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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 -
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 -
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 -
Annales de Biologie Clinique 2003Matrix metalloproteinases (MMPs) play a key role in the physiology of connective tissue development, morphogenesis and wound healing, but their unregulated activity has... (Comparative Study)
Comparative Study Review
Matrix metalloproteinases (MMPs) play a key role in the physiology of connective tissue development, morphogenesis and wound healing, but their unregulated activity has been implicated in numerous disease processes including arthritis, tumor cell metastasis and atherosclerosis. MMP family consists of at least 20 members; MMPs are produced by the different cell types (vascular smooth muscle cells, monocytes, endothelial cells) involved in the atheromatous plaque formation and participate to extracellular matrix remodelling and cell infiltration or migration. Since excessive tissue remodelling and increased matrix metalloproteinase activity have been demonstrated during atherosclerotic lesion progression (including plaque disruption), MMPs represent a potential target for therapeutic intervention to modify vascular pathology, by restoring the MMP/TIMP physiological equilibrium. This review highlights the structures of MMPs and their physiological inhibitors, the Tissue Inhibitors of MMPs (TIMPs), and describes the current developments in pharmacological MMP inhibition.
Topics: Animals; Anti-Bacterial Agents; Antineoplastic Agents; Aortic Diseases; Arteriosclerosis; Case-Control Studies; Clinical Trials as Topic; Coronary Artery Disease; Doxycycline; Humans; Hydroxamic Acids; Hyperlipidemias; Hypolipidemic Agents; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Metalloendopeptidases; Organic Chemicals; Phenylalanine; Polymorphism, Genetic; Prospective Studies; Rats; Risk Factors; Thiophenes; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinase-3; Tissue Inhibitor of Metalloproteinases
PubMed: 12702469
DOI: No ID Found -
Osteoarthritis and Cartilage Dec 2004To identify and characterize a cartilage degradation mechanism that is independent of the proteolytic cleavages by matrix metalloproteinases (MMPs) and aggrecanases.
OBJECTIVE
To identify and characterize a cartilage degradation mechanism that is independent of the proteolytic cleavages by matrix metalloproteinases (MMPs) and aggrecanases.
METHODS
The sensitivity of glycosaminoglycan (GAG) release and collagen release to an MMP/aggrecanase inhibitor, AG3340, was compared using a bovine nasal cartilage explant culture. The release of matrix proteins and hyaluronan (HA) from the culture was analyzed by immunoblotting and radioimmunoassay, respectively. Induction of HA-degrading activity by retinoic acid was examined using the cartilage explant culture and a primary culture of chondrocytes. Degradation of the matrix components of cartilage was also characterized in vivo using an acute arthritis model induced by an intra-articular injection of interleukin 1alpha (IL-1alpha).
RESULTS
AG3340 did not effectively inhibit GAG release at a concentration of more than 10muM, while 10nM of the inhibitor completely suppressed collagen degradation. Retinoic acid induced the release of the aggrecan G1 domain, link protein and HA into the culture medium, and the release of these molecules was not completely inhibited by 10muM of AG3340. The molecules were released as ternary complexes. Retinoic acid induced HA degradation in the explant culture and hyaluronidase activity in the primary culture of chondrocytes. The release of the G1 domain of aggrecan and link protein into the synovial fluid was also observed in the IL-1alpha-induced acute arthritis model.
CONCLUSION
A novel mechanism by chondrocyte-derived hyaluronidase(s) is involved in the release of the matrix components from cartilage, and the hyaluronidase(s) and MMPs/aggrecanases act in a coordinated manner in cartilage degradation.
Topics: Aggrecans; Animals; Cartilage; Cattle; Collagen Type II; Endopeptidases; Extracellular Matrix Proteins; Glycosaminoglycans; Interleukin-1; Lectins, C-Type; Matrix Metalloproteinase Inhibitors; Oncostatin M; Organic Chemicals; Peptides; Proteoglycans; Rabbits; Stimulation, Chemical; Synovial Fluid; Tissue Culture Techniques; Tretinoin
PubMed: 15564068
DOI: 10.1016/j.joca.2004.09.003 -
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 -
Technology in Cancer Research &... Aug 2004Macromolecular contrast medium-enhanced magnetic resonance imaging was applied to monitor the effect of matrix metalloprotease (MMP) inhibition on microvascular...
Macromolecular contrast medium-enhanced magnetic resonance imaging was applied to monitor the effect of matrix metalloprotease (MMP) inhibition on microvascular characteristics of human breast cancers implanted in athymic rats. Twice-daily intraperitoneal administration of Prinomastat over 1.5 days induced significant declines in MRI-assayed microvascular permeabilities (p<0.05); but this leak suppression effect had extinguished by the 10(th) day of MMP treatment using the same dose and time schedule. Results demonstrate that Prinomastat produces a rapid but transient decrease in tumor vascular permeability. Contrast-enhanced MRI using macromolecular contrast medium may prove useful as a biomarker for the dynamic MMP biological effect in cancers.
Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Capillary Permeability; Cell Division; Cell Line, Tumor; Contrast Media; Enzyme Inhibitors; Female; Homozygote; Humans; Magnetic Resonance Imaging; Matrix Metalloproteinase Inhibitors; Microcirculation; Neoplasm Transplantation; Neoplasms; Organic Chemicals; Rats; Rats, Nude; Statistics as Topic; Time Factors
PubMed: 15270589
DOI: 10.1177/153303460400300408 -
American Journal of Physiology. Lung... Aug 2004Murine Mycoplasma pulmonis infection induces chronic lung and airway inflammation accompanied by profound and persistent microvascular remodeling in tracheobronchial...
Murine Mycoplasma pulmonis infection induces chronic lung and airway inflammation accompanied by profound and persistent microvascular remodeling in tracheobronchial mucosa. Because matrix metalloproteinase (MMP)-2 and -9 are important for angiogenesis associated with placental and long bone development and skin cancer, we hypothesized that they contribute to microvascular remodeling in airways infected with M. pulmonis. To test this hypothesis, we compared microvascular changes in airways after M. pulmonis infection of wild-type FVB/N mice with those of MMP-9(-/-) and MMP-2(-/-)/MMP-9(-/-) double-null mice and mice treated with the broad-spectrum MMP inhibitor AG3340 (Prinomastat). Using zymography and immunohistochemistry, we find that MMP-2 and MMP-9 rise strikingly in lungs and airways of infected wild-type FVB/N and C57BL/6 mice, with no zymographic activity or immunoreactivity in MMP-2(-/-)/MMP-9(-/-) animals. However, microvascular remodeling as assessed by Lycopersicon esculentum lectin staining of whole-mounted tracheae is as severe in infected MMP-9(-/-), MMP-2(-/-)/MMP-9(-/-) and AG3340-treated mice as in wild-type mice. Furthermore, all groups of infected mice develop similar inflammatory infiltrates and exhibit similar overall disease severity as indicated by decrease in body weight and increase in lung weight. Uninfected wild-type tracheae show negligible MMP-2 immunoreactivity, with scant MMP-9 immunoreactivity in and around growing cartilage. By contrast, MMP-2 appears in epithelial cells of infected, wild-type tracheae, and MMP-9 localizes to a large population of infiltrating leukocytes. We conclude that despite major increases in expression, MMP-2 and MMP-9 are not essential for microvascular remodeling in M. pulmonis-induced chronic airway inflammation.
Topics: Animals; Epithelial Cells; Gene Expression Regulation, Enzymologic; Immunohistochemistry; Leukocytes; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Neovascularization, Pathologic; Pneumonia, Mycoplasma; Pulmonary Circulation; Trachea
PubMed: 15075248
DOI: 10.1152/ajplung.00404.2003 -
Investigative Ophthalmology & Visual... Feb 2007The efficacy of three matrix metalloproteinase (MMP) inhibitors with various selectivities (Ro-31-9790, AG3340, and DPC-A37668) was investigated in a rat model of...
PURPOSE
The efficacy of three matrix metalloproteinase (MMP) inhibitors with various selectivities (Ro-31-9790, AG3340, and DPC-A37668) was investigated in a rat model of retinopathy of prematurity, to examine the roles of MMP-2 and -9 in retinal neovascularization. The susceptibilities of MMP-2(-/-) and -9(-/-) mice to preretinal neovascularization were investigated in a mouse model of oxygen-induced retinopathy.
METHODS
Sprague-Dawley newborn rats were exposed to alternating episodes of 50% and 10% oxygen (variable oxygen exposure) to induce retinal neovascularization. Three MMP inhibitors with various selectivity profiles were administered to variable oxygen-exposed rats via local or systemic routes. Antineovascular efficacy was determined in drug-treated versus vehicle-treated rat pups by computerized imaging of adenosine diphosphatase (ADPase)-stained retinal flatmounts. Wild-type C57BL/6J and isogenic MMP-2(-/-) and -9(-/-) mice were exposed to 75% oxygen followed by normoxia. The mice were killed immediately before or after the normoxic exposure, and eyes were either harvested for retinal dissection and flatmounting or were paraffin embedded and sectioned. Retinal vascular area and retinal neovascularization were assessed by adenosine diphosphatase staining of retinal flatmounts and by counting preretinal nuclei of hematoxylin and eosin-stained retinal sections, respectively.
RESULTS
Ro-31-9790, AG3340, and DPC-A37668 had no effect on normal development of the rat retinal vasculature, regardless of dose or route of administration. Intravitreal injection of Ro-31-9790 (broad-spectrum) immediately after variable-oxygen exposure and 2 days after exposure resulted in 78% and 82% inhibition of retinal neovascularization, respectively. AG3340 (MMP-2- and -9-selective inhibitor) and DPC-A37668 (MMP-2-selective inhibitor) resulted in 65% and 52% inhibition, respectively, when administered by intravitreal injection immediately after variable-oxygen exposure. Intraperitoneal injection of 5, 15, and 50 mg/mL AG3340 or DPC-A37668 for 6 days after variable oxygen exposure resulted in 22% to 39% and 0% to 31% inhibition of neovascularization, respectively. AG3340 and DPC-A37668 administered by oral gavage at doses of 3, 10, or 30 mg/mL provided up to 42% and 86% inhibition of neovascularization, respectively. The average vascular areas of retinas from MMP-2(-/-) or -9(-/-) mice at postnatal day 12 were not significantly different from the wild-type control. There was a 75% (P < 0.001) and 44% (P < 0.01) reduction in preretinal neovascularization in oxygen-exposed MMP-2(-/-) and -9(-/-) mice at postnatal day 19, respectively, compared with wild-type control mice.
CONCLUSIONS
The results of this study suggest that MMP-2 plays a predominant role in retinal angiogenesis in both the mouse and rat models of oxygen-induced retinopathy. Furthermore, MMP-2 inhibition may be a viable therapeutic approach for ocular diseases characterized by retinal neovascularization.
Topics: Animals; Animals, Newborn; Apyrase; Disease Models, Animal; Enzyme Inhibitors; Gene Silencing; Humans; Hydroxamic Acids; Infant, Newborn; Injections; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred C57BL; Organic Chemicals; Oxygen; Pyridines; Rats; Rats, Sprague-Dawley; Retina; Retinal Neovascularization; Retinopathy of Prematurity; Vitreous Body
PubMed: 17251494
DOI: 10.1167/iovs.06-0082 -
Journal of Ocular Pharmacology and... Jun 2004AG3340 (prinomastat) is a nonpeptidic, small-molecular-weight, synthetic matrix metalloproteinase inhibitor (MMPI) with selective inhibitory action of MMP-2, MMP-9,... (Comparative Study)
Comparative Study
PURPOSE
AG3340 (prinomastat) is a nonpeptidic, small-molecular-weight, synthetic matrix metalloproteinase inhibitor (MMPI) with selective inhibitory action of MMP-2, MMP-9, MMP-3, and MT-MMP1. We evaluated AG3340 injected intravitreally to treat choroidal neovascularization in a laser induced rat CNV model.
METHODS
In the pretreatment group, the drug was injected the same day after induction of choroidal neovascularization by diode laser. In the treatment group, the drug was injected 2 weeks after induction of choroidal neovascularization (CNV). Fluorescein and indocyanine green angiography were performed to evaluate CNV. ERG recordings and histology were performed to assess toxicity and the CNV lesions.
RESULTS
When used at the time of CNV induction, 62.8% of lesions in control versus 22.8% of the laser lesions in treated eyes developed CNV (p < 0.0001). The invading fibrovascular complex was thicker in the control eyes than that in the treated eyes. No signs of toxicity were detected. When used to treat established CNV, the percentage of leakage in treated and control eyes were 54.1% and 58.9% respectively (p > 0.05). Prinomastat was effective when given at the time of induction of CNV in the rat model. Administration of prinomastat 2 weeks after laser induction did not show efficacy.
CONCLUSION
Prinomastat was active in the earliest stages of experimental CNV. It might be best used in combination with photodynamic therapy to inhibit recurrence of CNV from temporarily closed new vessels.
Topics: Animals; Choroidal Neovascularization; Disease Models, Animal; Electroretinography; Enzyme Inhibitors; Female; Fluorescein Angiography; Injections; Laser Coagulation; Matrix Metalloproteinase Inhibitors; Organic Chemicals; Rats; Rats, Inbred BN; Retinal Hemorrhage; Time Factors; Vitreous Body
PubMed: 15279727
DOI: 10.1089/1080768041223657