expensive, especially for researchers based in LMIC where snakebite is prevalent.
This could be addressed by companies either producing cost-effective detection kits in line with demand as the use of these kits expands, or by exploring the use of alternative, more affordable, PLA 2 substrates, which could then be incorporated into newly validated laboratory methodologies alongside use of appropriate home-made buffers.
In conclusion, we have successfully miniaturised a commercial PLA 2 assay for high throughput screening and validated it for snakebite drug discovery purposes using snake venom.
We show that the method is robust and applicable for screening of PLA 2 inhibitors across various distinct snake venoms, but that it also has the potential for wider applications, including exploring the inhibitory potential of other therapeutic molecules for snakebite i.
In addition, the HTS approach and platform have also been successfully used in our lab to screen for inhibitors of other toxin families SVMPs, Clare et al. The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.
L-OA: Methodology, Writing—original draft, Investigation. AW: Investigation, Methodology, Writing—original draft. RC: Methodology, Funding acquisition, Writing—review and editing.
CW: Methodology, Writing—review and editing, Investigation. NJ: Investigation, Methodology, Writing—review and editing.
JK: Writing—review and editing, Conceptualization, Funding acquisition. NB: Conceptualization, Funding acquisition, Writing—review and editing, Methodology.
NC: Conceptualization, Funding acquisition, Methodology, Writing—original draft. This research was funded in part by the Wellcome Trust. For the purpose of open access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.
We thank Paul Rowley and Edouard Crittenden for maintenance and husbandry of the snake collection at LSTM and for performing venom extractions. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
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PLOS Inhibitioon Tropical Diseases 16 6 : e Bioavailable energy supplement is a neglected Nitric oxide and inflammation reduction disease that venm high global rates of mortality and morbidity. Antivenoms Snalebite the Snakebite venom inhibition therapeutic for treating the toxic Snakebite venom inhibition of snakebite, Snakebitf despite vehom thousands of lives annually, these therapies are inhibtion with limited cross-snake species efficacy due to venom variation, which ultimately inhigition their therapeutic utility to particular geographical regions. In this study we explored the feasibility of generating globally effective pathology-specific antivenoms to counteract the haemotoxic signs of snakebite envenoming. Despite variability of the immunogen mixtures, both experimental antivenoms exhibited broadly comparable in vitro venom binding and neutralisation profiles against the individual venom immunogens in immunological and functional assays. However, in vivo assessments using a murine preclinical model of antivenom efficacy revealed substantial differences in venom neutralisation. The experimental antivenom generated from the seven venom immunogen mixture outperformed the comparator, by providing protective effects against venom lethality caused by seven of the eight geographically diverse venoms tested, including three distinct venoms that were not used as immunogens to generate this antivenom.
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