Current Topics in Medicinal Chemistry - Volume 11, Issue 20, 2011

Snakebite is a medical emergency and considered as an integral occupational hazard for farming and agricultural workers of the rural population worldwide. The human sufferings, morbidity and mortality rate is becoming increasingly high in South and Southeast Asian countries due to snakebite. Death or the physical deformities due to snakebite generally affects the young and healthy bread winners of the family that results in the devastating socio-economic balance. World Health Organization for the first time in 2009 declared snakebite as the neglected tropical disease. Although the available epidemiology data is highly elusive, a daunting value of about 5.5 million people is estimated to be affected by snakes, of which about 400000 amputations and between 20000 and 125 000 deaths each year. Snakebite results in simultaneous lethal assault on vital functions including nervous, respiratory, cardiovascular and haemostatic functions. In addition, the bite in most cases causes intimidating necrosis at the bite site. Timely administration of the anti-venom by intravenous route is the effective and preferred therapy available as of now to save the victim. A range of antivenoms, monospecific, polyspecific, F(ab)2 and Fab are produced worldwide and all of them are found to be effective nearly to a similar extent. Serum sickness due to over dosage of foreign proteins and inability to protect local tissue degradation are the major limitations of this therapy. In several cases, persistent tissue necrosis even after successful neutralization of fatal systemic toxicity is a serious concern. This morbidity might lead to permanent disfigurement, crippling or amputation of the affected limb in a healthy individual. In the last 50 years of snake venom research, there has been a commendable achievement made towards understanding the structure-activity relationships of target specific toxins, exploring them as tools to understand many complicated mechanisms of Biology and to design lead therapeutics. Despite, studies' addressing the complications of snake bite therapy such as designing diagnostic kits to identify the bitten species, improving the efficacy of anti-venom therapy or inhibition of local toxicity has remained nearly stand still. Nonetheless, only a few studies are attempting to find new therapeutics from plants and snake plasma and also generated synthetic molecules that would complement the anti-venom therapy in better neutralization and management of both systemic and local toxicity of snakebite. In contrast to the well understood systemic toxicity, the pharmacology of local toxicity (tissue destruction at the bitten region) is comparatively less understood. The latter is not usually associated with fatality, and this could be the reason for less interest shown. The local toxicity is characterized by edema, hemorrhage, blistering and dermo- and myonecrosis. In spite of myriad data on venom pharmacology, the precise molecular mechanism attributable for tissue necrosis is obscure and elusive. Although venom induced long term inflammatory response cannot be ignored, an array of hydrolytic enzymes including matrix degrading metalloproteases and hyaluronidases, phospholipases A2 and non-enzymatic myotoxins appears to be the key factors. It has generally conceived that the tissue damage at the bite site is associated with viperine and crotaline bites; however, several species of spitting cobra such as Naja asher, N. nigricollis, and N. siamensis also cause extensive tissue damage. In some cases, the venom is non-lethal but, cause violent local toxicity, for example, the Indian Trimeresurus malabaricus venom cause terrifying tissue necrosis at the site of injection. As of now, no remedial measures are available to treat such incidents. Thus, the snake bite (systemic and local toxicities) and its efficient management not only an issue of greater concern restricted to developing, tropical, subtropical or southeast Asian countries but, a global issue that needs special attention. Hence, there is an absolute demand for the culmination of research output from researchers and medical practitioners, who are directly or indirectly, associated with snakebite and its management worldwide. Thus, this special issue is focused on addressing some of the burning problems associated with snake bite, including long term complications and the possible therapeutics that might complement the anti-venom therapy which would enhance its efficacy and also effective management.

Snakebite is a medical emergency in many parts of the world, particularly in the temperate regions. According to 2007 World Health Organization (WHO) report, there are about 5 million snakebite incidences resulting in 2.5 million envenoming, and 125,000 deaths occur annually. Most affected are the healthy individuals like children and farming populations with resource poor settings and away from health care centers in low-income countries of Africa, Asia and Latin America. In view of this, the WHO has declared snakebite as an ignored health crisis and a tropical disease. Although the death rate has reduced markedly due to anti-venom regiment, several limitations of it offer scope for better understanding of various ignored issues. Currently, snakebite therapeutics facing plethora of scientific, technological and public health challenges, including secondary/long term complications that have not been given importance so far. Because of dearth of knowledge, venom researchers and medical practitioners from affected countries worldwide should join together to accomplish this scenario. In view of this, the present review provides a broader perspective on the possible production and application of highly effective therapeutic master anti-venom, designing master diagnostic kit and also to deal with the inefficacy of anti-venom therapy against local manifestations and secondary complications of snakebite. The review demands thorough understanding of venom pharmacology, inculcating new strategies to handle and to enhance the efficacy of snakebite management and urge the governing systems of affected countries to take steps to curtail accidental debilitation and death rate of healthy individuals due to snakebite.

Phospholipases A2 (PLA2s) from snake venoms comprise a group of 14-18 kDa proteins, responsible for several toxic effects induced by the whole venom. Considering this, studies aiming at the search for natural inhibitors of these proteins are very important. The present work had as objectives the isolation and functional/structural characterization of a γ-type phospholipase A2 inhibitor (PLI) from Bothrops jararacussu snake plasma, named γBjussuMIP. This acidic glycoprotein was isolated in a high purity level through affinity chromatography on CNBr-Sepharose 4B coupled with BthTXII, showing a pI ∼ 5.5 and molecular weight of 23,500 for the monomer (determined by SDS-PAGE), and 160,000 for the oligomer (determined by molecular exclusion chromatography on Sephacryl S-200). The interaction between γBjussuMIP (MIP) and Phospholipase A2 (PLA2) was confirmed using circular dichroism (CD) and emission fluorescence techniques. The helical content of the 1:1 molar mixture was higher than that calculated for the addition of the spectra of the unbound proteins indicating binding. The emission fluorescence experiments pointed that Trp residues in PLA2 participate in proteins interaction as blue shift of 4 nm was observed. The γBjussuMIP cDNA, obtained by PCR of the liver of B. jararacussu snake, revealed 543 bp codifying for a mature protein of 181 amino acid residues. Alignment of its amino acid sequence with those of other snake γPLIs showed 89-94% of similarity. γBjussuMIP mainly inhibited the pharmacological properties of Asp49 PLA2s, such as phospholipase, anticoagulant, myotoxic, edema inducing, cytotoxic, bactericidal and lethal activities. In addition, it showed to be able to supplement Bothrops antivenom, potentiating its antimyotoxic effect. The aspects broached in this work will be able to provide complementary information on possible mechanisms of action, relating structure and function, which could result in a better understanding of the inhibitory effects induced by γBjussuMIP.

Despite a long history on treatment and management of snakebite, as of now, no satisfactory cure exists to treat local toxicity, including anti-venom therapy. Several natural compounds from plants and their synthetic analogs have shown to be protective. In this study 3, 4, 5-tri-hydroxy benzoic acid, the gallic acid (GA) was tested against the local toxicity of Daboia russelli (DR) venom and its purified hemorrhagic complex (HC). GA inhibited in vitro proteolytic activity of both DR venom and HC but, it did not inhibit phospholipase activity of DR venom. GA inhibited hemorrhage, edema forming, dermo- and myonecrotic activities of both HC and DR venom in in vivo experiments. GA was particularly effective against hemorrhagic activity but, GA inhibition had a greater effect on HC when compared to DR venom. The inhibition was likely due to GA induced structural changes in HC as revealed by alterations in fluorescence emission and CD spectral properties. However, the inhibition was not due to chelating property of GA as suggested by UV-visible spectral studies. Inhibition of collagen type IV, laminin and fibronectin degradation essentially provided the biochemical basis for GA which inhibited local effects of HC as well as DR venom. Thus, the study appears highly promising to explore GA and its generics against ruthless local effects and perhaps systemic hemorrhage of DR and other snake bites as well. Further, these agents will possibly find an immense value in the regulation of matrix metalloproteases (MMPs) in processes such as wound healing, inflammation and in the treatment of cancer.

L-ascorbic acid upon condensation with palmitic acid in the presence of sulphuric acid results in L-ascorbic acid-6-palmitate (AP). The effect of L-ascorbic acid derivative, AP on the pharmacological activities of purified basic multi-toxic PLA2 enzyme, VRV-PL-VIIIa from Vipera russelli snake venom along with in vitro activities is described. AP inhibited VRV-PL-VIIIa enzyme activity in a concentration dependent manner with IC50 value of 48.85 μM and the inhibition is found to be independent of substrate and calcium concentration. Upon investigating the in vivo pharmacological activities, it has been found that AP inhibited VRV-PL-VIIIa induced mouse paw edematogenic activity in a dose dependant manner. Intramuscular co-injection of AP with VRV-PL-VIIIa (1:10 w:w) neutralized the VRV-PL-VIIIa induced myotoxocity. Sections of mouse thigh muscle showed normal intact musculature with normal levels of serum creatine kinase and lactate dehydrogenase. Histopathological studies showed that administration of VRV-PL-VIIIa (i.p) along with AP mixture inhibited VRV-PL-VIIIa induced lung haemorrhage in mouse indicated that enzyme activity is responsible for all these observed pathological and pharmacological activities. The biophysical interaction studies showed that AP interacted directly with the enzyme and decreased the relative intrinsic fluorescence intensity. CD spectral analysis showed an apparent shift in the far UV-CD spectra of VRV-PL-VIIIa with AP. Docking study also confirmed the interaction of AP with enzyme directly. These results demonstrate that AP neutralizes VRV-PL-VIIIa induced pharmacological activities by inhibiting the enzyme with direct interactions. This compound along with other inhibitors of snake venom hydrolytic enzymes might be of use to neutralize local toxicity of V. russelli venom where antivenoms have failed.

The CaTx-I (PLA2) toxin of Crotalus adamanteus venom is responsible for most of the symptoms observed during envenomation. Synthetic peptides were designed and screened for venom (0.8 μg/ml) and CaTx-I (0.1 μM) inhibition at varying doses of the peptide (10000- 0.0001 μM) using a Cayman chemical human secretory phospholipase A2 (sPLA2, Type II) assay kit. Further, in vitro neutralization studies were evaluated by a fixed dose of peptide (1 μM) against venom (0.8 μg/ml) and toxin (0.1 μM). Among the linear peptides (PIP-18, cyclic C and PIP59-67) that showed potent neutralizing effects against the venom/toxin of C. adamanteus. PIP-18 [IC50, 1.23 μM] and cyclic C [IC50, 1.27 μM] peptides possessed the strongest inhibitory effect against CaTx-I. A fixed dose of CaTx-I (75 μg/kg) was administered intraperitoneally (i.p.) into mice followed by an i.p. injection of peptides PIP-18 and cyclic C at (6 μg/mouse), venom (150 μg/kg) and toxin CaTx-I alone served as references. Mice treated with PIP-18 and cyclic C showed a very strong neutralizing effect and markedly reduced mortality compared to the control after 24 h. The CA venom and CaTx-I injected mice showed severe toxicity after 24 h. Peptides PIP-18 and cyclic C were non-hemolytic at 100 μM. They produced a significant decrease in lipid peroxidase (LPx) and enhancement of superoxide dismutase (SOD), catalase (CAT) and Glutathione-s-transferase (GST) levels indicating their antioxidant property against venom-induced changes in mice. This study confirmed the potent snake venom neutralizing properties of peptides.

The present study describes the purification and characterization of a hyaluronidase (DRHyal-II) from Daboia/Vipera russelli venom and its inhibition by β-3-(3-hydroxy-4-oxopyridyl) α-amino-propionic acid, the mimosine. Gel permeation and ion exchange chromatography were employed to isolate DRHyal-II. The molecular mass by MALDITOF mass spectrometry was found to be 28.3 kDa. Single band in reduced SDS-PAGE suggested the monomeric nature. It was optimally active at pH 5.5 and at 37°C and require 150 mM NaCl in the reaction mixture. It was specific to hyaluronan substrate and belongs to class-I or the neutral active enzymes. DRHyal-II was non-toxic by itself but, it potentiated the myotoxicity of VRV-PL-VIII myotoxin and hemorrhagic activity of hemorrhagic complex (HC). In in vitro experiments, mimosine inhibited the activity of DRHyal-II and the hyaluronidase activity of whole venom dose dependently. In in vivo experiments, mimosine inhibited the DRHyal-II potentiated myotoxicity of VRV-PL-VIII myotoxin and hemorrhagic activity of HC. The inhibition was due to the formation of DRHyal-II-mimosine inhibitory complex that resulted in significant structural changes at secondary and tertiary levels as evidenced by fluorescence emission and CD spectral studies. Hence, in this study an attempt was made to establish the possible role of hyaluronidase activity in the pathology of Daboia/Vipera russelli venom and the beneficial effects of its inhibition with special emphasis on the management of local toxicity.

Four compounds (isoquercitrin, myricetin-3-O-glucoside, catechin and gallocatechin) were isolated from lyophilized aqueous extract of Schizolobium parahyba leaves by chromatography on Sephadex LH-20, followed by semipreparative HPLC using a C-18 column, and identified by 1H and 13C NMR. The compounds were then, tested against hemorrhagic and fibrinogenolytic activities of Bothrops crude venoms and isolated metalloproteinases. The inhibitors neutralized the biological and enzymatic activities of Bothrops venoms and toxins isolated from B. jararacussu and B. neuwiedi venoms. The results showed that gallocatechin and myricetin-3-O-glucoside are good inhibitors of hemorrhagic and fibrinogenolytic activities of metalloproteinases, respectively. Gallocatechin also inhibited the myotoxic activity of both B. alternatus venom and BnSP-6 (Lys49 PhospholipaseA2 from B. neuwiedi). Circular dichroism and docking simulation studies were performed in order to investigate the possible interaction between BnSP-6 and gallocatechin. This is the first time these compounds and their anti-ophidian properties are reported for S. parahyba species. Forthcoming studies involving X-ray co-crystallization, will be of great importance for the development of new therapeutic agents for the treatment of ophidian accidents and for the better understanding of the structure/function relationship of venom toxins.

A major myonecrotic zinc containing metalloprotease ‘malabarin’ with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes Aα followed by Bβ subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

The mortality rate due to snakebite is reduced markedly by the use of anti-venoms, which are the only medically approved remedial agents available. The anti-venoms effectively neutralize the systemic toxicity but offer no protection towards local tissue degradation. In viperid snake envenomations, SVMPs and SVHYs are the major agents responsible for brutal local tissue damage as they degrade ECM and basement membrane surrounding the blood vessels. Thus, the usage of inhibitor(s) against ECM degrading enzymes in the treatment of viper bites is an affirmative therapeutic choice. The present study assessed the efficacy of N-acetyl cysteine (NAC) to inhibit gelatinase, hyaluronidase, hemorrhagic and defibrinogenating activities of Vipera russelli and Echis carinatus venoms. NAC inhibited these activities dosedependently, but it did not inhibit the PLA2, 5' nucleotidase, procoagulant and edema inducing activities of both the venoms. NAC showed complete inhibition of hemorrhagic activity when incubated with venom prior to testing. Whereas little inhibition was observed when venom and NAC were injected independently. Inhibition of the basement membrane degradation and accumulation of inflammatory leukocytes at the site of venom injection in histological sections further corroborate the inhibitory property of NAC. The observed inhibition of hemorrhage was likely due to zinc chelation as supported by spectral studies. Further, docking predictions suggested the role of -SH and -NH-CO-CH3 groups of NAC in the inhibition of SVMPs and SVHYs. Future studies related to the protective role of NAC against the venom induced systemic hemorrhage and secondary complications are highly exciting.