Snake Venom Proteins: Development Into Antimicrobial and Wound Healing Agents

Send Orders for Reprints to [email protected] 4 Mini-Reviews in Organic Chemistry, 2014, 11, 4-14 Snake Venom Proteins: Development into Antimicrobial and Wound Healing Agents

1,2 3 4 5 Ramar Perumal Samy *, Jayapal Manikandan , Gautam Sethi , Octavio L. Franco , Josiah C. Okonkwo6, Bradley G. Stiles7,#, Vincent T.K. Chow1, Ponnampalam Gopalakrishnakone2 and Mohammed Al Qahtani3

1Infectious Diseases Programme, Department of Microbiology, 2Venom and Toxin Research Programme, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore - 117597; 3Center of Excellence in Genomic Medicine Research, King Abdulaziz University, PO Box 80216, Jeddah 21589, Kingdom of Saudi Arabia; 4Department of Pharmacology, Clinical Research Centre, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System (NUHS), Singapore – 117597; 5Universidade Católica de Brasília, Centro de Análises Proteômicas e Bioquímicas, Pós-Graduação em Ciências Genômicas e Biotecnologia UCB, Brasília-DF, Brazil; 6Department of Animal Science and Technology, Nnamdi Azikiwe University, PMB 5025 Awka, Anambra, Nigeria; 7Integrated Toxicology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702-5011, USA; #Department of Biology, Wilson College, 1015 Philadelphia Avenue, Chambersburg, Pennsylvania 17201, USA

Abstract: Infectious diseases are a significant cause of morbidity and mortality worldwide, accounting for approximately 50% of all deaths in tropical countries and as much as 20% of deaths in the USA. The emergence of multi-drug resistant (MDR) strains makes the risk of these infections even more threatening and an important public health problem thereby increasing need of new agents for fighting pathogens. In this review, the remarkable antibacterial properties possessed by various snake venoms (Crotalide, Elapidae, and Viperidae families) were discussed and in particular phospholipase A2s (PLA2s) that have emerged from various studies as potential in the last few years. Group IIA PLA2s are the most potent among the snake venom (sv)PLA2s against various types of bacteria. Further, antibacterial derivatives from PLA2s, e.g. peptides derived from the C-terminal sequence of Lys49-PLA2s (amino acids 115-129), kill bacteria and cause severe membrane-damaging effects. Mechanisms of binding to the bacterial surface and subsequent killing by peptides are based on positive charge, hydrophobicity, and length. These peptide candidates are easy to design and synthesize in pure form (~95% purity). Such peptides may be potentially useful in the clinic as new antimicrobials for combating infections due to antibiotic-resistant bacteria that include methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus in the near future.

Keywords: Methicillin-resistant Staphylococcus aureus (MRSA), snake venom protein, endogenous antibiotics, Lys49 & Asp49-PLA2, inflammatory cytokines, growth factors, skin, wound healing, transcription factors NF-kB, Cys-rich protein.

1. INTRODUCTION infections. SA pneumonia has increased in frequency over the years and most have been caused by community-acquired Scabies cause discomfort to the patient and serious MRSA and in particular USA 300 isolates. Viral co-infection complication as a results of secondary bacterial infection with S. aureus in 15% of the cases is associated with caused by Streptococcus pyogenes (GAS) or Staphylococcus respiratory failure [4]. Bacterial infections involving multi- aureus [1]. Recently, a high burden of skin and soft tissue drug-resistant (MDR) strains are one of the ten leading infections (a total of 7252 isolates), and 55% of methicillin- causes of death world-wide according to the WHO [5]. The resistant S. aureus (MRSA) bloodstream infections were emergence of MDR strains makes the risk of these infections reported and spread out in hospitals across different regions more threatening now [6] and an important health problem in of the world. [2]. Particularly, cellulitis (up to 38% of need of new antibacterial strategies. Besides, antimicrobial patients affected by these diseases) is an important serious resistance is recognized as one of the greatest threats to skin infections and increasing problem in children [3]. The human health in worldwide [7]. The reasons for this rate of community-acquired S. aureus (SA) pneumonia per resistance include a reduction in the new antibacterial drugs 10,000 admissions increased from 4.81% hospitalizations in in the pharmaceutical pipeline, an increase in antimicrobial year 2001 to 2009. MRSA caused 74% and methicillin- resistance, and the need of treatments for newly discovered susceptible (MSSA) caused 26% of these pneumonia pathogens.

Venoms from snakes in the Viperidae, Crotadiae and *Address correspondence to this author at the Venom and Toxin Research Elapidae families are known to consist of complex mixtures Programme, Department of Anatomy, Yong Loo Lin School of Medicine, of proteins and peptides responsible for various toxic effects. National University of Singapore, Singapore 117597; Tel: 65-65168733; Fax: +65-67787643; E-mail: [email protected] Snake venom contains numerous components of biological

Vancomycin Quinolone

Tetracycline Cell wall synthesis

Protein DNA (A) synthesis gyrase Bacteria Sulfanomide Folic acid DNA directed RNA metabolism polymorphism Susceptible Bacteria Rifampicin

Mutation Foreign DNA

(Chromosomal) (Acquisition) Antibiotic Selection Pressure

Aminoglycosides by enzyme Modification Inactivation/ Antibiotic

Target mutation

Efflux pump

Reduced permeability Resistant Bacteria

Imipenem Tetracycline Rifampicin

Fig. (1). Possible mechanisms of susceptibility by various antibiotics and representation of the emergence of drug-resistant bacteria. interest. Venom is a complex mixture that varies in addition, PLA2s can have a significant antibacterial effect not proportions and characteristics among different species, strictly dependent on its enzymatic activity [19]. Furthe- containing non-protein and protein components with varying rmore, a PLA2-derived peptide exhibits fungicidal activity structures and biochemical activities. Major protein com- against Candida albicans [20]. An antimicrobial peptide ponents are neurotoxins, cardiotoxins, lectins, disintegrins, derived from the C-terminus of (B. asper) myotoxin II natriuretic peptides, proteases, phospholipases, phospho- interacts with phospholipid bilayers [21]. Another report diesterases, nucleotidases, and L-amino acid oxidases [8]. In shows that cationic peptides designed from a Lys49 PLA2 of addition, venom enzymes exert a variety of pharmacological snake venom possesses bactericidal and anti-endotoxic effects such as neurotoxicity, myotoxicity, cardiotoxicity, properties [22]. However, those peptides (amino acids 115- hemorrhage, pro- and anti-coagulation, etc. [9, 10]. Venoms 129) derived from the carboxy terminus are devoid of and more specifically their phospholipase A2s (PLA2s) have toxicity and are more bactericidal than the parent molecules. demonstrated antibacterial activity [11-13]. Type-IIA These peptide derivatives of snake venom (sv)PLA2s, and secretory (s) PLA2 is an endogenous antibiotic-like protein other biologically active proteins from venoms, may lead to of the host [14-17]. Lys49 and Asp49 myotoxic PLA2s from the production of new drugs of potential therapeutic value in Bothrops asper also possess bactericidal activity [18]. In the near future (Fig. 1) [23, 24]. 6 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 Samy et al.

2. BIOLOGICAL AND CELLULAR FUNCTIONS OF events regulating cellular functions such as direct killing svPLA2 of bacteria and modulation of inflammatory responses (Fig. 2A) [33]. Most PLA2s catalyze hydrolysis of glycerophospholipids at the sn-2 position to release free fatty acids and lysophos- 4. BACTERICIDAL ACTIVITY OF SNAKE VENOMS pholipids, that plays an important role in biosynthesis of biologically active lipid mediators [25]. A recent study Antimicrobial agents are used to treat infections by reported a variety of membrane and soluble proteins that microbes, which include different classes of pathogenic bind to sPLA2s, suggesting that sPLA2s may also function as organisms-viruses, rickettsia, mycoplasma, chlamydia, high affinity ligands [26]. Collectively, venom-derived protozoa, bacteria and fungi. Bacteria are the largest and sPLA2s bind to membrane and soluble mammalian proteins most diverse group of pathogenic microorganisms [34]. that include the C-type lectin superfamily (M-type sPLA2 There are a number of antimicrobial agents normally used to receptors and surfactant proteins), pentraxin and reticulo- treat infections caused by bacteria, on which they have a calbin, factor Xa, and N-type receptors [25]. Otherwise, selective toxic action. Bacteriostatic agents act primarily by mammalian group IB and IIA sPLA2s can bind to the M-type arresting growth (e.g. sulphonamides, tetracycline, chlora- receptor, and group IIA sPLA2 can associate with lung mphenicol), as compared to the bactericidal agents, which surface proteins, factor Xa and proteoglycans including act primarily by killing cells through disruption of cell glypican and decorin, a mammalian protein containing a wall/membrane function [35]. Several in vitro studies clearly leucine-rich repeat. sPLA2-IIA behaves as a ligand for a demonstrate that the bactericidal activity of Bothrops receptor group within the C-type multi-lectin, mannose alternatus venom is higher against Escherichia coli and S. receptor family. sPLA2-IIA also interacts with heparin aureus versus Pseudomonas aeruginosa and Enterobacter sulfate proteoglycans such as glypican as well as dermatan/ faecalis [36]. These studies provide further experimental chondroitin sulfate-rich decorin and versican, which in turn evidence that snake venom proteins trigger antibacterial enables sPLA2-IIA to become internalized and traffic to functions that may vary between different bacteria. Snake specific compartments within the cell to produce a biological venoms have antibacterial properties that are mainly responses [27]. dependent on the venom components and bacterial types [37]. Crotamine-myotoxin from venom of the South Fatty acids (FAs) and other lipid molecules are important American rattlesnake is structurally related to beta-defensin for many cellular functions. Antimicrobial peptides (AMPs) antimicrobial peptides (AMPs) found in vertebrate animals can activate the protein kinase/malonyl-CoA/long-chain (Fig. 2B) [38]. acyl-CoA (LC-CoA) signaling network. Glucose, with other anaplerotic fuels, increase cytosolic malonyl-CoA, which a. Crotalidae inhibits FA partitioning into oxidation that in turn increases availability of LC-CoA for signaling [28]. Besides, free fatty Several studies report the structural, functional and acid (FFA) stimulation of a G-protein-coupled receptor evolutionary relationships between defensins and other host (GPR40) and membrane free fatty acid receptor (FFAR1) defense or offense molecules such as toxins. Similar to AMPs, causes glucose-stimulated accumulation of cytosolic Ca2+ numerous toxins are small, cysteine-stabilized and cationic and consequently insulin secretion. It is possible that FFA with a striking degree of conservation with host defense released by the lipolysis of triglyceride (TG)/FFA cycling is peptides containing a γ-core motif [39]. Interestingly, the partly secreted via autocrine and paracine mechanisms, is crotamine-myotoxin family of toxins from South American additive to exogenous FFAs in activating the FFAR1. rattlesnake venom is quite homologous to β-defensin (60- Glucose-stimulated release of arachidoinc acid from 80% of homology) [40, 41]. In addition, a recent hypothesis 2+ phospholipids by a Ca -independent PLA2 and/or TG/FFA suggests that antimicrobial and cytotoxic polypeptides have cycling may also be involved. PLA2 receptor (PLA2R) an ancestral base linked to structure and function; however, regulates a variety of biological responses elicited by over time these molecules have evolved to preferentially specific types of sPLA2s. Group IB sPLA2 (sPLA2-IB) acts target respective microbial versus mammalian ion channels an endogenous PLA2R ligand to induce cell proliferation, via residue-specific interactions. These insights may cell migration, and lipid mediator production [29]. PLA2R is accelerate the development of anti-infective or therapeutic also involved in the clearance of sPLA2s, including group X peptides that selectively target microbial or host cells [42, which is a particular type that becomes enzymatically 43]. Talan and coworker reported that venoms from crotalid suppressed by this receptor [30]. species have variable activity against aerobic bacteria at 5-20 µg/ml doses. These venoms have the greatest activity with 3. STRUCTURE OF sPLA s MICs of 80 µg/ml against S. aureus, P. aeruginosa, 2 Enterobacter species, Citrobacter species, Proteus vulgaris sPLA2 enzymes are found in many biological sources, and Morganella species. The inhibitory activity was lost including snake venoms [31]. Several studies report with prolonged incubation for several Gram-negative enzymatic and structural characterization of various snake bacteria. It may be due to the degradation of the venom venom sPLA2s [19, 32]. For example, sPLA2 from the white protein by bacterial proteases. Furthermore, Crotalidae venom of Crotalus durissus ruruima has a high alpha helix venoms can be broadly active against aerobic Gram (-/+) and beta-turn content (45.7 and 35.6% of total protein bacteria. For example, crotamine inhibits several strains of structure, respectively) [31]. sPLA2 provides an array of E. coli with MIC ranges of 25-100 µg/ml via membrane potential biological functions. These enzymes vary in their permeablilization [38]. Importantly, Crotacetin is a novel tissue distribution, hydrolytic activity and phospholipid snake venom C-type lectin that shares homology with substrate specificity. sPLA2s trigger various cell-signaling convulxin and possesses antimicrobial activity against Gram Snake Venom Proteins: Development into Antimicrobial and Wound Healing Agents Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 7

(A) Crotamine (Myotoxin)

Fig. (2A). Structure of PLA2 belonging to the Crotalidae family of snakes. Structure resolving the three disulfide bonding patterns (Cys4- Cys36, Cys11-Cys30 and Cys18-Cys37) of crotamine, myotoxin from Crotalus durissus terrificus snake venom (PDB id: 2PYC). The ++ svPLA2 structure consists of the following: (i) substrate binding domain, (ii) catalytic site, (iii) the catalytic network and Ca binding domain etc.

(B) Bonding Patterns (Myotoxin)

β-Wing

C-Terminus Hydrogen bonds

α -helix

S-S bonds N-terminus

Fig. (2B). Three dimensional structure of presynaptic neurotoxic PLA2 of notexin from snake venom. Significant differences from PLA2s from bovine pancreas and rattlesnake occur in the stretches 56-80 and 85-89. Residue 69, that shown to be important for phospholipase binding, no Ca2+ ions instead of a water molecule occupies the calcium site. C-alpha positions for residues 86-88 differ by about 6 A from both the enzymes (PDB: 1AE7). (-/+) bacteria that is comparable to crude C. durissus number of AMPs have been elucidated from a wide-variety terrificus venom [44]. of animal sources, particularly snake venoms. Naja naja venom as well as purified peptides possess potent b. Elapidae antibacterial action against Gram -/+ bacteria such as E. coli, P. aeruginosa, Vibrio cholerae, S. aureus, and Bacillus Like many other snake venoms, those from the Elapidae subtilis. The most potent activity targeted Gram - bacteria at family are also known to consist of complex mixtures of 100 µg/ml concentrations [51]. Based on the evidence, toxins and enzymes responsible for a multitude of biological Elapidae venoms may represent valuable resources for future activities. The bactericidal action of various venoms has development of novel human therapeutics [52]. been examined in crude forms from several species [45-48]. In addition, venom of Naja mossambica, Notechis scutatus c. Viperidae scutatus, Acanthophis antarcticus and Pseudechis australis did not inhibit the S. aureus [45]. Snake venoms of thirty Viper venoms from the Elapidae, Hdrophiidae, different species were tested against Gram (-/+) bacteria, and Atractaspididae, Viperidae and Colubridae families contain number of venoms gave a zone of inhibition against bacteria at least 25 separate classes of biologically active compounds using the classic Kirby Bauer disk diffusion assay. Other including enzymatic and non-enzymatic molecules [53]. authors have also report the various biological activities of Nomenclature standardization of these by structure and venom components from Pseudechis australis [47, 49, 50]. function has been reported for prothrombin activators. Crude An antibacterial component (L-amino acid oxidase) from venoms from Viperidae species generate significant Pseudechis australis venom is 17.5 times more effective inhibition zones, with Calloselasma rhodostoma showing the than tetracycline against Aeromonas hydrophila on a molar largest against S. aureus [54]. However, Viperidae venoms basis [45]. A recent study reveals that at of 14 Elapidae and represent an enormous source of proteins/peptides that have 8 Viperidae venoms, Bothrops moojeni and B. jararacussu not been fully explored for this inherent biological activities. effectively inhibit the growth of Streptococcus mutans, Ferreira et al. [55] studied the antibacterial profile of four which is a principal agent involved in dental caries [48]. A different viperid venoms (Agkistrodon rhodostoma, Bothrops 8 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 Samy et al. atrox, B. jararaca and Lachesis muta) against ten drug bactericidal against Gram-positive and negative bacteria resistant Gram -/+ bacteria. Results show effects of A. activity was isolated and biochemically characterized, clearly rhodostoma and B. atrox venoms against S. epidermidis and suggesting a secondary protective function. Nevertheless, E. faecalis (MIC = 4.5 µg/ml), while B. jararaca inhibited S. none cytotoxic activities against macrophages and ery- aureus growth (MIC = 13 µg/ml). This data was compared to throcytes were obtained. Finally, three small LAAO that of commercial antibiotics with MICs (1-32 µg/ml). In fragments were produced showing enhanced antimicrobial addition, one of our previous studies reported that venoms of activity. The fragments reported here could be promising D. russelli russelli, C. adamanteus, and D. russelli siamensis candidates in the new antibiotics design [68]. also demonstrate significant antibacterial activity [47]. D. russelli russelli venom exhibited the most potent activity 5. BACTERICIDAL PROPERTIES OF SNAKE against S. aureus with MICs of 160-25 µg/ml. Stronger VENOM PROTEINS effects were noted with the Viperidae venoms (20 µg/ml) when compared to standard drugs such as chloramphenicol, Snake venoms are complex mixture of proteins among streptomycin and penicillin. Many naturally occurring which both basic and acidic PLA2s can be found. Basic proteins/peptides from various snake venomspossess PLA2s are usually responsible for major toxic effects antimicrobial activity in vitro that rivals currently used induced by snake venoms, while acidic PLA2s tend to have a antibiotics [45-47, 56]. Myotoxic PLA2 enzymes also have lower toxicity. Venom PLA2 (basic) isolated from Crotalus bactericidal activity against E. coli and S. aureus [18]. The durissus terrificus has high bactericidal effects against Gram-positive and -negative bacteria [69]. An acidic PLA bactericidal effect of PLA2 isolated from Bothrops snake 2 venoms have been suggested to be due to its catalytic from Porthidium nasutum venom has bactericidal activity activity [57] but according to Lomonte et al. [58], the against S. aureus in a dose-dependant manner with MIC value of 32 µg/ml [70]. Interestingly, a myotoxic PLA catalytically inactive Lys49 PLA2 also has antibactericidal 2 effects. (MjTX-II) from Bothrops moojeni has antimicrobial activity against E. coli [68]. A cationic protein from venom of the d. Antimicrobial Action by L-amino Acid Oxidase (LAAO) inland taipan (Oxyuranus microlepidotus) displays dose- dependent antibacterial activity mainly against Gram- Snake venom LAAO (L-amino acid: O2 oxidoreductase) positive bacteria with MIC ranges of 2-10 µg/ml. Anti- has been well-studied by several groups. LAAO is a bacterial effect of this snake venom protein was salt tolerated flavonenzyme which catalyzes the oxidative deamination of up to 35 mM of sodium chloride [12]. In our previous study, L-amino acids into alpha-keto acid, ammonia (NH3), and a Saw-sealed viper PLA2 (EcTx-I) has a significant hydrogen peroxide (H2O2). LAAO is the only FAD- bacteriostatic and bactericidal effect of with MICs of less dependent oxidase found in snake venom, and venom than 15 µg/ml against B. pseudomallei (KHW) and 30 µg/ml toxicity may be related to the H2O2 generated during against E. aerogenes (Fig. 3A-B). Additionally, EcTx-I reoxidation of the transiently reduced flavin cofactor by displayed a novel mechanism for damaging membranes [13]. molecular oxygen [59, 60]. A number of authors have High dose of SVPLA2 (EcTx-I) induced toxic in mice (Fig. observed that the antimicrobial action of this enzyme is 4A-D) sPLA2 (Echis carinatus) has not only effective significantly reduced by catalase, suggesting the importance antibacterial activity [11], but also potent inhibitory effects of enzymatically-produced H2O2 for biological activity [45, against HIV-1 by blocking virus entry into cells [71]. A 61, 62]. In addition, the crude venom of Bothrops moojeni novel PLA2 named as PnPLA2s isolated from the venom of and its LAAO are significantly active against the Porthidium nasutum with molecular mass of 15,802.6 Da as promastigote forms of Leishmania amazonensis, L. chagasi determined by electrospray ionization mass spectrometry and L. panamensis, with IC50 values at 1 µg/ml [64]. (ESI-MS). The N-terminal sequencing has homology with Hydrogen peroxide generated from LAAO activity strongly other acidic PLA2s from viperid venoms. PnPLA2s have induces apoptosis, causing oxidative stress initiating bactericidal activity against S. aureus in a dose-dependent disruption of membranes and cytoplasm and consequently manner with a MIC and MBC of 32 µg/ml [70]. leads to cell-death [64]. One study reports that Bothrops leucurus L-amino acid oxidase (BleuLAAO) has no a. Bactericidal Properties of svPLA2 Enzymes and Active cytotoxic effects on microorganism or macrophages, Site suggesting that these fractions do not produce H2O2 in Type-IIA sPLA2 is an endogenous antibiotic-like protein sufficient quantities at the studied doses of enzyme [65]. The of the host (mammals) [14] [15-17], whereas Lys49 and venom of B. leucurus inhibits S. aureus growth in a dose- Asp49 myotoxic PLA2 from Bothrops asper showed dependent manner, with a MIC of 25 µg/ml. Apart from the bactericidal activity [18]. On the other hand, svPLA2s has B. leucurus (BleuLAAO) exception, typically snake venom also showed a significant antibacterial effect not strictly LAAO do possess varying levels of antimicrobial activity dependent on enzymatic activity [19]. More interestingly, against a variety of microorganism. LAAOs from Crotalus PLA2 derived peptide has fungicidal activity against Candida adamanteus and Bothrops asper exert antibacterial activity albicans [20]. Whereas, the antimicrobial peptide derived against S. aureus and Proteus mirabilis. svLAAO from from the C-terminus of myotoxin II interacts with Bothrops venoms also display antimicrobial effects [64, 66, phospholipid bilayer [21]. Another report shows that cationic 67]. Another svLAAO from B. pirajai controls the growth of peptides designed from a Lys49 PLA2 of snake venom Pseudomonas aeruginosa and Escherichia coli [66]. LAAO possesses bactericidal and antiendotoxic properties [22]. from B. jararacussu has significant activity against However, peptides from residues 115-129 are devoid of Leishmania species [63]. Finally and not less im-portant a toxic effects and increasing bactericidal potency versus the LAAO from Bothrops mattogrosensis (BmLAAO) with parent molecule. Snake Venom Proteins: Development into Antimicrobial and Wound Healing Agents Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 9

(A) (B) 40 40 35 24 h 35 * * * 24 h 30 * 30 25 25 20 20 15 15 10 10 Inhibition zones (mm) zones Inhibition Inhibition zones (mm) zones Inhibition 5 5 0 0 AKH-V AgTx-I AKH-V AgTx-I CF Ec-V EcTx-I Ec-V EcTx-I CF Samy et al., 2010 B. pseudomallei KHW (-) TES (-) KHW (-) TES (-) Perumal Samy et al., 2008 Fig. (3). Antimicrobial activity of snake venom proteins. Saw-scaled viper (Echis carinatus) toxin-I (EcTx-I - panel A) and Chinese viper pallas (Agkistrodon halys) toxin-II (AgTx-II - panel B) showed potent inhibitory action against a Gram-negative multi-drug resistant bacterium isolated from two different patients (Burkholderia pseudomallei strains KHW and TES). Symbols: Ec-V - Echis carinatus venom, AKH-V - Agkistrodon halys venom, CF-Ceftazidime. b. Membrane Damaging Effects of Venom Proteins 135 residues and finally a mature AMP of 34 residues following proteolysis [78]. The deduced mature cathelicidin Mode of action antibacterial peptides is mainly the from king cobra venom has been chemically synthesized and disruption of the lipoid plasma membrane. By using model exhibits potent antimicrobial action on different types of phospholipid membranes, peptides which act preferentially bacteria, with MICs of 1-20 µg/ml. The same peptide shows on bacteria are able to interact with and permeate efficiently hemolytic activity on human RBC at a high dose of 200 anionic phospholipids. Chiefly, the target membrane depends µg/ml [78] hemolysis. on the structure, length, and complexity of the hydrophilic polysaccharide found in its layer. This affects peptide diffusion through the outer barrier and its ability to reach the d. Modes of Action for Snake Venom Proteins and cytoplasmic plasma membrane [72]. For example, cobra Peptides toxin γ (Naja nigricollis) has bactericidal killing effects on S. Naja nigricollis toxin γ has a similar inhibitory activity aureus and E. coli bacteria, increasing membrane per- on select Gram-positive (S. aureus) and Gram-negative meability [73] but does not affect membrane transport (Escherichia coli) bacteria. The antibacterial activity of toxin system and ATPs generation. Besides disrupting bacterial γ correlates positively with increased membrane perme- membranes, toxin binds to lipopolysaccharide (LPS) and ability of bacteria. Morphological exami-nation shows that lipoteichoic acid (LTA), which are respectively major toxin γ disrupts the integrity of the bac-terial membrane. membrane constituents for Gram negative and positive Toxin γ binds similarly to Gram-negative specific LPS and bacteria. Destabilization of LPS layer and inhibition of LTA Gram-positive specific LTA, causing destabilization of the biosynthesis causes bactericidal effects [73]. Differential LPS layer and inhibits LTA biosynthesis on the cell wall binding to phospholipid bilayers modulates membrane- [79]. Cardiotoxin 3 (CTX3) obtained from Naja naja atra damaging effects attributed to a cardiotoxin from N. naja (Taiwan cobra) possesses greater inhibitory action on S. atra venom [74]. The positively charged residues of car- aureus relative to that of E. coli via disruption of bacterial diotoxin play an important role in damaging anionic and membranes in similar fashion as toxin γ [79]. Guanidination zwitterionic, but not cationic, phospholipid vesicles. These of Naja naja atra cardiotoxin 3 (CTX3) and selective findings data reflects that membrane-damaging activity of trinitrophenylation of the N-terminal alpha-amino group toxin is affected by phospholipid compositions [75]. Studies enhances notably the membrane-damaging activity on egg with a cationic peptide (myotoxin II from B. asper venom) yolk phosphatidyl choline (EYPC) and egg yolk further prove that electrostatic interactions play a vital role in sphingomyelin (EYSM) vesicles [75]. the initial recognition, binding, and membrane rupturing effects [21]. CTXs are polypeptides that consist of 62 and 60 amino acid residues adopt a three-finger structure containing a beta c. Snake Cathelicidins as AMPs sheet fold. CTX membrane-damaging activity is mediated through the formation of an oligomeric pore structure or AMPs play an important role in preventing microor- inducing membrane fusion. Moreover, the ability of CTXs to ganism infections. The majority of cathelicidins, a subgroup induce membrane permeability critically depends on the of AMPs found in lysosomes of cells in the immune system, lipid compositions and membrane interaction mode. are 10-15 amino acids in length and important for innate Positively charged residues of CTX3 play a distinctive role immunity. Many cathelicidin AMPs are found in mammals, in damaging anionic and zwitterionic phospholipid vesicles, birds and others. Recently, cathelicidin AMPs were thus reflecting that membrane-damaging activity of CTX3 is identified from snake venoms 60-90% do these share affected by phospholipid composition. Phospholipid-binding homology with other cathelicidins from other species [76, capability and oligomeric assembly upon binding with lipid 77]. cDNA sequences coding for elapid cathelicidins (Naja vesicles does not closely correlate with membrane-damaging atra, Bungarus fasciatus and Ophiophagus hannah) have a potency of native and modified CTX3 [74]. 22 amino acid signal peptide, a conserved cathelin domain of 10 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 Samy et al.

Histopathology (H & E)

(A) if (B) vc if nc nc vc Liver

Kidney nc nc nc

if Lung mn Muscle mn if if if

svPLA (14 µg/mouse) 2

Fig. (4). Each mouse received 14 µg of svPLA2 (Echis carinatus) which led to severe nephrotoxicity (A) and hepatotoxicity (B) within six hours, accompanied by gradual weight loss. (C). Myonecrotic effect induced by svPLA2 in the muscle. (D) Lung tissue showed severe necrotic effects caused by the svPLA2 that included heavy accumulation of infiltrated cells. Abbreviations include: vc-vacoules; if-infiltrates; nc-necrotic; mc-myonecrosis; ctrl-control (Original magnification x10).

CTX3 showed effective antibacterial activity against 6. SNAKE VENOM PROTEINS ACCELERATE Gram-positive bacteria S. aureus, Enterococcus faecalis and WOUND REPAIR Bacillus subtilis with minimum inhibitory concentrations of Wound repair is a complex biological process consisting 31.25, 62.25 and 125 µg/ml respectively. Bothrops leucurus of sequential events like hemostasis, inflammation, snake venom contains a galactoside-binding lectin with proliferation and remodeling of tissue. Wound healing is antibacterial activity. CTX3 membrane-damaging activity controlled by different types of cytokines and growth factors was inhibited by LPS and LTA, while increasing the CTX3 i.e. epidermal growth factor (EGF) and transforming growth concentration counteracted the inhibitory action of LPS and factor-alpha (TGF-alpha), stimulates keratinocytes, causes LTA [80]. The effect of a short synthetic cationic peptide, dermal fibroblast migration [83], cell proliferation, regulates pEM-2 (KKWRWWLKALAKK), derived from the C- angiogenesis [84], and promotes re-epithelialisation [85] terminus of myotoxin II from B. asper venom shows that which are all together essential for wound repair [86, 87]. A electrostatic interactions play a significant role in the initial group of small integrin binding proteins called disintegrins recognition and binding of pEM-2 to the cell membrane. found in various snake venoms strongly inhibit integrin- However, membrane rupturing activity of the peptide mediated cell adhesion and migration [88]. These proteins depends on interactions other than simple ionic attraction bind to integrins with high affinity, therefore inhibiting cell [21]. The mode of action of many antibacterial peptides is adhesion and migration. A group of small integrin-binding believed to be via disruption of lipidic plasma membrane. proteins called disintegrins have been previously reported Therefore, model phospholipid membranes have been used from snake venoms and powerfully inhibit integrin-mediated to study the mode of action of antimicrobial peptides [81]. cell adhesion and migration [89]. For example, alternagin-C These studies demonstrate that peptides acting preferentially is a protein identified from the venom of a Brazilian snake on bacteria interact with, and permeate efficiently, anionic (Bothrops alternatus) and belonging to the PIII group of phospholipids [82]. In contrast, peptides that lyse snake venom metalloproteinases (SVMP). However, this mammalian cells bind and permeate efficiently both acidic SVMP is a disintegrin-like cysteine-rich domain released and zwitterionic phospholipid membranes, mimicking the from alternagin following proteolysis that potently inhibits plasma membranes of eukaryotic cells. Regarding target collagen-induced adhesion by blocking ά2β1 integrin [90]. membranes, additional considerations include the structure The ά2β1 integrin is a major collagen receptor which plays type, overall length, and complexity of the hydrophilic an important role in cell adherence to extracellular matrix polysaccharide found in its outer layer. These parameters [91]. Previous reports demonstrate that alternagin-C affect peptide diffusion through the cells outer barrier and powerfully induces human endothelial cell (HUVEC) reaching its cytoplasmic membrane [72]. Snake Venom Proteins: Development into Antimicrobial and Wound Healing Agents Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 11 proliferation by up-regulating the expression of growth coagulation. On the other hand, some venom, toxins [24, 52, factors and their receptors such as vascular endothelial 105] and many peptides from several natural sources [106- (VEGF) and VEGF receptor 2 (VEGFR-2) [92, 93]. Besides, 108] are important therapeutic alternatives to existing pain it has been confirmed that alternagin-C induced myoblast killer. For example, Lys-49 PLA2 homologues constitute a proliferation changes their gene expression pattern for major large family of toxins present in the venoms of Viperidae histocompatibility complex (MHC) and modulates matrix snake species, that lacking catalytic activity cause important metalloprotease (MMP) activity in a skeletal muscle skeletal muscle necrosis [109]. Structural determinants of regeneration model [94]. this toxic effect experimentally mapped in their C-terminal region (115-129) which combines cationic and hydrophobic A peptide derived from alternagin-C induces angiogenesis aromatic amino acid residues. Further modification of their in a rat wound model [95]. In addition, prior studies indicate the role of ά2β1 integrin during wound healing which may sequence enhances their effects on bacterial cells, while significantly decreasing toxicity for eukaryotic cells [109]. be useful for developing novel therapeutics for skin wound These biomimetic actions exhibited by the peptides highlight regeneration. Alternagin-C 60- (10, 60 and 100 ng of ALT-C ® their potential value as a molecular tool that can lead to protein in the gel) associated with gel (natrozol ) was used several biomedical applications. On the other hand snake to treat rat skin wounds for 7 days. After treatment, venom-derived peptides [11], may offer an effective and alternagin-C influences type I collagen deposition, fibroblast density, and accelerates the inflammatory process which viable therapeutic option for drug development. ultimately results in accelerated skin regeneration. Type I collagen is the major collagen component in skin, where 8. CONCLUSION collagen type III intercalates into the collagen type I fibrils Secreted PLA2s may be potentially useful, novel and produces smaller and less oragnised fibrils [96]. antimicrobials to combat infections including those caused Whereas, previous human wound healing studies prove that by antibiotic-resistant bacteria such as MRSA and a proportion of collagen I increases significantly up to 6 vancomycin-resistant Enterococci. Type-IIA sPLA2 is an weeks after injury [97] and continues up to 6 months. They endogenous antibiotic-like protein and peptides derived from reported that the alternagin-C could be benenficial to skin Lys49 and Asp49 myotoxic PLA2s from Bothrops asper wound repair and influenced deposition of type I collagen as possess interesting bactericidal activity. Structural deter- well as fibroblast density. Fibroblasts are important cells for minants of these PLA2s have been mapped to their C- wound healing, secreting provisional matrix and growth terminal region (115-129) which combines cationic and factors that are indispensable for initiating repair and hydrophobic aromatic amino acids. All of the above progressing into a healing state [82]. On the other hand, evidence clearly proves that some of this snake protein alternagin-C enhances VEGF expression in fibroblasts and promotes wound healing and tissue regeneration. endothelial cell proliferation [92], as well as induces in vivo angiogenesis in nude mice and up-regulates the expression of CONFLICT OF INTEREST VEGFR-2 (KDR) in HUVEC cells [93]. All of the above evidence clearly proves that this particular snake protein The authors confirm that this article content has no promotes wound healing and tissue regeneration. conflict of interest.

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Received: March 17, 2013 Revised: May 14, 2013 Accepted: June 25, 2013