Anti-inflammatory Activity of PLA2 Inhibitory Saccharumoside-B.

BACKGROUND: Saccharumoside-B and its analogs were found to have anticancer potential in vitro. The present study reports acute toxicity, molecular docking, ADMET profile analysis, and in vitro and in vivo anti-inflammatory activity of saccharumoside-B for the first time. METHODS: The in vitro enzyme inhibitory activity of saccharumoside-B on PLA2, COX-1, COX-2, and 5-LOX enzymes was evaluated by the cell-free method, and its effect on TNF-α, IL1ß, and IL- 6 secretion levels in LPS stimulated THP-1 human monocytes was determined by ELISA-based methods. The anti-inflammatory activity was evaluated in vivo by carrageenan-induced rat paw edema model. To test its binding affinity at the active site pockets of PLA2 enzymes and assess drug-like properties, docking experiments and ADMET studies were performed. RESULTS: Saccharumoside-B showed selective inhibition of the sPLA2 enzyme (IC50 = 7.53 ± 0.232 µM), and thioetheramide-PC was used as a positive control. It showed significant inhibition (P ≤ 0.05) of TNF-α, IL-1ß, and IL-6 cytokines compared to the positive control dexamethasone. Saccharumoside-B showed a dose-dependent inhibition of carrageenan-induced rat paw edema, with a maximum inhibition (76.09 ± 0.75) observed at 3 hours after the phlogistic agent injection. Saccharumoside-B potentially binds to the active site pocket of sPLA2 crystal protein (binding energy -7.6 Kcal/Mol). It complies with Lipinski's Rule of Five, showing a promising safety profile. The bioactivity scores suggested it to be a better enzyme inhibitor. CONCLUSION: Saccharumoside-B showed significant PLA2 inhibition. It can become a potential lead molecule in synthesizing a new class of selective PLA2 inhibitors with a high safety profile in the future.

BoaγPLI from Boa constrictor Blood is a Broad-Spectrum Inhibitor of Venom PLA2 Pathophysiological Actions.

The use of venom in predation exerts a corresponding selection pressure for the evolution of venom resistance. One of the mechanisms related to venom resistance in animals (predators or prey of snakes) is the presence of molecules in the blood that can bind venom toxins, and inhibit their pharmacological effects. One such toxin type are venom phospholipase A2s (PLA2s), which have diverse effects including anticoagulant, myotoxic, and neurotoxic activities. BoaγPLI isolated from the blood of Boa constrictor has been previously shown to inhibit venom PLA2s that induced myotoxic and edematogenic activities. Recently, in addition to its previously described and very potent neurotoxic effect, the venoms of American coral snakes (Micrurus species) have been shown to have anticoagulant activity via PLA2 toxins. As coral snakes eat other snakes as a major part of their diet, neonate Boas could be susceptible to predation by this sympatric species. Thus, this work aimed to ascertain if BoaγPLI provided a protective effect against the anticoagulant toxicity of venom from the model species Micrurus laticollaris in addition to its ability shown previously against other toxin types. Using a STA R Max coagulation analyser robot to measure the effect upon clotting time, and TEG5000 thromboelastographers to measure the effect upon clot strength, we evaluated the ability of BoaγPLI to inhibit M. laticollaris venom. Our results indicate that BoaγPLI is efficient at inhibiting the M. laticollaris anticoagulant effect, reducing the time of coagulation (restoring them closer to non-venom control values) and increasing the clot strength (restoring them closer to non-venom control values). These findings demonstrate that endogenous PLA2 inhibitors in the blood of non-venomous snakes are multi-functional and provide broad resistance against a myriad of venom PLA2-driven toxic effects including coagulotoxicity, myotoxicity, and neurotoxicity. This novel form of resistance could be evidence of selective pressures caused by predation from venomous snakes and stresses the need for field-based research aimed to expand our understanding of the evolutionary dynamics of such chemical arms race.

BoaγPLI from Boa constrictor blood is a broad-spectrum inhibitor of venom PLA2 pathophysiological actions

The use of venom in predation exerts a corresponding selection pressure for the evolution of venom resistance. One of the mechanisms related to venom resistance in animals (predators or prey of snakes) is the presence of molecules in the blood that can bind venom toxins, and inhibit their pharmacological effects. One such toxin type are venom phospholipase A2s (PLA2s), which have diverse effects including anticoagulant, myotoxic, and neurotoxic activities. BoaγPLI isolated from the blood of Boa constrictor has been previously shown to inhibit venom PLA2s that induced myotoxic and edematogenic activities. Recently, in addition to its previously described and very potent neurotoxic effect, the venoms of American coral snakes (Micrurus species) have been shown to have anticoagulant activity via PLA2 toxins. As coral snakes eat other snakes as a major part of their diet, neonate Boas could be susceptible to predation by this sympatric species. Thus, this work aimed to ascertain if BoaγPLI provided a protective effect against the anticoagulant toxicity of venom from the model species Micrurus laticollaris in addition to its ability shown previously against other toxin types. Using a STA R Max coagulation analyser robot to measure the effect upon clotting time, and TEG5000 thromboelastographers to measure the effect upon clot strength, we evaluated the ability of BoaγPLI to inhibit M. laticollaris venom. Our results indicate that BoaγPLI is efficient at inhibiting the M. laticollaris anticoagulant effect, reducing the time of coagulation (restoring them closer to non-venom control values) and increasing the clot strength (restoring them closer to non-venom control values). These findings demonstrate that endogenous PLA2 inhibitors in the blood of non-venomous snakes are multi-functional and provide broad resistance against a myriad of venom PLA2-driven toxic effects including coagulotoxicity, myotoxicity, and neurotoxicity. This novel form of resistance could be evidence of selective pressures caused by predation from venomous snakes and stresses the need for field-based research aimed to expand our understanding of the evolutionary dynamics of such chemical arms race.

Biochemical and functional characterization of a new recombinant phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum.

Phospholipase A2 plays an important role in many diseases. Thus, the production of bioactive molecules, which can modulate PLA2 activity, became an important target for the pharmaceutical industry. Previously, we demonstrated the inhibitory and anti-angiogenic effect of γCdcPLI, the natural PLA2inhibitor from Crotalus durissus collilineatus. The aim of the present study was to recombinantly express the γCdcPLI inhibitor and analyze its biochemical and functional characteristics. Based on the amino acid sequence from the natural protein, we designed a synthetic gene for production of a non-tagged recombinant recγCdcPLI using the pHis-Parallel2 vector. To enable disulfide bond formation, protein expression was performed using E. coli Rosetta-gamiB. The protein was purified by anion and affinity chromatography with a yield of 5 mg/L. RecγCdcPLI showed similar secondary structure in CD and FTIR, revealing predominately ß-strands. Analogous to the natural protein, recγCdcPLI was able to form oligomers of ~5.5 nm. The inhibitor was efficiently binding to PLA2 from honeybee (Kd = 1.48 µM) and was able to inhibit the PLA2 activity. Furthermore, it decreased the vessel formation in HUVEC cells, suggesting an anti-angiogenic potential. Heterologous production of recγCdcPLI is highly efficient and thus enables enhanced drug design for treatment of diseases triggered by PLA2 activity.

Biochemical and functional characterization of a new recombinant phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum

Phospholipase A2 plays an important role in many diseases. Thus, the production of bioactive molecules, which can modulate PLA2 activity, became an important target for the pharmaceutical industry. Previously, we demonstrated the inhibitory and anti-angiogenic effect of γCdcPLI, the natural PLA2inhibitor from Crotalus durissus collilineatus. The aim of the present study was to recombinantly express the γCdcPLI inhibitor and analyze its biochemical and functional characteristics. Based on the amino acid sequence from the natural protein, we designed a synthetic gene for production of a non-tagged recombinant recγCdcPLI using the pHis-Parallel2 vector. To enable disulfide bond formation, protein expression was performed using E. coli Rosetta-gamiB. The protein was purified by anion and affinity chromatography with a yield of 5 mg/L. RecγCdcPLI showed similar secondary structure in CD and FTIR, revealing predominately β-strands. Analogous to the natural protein, recγCdcPLI was able to form oligomers of ~5.5 nm. The inhibitor was efficiently binding to PLA2 from honeybee (Kd = 1.48 μM) and was able to inhibit the PLA2 activity. Furthermore, it decreased the vessel formation in HUVEC cells, suggesting an anti-angiogenic potential. Heterologous production of recγCdcPLI is highly efficient and thus enables enhanced drug design for treatment of diseases triggered by PLA2 activity.

Endogenous phospholipase A2 inhibitors in snakes: a brief overview.

The blood plasma of numerous snake species naturally comprises endogenous phospholipase A2 inhibitors, which primarily neutralize toxic phospholipases A2 that may eventually reach their circulation. This inhibitor type is generally known as snake blood phospholipase A2 inhibitors (sbPLIs). Most, if not all sbPLIs are oligomeric glycosylated proteins, although the carbohydrate moiety may not be essential for PLA2 inhibition in every case. The presently known sbPLIs belong to one of three structural classes - namely sbαPLI, sbßPLI or sbγPLI - depending on the presence of characteristic C-type lectin-like domains, leucine-rich repeats or three-finger motifs, respectively. Currently, the most numerous inhibitors described in the literature are sbαPLIs and sbγPLIs, whereas sbßPLIs are rare. When the target PLA2 is a Lys49 homolog or an Asp49 myotoxin, the sbPLI is denominated a myotoxin inhibitor protein (MIP). In this brief overview, the most relevant data on sbPLIs will be presented. Representative examples of sbαPLIs and sbγPLIs from two Old World - Gloydius brevicaudus and Malayopython reticulatus - and two New World - Bothrops alternatus and Crotalus durissus terrificus - snake species will be emphasized.

Endogenous phospholipase A2 inhibitors in snakes: a brief overview

The blood plasma of numerous snake species naturally comprises endogenous phospholipase A2 inhibitors, which primarily neutralize toxic phospholipases A2 that may eventually reach their circulation. This inhibitor type is generally known as snake blood phospholipase A2 inhibitors (sbPLIs). Most, if not all sbPLIs are oligomeric glycosylated proteins, although the carbohydrate moiety may not be essential for PLA2 inhibition in every case. The presently known sbPLIs belong to one of three structural classes - namely sbαPLI, sbβPLI or sbγPLI - depending on the presence of characteristic C-type lectin-like domains, leucine-rich repeats or three-finger motifs, respectively. Currently, the most numerous inhibitors described in the literature are sbαPLIs and sbγPLIs, whereas sbβPLIs are rare. When the target PLA2 is a Lys49 homolog or an Asp49 myotoxin, the sbPLI is denominated a myotoxin inhibitor protein (MIP). In this brief overview, the most relevant data on sbPLIs will be presented. Representative examples of sbαPLIs and sbγPLIs from two Old World - Gloydius brevicaudus and Malayopython reticulatus - and two New World - Bothrops alternatus and Crotalus durissus terrificus - snake species will be emphasized.(AU)

Suppression of severe lesions, myonecrosis and hemorrhage, caused by Protobothrops flavoviridis venom with its serum proteins.

Protobothrops flavoviridis serum proteins precipitated with ammonium sulfate were chromatographed on a DEAE-Toyopearl 650M column at pH 7.5 with stepwise increase or with linear gradient of NaCl concentration. Peaks 3 and 4 serum proteins, obtained by linear gradient elution and named Fr(de3) and Fr(de4), contained Habu serum factors (HSF) and phospholipase A2 (PLA2) inhibitors (PfPLI), respectively. The serum proteins eluted at 0.2 M NaCl by stepwise elution, named Fr(0.2NaCl), effectively suppressed myonecrosis and hemorrhage caused by P. flavoviridis venom in rat or mouse thigh muscles. The Fr(0.2NaCl) were fractionated by HPLC and the fractions, after SDS-PAGE, underwent far-western blot analysis with PLA2 ([Asp(49)]PLA2) and BPI ([Lys(49)]PLA2) as the probes. Four PfPLIs, namely, PfαPLI-A, PfαPLI-B, PfγPLI-A and PfγPLI-B, were identified together with their selective binding specificities to PLA2 species. In addition, a new 9 kDa protein, which is specifically bound to BPI, was found. Suppression of P. flavoviridis venom-induced severe lesions, such as myonecrosis, hemorrhage and edema, with its serum proteins was histopathologically observed in the present work for the first time. The cooperative use of P. flavoviridis antivenom and its serum proteins as medication for P. flavoviridis snake bites is discussed.

Bioactive indole derivatives from the South Pacific marine sponges Rhopaloeides odorabile and Hyrtios sp.

Indole derivatives including bromoindoles have been isolated from the South Pacific marine sponges Rhopaloeides odorabile and Hyrtios sp. Their structures were established through analysis of mass spectra and 1D and 2D NMR spectroscopic data. Their potential inhibitory phospholipase A2 (PLA2), antioxidant and cytotoxic activities were evaluated. The new derivative 5,6-dibromo-L-hypaphorine (9) isolated from Hyrtios sp. revealed a weak bee venom PLA2 inhibition (IC50 0.2 mM) and a significant antioxidant activity with an Oxygen Radical Absorbance Capacity (ORAC) value of 0.22. The sesquiterpene aureol (4), also isolated from Hyrtios sp., showed the most potent antioxidant activity with an ORAC value of 0.29.