Virtual analysis of structurally diverse synthetic analogs as inhibitors of snake venom secretory phospholipase A2

Due to the toxic pathophysiological role of snake venom phospholipase A₂ (PLA₂), its compelling limitations to anti‐venom therapy in humans and the need for alternative therapy foster considerable pharmacological interest towards search of PLA₂ specific inhibitors. In this study, an integrated approach involving homology modeling, molecular dynamics and molecular docking studies on VRV‐PL‐V (Vipera russellii venom phospholipase A₂ fraction—V) belonging to Group II‐B secretory PLA₂ from Daboia russelli pulchella is carried out in order to study the structure‐based inhibitor design. The accuracy of the model was validated using multiple computational approaches. The molecular docking study of this protein was undertaken using different classes of experimentally proven, structurally diverse synthetic inhibitors of secretory PLA₂ whose selection is based on IC₅₀ value that ranges from 25 μM to 100 μM. Estimation of protein–ligand contacts by docking analysis sheds light on the importance of His 47 and Asp 48 within the VRV‐PL‐V binding pocket as key residue for hydrogen bond interaction with ligands. Our virtual analysis revealed that compounds with different scaffold binds to the same active site region. ADME analysis was also further performed to filter and identify the best potential specific inhibitor against VRV‐PL‐V. Additionally, the e‐pharmacophore was generated for the best potential specific inhibitor against VRV‐PL‐V and reported here. The present study should therefore play a guiding role in the experimental design of VRV‐PL‐V inhibitors that may provide better therapeutic molecular models for PLA₂ recognition and anti‐ophidian activity. Copyright © 2015 John Wiley & Sons, Ltd.     

Molecular dynamics simulations reveal structural insights into inhibitor binding modes and functionality in human Group IIA phospholipase A2

Human Group IIA phospholipase A₂ (hGIIA) promotes inflammation in immune‐mediated pathologies by regulating the arachidonic acid pathway through both catalysis‐dependent and ‐independent mechanisms. The hGIIA crystal structure, both alone and inhibitor‐bound, together with structures of closely related snake‐venom‐derived secreted phospholipase enzymes has been well described. However, differentiation of biological and nonbiological contacts and the relevance of structures determined from snake venom enzymes to human enzymes are not clear. We employed molecular dynamics (MD) and docking approaches to understand the binding of inhibitors that selectively or nonselectively block the catalysis‐independent mechanism of hGIIA. Our results indicate that hGIIA behaves as a monomer in the solution environment rather than a dimer arrangement that is in the asymmetric unit of some crystal structures. The binding mode of a nonselective inhibitor, KH064, was validated by a combination of the experimental electron density and MD simulations. The binding mode of the selective pentapeptide inhibitor FLSYK to hGIIA was stipulated to be different to that of the snake venom phospholipases A₂ of Daboia russelli pulchella (svPLA₂). Our data suggest that the application of MD approaches to crystal structure data is beneficial in evaluating the robustness of conclusions drawn based on crystal structure data alone. Proteins 2017; 85:827–842. © 2016 Wiley Periodicals, Inc.     

Alpha1-adrenoceptors trigger the snake venom production cycle in secretory cells by activating phosphatidylinositol 4,5-bisphosphate hydrolysis and ERK signaling pathway

Loss of venom from the venom gland after biting or manual extraction leads to morphological changes in venom secreting cells and the start of a cycle of production of new venom. We have previously shown that stimulation of both α- and β-adrenoceptors in the secretory cells of the venom gland is essential for the onset of the venom production cycle in Bothrops jararaca. We investigated the signaling pathway by which the α-adrenoceptor initiates the venom production cycle. Our results show that the α₁-adrenoceptor subtype is present in venom gland of the snake. In quiescent cells, stimulation of α₁-adrenoceptor with phenylephrine increased the total inositol phosphate concentration, and this effect was blocked by the phospholipase C inhibitor U73122. Phenylephrine mobilized Ca²⁺ from thapsigargin-sensitive stores and increased protein kinase C activity. In addition, α₁-adrenoceptor stimulation increased the activity of ERK 1/2, partially via protein kinase C. Using RT-PCR approach we obtained a partial sequence of a snake α₁-adrenoceptor (260 bp) with higher identity with α_1D and α_1B-adrenoceptors from different species. These results suggest that α₁-adrenoceptors in the venom secreting cells are probably coupled to a G_q protein and trigger the venom production cycle by activating the phosphatidylinositol 4,5-bisphosphate and ERK signaling pathway.     

Isolation,enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A₂ (PLA₂). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 °C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca²⁺ and Mg²⁺) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA₂ through mice tissues. CdtHya1 (32 TRU/40 μL) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA₂, thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms.     

Triacontyl p-coumarate: An inhibitor of snake venom metalloproteinases

Snake venom metalloproteinases (SVMPs) participate in a number of important biological, physiological and pathophysiological processes and are primarily responsible for the local tissue damage characteristic of viperid snake envenomations. The use of medicinal plant extracts as antidotes against animal venoms is an old practice, especially against snake envenomations. Such plants are sources of many pharmacologically active compounds and have been shown to antagonize the effects of some venoms and toxins. The present study explores the activity of triacontyl p-coumarate (PCT), an active compound isolated from root bark of Bombacopsis glabra vegetal extract (Bg), against harmful effects of Bothropoides pauloensis snake venom and isolated toxins (SVMPs or phospholipase A₂). Before inhibition assays, Bg or PCT was incubated with venom or toxins at ratios of 1:1 and 1:5 (w/w; venom or isolated toxins/PCT) for 30 min at 37 °C. Treatment conditions were also assayed to simulate snakebite with PCT inoculated at either the same venom or toxin site. PCT neutralized fibrinogenolytic activity and plasmatic fibrinogen depletion induced by B. pauloensis venom or isolated toxin. PCT also efficiently inhibited the hemorrhagic (3MDH – minimum hemorrhagic dose injected i.d into mice) and myotoxic activities induced by Jararhagin, a metalloproteinase from B. jararaca at 1:5 ratio (toxin: inhibitor, w/w) when it was previously incubated with PCT and injected into mice or when PCT was administered after toxin injection. Docking simulations using data on a metalloproteinase (Neuwiedase) structure suggest that the binding between the protein and the inhibitor occurs mainly in the active site region causing blockade of the enzymatic reaction by displacement of catalytic water. Steric hindrance may also play a role in the mechanism since the PCT hydrophobic tail was found to interact with the loop associated with substrate anchorage. Thus, PCT may provide a alternative to complement ophidian envenomation treatments.     

Determination of the X-ray structure of the snake venom protein omwaprin by total chemical synthesis and racemic protein crystallography

The 50‐residue snake venom protein L ‐omwaprin and its enantiomer D ‐omwaprin were prepared by total chemical synthesis. Radial diffusion assays were performed against Bacillus megaterium and Bacillus anthracis; both L ‐ and D ‐omwaprin showed antibacterial activity against B. megaterium. The native protein enantiomer, made of L ‐amino acids, failed to crystallize readily. However, when a racemic mixture containing equal amounts of L ‐ and D ‐omwaprin was used, diffraction quality crystals were obtained. The racemic protein sample crystallized in the centrosymmetric space group P2₁/c and its structure was determined at atomic resolution (1.33 Å) by a combination of Patterson and direct methods based on the strong scattering from the sulfur atoms in the eight cysteine residues per protein. Racemic crystallography once again proved to be a valuable method for obtaining crystals of recalcitrant proteins and for determining high‐resolution X‐ray structures by direct methods.     

Inhibition of Nicotinic Acetylcholine Receptors,a Novel Facet in the Pleiotropic Activities of Snake Venom Phospholipases A2

Phospholipases A₂ represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A₂, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A₂ from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A₂ content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A₂, which add a new target to the numerous activities of these enzymes.     

Molluscicidal and Leishmanicidal Activity of the Leaf Essential Oil of Syzygium cumini (L.) Skeels from Brazil

The chemical composition and biological potential of the essential oil extracted from Syzygium cumini leaves collected in Brazil were examined. GC/MS Analyses revealed a high abundance of monoterpenes (87.12%) in the oil. Eleven compounds were identified, with the major components being α‐pinene (31.85%), (Z)‐β‐ocimene (28.98%), and (E)‐β‐ocimene (11.71%). To evaluate the molluscicidal effect of the oil, it was tested against Biomphalaria glabrata and the LC₅₀ obtained was 90 mg/l. The essential oil also showed significant activity against Leishmania amazonensis, with an IC₅₀ value equal to 60 mg/l. In addition, to evaluate its toxicity towards a non‐target organism, the essential oil was tested against Artemia salina and showed a LC₅₀ of 175 mg/l. Thus, the essential oil of S. cumini showed promising activity as a molluscicidal and leishmanicidal agent and might be valuable in combating neglected tropical diseases such as schistosomiasis and leishmaniasis. Further research is being conducted with regard to the purification and isolation of the most active essential‐oil compounds.     

Synthesis and Antioxidant Activity of New Norcantharidin Analogs

New norcantharidin analogs were designed and obtained as compounds with biological activity. As a starting material, exo‐7‐oxabicyclo[2.2.1]heptane‐2,3‐dicarboxylic acid anhydride was used. Three groups of compounds: dicarboximides, triazoles and thiazolidines were obtained in multistep reactions. The ¹H‐ and ¹³C‐NMR spectra were used to confirm the structures of all obtained products and they were in agreement with the proposed structure of substances. All derivatives were screened for their antioxidant activity. The most promising group was dicarboximides ( 1 – 4 , 6 ). Derivatives 2–4 displayed antioxidant activity with EC₅₀=7.75–10.89 μg/ml, which may be comparable to strong antioxidant Trolox (EC₅₀=6.13 μg/ml). Excellent activity with EC₅₀=10.75 μg/ml also presented norcantharidin analog with 1,2,4‐triazole system ( 12 ).     

Identification of continuous interaction sites in PLA2-based protein complexes by peptide arrays

Crotoxin (CA.CB) is a β-neurotoxin from Crotalus durissus terrificus snake venom that is responsible for main envenomation effects upon biting by this snake. It is a heterodimer of an acidic protein (CA) devoid of any biological activity per se and a basic, enzymatically active, PLA₂ counterpart (CB). Both lethal and enzymatic activities of crotoxin have been shown to be inhibited by CNF, a protein from the blood of C. d. terrificus snakes. CNF replaces CA in the CA.CB complex, forming a stable, non-toxic complex CNF.CB. The molecular sites involved in the tight interfacial protein–protein interactions in these PLA₂-based complexes have not been clearly determined. To help address this question, we used the peptide arrays approach to map possible interfacial interaction sites in CA.CB and CNF.CB. Amino acid stretches putatively involved in these interactions were firstly identified in the primary structure of CB. Further analysis of the interfacial availability of these stretches in the presumed biologically active structure of CB, suggested two interaction main sites, located at the amino-terminus and β-wing regions. Peptide segments at the carboxyl-terminus of CB were also suggested to play a secondary role in the binding of both CA and CNF.     

Eggs-Only Diet: Its Implications for the Toxin Profile Changes and Ecology of the Marbled Sea Snake (Aipysurus eydouxii)

Studies so far have correlated the variation in the composition of snake venoms with the target prey population and snakes diet. Here we present the first example of an alternative evolutionary link between venom composition and dietary adaptation of snakes. We describe a dinucleotide deletion in the only three finger toxin gene expressed in the sea snake Aipysurus eydouxii (Marbled Sea Snake) venom and how it may have been the result of a significant change in dietary habits. The deletion leads to a frame shift and truncation with an accompanying loss of neurotoxicity. Due to the remarkable streamlining of sea snake venoms, a mutation of a single toxin can have dramatic effects on the whole venom, in this case likely explaining the 50- to 100-fold decrease in venom toxicity in comparison to that of other species in the same genus. This is a secondary result of the adaptation of A. eydouxii to a new dietary habit — feeding exclusively on fish eggs and, thus, the snake no longer using its venom for prey capture. This was parallel to greatly atrophied venom glands and loss of effective fangs. It is interesting to note that a potent venom was not maintained for use in defense, thus reinforcing that the primary use of snake venom is for prey capture.Nucleotide sequence data reported here have been deposited in the GenBank database under accession number AY559317.Reviewing Editor: Dr. Martin Kreitman     

In vitro Leishmanicidal and Cytotoxic Activities of the Glycoalkaloids from Solanum lycocarpum (Solanaceae) Fruits

Leishmaniasis is an infection caused by a protozoan parasite of the genus Leishmania and is the second most prevalent parasitic protozoal disease after malaria in the world. We report the in vitro leishmanicidal activity on promastigote forms of Leishmania amazonensis and cytotoxicity, using LLCMK₂ cells, of the glycoalkaloids from the fruits of Solanum lycocarpum, determined by colorimetric methods. The alkaloidic extract was obtained by acid‐base extraction; solamargine and solasonine were isolated by silica‐gel chromatography, followed by reversed‐phase HPLC final purification. The alkaloidic extract, solamargine, solasonine, as well as the equimolar mixture of the glycoalkaloids solamargine and solasonine displayed leishmanicidal activity against promastigote forms of L. amazonensis, whereas the aglycone solasodine was inactive. After 24 and 72 h of incubation, most of the samples showed lower cytotoxicities (IC₅₀ 6.5 to 124 μM ) as compared to leishmanicidal activity (IC₅₀ 1.1 to 23.6 μM ). The equimolar mixture solamargine/solasonine was the most active with an IC₅₀ value of 1.1 μM , after 72 h. Likewise, solamargine was the most active after 24 h with an IC₅₀ value of 14.4 μM , both in comparison with the positive control amphotericin B.     

Rapid purification and molecular modeling of AaIT peptides from venom of Androctonus australis

As recombinant viruses expressing scorpion toxins are moving closer toward the market, it is important to obtain large amounts of pure toxin for biochemical characterization and the evaluation of biological activity in nontarget organisms. In the past, we purified a large amount of Androctonus australis anti-insect toxin (AaIT) present in the venom of A. australis with an analytical reversed-phase column by repeated runs of crude sample. We now report 20 times improved efficiency and speed of the purification by employing a preparative reversed-phase column. In just two consecutive HPLC steps, almost 1 mg of AaIT was obtained from 70 mg crude venom. Furthermore, additional AaIT was obtained from side fractions in a second HPLC run. Recently discovered insect selective toxin, AaIT5, was isolated simultaneously from the same venom batch. It shows different biological toxicity symptoms than the known excitatory and depressant insect toxins. AaIT5 gave 100% mortality with a dose of less than 1.3 μg against fourth-instar tobacco budworms Heliothis virescens 24 h after injection. During the purification process, we implemented mass spectrometry in addition to bioassays to monitor the presence of AaIT and AaIT5 in the HPLC fractions. Mass spectrometric screening can unambiguously follow the purification process and can greatly facilitate and expedite the downstream purification of AaIT and AaIT5 eliminating the number of bioassays required. Further, electrospray ionization was compared with matrix-assisted desorption/ionization and evaluated as a method of choice for mass spectrometric characterization of fractions from the venom purification for it provided higher mass accuracy and relative quantitation capability. Molecular models were built for AaIT5, excitatory toxin AaIT4, and depressant toxin LqhIT2. Three-dimensional structure of AaIT5 was compared with structures of the other two toxins, suggesting that AaIT5 is similar to depressant toxins. Arch. Insect Biochem. Physiol. 38:53–65, 1998. © 1998 Wiley-Liss, Inc.     

Phospholipases A2 isolated from snake venoms block acetylcholine-elicited currents in identified Lymnaea stagnalis neurons

Phospholipases A₂ (PLA₂s) are the most abundant family of snake venom proteins and play a significant role in prey envenomation. Their content in venoms is rather high. PLA₂s not only have enzyme activity but exhibit other types of biological activities including neurotoxicity. We have earlier shown that a protein bitanarin from the venom of the puff adder Bitis arietans is capable to block the responses of Lymnaea stagnalis neurons to acetylcholine and represents an active PLA₂ at the same time. Further investigation of PLA₂s isolated from the venoms of snakes of two families revealed their capability to interact with nicotinic acetylcholine receptors (nAChRs): PLA₂ from Vipera ursinii (Viperidae family), Naja kaouthia, and Bungarus fasciatus (Elapidae family) suppressed acetylcholine-induced current in identified neurons of L. staganlis. The effect was evident at PLA₂ concentration in the range of tens micromoles. The data obtained suggest the presence in a PLA₂ molecule of a site interacting with nAChR and a possible involvement of nAChR block in toxic action of PLA₂s.     

Agkihpin,a Distinct SVTLE from the Venom of Gloydius halys Pallas: Purification,Characterization and Structure–Activity Determination

Blood clots produced by snake‐venom thrombin‐like enzymes (SVTLEs) are cleared rapidly, which makes SVTLEs attractive as potential candidates for antithrombotic therapy. We isolated a SVTLE, agkihpin, from the venom of Gloydius halys Pallas . Agkihpin was confirmed to a single‐chain TLE with molecular mass of 25.5 kD, pI of 7.43, optimal pH of 8.0 (hydrolyzing TAME), linked carbohydrate absent, and weak fibrinogen clotting activity. It was also found that (i) G. halys might be the latest species in SVTLEs phylogenetic tree; (ii) different level of conservation was shown among the SVTLEs from the Viperidae snakes. Some of those site may account for different activities exhibited by those SVTLEs, especially position 181, at which a fibrinogenolytic activity increase was found when a basic and larger amino acid substituted by a neutral and smaller one; (iii) an extra α‐helix constructed with a ‘Pro + acidic amino acid + aromatic amino acid’ pattern was found in the SVTLEs from Gloydius and Agkistrodon snakes, although it does not necessarily imply an effect on the fibrinogenolytic activity of the SVTLEs. This study provided some new insight into the activity of SVTLE.     

Heterologous expression and purification of active L‐asparaginase I of Saccharomyces cerevisiae in Escherichia coli host

l ‐asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His)₆‐tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni²⁺‐charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc_ASNaseI with high specific activity (110.1 ± 0.3 IU mg^?1) were obtained. In addition, the use of FPLC‐IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 ± 0.01%) was achieved with a purification factor of 17‐fold. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416–424, 2017     

重组α-银环蛇毒素的纯化及活性分析

以表达重组α-银环蛇毒素的高效表达菌株BL21(PDZ04)为材料,研究重组α-银环蛇毒素(α-bungarotoxin,α-BgTx)的分离纯化.采取亲和色谱和离子交换色谱的方法都得到了重组α-银环蛇毒素的纯品.首先从天然的银环蛇毒干粉中分离纯化得到了天然α-银环蛇毒素的纯品.然后以天然α-银环蛇毒素为对照来检测重组α-银环蛇毒素的抗原性和毒性.ELISA结果显示其具有与天然α-银环蛇毒素相似的抗原性,以小鼠为动物模型,纯化的重组α-银环蛇毒素与天然α-银环蛇毒素相比,其腹腔注射的LD50也基本一致,约为0.22μg/g.结果表明利用基因工程的方法生产蛇神经毒素是可行的.     

Properties and applications of immobilized snake venom NAD glycohydrolase

The NAD glycohydrolase (NADase) from Bungarus fasciatus snake venom was adsorbed on concanavalin A-Sepharose, and demonstrated to retain both hydrolase and transglycosidase activities in the bound form. The matrix-bound enzyme was stable to repeated washing with buffer and storage at 4°C. The bound enzyme exhibited the same K_m value for hydrolysis of nicotinamide-1,N⁶-ethenoadenine dinucleotide as previously measured with the soluble, purified form of the enzyme. The bound NADase was used repeatedly for a preparative-scale synthesis of 3-acetylpyridine adenine dinucleotide. It was further demonstrated that the immobilized enzyme could be prepared directly from crude snake venom, thus avoiding the time required for purification. The application of the immobilized snake venom NADase for the preparation of pyridine nucleotide coenzyme analogs has many advantages over procedures used previously for analog synthesis.     

Purification and Characterization of Glucose 6‐Phosphate Dehydrogenase, 6‐Phosphogluconate Dehydrogenase,and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide,Digoxin, and Dopamine on the Enzymes Activities

The present study was aimed to investigate characterization and purification of glucose–6‐phosphate dehydrogenase, 6‐phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'‐ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS‐PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and K_i values with K_m and V_max values of the substrates and coenzymes were determined. According to the obtained K_i and IC₅₀ values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive.