Structural, Functional and Therapeutic Aspects of Snake Venom Metal- Loproteinases

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28 Mini-Reviews in Organic Chemistry, 2014, 11, 28-44 Structural, Functional and Therapeutic Aspects of Snake Venom Metal- loproteinases

P. Chellapandi*

Department of Bioinformatics, School of Life Sciences, Bharathidasan University, Tiruchirappalli-620024, Tamil Nadu, India

Abstract: Snake venoms are rich sources of metalloproteinases that are of biological interest due to their diverse molecular diversity and selective therapeutic applications. Snake venoms metalloproteinases (SVMPs) belong to the MEROPS peptidase family M12B or reprolysin subfamily, which are consisted of four major domains include a reprolysin catalytic domain, a disintegrin domain, a reprolysin family propeptide domain and a cysteine-rich domain. The appropriate structural and massive sequences information have been available for SVMPs family of enzymes in the Protein Data Bank and National Center for Biotechnology Information, respectively. Functional essentiality of every domain and a crucial contribution of binding geometry, primary specificity site, and structural motifs have been studied in details, pointing the way for designing potential anti-coagulation, antitumor, anti-complementary and anti-inflammatory drugs or peptides. These enzymes have been reported to degrade fibrinogen, fibrin and collagens, and to prevent progression of clot formation. An- giotensin-converting enzyme activity, antibacterial properties, haemorrhagic activity and platelet aggregation response of SVMPs have been studied earlier. Structural information of these enzymes together with recombinant DNA technology would strongly promote the construction of many recombinant therapeutic peptides, particularly fibrinogenases and vaccines. We have comprehensively reviewed the structure-function-evolution relationships of SVMPs family proteins and their advances in the promising target models for structure-based inhibitors and peptides design. Moreover, structure- function-evolution integrity of metalloproteinase from Gloydius halys venom was preliminarily analyzed herein that may provide a conceptual idea for the future of antibacterial peptide design.

Keywords: Reprolysin, Bioactive peptides, Family M12B, ADAMs, Adamalysin, Antimicrobial activity, Metalloproteinase, Snake venoms.

1. INTRODUCTION charged amino acid side chains are ligands for the metal ions in the binding packet. Side chain residues His, Glu, Asp or Proteinase is a hydrolytic enzyme that begins the Lys are the known metal ligands and at least one other hydrolytic breakdown of proteins and peptides, usually by residue (Glu) are required for catalysis [1]. splitting them into polypeptide chains. It occurs in all living kingdoms involving in major physiological phenomena. Proteomic study on snake venom proteins reported the Proteinase family can be classified into five major groups SVMPs belong to the MEROPS peptidase family M12B or based on the catalytic residue viz. serine, threonine, cysteine, reprolysin subfamily [10]. M12B reprolysin subfamily exists aspartate and metal-binding sites [1]. A significant pro- in a majority of snake venoms (Fig. 1). The current portion of serine and metalloproteinases present in snake bioinformatics data on reprolysin subfamily M12B pointed venoms proteins that are typically used in traditional out that there are 148, 145 and 122 species having reprolysin medicine or therapeutics [2-6]. Nevertheless, the majority of (PF01421), reprolysin propeptide (PF01562) and ADAMs snakes have metalloproteinases in the venoms for their (A Disintegrin And Metalloproteinase) cysteine-rich pathophysiological functions in the bitten preys [7]. (PF08516) subfamilies, respectively. Among Colubroidea superfamily (all snakes belong to this superfamily), a Snake venom metalloproteinases (SVMPs) are part of the predominant existence of reprolysin subfamily is reported in metzincin family of zinc-dependent metalloproteinases the venoms of Crotalinae family and then in the venoms of consisting of an identical zinc-dependent motif HEX- Elapidae family. A limited study has been done on the XHXXGXXH and of a methionine turn [8]. Matrixin, astacin reprolysin subfamily in the venoms of Atractaspididae and serralysin are other parts of metzincin family [9]. A family. The protein sequences deposited for reprolysin zincin-like fold is common in SVMPs so that Zn2+ ion takes subfamily in Viperinae family venoms are accounted to be a major role in the catalysis of the hydrolysis of internal, more than in the venoms of Crotalinae family. It suggests the alpha-peptide bonds in a polypeptide chain. One or two advantages of using SVMPs from Viperinae family showing metal ions hold the water molecule (as a nucleophile) and a vital importance in therapeutic and medical applications.

Brevilysin H6, acutolysin, bilitoxin, leucolysin, *Address correspondence to this author at the Department of Bioinformatics, jararhagin, bothrolysin, bothropasin, adamalysin, atrolysin A, School of Life Sciences, Bharathidasan University, Tiruchirappalli-620024, atroxase, basilysin, horrilysin, ruberlysin, trimerelysin, Tamil Nadu, India; Tel: +91-431-2407071; Fax:+91-431-2407045; E-mail: [email protected] russellysin, cobrin, najalysin and catrocollastatin are some

Fig. (1). Dispersion of snake venom metalloproteinases in Colubroidea superfamily. The above figure is represented the total number of species reported for the metalloproteinases and the below figure represents the total number protein sequence entries available in Pfam version 26.0 for Colubroidea superfamily. examples of SVMPs molecular diversity available in reprolysin subfamily has four different conserved domains: a MEROPS database [11]. Twenty eight structures of rep- reprolysin catalytic domain, a disintegrin domain, a reprolysin subfamily and 2 structures of ADAMs cysteine-rich rolysin family propeptide domain and an ADAMs cysteine- subfamily include with ligands have been deposited in the rich domain. A reprolysin is a main catalytic enzyme domain protein data bank (PDB) to date. Thus, overwhelming bioin- involving in Zn2+ ion binding and peptide cleavage activity. formatics resources, crystallographic structural information Disintegrin is a small domain typically with an Arg-Gly-Asp and literature records for SVMPs family of enzymes are recognition sequence on a disintegrin-loop that inhibits collectively support the identification, designing and platelet aggregation via integrin binding. Reprolysin family development of therapeutic compounds or peptides from the propeptide domain is the propeptide for members of snake venom sources. peptidase family M12B. The propeptide contains a sequence motif similar to the "cysteine switch" of the matrixins [13- 2. FUNCTIONAL DOMAINS 15]. The last domain, adamalysin (ADAMs), is membrane- bound zinc containing metalloproteinase, which is con- SVMPs are classified into three types (P-I to P-III) based sidered to be crucial modulators of physiological and patho- on their conserved domain architecture [5, 12]. M12B 30 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 P. Chellapandi

(A)

(B)

Fig. (2). Topological view of conserved domains identified in metalloproteinase of G. halys (2a) and WebLogo representation of the conserved functional motifs identified across the peptidase M12B family (2b). physiological processes [16]. The residues that are important the molecule [22, 23]. Such multiple functional motifs in the for stabilizing domains architecture are strictly conserved metalloproteinase of G. halys may influence its binding cleft throughout the primary structure among SVMPs [17]. orientation on bacterial membrane proteins. Reprolysin, disintegrin and ADAM cysteine-rich domains are common in the PDB structures with accession 3. STRUCTURAL EVALUATION number of 3K7L, 3DSL, 3HDB, 2ERQ, 2DW0 and 2E3X To elucidate their molecular structure and function, and other available PDB structures have only a reprolysin crystallographic studies have been carried out for the venoms domain. In addition to these domains, a reprolysin M12B of Naja atra, Bothrops moojeni, B. asper, B. jararaca, A. propeptide domain is predicted in the metalloproteinase of acutus, Crotalus adamanteus, C. atrox, Trimeresurus flavo- Gloydius halys (Agkistrodon halys) venom (Fig. 2a). We viridis, T. mucrosquamatus and Daboia russellii siamensis identified consensus motif HEXXHXUGUXHU and a bulky (Table 1). In general, SVMPs are structurally related to the hydrophobic core in the binding packets. Conserved domains mammalian membrane-bound ADAMs and other bacterial in those sequences were searched out with SMART 7.0 reprolysin subfamilies [24]. server [18] and NCBI-conserved domain database [19]. Catalytic site residue, Glu335 and 3 actives site residues, 3.1. Adamalysin II His334, His338 and His344, are located in a catalytic reprolysin domain of metalloproteinase from G. halys venom. The first crystallographic structure of adamalysin II In contrast, two highly conserved characteristic sequences from the snake venom of C. adamanteus was determined His142-Glu143-X-X-His146-X-X-Gly149-X-X-His152 and by Gomis-Ruth et al. [13]. Adamalysin II is mostly multi- Cys162-Ile163-Met164 are identified in acutolysin C [20]. domain protein, an ellipsoidal molecule with a shallow The sequentially conserved motifs across the subfamilies active-site cleft and composed of a -sheet and four - helices. A tetrahedral coordination is occurred between were discovered by MEME Suite 4.8.1 [21]. We identified a 2+ conserved motif, FSCSYLPCLI among reprolysin domains Zn ion and His residues (His142, His146 and His152), whereas motif, CAMCSDSAVMAHEHNLGI among the and a water molecule anchors to a catalytic residue (Glu143). It has shared a similar overall topology to other metal- PDB structures of SVMPs (Fig. 2b). A conserved motif, CYNGCPQCGVDCF is observed among adamalysin II lopro-teinases and exhibits a virtual identical zinc environ- domains. No significant motif has found in reprolysin ment. Based on the structural information, a phosphonate propeptide domain, many of them are showed a regular inhibitor, N-[(furan-2-yl)carbonyl]-(S)-leucyl-(R)-[1-amino- expression pattern among reprolysin subfamily. Identified 2(1H-indol-3-yl)ethyl]-phosphonic acid [25], peptido- functional motif sites have bulky hydrophobic residues metric inhibitors, Pol 647 and Pol 656, active site model of contributing in substrate recognition, in particular on bac- tumor necrosis factor -convertase (TACE) have been terial membrane proteins, at a binding cleft. Amino acid developed in earlier [13]. Zinc ion is ligated in an asym- substitutions, deletions, and insertions in structural motifs, metric bidentate mode by a phosphonate group and then often located in flexible loops, may significantly modify the S1 packet partly filled with the adjacent Trp indole system function of a SVMP without affecting structural stability of [25]. Pol 647 and Pol 656 inhibitors are similarly bound to Structural, Functional and Therapeutic Aspects of Snake Venom Metalloproteinases Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 31

Table 1. Crystallographic Structural Information of Snake Venom Metalloproteinases and Their Ligands/Inhibitors to Date

Snake Name PDB Molecule Inhibitors/Ligands/Metal Ions E.C. Pharmacological Properties References

A novel target for designing anti- Naja atra 3K7L Atragin -l-Fucose, -l-Fucose, Zn2+ and Ca2+ 3.4.24.- inflammatory and anti-metastatic [33] invasion drugs

Thrombolytic agent and biochemical Bothrops BmooMP- 3GBO Zn2+ and Ca2+ 3.4.24.- tool in coagulation research and [36] moojeni I diagnosis

Peptidomimetic inhibitor WR2 ((2R,3R)- N-1-[(1S)-2,2-dimethyl-1-(methylcar- Activation of complement system, Bothrops bamoyl)propyl]- N-4-hydroxy-2-(2- 2W12 BaP1 3.4.24.- neutrophil chemotaxis and role in [37] asper methylpropyl)-3-{[(1,3- thiazol-2- leucocyte recruitment ylcarbonyl)amino]methyl}butanediamide) and Zn2+

Activation of complement system, Bothrops 1ND1 BaP1 Zn2+ 3.4.24.- neutrophil chemotaxis and role in [23] asper leucocyte recruitment

Thrombolytic agent and biochemical Bothrops Bothropasi 3DSL Furoyl-leucine, Zn2+ and Ca2+ 3.4.24.49 tool in coagulation research and [41] jararaca n diagnosis

Agkistrodon A novel target recognizing model for 3HDB AaHIV Zn2+ and Ca2+ 3.4.24.- [35] acutus ADAM/reprolysin family

Agkistrodon Acutolysin A novel target for designing anti-tumor 1BUD Zn2+ and Ca2+ 3.4.24.- [29] acutus A metastasis and anti-arthritis drugs

Agkistrodon Acutolysin A novel target for designing anti-tumor 1QUA Zn2+ 3.4.24.- [20] acutus C metastasis and anti-arthritis drugs

Agkistrodon A therapeutic use against thrombotic 1YP1 FII Tri-peptide Lys-Asn-Leu and Zn2+ 3.4.24.- acutus occlusive diseases

Peptide phosphonate inhibitor FLX (N- A starting model for the rational Crotalus Adamalysin [(furan-2-yl)carbonyl]-(S)-leucyl-(R)-[1- design of TACE inhibitors for 4AIG 3.4.24.46 [25] adamanteus II amino-2(1H-indol-3-yl)ethyl]-phosphonic inflammation and joint damage in acid) and Zn2+ rheumatoid arthritis

A starting model for the rational Peptidomimetic inhibitor ( N-(furan-2- Crotalus Adamalysin design of TACE inhibitors for 2AIG ylcarbonyl)-L-leucyl-L-tryptophan), Zn2+ 3.4.24.46 [26] adamanteus II inflammation and joint damage in and Ca2+ rheumatoid arthritis

A starting model for the rational Crotalus Adamalysin design of TACE inhibitors for 1IAG Zn2+ and Ca2+ 3.4.24.46 [13] adamanteus II inflammation and joint damage in rheumatoid arthritis

BB94 (4-(N-hydroxyamino)-2r-isobutyl- Crotalus 2S-(2-thienylthiomethyl)succinyl- l- A novel target for designing anti-tumor 1DTH Atrolysin C 3.4.24.42 [28] atrox phenylalanine-N-methylamide), Zn2+ and metastasis and anti-arthritis drugs Ca2+

Synthetic inhibitor (SCH 47890) (O- Crotalus Methyl-N-[(2S)-4-methyl-2- A novel target for designing anti-tumor 1ATL Atrolysin C 3.4.24.42 [27] atrox (sulfanylmethyl)pentanoyl]- L-tyrosine), metastasis and anti-arthritis drugs Zn2+ and Ca2+

A molecular model for finding the Crotalus 2ERQ VAP 1 Zn2+ and Ca2+ 3.4.24.- physiological targets and adhesion [17] atrox mechanisms of ADAMs for shedding. 32 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 P. Chellapandi

Table 1. Contd…..

Snake Name PDB Molecule Inhibitors/Ligands/Metal Ions E.C. Pharmacological Properties References

GM6 (3-(N-Hydroxycarboxamido)- Inhibition of collagen-induced platelet Catrocollasta Crotalus atrox 2DW0 2-isobutylpropanoyl- trp- 3.4.24.- aggregation by binding to collagen via [32] tin methylamide), Zn2+ and Ca2+ its disintegrin-like domain.

Thrombolytic agent and biochemical Trimeresurus Trimerelysin 1WNI Zn2+ 3.4.24.53 tool in coagulation research and [31] flavoviridis II diagnosis

Endogenous inhibitors pyroGlu- Structural based designing of synthetic Trimeresurus 1KUG Atrolysin E Asn-Trp, pyroGlu-Gln-Trp, 3.4.24.44 inhibitor analogues for arthritis and [30] mucrosquamatus pyroGlu-Lys-Trp and Cd2+ tumor metastasis

Activation of coagulation system, GM6 (3-(N-Hydroxycarboxamido)- Daboia russellii factor X, with consumptive 2E3X Russellysin 2-isobutylpropanoyl- trp- 3.4.24.58 [34] siamensis coagulopathy, thrombotic occlusion methylamide), Zn2+ and Ca2+ and thrombopenia

VAP: Vascular apoptosis-inducing protein, TACE: Tumor necrosis factor-alpha-converting enzyme the S1 packet and established a mixed parallel-antiparallel 3.3. Acutolysin three-stranded -sheet [26]. Thus, the structural features of The crystallographic structure of acutolysin, a P-I class ADAMs would provide insights in rational drug designing SVMP, from the venom of A. acutus has two conserved with higher specificity as ADAMs are potential therapeutic targets. motifs including His142-Glu143-X-X-His146-X-X-Gly149- X-X-His152 and Cys162-Ile163-Met164 [20]. The crystallographic structures of all P-I class SVMPs have a conserved 3.2. Atrolysin C disulfide bridge Cys117-Cys195. A catalytic zinc ion is Atrolysin C inhibitors, generally derivatized peptides, localized in one of the sub domains, coordinated in a tetra- showed to have potential therapeutic value in a number of hedral manner with one catalytic water molecule anchoring pathologic conditions ranging from corneal ulceration to to an intermediate glutamic acid residue (Glu143) and three arthritis to cancer. Structural information on atrolysin C from imidazole Nepsilon2 atoms of His142, His146 and His152 C. atrox venom and its similarity to mammalian enzymes has in the conserved motif His142-Glu143-X-X-His146-X-X- been employed to identify the inhibitors PyroGlu-Asn-Trp Gly149-X-X-His152 [29]. The local conformation and and SCH 47890. These inhibitors may be useful to control coordination of the catalytic water molecules in acutolysin the diseases associated with aberrant regulation of collage- are somewhat differed from other P-I class SVMPs, but it nases [27]. The structure of atrolysin C has significant has similar conformations in the active-site cleft. Acutolysin characteristics; the active-site-helix (residues 132-147) with A (acidic) and acutolysin C (alkaline) were also crystallized Zn2+ ions (His142, His146) and ubiquitously conserved Met in two different physiological pH conditions [20]. Both turn. Zn ligation, occupancy of subsites along the extended proteins have a high homology with other zinc metallopro- binding site and optimal occupancy of the primary teinases of a low molecular weight. Acutolysin A is inactive specificity site (S1) are common binding features of atrolysin under acidic conditions due the polarization capability of the C. A large aromatic or extended moiety is located in the Glu143 carboxylate group to the catalytic water molecule dominant primary specificity site. It has hydrophobic active become weaker [29]. Moreover, the size of the active site in site. The extended binding site with charged groups at both acutolysin C is not correlated with the crystallization pH ends is defined by the antiparallel -strand and hydrophilic values or the proteolytic activities. Calcium ion may not groups occupied in the surface loci pointing ‘out’ into the affect the proteolytic activity or haemorrhagic activity directly because of no structural calcium ion in acutolysin hydrated surface. The hydroxamate group precludes creation [20]. Crystallographic studies revealed that P-I class SVMPs of inhibitors capable of binding to both S and S' loci in the are inactive or have a low activity under acidic conditions. extended binding site. Under physiological conditions, the No crystallography evidence for inhibitor complex of this hydroxamate moiety may be a better Zn ligand than the structure to date. methylamide group. However, in high salt buffer van der Waals interactions derived especially from the full insertion of the thiophene side chain into the cavernous S1 pocket can 3.4. Atrolysin E stabilize the formed complex of atrolysin C-batimastat Huang et al. have described the crystal structures of a (BB94). Hence, an unexpected binding geometry found to SVMP, TM-3, from T. mucrosquamatus venom which was exhibit in atrolysin C for BB94, a matrix metalloproteinase cocrystallized with the endogenous inhibitors pyroGlu-Asn- inhibitor, with the thiophene ring deeply inserted into the Trp, pyroGlu-Gln-Trp or pyroGlu-Lys-Trp [30]. As com- primary specificity [28]. Such binding geometry implied the pared to adamalysin II structure, the binding modes of inhi- significance of the cavernous primary specificity site, bitors (to fill the S1 pocket) are almost same in atrolysin E. pointing the way for designing potential antitumor drugs. Upon inhibitor binding, some residues around its inhibitor- Structural, Functional and Therapeutic Aspects of Snake Venom Metalloproteinases Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 33 binding pocket are slightly move away from active-site pasin [41]. Structural alignment (Fig. 3b) and superim- center and displaced two metal-coordinated water molecules position (Figs. 3c and d) of the model with respective PDB by the C-terminal carboxylic group of the inhibitors. The Trp structures were performed with 3D-SS server [42]. There indole ring of the inhibitors is stacked against the imidazole was no significant conformational change between a metal- of His143 in the S1 site. The primary specificity of Trp loproteinase model and a crystallographic structure (pdb id: residue of the inhibitors at the P1 site corroborates the 3had) of metalloproteinase from A. acutus. A few slight stacking effect. A close correlation is found between the changes are also observed in the flexible residue side-chains inhibitory activity of an inhibitor and its ability to fill the S1 on the molecular surface as similar to earlier study [29]. pocket of this enzyme that may provide insights into the However, a major conformational change is noted to occur in rational design of small molecules. the terminal residues and active-site cleft when model superimposed with other crystallographic structure of 3.5. Molecular Mechanisms of Target Recognition SVMPs. Methionine-containing turn is structurally conserved in H2-proteinase from T. ravoviridis venom, suggesting the 4. SUBSTRATE SPECIFICITIES disparity in substrate recognition [31]. Vascular apoptosis- Knowing substrate specificity of SVMPs is required to inducing protein 1 [17], vascular apoptosis-inducing protein design the peptides based on their identified cleavage sites. 2B [32] from C. atrox venom and cysteine-rich domain of Bothropasin (EC 3.4.14.49) cleaves the His5-Leu, His10- kaouthiagin-like (atragin) from N. atra venom [33] were Leu, Ala14-Leu, Tyr16-Leu and Phe24-Phe in insulin B studied to understand the role of hyper-variable region in chain [41, 43]. Cleavage of Phe1-Val, His5-Leu, His10-Leu, protein-protein adhesive interface. Modular architecture of Ala14-Leu, Leu15-Tyr, and Tyr16-Leu can be catalyzed by these structures in this region may be involved in a cell- adamalysin II (EC 3.4.24.46) [13, 24-26, 44]. Trimerelysin II migration activity and molecular recognition. or H2-proteinase (EC 3.4.24.53) can cleave the Asn3-Gln, His10-Leu and Ala14-Leu in the insulin B chain, and the Russell's viper venom factor X activator, a heterotrimeric metalloproteinase, is a hook-spanner-wrench-like architec- bond Z-Gly-Pro-Leu-Gly-Pro in the microbial collagenase [31, 45]. Enzymatic studies on adamalysin in the venom of ture and the lectin-like domains constitute a handle. This is a C. adamanteus by Kress and Catanese found that Glu37- typical example of the molecular evolution of multi-subunit Gln38, Ala375-Ser376 and Ala378-Val379 are cleavage sites proteins and insights into the molecular basis of a target in the presence of heparin whereas Ser383-Ala384 is in the recognition and proteolysis [34]. A novel target recognition absence of heparin [46]. mechanism was recently elucidated by Zhu et al., for the structure of AaHIV, purified from A. acutus venom [35]. Atrolysin C (EC 3.4.24.42) reported to cleave His5-Leu, Understanding their stereo-specificity and action mechanism His10-Leu, Ala14-Leu, Tyr16-Leu and Gly23-Phe bonds is also instrumental for designing metalloproteinase. Crystal- [27, 28]. In contrast, atrolysin E (EC 3.4.24.44) hydrolyzes lographic studies on BmooMPalpha-I structure (B. moojeni) Asn3-Gln, Ser9-His and Ala14-Leu bonds in insulin B chain have revealed that catalytic zinc ion displayed an unusual and Tyr14-Gln and Thr8-Ser in insulin A chain. Also, it octahedral coordination is formed by His142, His146 and cleaves the type IV collagen at Ala7-Gln in 1 (IV) collagen His152 residues and by three water molecules [36]. and at Gly7-Leu in 2 (IV) [30, 47]. Russellysin (EC Structure-activity relationship of the bound inhibitor form 3.4.24.58) specifically activates coagulation factor X and IX (BaP1-peptidomimetic inhibitor) and a flexible loop region of the blood clotting system and has no action on insulin B supported as a starting point for the design of drugs for the chain [34, 48]. However, many of the SVMPs have no clear treatment of local pathological effects caused by snake bites, cleavage specificities on respective peptides, but assigned as particularly B. asper [37]. Structural variations between the unclassified peptide cleavage (EC 3.4.24) [20, 23, 29, 33, C-shaped topology in atragin and I-shaped conformation in 35-37, 49]. kaouthiagin-like highlight the disulfide bond patterns in the disintegrin-like domain of SVMPs family proteins [33]. As a 5. INHIBITOR SPECIFICITIES result, both SVMPs showed to exhibit an enzymatic specificity toward pro-TNF (tumor necrosis factor) with Many enzyme kinetics studies on SVMPs have been less inhibition of cell migration in opposite manner. conducted to reveal their catalytic mechanisms and to calculate the inhibition constants. Adamalysin II may be inhibited by 2-mercaptoethanol [44], EDTA [44, 46], 3.6. Comparative Modeling cysteine and glutathione-SH [14, 44]. The peptide inhibitors Most recently, computational modeling has gained a such as acetyl-Trp-Cys-Gly-Pro-NH2, Lys-Pro-Arg-Cys-Gly- better understanding about the binding properties of various Val-NH2, Pro-Lys-Met-Cys-Gly-Val-NH2, Pro-Lys-Met-D- ligands to SVMPs. Free energy calculation on BaP1 has Cys-Gly-Val-NH2, Pro-Lys-Met-Ser-Gly-Val-NH2, Lys-Thr- performed to study the binding properties of various ligands Phe-Thr-Ser-Cys [14], PyroGlu-Asn-Trp [13], Fur-Leu-Trp- and made to guide future synthesis efforts for novel inhibi- OH, N-(furan-2-yl) carbonyl-L-leucyl-D-phosphotryptop- tors [38]. Three dimensional structure of metalloproteinase han,N-(furan-2-yl)carbonyl-L-leucyl-L-phosphotryptophan from G. halys was modeled from its sequence (accession: and N-(furan-2-yl)carbonyl-Leu-Trp(P)-(OH)2 [50] are known AAD02652) by Swiss-Model server [39] and then energy to retard the proteolysis of adamalysin II. Physiological constraints minimized with ModRefiner server [40]. Disin- inhibitor, alpha2-macroglobulin [51] and selective synthetic tegrin domain of a modeled protein is composed mostly of inhibitors [27, 52, 53] are described to inhibit their enzyme loops stabilized by seven disulfide bonds (Fig. 3a); therefore activities. The SCH 47890 [23], BB94 [28], peptide phos- there is no distinct secondary structure as similar to bothro- phonate [25], N-(furan-2-ylcarbonyl)-L-leucyl-L-tryptophan 34 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 P. Chellapandi

3a. Strcutural view of modeled protein 3b. Structural alignment of modeled protein with crystallographic structures (pdb id: 3HAD and 3DSL)

3c. Structural superimposition of modeled protein Vs 3HAD

3d. Structural superimposition of modeled protein Vs 2DWO, 3DSL, 3HAD Fig. (3). Graphical representation (in MolGro Molecular viewer) of modeled structure of metalloproteinase with antibacterial property from G. halys (accession: AAD02652) denoted with identified domains of reprolysin, cysteine-rich and disintegrin (3a) and of its multiple structural alignment (3b) and superimposition with a crystallographic structure (pdb id: 3HDB) of AaHIV from A. acutus (3c) and other structures (3d). Glu333 is catalytic site whereas His334, His338, His344 are active sites of this structure. A helix just near to a loop structure at the end of the reprolysin domain is noted to be interacting with a target protein. Structural deviated residues in this model are identified as I241, G245, A292 and S350 by its superimposition with a crystallographic structure (pdb id: 3HDB). This model is structurally deviated at a great extent from other crystallographic structures (pdb id: 2DWO, 3DSL) in the residue positions of 185-425 and 490-610 (3d). [28], pyroGlu-Asn-Trp [30], GM6 [32, 34], furoyl-leucine The proteolytic activity of FII is inhibited by a tri-peptide [41] and WR2 [37] are potential inhibitors that were Lys-Asn-Leu contributing four hydrogen bonds with Ala106, identified on the basis of structure and functions of SVMPs- Val108, Pro167 and Leu169 [51]. Furoyl-Leucine interacts inhibitor complexes. with the side chain residues of Pro109, Ile111, His145, Structural, Functional and Therapeutic Aspects of Snake Venom Metalloproteinases Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 35

Gly170 and Pro171 in the bothropasin [41]. Alpha-L-fucose Leu170) of the cleavage site adopting a retro-binding mode inhibits the activity of atragin by interacting with the side in Adamalysin II [25]. Similarly, N-(furan-2-ylcarbonyl)-L- chain residues of Asn319 and Glu329 while beta-L-fucose leucyl-L-tryptophan interacts with the same residues in will recognize the Asn490 residue to repress its activity [33]. addition to the Glu143 [26]. Endogenous inhibitor, pyroGlu- Atrolysin and batimastat (BB94), a potent matrix Asn-Trp, identified by Huang et al. to inhibit the Atrolysin E metalloproteinase inhibitor complex may be stabilized by the by interacting with the side chain residues of Arg107, formation of six hydrogen bonds with the side chains of Asn108, Ile110, His144, His154 and Ser168 [30]. The 3-(N- Gly109, Thr139, His142, Glu143, His152 and Pro168 [28]. hydroxycarboxamido)-2-isobutylpropanoyl-trp-methylamide Atrolysin may also be inhibited by SCH 47890, a synthetic is a known synthetic inhibitor that can be interacted with inhibitor interacting with Leu108, His152 and Leu170 Ile299, Gly300, His333, Glu334, His337, His343, Pro359, residues in the binding cleft [27]. Ile361 residues in the binding cleft of catrocollastatin [32] and with Asn109, Leu111, His145, His149, His155 residues A peptide phosphonate inhibitor, FLX, occupies the of russellysin [34]. As shown in (Fig. 4), a peptidomimetic primed region (Lys106, Ile108, His152, Asg167 and

Fig. (4). LigPlot representations showing the hydrogen bonding between selected crystallographic structures of SVMPs and ligands in the binding sites (see more details in Table 1). Ligands are represented in brown color whereas protein represented in black color. Hydrogen bonding is represented as dotted green line and measured as angstrom. (GM6: 3-(N-Hydroxycarboxamido)-2-isobutylpropanoyl- trp- methylamide; WR2: (2R,3R)-N-1-[(1S)-2,2-dimethyl-1-(methylcarbamoyl)propyl]- N-4-hydroxy-2-(2-methylpropyl)-3-[(1,3- thiazol-2- ylcarbonyl)amino]methyl}butanediamide; PCA-Asn-Trp: Pyroglutamic acid-Asparagine-Tryptophan). 36 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 P. Chellapandi inhibitor WR2-BaP1 complex is formed by contributing as a template for homology modeling. We also identified several hydrogen bonding between the side chains of meltrin-L precursor from Homo sapiens hypothetical protein Asn106, Leu170, Ser168, Thr107, Ile108, Gly109, Ile111, RCJMB04_17j13 from Gallus gallus (Chicken) and Glu143, His145, His146, His152, Gly170 and Pro171 of disintegrin and metalloproteinase domain-containing protein BaP1 and inhibitor molecule [37]. 8 precursors from Danio rerio (Dog) as homologous proteins to metalloproteinase of G. halys venom (Fig. 5). Hence, the 6. EVOLUTIONARY MECHANISMS evolutionary significance of SVMPs family of enzymes may urge the researchers to select the homologous targets from Snake venom protein superfamilies are mostly recruited mammals, and to study the molecular functional diversity. from the body proteins, some of them have evolved from non-toxic to most toxic proteins in the venom glands [4]. 7. SNAKE VENOM BIOACTIVE PEPTIDES Snake venom toxin types still process the bioactive of ancestral proteins that are gradually flourished into functionally Snake venom bioactive peptides have been utilized in diverse and multigene families [54]. Phylogenetic distri- studies of molecular enzymology, structural biology and bution is observed among SVMPs, which leads to diverse pharmaceutical biotechnology [3, 10, 69]. The development into sub-families and domains. Much more published of novel human therapeutics may be achieved from the evidences have been focused on the phylogenetic diversity bioactive peptides of SVMPs because of their haemorrhage, and distribution of snakes from different geographical areas, coagulopathy, and inflammatory responses [3-4, 70, 71]. but a very little is known about the evolutionary significance Construction of cDNA library and combinatorial peptide of SVMPs family of enzymes. library are general approaches used to identify the bioactive peptides from SVMPs [72-74]. A functional proteomic study Structural resemblance of SVMPs may be explained by has carried out to explore the bioactive protein components ancestral Zn-binding motif and conservation of the proteo- from Chinese G. shedaoensis snake venom [75]. These lytic motifs during the divergence of the proteins rather than attempts would manifest the elucidation of structure-function through convergent evolution [55]. A common ADAMs relationships in order to discover the peptides of scaffold may be acted as an origin for evolving disintegrin biotechnological interest. subfamilies [56]. Phylogenetic analysis of SVMPs revealed that subtype-specific amino-acid substitutions in the C- SVMPs have a potential of degrading fibrinogen, fibrin, terminal regions determine their structure-activity relation- type I collagen, fibronectin, laminin, collagens and ships [57]. A new type of P-II class jerdonitin has two proteoglycans published in many literatures [76-79]. The additional cysteines (Cys219 and Cys238) located in a spacer efficacy of recombinant fibrolase was investigated to lyse an domain and a disintegrin domain, respectively, which was occlusive thrombus formed in the carotid artery of the phylogenetically diverged from other types of P-II class [58]. anesthetized dog [80]. Prothrombin activators including Conserved Asp148 and Ser176 residues in P-I class are ecarin and carinactivase-1 are identified from Echis taking a role in the stabilization of the active site [59]. carinatus venom, which primarily recognized the Ca2+- Phylogenetic conservation of P-III class may be determined bound conformation of the Gla domain in prothrombin and by the presence of large hydrophobic areas and some conser- subsequently converted to active thrombin [81]. Chelating ved surface charge-positive residues [60]. Structural motifs agent CaNa2EDTA and BB94 at the site of venom injection include daborhagin-M and daborhagin-K in the PIII class has found to inhibit the proteolytic, hemorrhagic and SVMPs are responsible for their phylogenetic subtyping of dermonecrotic effects, and partially reduced edema-forming Russell's viper venoms [61]. A multigene protein family of activity induced by B.asper snake venom [82]. Aspercetin SVMPs is, therefore, evolved due to posttranslational modi- from the venom of B. asper mediates platelet aggregation by fications, accelerated evolution by positive selection and the interaction of von Willebrand factor with platelet multiple episodes of domain alteration, particularly domains receptor GPIb [83]. Mikarin, a group I prothrombin of cysteine-rich and disintegrin [62, 63]. Their conserved activator, isolated from Micropechis ikaheka venom has structure would thus confer extensive immunological cross- exhibited Ca2+-independent prothrombin activation, but no reactivity to toxin-specific antibody [64, 65], suggestive of effects on blood coagulation factor-X and fibrinogen [84]. in-depth knowledge of a target molecule is required for A novel fibrinolytic metalloproteinase, alfimeprase, antivenom development. derived from A. contortrix contortrix venom breaks down the The sequence similarity hits for metalloproteinase of G. fibrin-rich clots and prevents progression of clot formation halys venom were obtained from the NCBI-non-redundant [85]. Its preliminary clinical experience in subjects with protein database using the BLASTp program [66]. Multiple acute peripheral arterial occlusion and central venous access sequence alignment was conducted with the ClustalX 2.0.11 device occlusion has been reviewed by Deitcher and Toombs software [67]. All the aligned sequences were iterated at [86]. Eptifibatide (integrelin) modeled on the active site of each alignment step, corrected multiple substitutions and barbuorin, a lysine-glycine-aspartate containing disintegrin then manually inspected for a proper alignment. Phylogeny [87]. Tirofiban (Aggrastat) designed from echistatin is a was inferred from aligned sequences by the MEGA 5.05 synthetic compound that mimics arginine-glycine-aspartate software [68] with 1000 bootstrapping and visualized in the containing disintegrin [88]. Both are the approved MEGA. Phylogenetic analysis of our study revealed that therapeutic drugs for treating acute coronary ischaemic metalloproteinase from G. halys is evolutionarily related syndrome and the prevention of thrombotic complications with AaHIV from A. acutus and then with bothropasin from [89, 90]. A non-toxic metalloprotease, NN-PF3, from the N. B. jararaca and catrocollastatin from C. atrox. Since, a naja venom showed to hydrolyze blood and plasma clot so crystallographic structure (pdb id: 3HDB) of AaHIV selected that it can be used as an anti-coagulant agent [91]. Viper Structural, Functional and Therapeutic Aspects of Snake Venom Metalloproteinases Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 37

74 AaHIV (3HDB) Agkistrodon acutus 63 Metalloprotease (AAD02652) Gloydius halys 99 Bothropasin (3DSL) Bothrops jararaca 41 Catrocollastatin (2DW0) Crotalus atrox

46 Acutolysin A (1BUD) Agkistrodon acutus Jerdonitin (P83912) Protobothrops jerdonii

27 Russellysin (2E3X) Daboia russellii siamensis 73 SVMP group III (CAJ01687) Echis ocellatus 74 99 Acutolysin C (1QUA) Agkistrodon acutus

58 FII (1YP1) Agkistrodon acutus Trimerelysin II (1WNI) Trimeresurus flavoviridis 100 Atrolysin E (1KUG) Trimeresurus mucrosquamatus

42 99 BmooMPalfa-I (3GBO) Bothrops moojeni BaP1 (2W12) Bothrops asper 86 Adamalysin II (4AIG) Crotalus adamanteus 99 Atrolysin C (1DTH) Crotalus atrox Atragin (3K7L) Naja atra 45 VAP1 (2ERQ) Crotalus atrox Hypothetical protein RCJMB04_17j13 (CAH65319) Gallus gallus

99 Protein 8 precursor (NP_956931) Danio rerio Vertebrates 99 Meltrin-L precursor (AAC08702)| Homo sapiens

0.2 Fig. (5). Phylogenetic tree of metalloproteinase present in G. halys venom constructed by Neighbor Joining algorithm using closely related sequences to the PDB structures. Metalloproteinase with antibacterial activity (accession: AAD02652) is represented as purple color in the tree. venom-induced haemorrhagic activity is also inhibited by and for blood pressure regulation, and hemostasis [103]. 1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-3-oxo-1,3- Salmosin [104], and contortrostatin [105] derived from dihydroisobenzofuran-5-carbonitrile designed from the SVMPs may be considered as alternative drugs to the venoms of E. carinatus, E. ocelatus and C. atrox [92]. Hsu et humanized monoclonal antibodies used in cancer therapy. al. evidenced that a P-I SVMP, kistomin can be used as a Trummal et al. investigated a metalloprotease from V. new anti-thrombotic agent because of it inhibits the lebetina venom that found to induce human endothelial cell interaction between collagen and platelet glycoprotein VI apoptosis [106]. Saxatilin is a disintegrin known to suppress through its proteolytic activity on glycoprotein VI [93]. TNF-alpha-induced ovarian cancer cell invasion at 50nm BmHF-1 from B. marajoensis venom has weak hemorrhagic concentration [107]. Jararhagin has a role in proinflam- activity and also reported to release pharmacologically active matory pathogenesis and pro-apoptotic host response [108, mediators from protein precursors due to its enzymatic 109]. It also induces changes in the morphology and viability action [94]. Upon comparing 28 snake venom profiles, of the Skmel-28 [110] and SK-Mel-28 human melanoma Russell's viper venom-factor X activator identified to have cells [111]. HUVEC apoptosis inducing heterodimeric procoagulant and anticoagulant activities [95]. Halysase metalloproteinase from V. lebetina venom proved to inhibit [96], ammodytase from Vipera ammodytes ammodytes the viability of cancer cells and platelet aggregation [112]. venom [97], alsophinase from Alsophis portoricensis venom Since, studies on structure-function integrity of SVMPS [98], jararhagin from B.jararaca venom [99] and moojenin would manifest the designing and development of a potential from B. moojeni venom [100] recently identified for - anti-cancer drug. fibrinogenolytic and hemorrhagic activity suggests their Hemorrhagins purified from crude B. atrox venom is clinical use as antithrombotic agents. Costa Jde et al. done involved in acute inflammation characterized by edema, structure and function comparison analysis of fibrinogeno- accumulation of polymorphonuclear leucocytes and hemor- lytic metallo proteases, which was potential for clinical use rhage [113]. Silva et al., reported the activation of phago- in the treatment of hemostatic disorders, particularly cytosis via alpha (M) beta (2) integrin by a metalloproteinase cardiovascular disorders [101]. containing disintegrin-like/cysteine-rich domains [114]. The TACE active site model can be a starting model for Anti-complementary and edema-inducing activities are the rational design of inhibitors for TACE-involved inflam- exhibited by atrase B, which was purified from N. atra matory diseases [24-26]. Bradykinin-potentiating peptides venom [115]. identified in the snake venoms are valuable sources to BaP1 inhibitor is served as an anti-arthritis drug because prevent the angiotensin-converting enzyme activity [102] of BaP1 is involved in inflammatory joint hypernociception 38 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 P. Chellapandi and induced cyclooxygenase expression [116]. The hyper- cleft of this metalloprotease model (Fig. 6). The interfacing variable region of the cysteine-rich domain [117] and residues between this metalloprotease model and those metalloproteases from B. jararaca [118] venom have a membrane proteins are not still understood and further provital role in triggering pro-inflammatory effects mediated studies on designing synthetic peptides based the interfacing by integrins. Six monoclonal antibodies against BaP1 residues are under progress for antimicrobial therapy. produced, which may become important tools to understand its structure-function relationships [119]. Patagonfibrase, a 7.2. Recombinant Peptides and Vaccines P-III SVMP, from Philodryas patagoniensis venom exhibited to elicit the proinflammatory effects mainly mediated Many active recombinant proteins have been produced by its catalytic activity [120]. Jararhagin also reported to from the metalloproteinase encoding genes in the venom enhance the rolling of circulating leukocytes, nitric oxide glands of snakes by expressing in heterologous hosts. For the generation, prostacyclin production and pro-inflammatory inhibition of platelet aggregation and fibrinolytic activity, cytokines release, but it decreased endothelial cells viability recombinant atrolysin A [129], trimucytin from T. mucros- inducing apoptosis [121]. BJ-PI2 from B. jararaca venom is quamatus [130], pro-ACLF from A. contortrix laticinctus a non-hemorrhagic, non-myonecrotic, non-coagulant that [131], fibrinogenase IV (rFIVa) from A. acutus [132, 133] contributes to enhanced vascular permeability and inflam- and AplVMP2 from A. piscivorus leucostoma [134] have matory cell migration [122]. On account of blocking P- been evaluated earlier. Moreover, recombinant albolatin selectin/P-selectin glycoprotein ligand-1 interaction, mocar- from T. albolabris [135], jararhagin fragments [136], ahpfi- hagin from the venom of N. mocambique mocabique is brase from G. halys [137] and albocollagenase from Cryptel- desirable to treat inflammatory disease caused by P-selectin ytrops albolabris [138] have been produced to inhibit (Patent No. EP0842269A1). Stejnihagin from T. stejnegeri collagen-induced platelet aggregation. Recombinant fibrino- venom found to inhibit the L-type Ca2+ channels in A7r5 genase (rFII) from A. acutus exhibits for cleaving fibrin cells and simultaneously blocked K+-induced vessel contr- without plasminogen activation and suppressing ADP- action, and thus provided a clue to study the structure- induced platelet aggregation [131]. function relationship SVMPs and voltage-gated Ca2+ Addition to the inhibition of platelet aggregation, channel [123]. recombinant contortrostatin from A. contortrix contortrix blocks cancer cell adhesion to fibronectin and vitronectin 7.1. Antibacterial Properties and prevent invasion of cancer cells through a Matrigel barrier [139]. Jerdonitin, a PII SVMP, found to retard the Phospholipase A2, L-amino acid oxidase and lectin are growth of human liver cancer cells, leukemia cells and known potential antibacterial enzymes present in snake gastric carcinoma cells, suggesting further functional and venoms [6, 94]. The molecular mechanisms of these structural studies of recombinant Jerdonitin [140]. enzymes and their recognition on corresponding bacterial Recombinant disintegrin (leucurogin) derived from B. protein targets have been evidenced in many publications. leucurus venom glands reported to inhibit tumor growth Recently, metalloproteinase (23.1 kDa protein) from Agkis- [141]. Recombinant fibrinogenase II (rFII) venom has been trodon halys (Gloydius halys) venom reported to inhibit the found to protect against taurocholate-induced severe acute growth of some pathogenic bacteria that may be the reasons pancreatitis in rats due to its degradation potency of rat TNF- of altering membrane packing proteins and restraining [142]. Harrison et al. conducted the first study to apply mechanosensitive proteins [124]. The potential antibacterial DNA-based methods (with DNA encoding the carboxy- effect of this enzyme observed against Gram-positive terminal JD9 domain of jararhagin) to preparation of bacterium, S. aureus and Gram-negative bacteria (Burkho- antivenom for neutralization of the main lethal component of lderia pseudomallei, Escherichia coli, Enterobacter aero- B.jararaca venom [143]. The recombinant BJ46a can inhibit genes, Proteus vulgaris, P. mirabilis, and Pseudomonas the invasion and metastasis of tumor cells by reducing aeruginosa). However, no crystallographic structures matrix metalloproteinase activities, indicating that rBJ46a reported for this activity and molecular interactions with may be a novel therapeutic agent for antimetastasis of tumor bacterial membrane proteins, but structures for other cells [144]. GeneGun DNA immunization from jararhagin activities are available in many literatures. Additional [64], a single synthetic multiepitope DNA immunogen from structural and functional information of that protein would hemorrhagins [143], DNA immunization based on carboxy- support the rational based peptides or inhibitors designing. terminal JD9 domain of Jararhagin [65] and P-II We have performed protein-protein docking for identi- metalloproteinase have been developed successfully using fication bacterial membrane proteins as antimicrobial targets, recombinant DNA techniques. DNA immunization also which are liable to be interacting with metalloprotease with offers a rational approach to the design of toxin-specific antibacterial activity from G. halys. Protein-protein docking immunotherapy. A combined approach of bioinformatics and was carried out with GRAMM-X 1.2.0 server [125] and multiepitope DNA immunization are employed to design visualized in MolGrow Molecular viewer. It revealed the antivenom for E. ocellatus bits using structural and modeled metalloprotease may able to interact with a large- functional information of hemorrhagins from the same snake conductance mechanosensitive channel MscL (pdb id: 3hzq) venom [145]. and membrane assembling proteins MetN2 (pdb id: 3ced), LukE (pdb id: 3roh) and GmpC (pdb id: 1p99) of Staphylococcus aureus [126-128]. This in silico evidence 8. CONCLUSION provides the importance of molecular structure and Snake venoms information can be retrieved from the recognition of metalloproteinase from G. halys venom for public domain databases such as Snake Venom Database antibacterial potential that may be resulted at the binding (http://svdb.org.in/), Venoms Database (http://www.atheris. Structural, Functional and Therapeutic Aspects of Snake Venom Metalloproteinases Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 39

MscL: Large-conductance mechanosensitive channel; MetN 2: Methionine import ATP-binding protein; LukE: Leukotoxin; PG110: Membrane-associated lipoprotein-9 GmpC Fig. (6). Molecular interaction view of modeled metalloproteinase (accession: AAD02652) with large-conductance mechanosensitive channel (pdb id: 3HZQ) and membrane assembling proteins (pdb id: 3ROH, 3CED, 1P99) of Staphylococcus aureus. Metalloproteinase model is represented in molecular surface view while target proteins represented in ball and stick model. Binding sites are represented with electrostatic potential (green sphere). com/ven_data.php), Venomdoc (www.venomdoc.com/), Snake venom bioactive peptides are widely utilized in the Natural Toxins Research Center-Snake Database studies of pharmaceutical biotechnology. Multiple structural (http://ntrc.tamuk.edu/cgi-bin/serpentarium/snake.query), and functional motifs in the SVMPs may influence their Australian Venom and Toxin Database (www.kingsnake binding cleft orientations on mammalian targets and exerts .com/) and MEROPS database [11]. Current online resources the pathophysiological effects. Thus, a conserved structure and X-crystallographic structural information are strength- confers immunological cross-reactivity to toxin-specific anti- ening the research activities towards developing therapeutics body, suggested that in-depth knowledge of a target mol- based on their available structures, functional domains and ecule is required for antivenom development in future. It can sequences. Computational modeling has also gained a better be achieved effectively by a combined approach of bioinfor- understanding about the SVMP-inhibitor complexes. The matics and multiepitope DNA immunization. The present potential inhibitors including N-[(furan-2-yl)carbonyl]-(S)- snake venom research is mainly focused on the cloning and leucyl-(R)-[1- amino-2(1H-indol-3-yl)ethyl]-phosphonic acid, expression of genes encoded for metalloproteinase for N-(furan-2-ylcarbonyl)-L-leucyl-L-tryptophan,4-(N-hydro- producing recombinant peptides and on finding the novel xyamino)-2r-isobutyl-2S-(2-thienylthiomethyl)succinyl- l- therapeutic peptides from their available structures. Al- phenylalanine-N-methylamide, O-methyl-N-[(2S)-4-methyl- though the potential therapeutic peptides for anti-coagu- 2-(sulfanylmethyl)pentanoyl]-L-tyrosine,3-(N-hydroxycarb- lation, antitumor, anti-arthritis, anti-complementary and anti- oxamido)-2-isobutylpropanoyl-trp-methylamide,(2R,3R)-N- inflammatory activities have been successfully, the structure 1-[(1S)-2,2-dimethyl-1-(methylcarbamoyl)propyl]-N-4-hyd- and molecular recognition of antimicrobial peptides derived roxy-2-(2-methylpropyl)-3-[(1,3-thiazol-2-ylcarbonyl)am- from the SVMPs has been locked to update. This preliminary ino]methyl}butanediamide, pyroGlu-Asn-Trp, pyroGlu-Gln- study on conserved domains and modeled structure of Trp, pyroGlu-Lys-Trp, Lys-Asn-Leu, furoyl-leucine, have metalloproteinase from Gloydius halys and its molecular been identified on the basis of structure-function integrity of interaction with bacterial membrane proteins may provide a SVMPs. conceptual idea to strength the venom research in order to 40 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 P. Chellapandi design and produce the future antibacterial peptides. This is [16] Seals, D.F.; Courtneidge, S.A. The ADAMs family of metallopro- the first SVMP model exhibiting antibacterial activity. It teases: multidomain proteins with multiple functions. Genes Dev., 2003, 17, 7-30. suggests the protein chemists to urge the determination of its [17] Takeda, S.; Igarashi, T.; Mori, H.; Araki, S. Crystal structures of structure and complexes, as predicted in this study, for VAP1 reveal ADAMs' MDC domain architecture and its unique C- further understanding of structure-function integrity and its shaped scaffold. EMBO J., 2006, 25, 2388-2396. catalytic mechanism. We conclude that future therapeutics [18] Letunic, I.; Doerks, T.; Bork, P. SMART 7: recent updates to the designing and development would be possible from snake protein domain annotation resource. Nucleic Acids Res., 2011, 40, D302-D305. venom metalloproteinases for multiple disorders. Structure- [19] Marchler-Bauer, A.; Lu, S.; Anderson, J.B.; Chitsaz, F.; Derby- function integrity with a deep understanding of molecular shire, M.K.; DeWeese-Scott, C.; Fong, J.H.; Geer, L.Y.; Geer, evolution has now a number of potential benefits for basic R.C.; Gonzales, N.R.; Gwadz, M.; Hurwitz, D.I.; Jackson, J.D.; Ke, research, clinical diagnosis and development of new research Z.; Lanczycki, C.J.; Lu, F.; Marchler, G.H.; Mullokandov, M.; Omelchenko, M.V.; Robertson, C.L.; Song, J.S.; Thanki, N.; Ya- tools, and drugs of potential clinical use. mashita, R.A.; Zhang, D.; Zhang, N.; Zheng, C.; Bryant, S.H. CDD: a Conserved Domain Database for the functional annotation CONFLICT OF INTEREST of proteins. Nucleic Acids Res., 2011, 39, D225-D229. [20] Zhu, X.; Teng, M.; Niu, L. Structure of acutolysin-C, a haemor- The author confirms that this article content has no rhagic toxin from the venom of Agkistrodon acutus, providing fur- conflict of interest. ther evidence for the mechanism of the pH-dependent proteolytic reaction of zinc metalloproteinases. Acta Crystallogr. D Biol. Crys- tallogr., 1999, 55, 1834-1841. ACKNOWLEDGEMENTS [21] Bailey, T.L.; Elkan, C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proceedings of the Life Science Research Board-Defense Research and Second International Conference on Intelligent Systems for Molecu- Development Organization (Sanction No. DLS/81/48222/ lar Biology, AAAI Press, Menlo Park, California; 1994, pp. 28-36. LSRB-249/BTB/2012), New Delhi, India, is duly acknow- [22] Overall, C.M. Molecular determinants of metalloproteinase sub- ledged for financial assistance. strate specificity. Matrix metalloproteinase substrate binding domains, modules and exosites. Mol. 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Received: March 01, 2013 Revised: April 24, 2013 Accepted: June 24, 2013