Purification and enzymatic characterization of a novel metalloprotease from Lachesis muta rhombeata snake venom

Cordeiro, Francielle Almeida; Coutinho, Bárbara Marques; Wiezel, Gisele Adriano; Bordon, Karla de Castro Figueiredo; Bregge-Silva, Cristiane; Rosa-Garzon, Nathália Gonsales da; Cabral, Hamilton; Ueberheide, Beatrix; Arantes, Eliane Candiani

doi:10.1186/s40409-018-0171-x

Cited by 15 publications

(10 citation statements)

References 43 publications

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“…In this study, EDTA has inhibited the metalloprotease activity of both the venom and CAMP-2; however, its use is problematic, and being orally bioavailable, marimastat is a far better clinical approach, especially considering the passing of stage II clinical trials. Similarly, ZnCl 2 has proven to be a successful inhibitor of SVMPs through its ability to cause stereochemical and structural instabilities of metalloproteases when used in excess [37,38]. ZnCl 2 has also shown a significant inhibitory effect on the metalloprotease activity of whole venom and CAMP-2 at many of the concentrations tested.…”

Section: Discussionmentioning

confidence: 99%

Repurposing Cancer Drugs Batimastat and Marimastat to Inhibit the Activity of a Group I Metalloprotease from the Venom of the Western Diamondback Rattlesnake, Crotalus atrox

Layfield

Williams

Ravishankar

et al. 2020

Toxins

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Snakebite envenomation causes over 140,000 deaths every year, predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with incredibly complex pathophysiology due to the vast number of unique toxins/proteins present in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a Group I (PI) metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox. Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity is completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 μM, while it is partially potentiated by calcium chloride. Molecular docking studies have demonstrated that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, Group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites.

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Section: Discussionmentioning

confidence: 99%

Repurposing Cancer Drugs Batimastat and Marimastat to Inhibit the Activity of a Group I Metalloprotease from the Venom of the Western Diamondback Rattlesnake, Crotalus atrox

Layfield

Williams

Ravishankar

et al. 2020

Toxins

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“…In Brazil, these snakes are represented by three main genera, i.e., Bothrops , Crotalus and Lachesis (Viperidae–Crotalinae), being responsible for more than 20,000 cases of snakebites per year in this country, as reported by Notifiable Diseases Information System of the Brazilian Ministry of Health (SINAN, Brasília, DF, Brazil). Lachesis snakes found in South America ( L. muta muta and L. m. rhombeata ) occasionally cause severe human envenomations ( Magalhães et al, 2019 ; Diniz-Sousa et al, 2020 ), which are characterized by pronounced local and systemic disorders, e.g., necrosis ( Damico et al, 2006 ; Ferreira et al, 2009 ; Damico et al, 2012 ), haemorrhage, coagulopathy ( Sánchez et al, 1987 ; Sánchez et al, 1991 ; Sánchez et al, 1995 ; Fuly et al, 1997 ; Rucavado et al, 1999 ; Estêvão-Costa et al, 2000 ; Torres-Huaco et al, 2013 ) and hypotension ( Dias et al, 2016a ; Dias et al, 2016b ), strongly associated with a variety of enzymatically active proteins such as snake venom metalloproteases, serine proteases, PLA 2 , C-type lectins and l -amino acid oxidases ( Weinberg et al, 2004 ; Junqueira-de-Azevedo et al, 2006 ; Bregge-Silva et al, 2012 ; Madrigal et al, 2012 ; Cordeiro et al, 2018 ; Diniz-Sousa et al, 2018 ; Wiezel et al, 2019 ) present in these venoms.…”

Section: Discussionmentioning

confidence: 99%

“…is characterized by intense local pain, oedema and necrosis (Damico et al, 2006;Ferreira et al, 2009;Damico et al, 2012), systemic myotoxicity (Fuly et al, 2000;Fuly et al, 2003;Damico et al, 2006), renal failure (Damico et al, 2007), haemorrhage and coagulopathy (Sánchez et al, 1987;Sánchez et al, 1991;Sánchez et al, 1995;Fuly et al, 1997;Rucavado et al, 1999;Estêvão-Costa et al, 2000;Torres-Huaco et al, 2013), including severe cardiovascular disorders (Diniz and Oliveira, 1992;Giovanni-De-Simone et al, 1997;Dias et al, 2016a;Dias et al, 2016b). Such effects have been associated predominantly with the presence of phospholipases A 2 (PLA 2 ) (Cordeiro et al, 2015;Diniz-Sousa et al, 2018), metalloproteases (Cordeiro et al, 2018) and serine proteases (Wiezel et al, 2019), including biologically active peptides (Graham et al, 2005;Soares et al, 2005;Sanz et al, 2008;Pla et al, 2013;Pinheiro-Júnior et al, 2018), in these venoms.…”

Section: Introductionmentioning

confidence: 99%

Action of Varespladib (LY-315920), a Phospholipase A2 Inhibitor, on the Enzymatic, Coagulant and Haemorrhagic Activities of Lachesis muta rhombeata (South-American Bushmaster) Venom

et al. 2022

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Varespladib (VPL) was primarily developed to treat inflammatory disturbances associated with high levels of serum phospholipase A2 (PLA2). VPL has also demonstrated to be a potential antivenom support agent to prevent PLA2-dependent effects produced by snake venoms. In this study, we examined the action of VPL on the coagulant, haemorrhagic and enzymatic activities of Lachesis muta rhombeata (South-American bushmaster) venom. Conventional colorimetric enzymatic assays were performed for PLA2, caseinolytic and esterasic activities; in vitro coagulant activities for prothrombin time (PT) and activated partial thromboplastin time (aPTT) were performed in rat citrated plasma through a quick timer coagulometer, whereas the dimensions of haemorrhagic haloes obtained after i.d. injections of venom in Wistar rats were determined using ImageJ software. Venom (1 mg/ml) exhibited accentuated enzymatic activities for proteases and PLA2in vitro, with VPL abolishing the PLA2 activity from 0.01 mM; VPL did not affect caseinolytic and esterasic activities at any tested concentrations (0.001–1 mM). In rat citrated plasma in vitro, VPL (1 mM) alone efficiently prevented the venom (1 mg/ml)-induced procoagulant disorder associated to extrinsic (PT) pathway, whereas its association with a commercial antivenom successfully prevented changes in both intrinsic (aPTT) and extrinsic (PT) pathways; commercial antivenom by itself failed to avoid the procoagulant disorders by this venom. Venom (0.5 mg/kg)-induced hemorrhagic activity was slightly reduced by VPL (1 mM) alone or combined with antivenom (antivenom:venom ratio 1:3 ‘v/w’) in rats, with antivenom alone producing no protective action on this parameter. In conclusion, VPL does not inhibit other major enzymatic groups of L. m. rhombeata venom, with its high PLA2 antagonize activity efficaciously preventing the venom-induced coagulation disturbances.

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“…Kayano et al [ 40 ] isolated P-I metalloproteinases from B. brazili that showed activity at different concentrations and were inhibited by EDTA and DTT, but not by PMSF. Metalloproteinases isolated from Lachesis muta rhombeata snake venom showed maximum activity on azocasein at pH 7.0 - 9.0 and were also inhibited by EDTA but no effect with PMSF [ 46 ]. P-I metalloproteinases isolated from B. moojeni [ 47 , 48 ], B. asper [ 49 ] and B. atrox [ 43 ] cleaved Aα chain of fibrinogen.…”

Section: Discussionmentioning

confidence: 99%

P-I metalloproteinases and L-amino acid oxidases from Bothrops species inhibit angiogenesis

Bhat

Joshi

Vasishta

et al. 2021

J. Venom. Anim. Toxins incl. Trop. Dis

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Background: Snake venoms are composed of pharmacologically active proteins that are evolutionarily diverse, stable and specific to targets. Hence, venoms have been explored as a source of bioactive molecules in treating numerous diseases. Recent evidences suggest that snake venom proteins may affect the formation of new blood vessels. Excessive angiogenesis has been implicated in several pathologies including tumours, diabetic retinopathy, arthritis, inter alia . In the present study, we have examined the effects of P-I metalloproteinases isolated from Bothrops moojeni (BmMP-1) and Bothrops atrox (BaMP-1) and L-amino acid oxidases (LAAO) isolated from B. moojeni (BmLAAO) and B. atrox (BaLAAO) on biochemical and functional aspects of angiogenesis. Methods: P-I metalloproteinases and LAAO were purified from venom by molecular size exclusion and ion-exchange chromatography and subsequently confirmed using mass spectrometry. The P-I metalloproteinases were characterized by azocaseinolytic, fibrinogenolytic and gelatinase activity and LAAO activity was assessed by enzyme activity on L-amino acids. Influence of these proteins on apoptosis and cell cycle in endothelial cells was analysed by flow cytometry. The angiogenic activity was determined by in vitro 3D spheroid assay, Matrigel tube forming assay, and in vivo agarose plug transformation in mice. Results: P-I metalloproteinases exhibited azocaseinolytic activity, cleaved α and partially β chain of fibrinogen, and displayed catalytic activity on gelatin. LAAO showed differential activity on L-amino acids. Flow cytometry analysis indicated that both P-I metalloproteinases and LAAO arrested the cells in G0/G1 phase and further induced both necrosis and apoptosis in endothelial cells. In vitro, P-I metalloproteinases and LAAO exhibited significant anti-angiogenic properties in 3D spheroid and Matrigel models by reducing sprout outgrowth and tube formation. Using agarose plug transplants in mice harbouring P-I metalloproteinases and LAAO we demonstrated a marked disruption of vasculature at the periphery. Conclusion: Our research suggests that P-I metalloproteinases and LAAO exhibit anti-angiogenic properties in vitro and in vivo .

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Purification and enzymatic characterization of a novel metalloprotease from Lachesis muta rhombeata snake venom

Cited by 15 publications

References 43 publications

Repurposing Cancer Drugs Batimastat and Marimastat to Inhibit the Activity of a Group I Metalloprotease from the Venom of the Western Diamondback Rattlesnake, Crotalus atrox

Repurposing Cancer Drugs Batimastat and Marimastat to Inhibit the Activity of a Group I Metalloprotease from the Venom of the Western Diamondback Rattlesnake, Crotalus atrox

Action of Varespladib (LY-315920), a Phospholipase A2 Inhibitor, on the Enzymatic, Coagulant and Haemorrhagic Activities of Lachesis muta rhombeata (South-American Bushmaster) Venom

P-I metalloproteinases and L-amino acid oxidases from Bothrops species inhibit angiogenesis

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