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Toxins Oct 2022Envenomation by elapid snakes primarily results in neurotoxic symptoms and, consequently, are the primary focus of therapeutic research concerning such venoms. However,...
Envenomation by elapid snakes primarily results in neurotoxic symptoms and, consequently, are the primary focus of therapeutic research concerning such venoms. However, mounting evidence suggests these venoms can additionally cause coagulopathic symptoms, as demonstrated by some Asian elapids and African spitting cobras. This study sought to investigate the coagulopathic potential of venoms from medically important elapids of the genera (true cobras), (rinkhals), and (mambas). Crude venoms were bioassayed for coagulant effects using a plasma coagulation assay before RPLC/MS was used to separate and identify venom toxins in parallel with a nanofractionation module. Subsequently, coagulation bioassays were performed on the nanofractionated toxins, along with in-solution tryptic digestion and proteomics analysis. These experiments were then repeated on both crude venoms and on the nanofractionated venom toxins with the addition of either the phospholipase A (PLA) inhibitor varespladib or the snake venom metalloproteinase (SVMP) inhibitor marimastat. Our results demonstrate that various African elapid venoms have an anticoagulant effect, and that this activity is significantly reduced for cobra venoms by the addition of varespladib, though this inhibitor had no effect against anticoagulation caused by mamba venoms. Marimastat showed limited capacity to reduce anticoagulation in elapids, affecting only and venom at higher doses. Proteomic analysis of nanofractionated toxins revealed that the anticoagulant toxins in cobra venoms were both acidic and basic PLAs, while the causative toxins in mamba venoms remain uncertain. This implies that while PLA inhibitors such as varespladib and metalloproteinase inhibitors such as marimastat are viable candidates for novel snakebite treatments, they are not likely to be effective against mamba envenomings.
Topics: Animals; Dendroaspis; Anticoagulants; Proteomics; Elapid Venoms; Elapidae; Snake Venoms; Phospholipases A2; Biological Assay; Metalloproteases; Antivenins
PubMed: 36355986
DOI: 10.3390/toxins14110736 -
Toxicon : Official Journal of the... Oct 2022Venoms are evolutionary novelties that have real-world implications due to their impact upon human health. However, relative to the abundant studies of elapid and...
Venoms are evolutionary novelties that have real-world implications due to their impact upon human health. However, relative to the abundant studies of elapid and viperid snake venoms, fewer investigations have been undertaken on those of rear-fanged snakes as they are more problematic for obtaining venom. While most rear-fanged venomous snakes are not considered to be of great medical importance, several species are capable of producing fatalities. Most notable among these are snakes from the genus Rhabdophis, the Asian "keelback" snakes. Prior work have described potent procoagulant toxicity suggesting Factor X and prothrombin activation, but did not investigate the ability to activate other clotting factors. Here we show that in addition to activating both Factor X and prothrombin (with prothrombin twice that of FX), the venom of Rhabdophis subminiatus is able to more potently activate Factor VII (ten times that of prothrombin), while also activating FXII and FIX equipotently to prothrombin, and with FXI also activated but at a much lower level. The ability to activate FVII represents a third convergent evolution of this trait. The Australian elapid clade of [Oxyuranus (taipans) + Pseudonaja (brown snakes)] was the first identified to have evolved this trait. and only recently was it shown to be independently present in another lineage (the Central American viperid species Porthidium volcanicum). In addition, the abilities to activate FXI and FXII are also convergent between R. subminiatus and P. volcanicum, but with R. subminiatus being much more potent. By testing across amphibian, avian, and mammalian plasmas we demonstrate that the venom is potently procoagulant across diverse plasma types. However, consistent with dietary preference, R. subminiatus venom was most potent upon amphibian plasma. While a Rhabdophis antivenom is produced in Japan to treat R. tigrinus envenomings, it is scarce even within Japan and is not exported. As this genus is very wide-ranging in Asia, alternate treatment options are in need of development. Hence we tested the ability of candidate, broad-spectrum enzyme inhibitors to neutralize R. subminiatus venom: marimastat was more effective than prinomastat but both marimastat and prinomastat were significantly more effective than DMPS (2,3-Dimercapto-1-propanesulfonic acid). The findings of this study shed light on the evolution of these fascinating rear-fanged snakes as well as explored their systemic effects upon blood coagulation and point to potential treatment options for the rare, but potentially lethal encounters.
Topics: Animals; Antivenins; Australia; Blood Coagulation; Blood Coagulation Factors; Colubridae; Elapidae; Factor VII; Factor X; Humans; Hydroxamic Acids; Mammals; Organic Chemicals; Prothrombin; Snake Venoms; Unithiol
PubMed: 36057394
DOI: 10.1016/j.toxicon.2022.08.017 -
Biosensors Aug 2022We have previously shown that human melanoma cells rapidly decrease human brain endothelial barrier strength. Our findings showed a fast mechanism of melanoma mediated...
We have previously shown that human melanoma cells rapidly decrease human brain endothelial barrier strength. Our findings showed a fast mechanism of melanoma mediated barrier disruption, which was localised to the paracellular junctions of the brain endothelial cells. Melanoma cells are known to release molecules which cleave the surrounding matrix and allow traversal within and out of their metastatic niche. Enzymatic families, such as matrix metalloproteinases (MMPs) and proteases are heavily implicated in this process and their complex nature in vivo makes them an intriguing family to assess in melanoma metastasis. Herein, we assessed the expression of MMPs and other proteases in melanoma conditioned media. Our results showed evidence of a high expression of MMP-2, but not MMP-1, -3 or -9. Other proteases including Cathepsins D and B were also detected. Recombinant MMP-2 was added to the apical face of brain endothelial cells (hCMVECs), to measure the change in barrier integrity using biosensor technology. Surprisingly, this showed no decrease in barrier strength. The addition of potent MMP inhibitors (batimastat, marimastat, ONO4817) and other protease inhibitors (such as aprotinin, Pefabloc SC and bestatin) to the brain endothelial cells, in the presence of various melanoma lines, showed no reduction in the melanoma mediated barrier disruption. The inhibitors batimastat, Pefabloc SC, antipain and bestatin alone decreased the barrier strength. These results suggest that although some MMPs and proteases are released by melanoma cells, there is no direct evidence that they are substantially involved in the melanoma-mediated disruption of the brain endothelium.
Topics: Blood-Brain Barrier; Brain; Endothelial Cells; Endothelium; Humans; Matrix Metalloproteinase 2; Melanoma; Peptide Hydrolases
PubMed: 36005056
DOI: 10.3390/bios12080660 -
Toxins Jul 2022Within Neotropical pit-vipers, the Mexican/Central-American clade consisting of , , , and is a wide-ranging, morphologically and ecologically diverse group of snakes....
Within Neotropical pit-vipers, the Mexican/Central-American clade consisting of , , , and is a wide-ranging, morphologically and ecologically diverse group of snakes. Despite their prevalence, little is known of the functional aspects of their venoms. This study aimed to fill the knowledge gap regarding coagulotoxic effects and to examine the potential of different therapeutic approaches. As a general trait, the venoms were shown to be anticoagulant but were underpinned by diverse biochemical actions. Pseudo-procoagulant activity (i.e., thrombin-like), characterized by the direct cleavage of fibrinogen to form weak fibrin clots, was evident for , , and In contrast, other venoms cleaved fibrinogen in a destructive (non-clotting) manner, with and being the most potent. In addition to actions on fibrinogen, clotting enzymes were also inhibited. FXa was only weakly inhibited by most species, but and were extremely strong in their inhibitory action. Other clotting enzymes were more widely inhibited by diverse species spanning the full taxonomical range, but in each case, there were species that had these traits notably amplified relatively to the others. and were the most potent amongst those that inhibited the formation of the prothrombinase complex and were also amongst the most potent inhibitors of Factor XIa. While most species displayed only low levels of thrombin inhibition, potently inhibited this clotting factor. The regional polyvalent antivenom produced by Instituto Picado Clodomiro was tested and was shown to be effective against the diverse anticoagulant pathophysiological effects. In contrast to the anticoagulant activities of the other species, was uniquely procoagulant through the activation of Factor VII and Factor XII. This viperid species is the first snake outside of the elapid snake clade to be shown to activate FVII and the first snake venom of any kind to activate FXII. Interestingly, while small-molecule metalloprotease inhibitors prinomastat and marimastat demonstrated the ability to prevent the procoagulant toxicity of , neither ICP antivenom nor inhibitor DMPS showed this effect. The extreme variation among the snakes here studied underscores how venom is a dynamic trait and how this can shape clinical outcomes and influence evolving treatment strategies.
Topics: Animals; Anticoagulants; Antivenins; Crotalid Venoms; Crotalinae; Elapid Venoms; Elapidae; Fibrinogen; Snake Venoms; Thrombin; Viperidae
PubMed: 35893753
DOI: 10.3390/toxins14080511 -
Oral Oncology Sep 2022Locoregional and lymphovascular involvement of invasive head and neck squamous cell carcinoma (HNSCC) complicates curative treatment. Matrix metalloproteinase (MMP) 9 is...
OBJECTIVES
Locoregional and lymphovascular involvement of invasive head and neck squamous cell carcinoma (HNSCC) complicates curative treatment. Matrix metalloproteinase (MMP) 9 is a negative prognostic marker in HNSCC and targets multiple extracellular matrix (ECM) substrates, where it contributes to breaching basement membrane and stromal barriers enabling invasive spread. Andecaliximab (ADX) is a second-generation MMP9 inhibitor well tolerated in clinical trials of gastric and pancreatic adenocarcinoma. The impact of selective MMP9 targeting by ADX in HNSCC has not been evaluated.
MATERIALS AND METHODS
Established and patient-derived xenograft (PDX) cell lines were utilized in HNSCC invasion assays to determine the inhibitory ability of MMP9-mediated invasion by ADX. MMP9 expression was confirmed using immunohistochemistry (IHC) and immunoblotting. ECM degradation was evaluated with confocal microscopy. Cell invasion from tumor spheroids was monitored by phase microscopy. Histological evaluation was used to determine ADX efficacy in three-dimensional organotypic cultures containing cancer associated fibroblasts (CAFs).
RESULTS
MMP9 was expressed in all established and PDX-derived cell lines. While the broad spectrum clinical MMP inhibitor marimastat (BB2516) blocked HNSCC invadopodia function and tumor spheroid invasion, ADX treatment failed to inhibit invadopodia-based matrix degradation, tumor cell or fibroblast-driven ECM invasion in collagen I-based matrices.
CONCLUSION
ADX monotherapy was ineffective at blocking initial MMP-dependent events of HNSCC invasion, likely due to redundant functions of additional non-targeted MMPs produced by tumor cells and microenvironment. Combination of ADX with existing and emerging therapies targeting additional MMP activation pathways may warrant future investigation.
Topics: Adenocarcinoma; Antibodies, Monoclonal, Humanized; Carcinoma, Squamous Cell; Head and Neck Neoplasms; Humans; Matrix Metalloproteinase 9; Matrix Metalloproteinases; Pancreatic Neoplasms; Squamous Cell Carcinoma of Head and Neck; Tumor Microenvironment
PubMed: 35803110
DOI: 10.1016/j.oraloncology.2022.106008 -
Biochemical and Biophysical Research... Sep 2022Marimastat is one of the potent inhibitors of MMP (MMPIs) with few side effects. The impact of marimastat on cellular senescence remains unexplored. Our study evaluated...
Marimastat is one of the potent inhibitors of MMP (MMPIs) with few side effects. The impact of marimastat on cellular senescence remains unexplored. Our study evaluated the marimastate effect on oxidative stress-induced cell senescence using NIH3T3 cells. Marimastate administration was found to suppress senescence-β-galactosidase (SA-β-gal) activity and development linked with aging. Furthermore, we observed that this effect of marimastat was closely linked with the recovery of autophagy dysfunction and mTOR suppression in HO-treated cells. Notably, this study demonstrated the marimastat effect on senescence inhibition for the first time.
Topics: Animals; Autophagy; Cellular Senescence; Hydrogen Peroxide; Hydroxamic Acids; Mice; NIH 3T3 Cells; Oxidative Stress
PubMed: 35780580
DOI: 10.1016/j.bbrc.2022.06.075 -
MBio Aug 2022The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently, virus variants...
The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently, virus variants that can evade the immunity in a host achieved through vaccination have emerged. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread, are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner and is TMPRSS2 independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific small interfering RNAS (siRNAs) revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytium formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosomal pathways could be an effective strategy by which to cure COVID-19 in the future. To develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2. SARS-CoV-2 binds to the cell surface receptor ACE2 via the spike protein, and then the spike protein is cleaved by host proteases to enable entry. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection in addition to the TMPRSS2-mediated pathway and the endosomal pathway. The metalloproteinase-mediated pathway requires both the prior cleavage of spike into two domains and a specific sequence in the second domain, S2, conditions met by SARS-CoV-2 but lacking in the related human coronavirus SARS-CoV. Besides the contribution of metalloproteinases to SARS-CoV-2 infection, inhibition of metalloproteinases was important in preventing cell death, which may cause organ damage. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.
Topics: COVID-19; Humans; Metalloproteases; SARS-CoV-2; Serine Endopeptidases; Spike Glycoprotein, Coronavirus; Virus Internalization
PubMed: 35708281
DOI: 10.1128/mbio.00519-22 -
Toxicon: X Jun 2022Snakebite envenoming affects more than 250,000 people annually in sub-Saharan Africa. Envenoming by (boomslang) results in venom-induced consumption coagulopathy...
Snakebite envenoming affects more than 250,000 people annually in sub-Saharan Africa. Envenoming by (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 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 and preclinical efficacy of four SVMP inhibitors to neutralise the effects of venom; the matrix metalloproteinase inhibitors marimastat and prinomastat, and the metal chelators dimercaprol and DMPS The venom of exhibited an SVMP-driven procoagulant phenotype . Marimastat and prinomastat demonstrated equipotent inhibition of the SVMP-mediated procoagulant activity of the venom , 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 snakebite envenoming. These two drugs have been previously shown to be effective against 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.
PubMed: 35321116
DOI: 10.1016/j.toxcx.2022.100118 -
Molecules (Basel, Switzerland) Mar 2022Snakebite remains a significant public health burden globally, disproportionately affecting low-income and impoverished regions of the world. Recently, researchers have...
Snakebite remains a significant public health burden globally, disproportionately affecting low-income and impoverished regions of the world. Recently, researchers have begun to focus on the use of small-molecule inhibitors as potential candidates for the neutralisation of key snake venom toxins and as potential field therapies. vipers represent some of the most medically important as well as frequently encountered snake species in Africa, with a number of species possessing anticoagulant phospholipase A (PLA) toxins that prevent the prothrombinase complex from inducing clot formation. Additionally, species within the genus are known to exert pseudo-procoagulant activity, whereby kallikrein enzymatic toxins cleave fibrinogen to form a weak fibrin clot that rapidly degrades, thereby depleting fibrinogen levels and contributing to the net anticoagulant state. Utilising well-validated coagulation assays measuring time until clot formation, this study addresses the in vitro efficacy of three small molecule enzyme inhibitors (marimastat, prinomastat and varespladib) in neutralising these aforementioned activities. The PLA inhibitor varespladib showed the greatest efficacy for the neutralisation of PLA-driven anticoagulant venom activity, with the metalloproteinase inhibitors prinomastat and marimastat both showing low and highly variable degrees of cross-neutralisation with PLA anticoagulant toxicity. However, none of the inhibitors showed efficacy in neutralising the pseudo-procoagulant venom activity exerted by the venom of . Our results highlight the complex nature of snake venoms, for which single-compound treatments will not be universally effective, but combinations might prove highly effective. Despite the limitations of these inhibitors with regards to in vitro kallikrein enzyme pseudo-procoagulant venom activity, our results further support the growing body of literature indicating the potential use of small molecule inhibitors to enhance first-aid treatment of snakebite envenoming, particularly in cases where hospital and thus antivenom treatment is either unavailable or far away.
Topics: Animals; Viperidae
PubMed: 35268832
DOI: 10.3390/molecules27051733 -
Life (Basel, Switzerland) Jan 2022Patients with diabetes suffer from poor fracture healing. Molecular reasons are not fully understood and our previous gene expression microarray analyses of regenerating...
Patients with diabetes suffer from poor fracture healing. Molecular reasons are not fully understood and our previous gene expression microarray analyses of regenerating bones from mice with type 2 diabetes (db/db) revealed accelerated activation of pathways concerning matrix metalloproteases (MMPs). Thus, we picked out the pathological MMP acceleration as a target for profound gene expression analyses and additional therapeutic intervention in the present study. In the first part, gene expression of ECM degrading proteinases and inhibitors was investigated three and seven days postoperatively. , , and gene expression of MMP inhibitor was significantly higher in regenerating bone fractures of db/db compared to wild type animals. and metalloproteinase showed no differences. In the second part, we locally applied a single dose (1 µL of 5 µM solution) of the broad-spectrum molecular MMP inhibitor Marimastat on tibial defects in db/db. We performed immunohistochemical and histological stainings seven days post operation. Impaired bone healing, collagen content, angiogenesis, and osteoclast invasion in db/db were restored significantly by application of Marimastat compared to PBS controls ( = 7/group). Hence, local intervention of bone defects by the molecular MMP inhibitor Marimastat might be an alternative therapeutic intervention for bone healing in diabetes.
PubMed: 35207422
DOI: 10.3390/life12020134