In vitro and in vivo preclinical venom inhibition assays identify metalloproteinase inhibiting drugs as potential future treatments for snakebite envenoming by Dispholidus typus

Stefanie K. Menzies; Rachel H. Clare; Chunfang Xie; Adam Westhorpe; Steven R. Hall; Rebecca J. Edge; Jaffer Alsolaiss; Edouard Crittenden; Amy E. Marriott; Robert A. Harrison; Jeroen Kool; Nicholas R. Casewell

doi:10.1016/j.toxcx.2022.100118

In vitro and in vivo preclinical venom inhibition assays identify metalloproteinase inhibiting drugs as potential future treatments for snakebite envenoming by Dispholidus typus

Stefanie K. Menzies^*, Rachel H. Clare, Chunfang Xie, Adam Westhorpe, Steven R. Hall, Rebecca J. Edge, Jaffer Alsolaiss, Edouard Crittenden, Amy E. Marriott, Robert A. Harrison, Jeroen Kool, Nicholas R. Casewell

^*Corresponding author for this work

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

Snakebite envenoming affects more than 250,000 people annually in sub-Saharan Africa. Envenoming by Dispholidus typus (boomslang) results in venom-induced consumption coagulopathy (VICC), whereby highly abundant prothrombin-activating snake venom metalloproteinases (SVMPs) consume clotting factors and deplete fibrinogen. The only available treatment for D. typus envenoming is the monovalent SAIMR Boomslang antivenom. Treatment options are urgently required because this antivenom is often difficult to source and, at US$6000/vial, typically unaffordable for most snakebite patients. We therefore investigated the in vitro and in vivo preclinical efficacy of four SVMP inhibitors to neutralise the effects of D. typus venom; the matrix metalloproteinase inhibitors marimastat and prinomastat, and the metal chelators dimercaprol and DMPS. The venom of D. typus exhibited an SVMP-driven procoagulant phenotype in vitro. Marimastat and prinomastat demonstrated equipotent inhibition of the SVMP-mediated procoagulant activity of the venom in vitro, whereas dimercaprol and DMPS showed considerably lower potency. However, when tested in preclinical murine models of envenoming using mixed sex CD1 mice, DMPS and marimastat demonstrated partial protection against venom lethality, demonstrated by prolonged survival times of experimental animals, whereas dimercaprol and prinomastat failed to confer any protection at the doses tested. The preclinical results presented here demonstrate that DMPS and marimastat show potential as novel small molecule-based therapeutics for D. typus snakebite envenoming. These two drugs have been previously shown to be effective against Echis ocellatus VICC in preclinical models, and thus we conclude that marimastat and DMPS should be further explored as potentially valuable early intervention therapeutics to broadly treat VICC following snakebite envenoming in sub-Saharan Africa.

Original language	English
Article number	100118
Pages (from-to)	1-10
Number of pages	10
Journal	Toxicon: X
Volume	14
Early online date	18 Mar 2022
DOIs	https://doi.org/10.1016/j.toxcx.2022.100118
Publication status	Published - Jun 2022

Bibliographical note

Funding Information:
This research was funded in whole, or in part, by the Wellcome Trust , grant numbers as detailed below. For the purpose of open access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. R.H.C. acknowledges funding support from the Director's Catalyst Fund at LSTM [supported by Wellcome Institutional Strategic Support Fund 3 ( 204806/Z/16/Z ) and LSTM Internal Funding]. N.R.C. acknowledges a UK Medical Research Council research grant ( MR/S00016X/1 ) and a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by Wellcome and the Royal Society . N.R.C and J.K. acknowledge funding provided by a Wellcome project grant ( 221712/Z/20/Z ). C.X. acknowledges funding support from the China Scholarship Council ( CSC ) fellowship ( 201706250035 ).

Funding Information:
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rachel H Clare reports financial support was provided by Wellcome Trust. Jeroen Kool reports financial support was provided by Wellcome Trust . Nicholas R Casewell reports financial support was provided by Wellcome Trust . Nicholas R Casewell reports financial support was provided by Medical Research Council . Nicholas R Casewell reports financial support was provided by The Royal Society. Chunfang Xie reports financial support was provided by China Scholarship Council .

Funding Information:
This research was funded in whole, or in part, by the Wellcome Trust, grant numbers as detailed below. For the purpose of open access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. R.H.C. acknowledges funding support from the Director's Catalyst Fund at LSTM [supported by Wellcome Institutional Strategic Support Fund 3 (204806/Z/16/Z) and LSTM Internal Funding]. N.R.C. acknowledges a UK Medical Research Council research grant (MR/S00016X/1) and a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by Wellcome and the Royal Society. N.R.C and J.K. acknowledge funding provided by a Wellcome project grant (221712/Z/20/Z). C.X. acknowledges funding support from the China Scholarship Council (CSC) fellowship (201706250035).The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rachel H Clare reports financial support was provided by Wellcome Trust. Jeroen Kool reports financial support was provided by Wellcome Trust. Nicholas R Casewell reports financial support was provided by Wellcome Trust. Nicholas R Casewell reports financial support was provided by Medical Research Council. Nicholas R Casewell reports financial support was provided by The Royal Society. Chunfang Xie reports financial support was provided by China Scholarship Council.We thank the staff in the Biomedical Service Unit of the University of Liverpool for their support in the maintenance and care of the study mice. We are grateful to Laura-Oana Albulescu for discussions regarding this work and comments on the manuscript.

Publisher Copyright:
© 2022 The Authors

Funding

This research was funded in whole, or in part, by the Wellcome Trust , grant numbers as detailed below. For the purpose of open access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. R.H.C. acknowledges funding support from the Director's Catalyst Fund at LSTM [supported by Wellcome Institutional Strategic Support Fund 3 ( 204806/Z/16/Z ) and LSTM Internal Funding]. N.R.C. acknowledges a UK Medical Research Council research grant ( MR/S00016X/1 ) and a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by Wellcome and the Royal Society . N.R.C and J.K. acknowledge funding provided by a Wellcome project grant ( 221712/Z/20/Z ). C.X. acknowledges funding support from the China Scholarship Council ( CSC ) fellowship ( 201706250035 ). The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rachel H Clare reports financial support was provided by Wellcome Trust. Jeroen Kool reports financial support was provided by Wellcome Trust . Nicholas R Casewell reports financial support was provided by Wellcome Trust . Nicholas R Casewell reports financial support was provided by Medical Research Council . Nicholas R Casewell reports financial support was provided by The Royal Society. Chunfang Xie reports financial support was provided by China Scholarship Council . This research was funded in whole, or in part, by the Wellcome Trust, grant numbers as detailed below. For the purpose of open access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. R.H.C. acknowledges funding support from the Director's Catalyst Fund at LSTM [supported by Wellcome Institutional Strategic Support Fund 3 (204806/Z/16/Z) and LSTM Internal Funding]. N.R.C. acknowledges a UK Medical Research Council research grant (MR/S00016X/1) and a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by Wellcome and the Royal Society. N.R.C and J.K. acknowledge funding provided by a Wellcome project grant (221712/Z/20/Z). C.X. acknowledges funding support from the China Scholarship Council (CSC) fellowship (201706250035).The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rachel H Clare reports financial support was provided by Wellcome Trust. Jeroen Kool reports financial support was provided by Wellcome Trust. Nicholas R Casewell reports financial support was provided by Wellcome Trust. Nicholas R Casewell reports financial support was provided by Medical Research Council. Nicholas R Casewell reports financial support was provided by The Royal Society. Chunfang Xie reports financial support was provided by China Scholarship Council.We thank the staff in the Biomedical Service Unit of the University of Liverpool for their support in the maintenance and care of the study mice. We are grateful to Laura-Oana Albulescu for discussions regarding this work and comments on the manuscript.

Funders	Funder number
Sir Henry Dale Fellowship
Commonwealth Scholarship Commission
University of Liverpool
Liverpool School of Tropical Medicine
Wellcome Trust	200517, 204806, 204806/Z/16/Z, 221712
UK Research and Innovation	MR/S00016X/1
Royal Society	221712/Z/20/Z
China Scholarship Council	201706250035
Medical Research Council	200517/Z/16/Z, MR/S00016X/1

Keywords

Boomslang
Drugs
Small molecules
Snakebite
SVMP

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10.1016/j.toxcx.2022.100118

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Cite this

Menzies, S. K., Clare, R. H., Xie, C., Westhorpe, A., Hall, S. R., Edge, R. J., Alsolaiss, J., Crittenden, E., Marriott, A. E., Harrison, R. A., Kool, J., & Casewell, N. R. (2022). In vitro and in vivo preclinical venom inhibition assays identify metalloproteinase inhibiting drugs as potential future treatments for snakebite envenoming by Dispholidus typus. Toxicon: X, 14, 1-10. Article 100118. https://doi.org/10.1016/j.toxcx.2022.100118

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title = "In vitro and in vivo preclinical venom inhibition assays identify metalloproteinase inhibiting drugs as potential future treatments for snakebite envenoming by Dispholidus typus",

abstract = "Snakebite envenoming affects more than 250,000 people annually in sub-Saharan Africa. Envenoming by Dispholidus typus (boomslang) results in venom-induced consumption coagulopathy (VICC), whereby highly abundant prothrombin-activating snake venom metalloproteinases (SVMPs) consume clotting factors and deplete fibrinogen. The only available treatment for D. typus envenoming is the monovalent SAIMR Boomslang antivenom. Treatment options are urgently required because this antivenom is often difficult to source and, at US$6000/vial, typically unaffordable for most snakebite patients. We therefore investigated the in vitro and in vivo preclinical efficacy of four SVMP inhibitors to neutralise the effects of D. typus venom; the matrix metalloproteinase inhibitors marimastat and prinomastat, and the metal chelators dimercaprol and DMPS. The venom of D. typus exhibited an SVMP-driven procoagulant phenotype in vitro. Marimastat and prinomastat demonstrated equipotent inhibition of the SVMP-mediated procoagulant activity of the venom in vitro, whereas dimercaprol and DMPS showed considerably lower potency. However, when tested in preclinical murine models of envenoming using mixed sex CD1 mice, DMPS and marimastat demonstrated partial protection against venom lethality, demonstrated by prolonged survival times of experimental animals, whereas dimercaprol and prinomastat failed to confer any protection at the doses tested. The preclinical results presented here demonstrate that DMPS and marimastat show potential as novel small molecule-based therapeutics for D. typus snakebite envenoming. These two drugs have been previously shown to be effective against Echis ocellatus VICC in preclinical models, and thus we conclude that marimastat and DMPS should be further explored as potentially valuable early intervention therapeutics to broadly treat VICC following snakebite envenoming in sub-Saharan Africa.",

keywords = "Boomslang, Drugs, Small molecules, Snakebite, SVMP",

author = "Menzies, {Stefanie K.} and Clare, {Rachel H.} and Chunfang Xie and Adam Westhorpe and Hall, {Steven R.} and Edge, {Rebecca J.} and Jaffer Alsolaiss and Edouard Crittenden and Marriott, {Amy E.} and Harrison, {Robert A.} and Jeroen Kool and Casewell, {Nicholas R.}",

note = "Funding Information: This research was funded in whole, or in part, by the Wellcome Trust , grant numbers as detailed below. For the purpose of open access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. R.H.C. acknowledges funding support from the Director's Catalyst Fund at LSTM [supported by Wellcome Institutional Strategic Support Fund 3 ( 204806/Z/16/Z ) and LSTM Internal Funding]. N.R.C. acknowledges a UK Medical Research Council research grant ( MR/S00016X/1 ) and a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by Wellcome and the Royal Society . N.R.C and J.K. acknowledge funding provided by a Wellcome project grant ( 221712/Z/20/Z ). C.X. acknowledges funding support from the China Scholarship Council ( CSC ) fellowship ( 201706250035 ). Funding Information: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rachel H Clare reports financial support was provided by Wellcome Trust. Jeroen Kool reports financial support was provided by Wellcome Trust . Nicholas R Casewell reports financial support was provided by Wellcome Trust . Nicholas R Casewell reports financial support was provided by Medical Research Council . Nicholas R Casewell reports financial support was provided by The Royal Society. Chunfang Xie reports financial support was provided by China Scholarship Council . Funding Information: This research was funded in whole, or in part, by the Wellcome Trust, grant numbers as detailed below. For the purpose of open access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. R.H.C. acknowledges funding support from the Director's Catalyst Fund at LSTM [supported by Wellcome Institutional Strategic Support Fund 3 (204806/Z/16/Z) and LSTM Internal Funding]. N.R.C. acknowledges a UK Medical Research Council research grant (MR/S00016X/1) and a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by Wellcome and the Royal Society. N.R.C and J.K. acknowledge funding provided by a Wellcome project grant (221712/Z/20/Z). C.X. acknowledges funding support from the China Scholarship Council (CSC) fellowship (201706250035).The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rachel H Clare reports financial support was provided by Wellcome Trust. Jeroen Kool reports financial support was provided by Wellcome Trust. Nicholas R Casewell reports financial support was provided by Wellcome Trust. Nicholas R Casewell reports financial support was provided by Medical Research Council. Nicholas R Casewell reports financial support was provided by The Royal Society. Chunfang Xie reports financial support was provided by China Scholarship Council.We thank the staff in the Biomedical Service Unit of the University of Liverpool for their support in the maintenance and care of the study mice. We are grateful to Laura-Oana Albulescu for discussions regarding this work and comments on the manuscript. Publisher Copyright: {\textcopyright} 2022 The Authors",

year = "2022",

month = jun,

doi = "10.1016/j.toxcx.2022.100118",

language = "English",

volume = "14",

pages = "1--10",

journal = "Toxicon: X",

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Menzies, SK, Clare, RH, Xie, C, Westhorpe, A, Hall, SR, Edge, RJ, Alsolaiss, J, Crittenden, E, Marriott, AE, Harrison, RA, Kool, J & Casewell, NR 2022, 'In vitro and in vivo preclinical venom inhibition assays identify metalloproteinase inhibiting drugs as potential future treatments for snakebite envenoming by Dispholidus typus', Toxicon: X, vol. 14, 100118, pp. 1-10. https://doi.org/10.1016/j.toxcx.2022.100118

In vitro and in vivo preclinical venom inhibition assays identify metalloproteinase inhibiting drugs as potential future treatments for snakebite envenoming by Dispholidus typus. / Menzies, Stefanie K.; Clare, Rachel H.; Xie, Chunfang et al.
In: Toxicon: X, Vol. 14, 100118, 06.2022, p. 1-10.

Research output: Contribution to Journal › Article › Academic › peer-review

TY - JOUR

T1 - In vitro and in vivo preclinical venom inhibition assays identify metalloproteinase inhibiting drugs as potential future treatments for snakebite envenoming by Dispholidus typus

AU - Menzies, Stefanie K.

AU - Clare, Rachel H.

AU - Xie, Chunfang

AU - Westhorpe, Adam

AU - Hall, Steven R.

AU - Edge, Rebecca J.

AU - Alsolaiss, Jaffer

AU - Crittenden, Edouard

AU - Marriott, Amy E.

AU - Harrison, Robert A.

AU - Kool, Jeroen

AU - Casewell, Nicholas R.

N1 - Funding Information: This research was funded in whole, or in part, by the Wellcome Trust , grant numbers as detailed below. For the purpose of open access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. R.H.C. acknowledges funding support from the Director's Catalyst Fund at LSTM [supported by Wellcome Institutional Strategic Support Fund 3 ( 204806/Z/16/Z ) and LSTM Internal Funding]. N.R.C. acknowledges a UK Medical Research Council research grant ( MR/S00016X/1 ) and a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by Wellcome and the Royal Society . N.R.C and J.K. acknowledge funding provided by a Wellcome project grant ( 221712/Z/20/Z ). C.X. acknowledges funding support from the China Scholarship Council ( CSC ) fellowship ( 201706250035 ). Funding Information: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rachel H Clare reports financial support was provided by Wellcome Trust. Jeroen Kool reports financial support was provided by Wellcome Trust . Nicholas R Casewell reports financial support was provided by Wellcome Trust . Nicholas R Casewell reports financial support was provided by Medical Research Council . Nicholas R Casewell reports financial support was provided by The Royal Society. Chunfang Xie reports financial support was provided by China Scholarship Council . Funding Information: This research was funded in whole, or in part, by the Wellcome Trust, grant numbers as detailed below. For the purpose of open access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. R.H.C. acknowledges funding support from the Director's Catalyst Fund at LSTM [supported by Wellcome Institutional Strategic Support Fund 3 (204806/Z/16/Z) and LSTM Internal Funding]. N.R.C. acknowledges a UK Medical Research Council research grant (MR/S00016X/1) and a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by Wellcome and the Royal Society. N.R.C and J.K. acknowledge funding provided by a Wellcome project grant (221712/Z/20/Z). C.X. acknowledges funding support from the China Scholarship Council (CSC) fellowship (201706250035).The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rachel H Clare reports financial support was provided by Wellcome Trust. Jeroen Kool reports financial support was provided by Wellcome Trust. Nicholas R Casewell reports financial support was provided by Wellcome Trust. Nicholas R Casewell reports financial support was provided by Medical Research Council. Nicholas R Casewell reports financial support was provided by The Royal Society. Chunfang Xie reports financial support was provided by China Scholarship Council.We thank the staff in the Biomedical Service Unit of the University of Liverpool for their support in the maintenance and care of the study mice. We are grateful to Laura-Oana Albulescu for discussions regarding this work and comments on the manuscript. Publisher Copyright: © 2022 The Authors

PY - 2022/6

Y1 - 2022/6

N2 - Snakebite envenoming affects more than 250,000 people annually in sub-Saharan Africa. Envenoming by Dispholidus typus (boomslang) results in venom-induced consumption coagulopathy (VICC), whereby highly abundant prothrombin-activating snake venom metalloproteinases (SVMPs) consume clotting factors and deplete fibrinogen. The only available treatment for D. typus envenoming is the monovalent SAIMR Boomslang antivenom. Treatment options are urgently required because this antivenom is often difficult to source and, at US$6000/vial, typically unaffordable for most snakebite patients. We therefore investigated the in vitro and in vivo preclinical efficacy of four SVMP inhibitors to neutralise the effects of D. typus venom; the matrix metalloproteinase inhibitors marimastat and prinomastat, and the metal chelators dimercaprol and DMPS. The venom of D. typus exhibited an SVMP-driven procoagulant phenotype in vitro. Marimastat and prinomastat demonstrated equipotent inhibition of the SVMP-mediated procoagulant activity of the venom in vitro, whereas dimercaprol and DMPS showed considerably lower potency. However, when tested in preclinical murine models of envenoming using mixed sex CD1 mice, DMPS and marimastat demonstrated partial protection against venom lethality, demonstrated by prolonged survival times of experimental animals, whereas dimercaprol and prinomastat failed to confer any protection at the doses tested. The preclinical results presented here demonstrate that DMPS and marimastat show potential as novel small molecule-based therapeutics for D. typus snakebite envenoming. These two drugs have been previously shown to be effective against Echis ocellatus VICC in preclinical models, and thus we conclude that marimastat and DMPS should be further explored as potentially valuable early intervention therapeutics to broadly treat VICC following snakebite envenoming in sub-Saharan Africa.

AB - Snakebite envenoming affects more than 250,000 people annually in sub-Saharan Africa. Envenoming by Dispholidus typus (boomslang) results in venom-induced consumption coagulopathy (VICC), whereby highly abundant prothrombin-activating snake venom metalloproteinases (SVMPs) consume clotting factors and deplete fibrinogen. The only available treatment for D. typus envenoming is the monovalent SAIMR Boomslang antivenom. Treatment options are urgently required because this antivenom is often difficult to source and, at US$6000/vial, typically unaffordable for most snakebite patients. We therefore investigated the in vitro and in vivo preclinical efficacy of four SVMP inhibitors to neutralise the effects of D. typus venom; the matrix metalloproteinase inhibitors marimastat and prinomastat, and the metal chelators dimercaprol and DMPS. The venom of D. typus exhibited an SVMP-driven procoagulant phenotype in vitro. Marimastat and prinomastat demonstrated equipotent inhibition of the SVMP-mediated procoagulant activity of the venom in vitro, whereas dimercaprol and DMPS showed considerably lower potency. However, when tested in preclinical murine models of envenoming using mixed sex CD1 mice, DMPS and marimastat demonstrated partial protection against venom lethality, demonstrated by prolonged survival times of experimental animals, whereas dimercaprol and prinomastat failed to confer any protection at the doses tested. The preclinical results presented here demonstrate that DMPS and marimastat show potential as novel small molecule-based therapeutics for D. typus snakebite envenoming. These two drugs have been previously shown to be effective against Echis ocellatus VICC in preclinical models, and thus we conclude that marimastat and DMPS should be further explored as potentially valuable early intervention therapeutics to broadly treat VICC following snakebite envenoming in sub-Saharan Africa.

KW - Boomslang

KW - Drugs

KW - Small molecules

KW - Snakebite

KW - SVMP

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In vitro and in vivo preclinical venom inhibition assays identify metalloproteinase inhibiting drugs as potential future treatments for snakebite envenoming by Dispholidus typus

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