Progressive hemorrhage and myotoxicity induced by echis carinatus venom in murine model: neutralization by inhibitor cocktail of n,n,n `,n `-tetrakis (2-pyridylmethyl) ethane-1,2-diamine and silymarin

Viperbite is often associated with severe local toxicity, including progressive hemorrhage and myotoxicity, persistent even after the administration of anti-snake venom (ASV). In the recent past, investigations have revealed the orchestrated actions of Zn2+ metalloproteases (Zn(2+)MPs), phospholipase A(2)s (PLA(2)s) and hyaluronidases (HYs) in the onset and progression of local toxicity from the bitten site. As a consequence, venom researchers and medical practitioners are in deliberate quest of potent molecules alongside ASV to tackle the brutal local manifestations induced by aforesaid venom toxins. Based on these facts, we have demonstrated the protective efficacy of inhibitor cocktail containing equal ratios of N,N,N', N'-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and silymarin (SLN) against progressive local toxicity induced by Echis carinatus venom (ECV). In our previous study we have shown the inhibitory potentials of TPEN towards Zn(2+)MPs of ECV (IC50: 6.7 mu M). In this study we have evaluated in vitro inhibitory potentials of SLN towards PLA(2)s (IC50: 12.5 mu M) and HYs (IC50: 8 mu M) of ECV in addition to docking studies. Further, we have demonstrated the protection of ECV induced local toxicity with 10 mM inhibitor cocktail following 15, 30 min (for hemorrhage and myotoxicity); 60 min (for hemorrhage alone) of ECV injection in murine model. The histological examination of skin and thigh muscle sections taken out from the site of ECV injection substantiated the overall protection offered by inhibitor cocktail. In conclusion, the protective efficacy of inhibitor cocktail is of high interest and can be administered locally alongside ASV to treat severe local toxicity

Combinatorial inhibition of Angiotensin converting enzyme, Neutral endopeptidase and Aminopeptidase N by N-methylated peptides alleviates blood pressure and fibrosis in rat model of dexamethasone-induced hypertension

Angiotensin converting enzyme (ACE), neutral endopeptidase (NEP) and aminopeptidase N (APN) are responsible for generation of vasoactive peptides that regulates vasoconstriction, vasodilation and natriuresis, which altogether regulate blood pressure. Cumulative inhibition of ACE, NEP and APN effectively blocks the progression of respective pathways. In this study, N-methylated peptide inhibitors F-N(Me)H-L, V-N(Me)F-R and R-N(Me)V-Y were synthesized against ACE, NEP and APN respectively, using their respective physiological substrates. F-N(Me)H-L inhibited ACE activity with an IC50 of 83 nmol/L, V-N(Me)F-R inhibited NEP activity with an IC50 of 1.173 mu mol/L and R-N(Me)V-Y inhibited APN activity with an IC50 of 3.94 nmol/L respectively. Further, the anti-hypertensive effect of N-methylated peptides was evaluated using rat model of dexamethasone-induced hypertension. Individual peptides and their cocktail treatment were started from day 6 of the study period and blood pressure was measured on every alternate day during 15 day study. Administration of F-N(Me) H-L (138 +/- 3 mmHg) and cocktail of all the three peptides at a dose of 100 mg/kg significantly reduced systolic blood pressure (SBP) compared to dexamethasone group (SBP of Groups-dexamethasone; (167 +/- 5 mmHg), F-N (Me)H-L (138 +/- 3 mmHg), and Cocktail (122 +/- 3 mmHg). Anti-hypertensive, anti-hypertrophic and anti-fibrotic effects of N-methylated peptides and cocktail was further reflected by the decreased levels of circulating Ang II and increased ANP levels in sera of hypertensive rats along with decrease in collagen deposition in heart and kidney. Though, ACE inhibition is adequate to reduce SBP, targeting NEP and APN along with ACE is beneficial in tackling hypertension and associated fibrosis of heart

Dimethyl ester of bilirubin exhibits anti-inflammatory activity through inhibition of secretory phospholipase A2, lipoxygenase and cyclooxygenase

Overproduction of arachidonic acid (AA) mediated by secretory phospholipase A2 group IIA (sPLA2IIA) is a hallmark of many inflammatory disorders. AA is subsequently converted into pro-inflammatory eicosanoids through 5-lipoxygenase (5-LOX) and cyclooxygenase-1/2 (COX-1/2) activities. Hence, inhibition of sPLA2IIA, 5-LOX and COX-1/2 activities is critical in regulating inflammation. We have previously reported unconjugated bilirubin (UCB), an endogenous antioxidant, as sPLA2IIA inhibitor. However, lipophilic UCB gets conjugated in liver with glucuronic acid into hydrophilic conjugated bilirubin (CB). Since hydrophobicity is pre-requisite for sPLA2IIA inhibition, conjugation reduces the efficacy of UCB. In this regard, UCB was chemically modified and derivatives were evaluated for sPLA2IIA, 5-LOX and COX-1/2 inhibition. Among the derivatives, BD1 (dimethyl ester of bilirubin) exhibited ∼ 3 fold greater inhibitory potency towards sPLA2IIA compared to UCB. Both UCB and BD1 inhibited human 5-LOX and COX-2 activities; however only BD1 inhibited AA induced platelet aggregation. Molecular docking studies demonstrated BD1 as better inhibitor of aforesaid enzymes than UCB and other endogenous antioxidants. These data suggest that BD1 exhibits strong anti-inflammatory activity through inhibition of AA cascade enzymes which is of great therapeutic importance

Predicted structure of ECVHY.

<p>(a) Target—template sequence alignment, (b) model validation, (c) target-template structure superposition and (d) conserved active site residues. The template structures—bee venom hyaluronidase (PDB ID: 1FCQ-template 1) and human hyaluronidase (PDB ID: 2PE4- template 2) showed 33.3% and 42% sequence identity and 92% and 70% query coverage with the target sequence—<i>Echis ocellatus</i> venom hyaluronidase (UniProt ID: A3QVN2).</p

(i) Serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels and (ii) histopathology of mice injected (i.m.) with ECV and its protection by inhibitor cocktail.

<p>Mice were injected with 5 μg ECV + different doses of inhibitor cocktail (independently after 30 min of ECV injection). After 3 h, mice were sacrificed and serum CK and LDH levels were assayed using AGAPPE kit. **, ^##<i>p</i> < 0.01 and ***, ^###<i>p</i> < 0.001 compared to ECV induced CK and LDH values. Further, dissected thigh muscles from the site of ECV injection were processed for hematoxylin and eosin staining and were observed at 200 X magnification. (a) Saline control showed characteristic muscular striations and intact myocytes. Five μg ECV injected sections dissected at different time points—30 min (c); and 180 min (b) showed disorganization in muscular striations and myocytes in time dependent fashion as evidenced by proportionate elevation of serum CK and LDH activities compared to control. On independent injection, inhibitor cocktail—(d), (e): 3 and 10 mM showed dose-dependent protection against ECV induced myotoxicity. 10 mM inhibitor cocktail alone-injected section (f) showed characteristic muscular striations and intact myocytes. The dark arrows show the damaged portion of muscle sections.</p

Inhibition of hyaluronidase activity of ECV by AP and SLN.

<p>Reaction mixture 300 μl contained 100 μg ECV in 100 mM acetate buffer pH 5.5, 150 mM NaCl and various concentrations of AP and SLN (0.1 nM-10 mM). The reactions were initiated by adding 50 μl substrate (hyaluronic acid) and incubated at 37°C for 2.5 h. After terminating the reaction, the contents were processed for color development.</p

Energetically favorable binding modes of AP and SLN calculated using Induced fit docking method.

<p>Glide score (calculated in kcal/mol) associated with best binding modes of AP and SLN with the active site of ECVPLA₂ (a) and modeled ECVHY (a1). The hydrogen bonding and hydrophobic interaction of AP and SLN with ECVPLA₂ (b, c) and modeled ECVHY (b1, c1) respectively are depicted using the LigPlot software.</p

Energetically favorable binding modes of CSS, SAH, and SLN calculated using IFD method.

<p>Glide score (a) and glide energy (b) (calculated in kcal/mol) associated with best binding modes of CSS, SAH, and SLN with the active site of modeled ECVHY. The hydrogen bonding and hydrophobic interactions between the enzyme and CSS (c), SAH (d), and SLN (e) respectively are depicted using the LigPlot software. CSS, SAH, and SLN are labeled using respective three letter codes with a common residue number 999(Z).</p

Hemorrhagic activity of ECV and its protection by inhibitor cocktail of TPEN and SLN upon independent injections.

<p>(i) Dorsal surface of mouse skin showing hemorrhagic spots. (ii) Area of hemorrhagic spots measured using graph paper. Mice were injected intradermally with constant 3 μg ECV (3 MHD dose) and various doses of inhibitor cocktail of TPEN and SLN (0.3 to 10 mM) at different time points (15 to 60 min) after venom injection. After 3 h, mice were sacrificed and hemorrhagic spots on the inner surface were examined for protection of ECV induced hemorrhage; a. negative control (30μl Saline); b. positive control (hemorrhagic spot appeared after 3 h of 3 μg ECV injection); c. hemorrhagic spot appeared after 15 min of 3 μg ECV injection; d, e, and f: 0.3, 3, and 10 mM inhibitor cocktail injected after 15 min of ECV injection; g. hemorrhagic spot appeared after 30 min of 3 μg ECV injection; h and i: 3 and 10 mM inhibitor cocktail injected after 30 min of ECV injection; j. hemorrhagic spot appeared after 60 min of 3 μg ECV injection; k and l: 3 and 10 mM inhibitor cocktail injected after 60 min of ECV injection; m, n and o: 0.3, 3 and 10 mM inhibitor cocktail alone (cocktail control). *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***, ^###<i>p</i> < 0.001 compared to ECV induced hemorrhage.</p