-
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 -
Blood Advances May 2022Acute myeloid leukemia (AML) is a blood cancer of the myeloid lineage. Its prognosis remains poor, highlighting the need for new therapeutic and precision medicine...
Acute myeloid leukemia (AML) is a blood cancer of the myeloid lineage. Its prognosis remains poor, highlighting the need for new therapeutic and precision medicine approaches. AML symptoms often include cytopenias linked to loss of healthy hematopoietic stem and progenitor cells (HSPCs). The mechanisms behind HSPC decline are complex and still poorly understood. Here, intravital microscopy (IVM) of a well-established experimental model of AML allows direct observation of the interactions between healthy and malignant cells in the bone marrow (BM), suggesting that physical dislodgment of healthy cells by AML through damaged vasculature may play an important role. Multiple matrix metalloproteinases (MMPs), known to remodel extracellular matrix, are expressed by AML cells and the BM microenvironment. We reason MMPs could be involved in cell displacement and vascular leakiness; therefore, we evaluate the therapeutic potential of MMP pharmacological inhibition using the broad-spectrum inhibitor prinomastat. IVM analyses of prinomastat-treated mice reveal reduced vascular permeability and healthy cell clusters in circulation and lower AML infiltration, proliferation, and cell migration. Furthermore, treated mice have increased retention of healthy HSPCs in the BM and increased survival following chemotherapy. Analysis of a human AML transcriptomic database reveals widespread MMP deregulation, and human AML cells show susceptibility to MMP inhibition. Overall, our results suggest that MMP inhibition could be a promising complementary therapy to reduce AML growth and limit HSPC loss and BM vascular damage caused by MLL-AF9 and possibly other AML subtypes.
Topics: Animals; Bone Marrow; Hematopoietic Stem Cells; Leukemia, Myeloid, Acute; Metalloproteases; Mice; Prognosis; Tumor Microenvironment
PubMed: 35157757
DOI: 10.1182/bloodadvances.2021004321 -
Frontiers in Immunology 2021African spitting cobras are unique among cobras for their potent anticoagulant venom activity arising from strong inhibition of Factor Xa. This anticoagulant effect is...
African spitting cobras are unique among cobras for their potent anticoagulant venom activity arising from strong inhibition of Factor Xa. This anticoagulant effect is exerted by venom phospholipase A (Group I PLA) toxins whose activity contributes to the lethality of these species. This anticoagulant toxicity is particularly problematic as it is not neutralized by current antivenoms. Previous work demonstrated this trait for , , and The present work builds upon previous research by testing across the full taxonomical range of African spitting cobras, demonstrating that , , and are also potently anticoagulant through the inhibition of Factor Xa, and therefore the amplification of potent anticoagulant activity occurred at the base of the African spitting cobra radiation. Previous work demonstrated that the enzyme-inhibitor varespladib was able to neutralize this toxic action for , , and venoms. The current work demonstrates that varespladib was also able to neutralize , , and . Thus varespladib is shown to have broad utility across the full range of African spitting cobras. In addition, we examined the cross-reactivity of the metalloprotease inhibitor prinomastat, which had been previously intriguingly indicated as being capable of neutralizing viperid venom PLA (Group II PLA). In this study prinomastat inhibited the FXa-inhibiting PLA toxins of all the African spitting cobras at the same concentration at which it has been shown to inhibit metalloproteases, and thus was comparably effective in its cross-reactivity. In addition we showed that the metalloprotease-inhibitor marimastat was also able to cross-neutralize PLA but less effectively than prinomastat. Due to logistical (cold-chain requirement) and efficacy (cross-reactivity across snake species) limitations of traditional antivenoms, particularly in developing countries where snakebite is most common, these small molecule inhibitors (SMIs) might hold great promise as initial, field-based, treatments for snakebite envenoming as well as addressing fundamental limitations of antivenom in the clinical setting where certain toxin effects are unneutralized.
Topics: Acetates; Africa; Animals; Antivenins; Enzyme Inhibitors; Hydroxamic Acids; In Vitro Techniques; Indoles; Keto Acids; Naja; Organic Chemicals; Snake Bites; Snake Venoms
PubMed: 34691069
DOI: 10.3389/fimmu.2021.752442 -
Frontiers in Immunology 2021Palearctic vipers are medically significant snakes in the genera , and which occur throughout Europe, Central Asia, Near and Middle East. While the ancestral condition... (Comparative Study)
Comparative Study
Palearctic vipers are medically significant snakes in the genera , and which occur throughout Europe, Central Asia, Near and Middle East. While the ancestral condition is that of a small-bodied, lowland species, extensive diversification has occurred in body size, and niche specialization. Using 27 venom samples and a panel of coagulation assays, we evaluated the relative coagulotoxic potency of Palearctic viper venoms and compared their neutralization by three antivenoms (Insoserp Europe, VIPERFAV and ViperaTAb) and two metalloprotease inhibitors (prinomastat and DMPS). We show that variation in morphology parallels variation in the Factor X activating procoagulant toxicity, with the three convergent evolutions of larger body sizes ( genus, genus, and uniquely within the genus) were each accompanied by a significant increase in procoagulant potency. In contrast, the two convergent evolutions of high altitude specialization (the genus and uniquely within the genus) were each accompanied by a shift away from procoagulant action, with the species being particularly potently anticoagulant. Inoserp Europe and VIPERFAV antivenoms were both effective against a broad range of species, with Inoserp able to neutralize additional species relative to VIPERFAV, reflective of its more complex antivenom immunization mixture. In contrast, ViperaTAb was extremely potent in neutralizing but, reflective of this being a monovalent antivenom, it was not effective against other species. The enzyme inhibitor prinomastat efficiently neutralized the metalloprotease-driven Factor X activation of the procoagulant venoms. In contrast, DMPS (2,3-dimercapto-1-propanesulfonic acid), which as been suggested as another potential treatment option in the absence of antivenom, DMPS failed against all venoms tested. Overall, our results highlight the evolutionary variations within Palearctic vipers and help to inform clinical management of viper envenomation.
Topics: Animals; Antivenins; Blood Coagulation; Blood Coagulation Tests; Evolution, Molecular; Humans; Immunoglobulin Fab Fragments; Matrix Metalloproteinase Inhibitors; Organic Chemicals; Snake Bites; Species Specificity; Time Factors; Unithiol; Viper Venoms; Viperidae
PubMed: 34177943
DOI: 10.3389/fimmu.2021.688802 -
Toxicology Letters Sep 2021Is snake venom activity influenced by size? This is a long-standing question that can have important consequences for the treatment of snake envenomation. Ontogenetic...
Is snake venom activity influenced by size? This is a long-standing question that can have important consequences for the treatment of snake envenomation. Ontogenetic shifts in venom composition are a well-documented characteristic of numerous snake species. Although snake venoms can cause a range of pathophysiological disturbances, establishing the coagulotoxic profiles related to such shifts is a justified approach because coagulotoxicity can be deadly, and its neutralisation is a challenge for current antivenom therapy. Thus, we aimed to assess the coagulotoxicity patterns on plasma and fibrinogen produced by B othrops jararacussu venoms from individuals of different sizes and sex, and the neutralisation potential of SAB (anti bothropic serum produced by Butantan Institute). The use of a metalloproteinase inhibitor (Prinomastat) and a serine proteinase inhibitor (AEBSF) enabled us to determine the toxin class responsible for the observed coagulopathy: activity on plasma was found to be metalloprotease driven, while the activity on fibrinogen is serine protease driven. To further explore differences in venom activity, the activation of Factor X and prothrombin as a function of snake size was also evaluated. All the venoms exhibited a potent procoagulant effect upon plasma and were less potent in their pseudo-procoagulant clotting effect upon fibrinogen. On human plasma, the venoms from smaller snakes produced more rapid clotting than the larger ones. In contrast, the venom activity on fibrinogen had no relation with size or sex. The difference in procoagulant potency was correlated with the bigger snakes being proportionally better neutralized by antivenom due to the lower levels of procoagulant toxins, than the smaller. Thus, while the antivenom ultimately neutralized the venoms, proportionally more would be needed for an equal mass of venom from a small snake than a large one. Similarly, the neutralisation by SAB of the pseudo-procoagulant clotting effects was also correlated with relative potency, with the smaller and bigger snakes being neutralized proportional to potency, but with no correlation to size. Thromboelastography (TEG) tests on human and toad plasma revealed that small snakes' venoms acted quicker than large snakes' venom on both plasmas, with the action upon amphibian plasma consistent with smaller snakes taking a larger proportion of anuran prey than adults. Altogether, the ontogenetic differences regarding coagulotoxic potency and corresponding impact upon relative antivenom neutralisation of snakes with different sizes were shown, underscoring the medical importance of investigating ontogenetic changes in order to provide data crucial for evidence-based design of clinical management strategies.
Topics: Animals; Antivenins; Blood Coagulation; Bothrops; Crotalid Venoms; Factor X; Female; Humans; Male; Snake Bites; Thrombelastography
PubMed: 34044056
DOI: 10.1016/j.toxlet.2021.05.005 -
Frontiers in Immunology 2021Rattlesnakes are a diverse clade of pit vipers (snake family Viperidae, subfamily Crotalinae) that consists of numerous medically significant species. We used validated...
Rattlesnakes are a diverse clade of pit vipers (snake family Viperidae, subfamily Crotalinae) that consists of numerous medically significant species. We used validated assays measuring venom-induced clotting time and strength of any clots formed in human plasma and fibrinogen to assess the coagulotoxic activity of the four medically relevant Mexican rattlesnake species , and . We report the first evidence of true procoagulant activity by Neotropical rattlesnake venom in . This species presented a strong ontogenetic coagulotoxicity dichotomy: neonates were strongly procoagulant Factor X activation, whereas adults were pseudo-procoagulant in that they converted fibrinogen into weak, unstable fibrin clots that rapidly broke down, thereby likely contributing to net anticoagulation through fibrinogen depletion. The other species did not activate clotting factors or display an ontogenetic dichotomy, but depleted fibrinogen levels by cleaving fibrinogen either in a destructive (non-clotting) manner or a pseudo-procoagulant mechanism. We also assessed the neutralization of these venoms by available antivenom and enzyme-inhibitors to provide knowledge for the design of evidence-based treatment strategies for envenomated patients. One of the most frequently used Mexican antivenoms (Bioclon Antivipmyn®) failed to neutralize the potent procoagulant toxic action of neonate venom, highlighting limitations in snakebite treatment for this species. However, the metalloprotease inhibitor Prinomastat substantially thwarted the procoagulant venom activity, while 2,3-dimercapto-1-propanesulfonic acid (DMPS) was much less effective. These results confirm that venom-induced Factor X activation (a procoagulant action) is driven by metalloproteases, while also suggesting Prinomastat as a more promising potential adjunct treatment than DMPS for this species (with the caveat that studies are necessary to confirm this potential clinical use). Conversely, the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) inhibited the direct fibrinogen cleaving actions of venom, thereby revealing that the pseudo-procoagulant action is driven by kallikrein-type serine proteases. Thus, this differential ontogenetic variation in coagulotoxicity patterns poses intriguing questions. Our results underscore the need for further research into Mexican rattlesnake venom activity, and also highlights potential limitations of current antivenom treatments.
Topics: Animals; Antivenins; Blood Coagulation; Blood Coagulation Factors; Blood Coagulation Tests; Coagulation Protein Disorders; Crotalid Venoms; Crotalus; Mexico; Neutralization Tests
PubMed: 33815366
DOI: 10.3389/fimmu.2021.612846 -
Osteoarthritis and Cartilage Open Jun 2021Arthropathy is a major clinical problem in patients with hemochromatosis, the most common genetic disorder of iron overload. The pathological features of hemochromatosis...
OBJECTIVE
Arthropathy is a major clinical problem in patients with hemochromatosis, the most common genetic disorder of iron overload. The pathological features of hemochromatosis arthropathy (HA) are heterogeneous and its specific nature remains unknown. One important drawback is the lack of proper models. The aim of the present study was to set up a model to investigate the biological response of cartilage to iron exposure.
DESIGN
Bovine articular cartilage explants were incubated with ferric citrate for up to 9 days. We evaluated chondrocyte viability, iron deposition, and biomarkers of cartilage degradation in the conditioned medium.
RESULTS
Iron accumulated within chondrocytes, which was associated with programmed cell death through chondroptosis. Iron treatment increased the release of sulfated glycosaminoglycans (sGAG), a component of the extracellular matrix, into the medium (p=0.0189). This was dependent on the presence of viable chondrocytes and was associated with increased activity of matrix-degrading metalloproteinases (MMP) (pro/active MMP-9, p=0.0317; pro MMP-2, p=0.0092; active MMP-2, p=0.0288). Co-treatment with the broad MMP/aggrecanase inhibitor prinomastat reduced iron-mediated sGAG release (0.02 μM, p=0.0425; 2 μM, p=0.0014), confirming that iron induces sGAG release via the activation of catabolic enzymes. Notably, iron-treated cartilage continued to release an increased amount of sGAG into the medium for 6 days after termination of the ferric citrate treatment (p=0.0259).
CONCLUSIONS
Iron triggers the early stages of cartilage degeneration. Removal of iron exposure does not prevent further damage to the cartilage, thus providing a possible explanation why HA is not prevented after iron depletion by phlebotomy treatment.
PubMed: 36474980
DOI: 10.1016/j.ocarto.2021.100145 -
Toxicology Letters Apr 2021Species within the viperid genus Macrovipera are some of the most dangerous snakes in the Eurasian region, injecting copious amounts of potent venom. Despite their...
Clinical implications of differential procoagulant toxicity of the palearctic viperid genus Macrovipera, and the relative neutralization efficacy of antivenoms and enzyme inhibitors.
Species within the viperid genus Macrovipera are some of the most dangerous snakes in the Eurasian region, injecting copious amounts of potent venom. Despite their medical importance, the pathophysiological actions of their venoms have been neglected. Particularly poorly known are the coagulotoxic effects and thus the underlying mechanisms of lethal coagulopathy. In order to fill this knowledge gap, we ascertained the effects of venom upon human plasma for Macrovipera lebetina cernovi, M. l. lebetina, M. l. obtusa, M. l. turanica, and M. schweizeri using diverse coagulation analysing protocols. All five were extremely potent in their ability to promote clotting but varied in their relative activation of Factor X, being equipotent in this study to the venom of the better studied, and lethal, species Daboia russelii. The Insoserp European viper antivenom was shown to be highly effective against all the Macrovipera venoms, but performed poorly against the D. russelii venom. Reciprocally, while Daboia antivenoms performed well against D. russelii venom, they failed against Macrovipera venom. Thus despite the two genera sharing a venom phenotype (Factor X activation) driven by the same toxin type (P-IIId snake venom metalloproteases), the surface biochemistries of the toxins differed significantly enough to impede antivenom cross- neutralization. The differences in venom biochemistry were reflected in coagulation co-factor dependence. While both genera were absolutely dependent upon calcium for the activation of Factor X, dependence upon phospholipid varied. The Macrovipera venoms had low levels of dependence upon phospholipid while the Daboia venom was three times more dependent upon phospholipid for the activation of Factor X. This suggests that the sites on the molecular surface responsible for phospholipid dependence, are the same differential sites that prevent inter-genera antivenom cross- neutralization. Due to cold-chain requirements, antivenoms may not be stocked in rural settings where the need is at the greatest. Thus we tested the efficacy of enzyme inhibitor Prinomastat as a field-deployable treatment to stabilise patients while being transported to antivenom stocks, and showed that it was extremely effective in blocking the Factor X activating pathophysiological actions. Marimastat however was less effective. These results thus not only shed light on the coagulopathic mechanisms of Macrovipera venoms, but also provide data critical for evidence-based design of snakebite management strategies.
Topics: Animals; Antivenins; Blood Coagulation; Factor X; Humans; Hydroxamic Acids; Matrix Metalloproteinase Inhibitors; Organic Chemicals; Phospholipids; Species Specificity; Viper Venoms; Viperidae
PubMed: 33412251
DOI: 10.1016/j.toxlet.2020.12.019 -
European Journal of Medicinal Chemistry Mar 2021Matrix metalloproteinases (MMPs) are involved in several pathological and physiological functions. Gelatinases (MMP-2 and -9) have significant attention as therapeutic... (Review)
Review
Matrix metalloproteinases (MMPs) are involved in several pathological and physiological functions. Gelatinases (MMP-2 and -9) have significant attention as therapeutic targets against cancer. Gelatinase inhibitors have demonstrated their effectiveness in several diseases including cancer. However, it is quite a challenging task to develop inhibitors as a therapeutic agent. This review summarizes the patent dedicated to the medicinal chemistry of gelatinase inhibitor reported over last decades. We examine the patent being pursued for gelatinase inhibitor development to highlight the key issues. The main aim is to provide the scientific community with an overview of the patented gelatinase inhibitors to allow further development. During early 2000s, some MMP inhibitors failed to pass the clinical trials. Hence, the lessons learned from early evidence and recent knowledge in that field will rejuvenate the development of selective inhibitors. Various studies and patents have continued in the recent years to expand knowledge. Continuously, our research team has been involved in the design of potent and selective gelatinase inhibitors for the past few years. This study is a part of our efforts. This study may be beneficial in the design and development of better gelatinase inhibitors in the future.
Topics: Animals; Antineoplastic Agents; Biphenyl Compounds; Diphosphonates; Drug Design; Humans; Hydroxamic Acids; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Models, Molecular; Organic Chemicals; Phenylbutyrates; Structure-Activity Relationship
PubMed: 33279289
DOI: 10.1016/j.ejmech.2020.113044 -
PloS One 2018Characterization of cytochrome P450 2D6 (CYP2D6) and the impact of the major identified allelic variants on the activity of one of the most dominating drug-metabolising...
Characterization of cytochrome P450 2D6 (CYP2D6) and the impact of the major identified allelic variants on the activity of one of the most dominating drug-metabolising enzymes is essential to increase drug safety and avoid adverse reactions. Microsecond molecular dynamics simulations have been performed to capture the dynamic signatures of this complex enzyme and five allelic variants with diverse enzymatic activity. In addition to the apo simulations, three substrates (bufuralol, veliparib and tamoxifen) and two inhibitors (prinomastat and quinidine) were included to explore their influence on the structure and dynamical features of the enzyme. Our results indicate that the altered enzyme activity can be attributed to changes in the hydrogen bonding network within the active site, and local structural differences in flexibility, position and shape of the binding pocket. In particular, the increased (CYP2D6*53) or the decreased (CYP2D6*17) activity seems to be related to a change in dynamics of mainly the BC loop due to a modified hydrogen bonding network around this region. In addition, the smallest active site volume was found for CYP2D6*4 (no activity). CYP2D6*2 (normal activity) showed no major differences in dynamic behaviour compared to the wild-type.
Topics: Alleles; Benzimidazoles; Catalytic Domain; Cytochrome P-450 CYP2D6; Dextromethorphan; Enzyme Inhibitors; Ethanolamines; Heme; Humans; Hydrogen Bonding; Inactivation, Metabolic; Molecular Dynamics Simulation; Substrate Specificity; Tamoxifen
PubMed: 30133539
DOI: 10.1371/journal.pone.0202534