Research Article Kadiyala Gopi et al. / Journal of Pharmacy Research 2011,4(4),1069-1072 ISSN: 0974-6943 Available online through http://jprsolutions.info Interaction of Andrographolide (diterpene derivative) ligands with selected venom toxins: Evidences from docking studies Kadiyala Gopi, Mrunalini Sarma, Arnold Emerson and Muthuvelan. B* School of Bio Sciences and Technology,VIT University, Vellore – 632014,Tamil Nadu, India Received on: 04-01-2011; Revised on: 17-02-2011; Accepted on:16-03-2011 ABSTRACT Snake envenomation still continues to be a major health problem, for which treatments are still in progress. The latest treatments include search of bio-active compounds as inhibitors for the venom. Preferably isolation of bio-active compounds (ligands) from herbal sources is the most preferred treatment, since they are likely not to cause any side effects, like the current anti-venom antibodies. In this study Andrographolide from Andrographis paniculata (Ligand 1) and its derivative isolated from Andrographis lineata (Ligand 2) have been studied as ligands of snake venom toxins (these two plant extracts are used to snake bites in rural India).Six varied toxins (Disintegrin, Aggretin, Echicetin, Acutolysin, Irditoxin & Haditoxin) from different snake venoms have been chosen to check the spectrum of these bio-active compounds. The docking study was performed by AutoDock 4.2 program. These simulation studies provided a preliminary view of whether the compounds (ligands) have activity against the toxins. In the results, binding energy of -11.92 Kcal/mol to -4.74 Kcal/mol was obtained, with the RMSD tolerance of 0.5 Å to 1.0 Å. Moreover, results indicate that overall both the bio-active compounds (ligands) have shown significant binding with five toxins except Acutolysin. Also amongst both ligands, Ligand 1 has shown more affinity towards the toxins. This may be due to the structural differences in both the ligands. However, once the reasons are confirmed, these findings can be used to modify the bio-active compounds and use them as drug candidates for snake venom neutralization.

Key words: Snake venom toxin; Andrographolide; docking; ligands; Andrographis lineata INTRODUCTION Snake bite throughout the world poses a serious health concern and is a very common cause Around five hundred grams of shade dried Andrographis paniculata was extracted with of morbidity and mortality [1- 3]. Estimates have been made that the snake envenomation cases could exceed more than 5 million per annum [4]. Even with such serious health risks methanol (1:2 ratios) and the solvent was com- O completely reliable treatments are not fully developed and absolutely effective treatments are pletely removed by rotary vacuum evaporator. The O OH still being explored. The most common treatments include anti-venoms, polyvalent and crude extract obtained (24.5 g) was stored in air monovalent, which is purified serum of horses immunized with venoms of poisonous snakes tight vial for a long time (around 25 days). After [5]. These anti-venoms are not completely effective given to reasons like cost and non- that 10g of the extract was dissolved in mixture of CH3 availability [6] and are also known to produce allergic reactions [7]. Most of the anti-venoms ethanol and tetrahydrofolate (1:1), and heated on H3C do help in restoring the non coaguable blood but they are ineffective against the local effect of water bath for five minutes. Then the extract was filtered in muslin cloth and allowed to form crys- HO snake bites and produce serious side effects [8]. So alternative treatments especially herbal CH3 tals until complete evaporation of solvents. Crys- medicines are being developed [9, 10]. Traditional herbal medicines for snake bites have been OH widely used by tribal and rural people for centuries. This is done by application of sap, leaves tals formed were separated from extract and washed Fig. 1 Structure of the two ligands: (a) or concoctions of these plants to the bite area, to mitigate the effect of the snake venom. These well with methanol to get pure form and subjected Bio- active compound isolated from herbal plants have been used to find an alternative treatment, which includes characterizing and for structural analysis using single crystal X-RD Andrographis paniculata. isolating different compounds from these plants that are known to neutralize or delay the action and the structure has been used for our study as O ligand 1 (Fig 1a). of snake venom. Among these, Andrographis lineata and Andrographis paniculata are HO O commonly used plants to treat snake bites in rural India. Furthermore Andrographis is also an important “cold property” herb used to rid the body of heat, as in fevers, and to dispel toxins Ligand 2 H O from the body etc. Another active compound Andrographolide form Andrographis paniculata which has already been Since the development of herbal medicines is being implemented and new drugs are being isolated and documented [11, 12] for an array of HO H explored, and the effectiveness of Andrographis paniculata is already proven, we have selected functions, and has been used as ligand 2 (Fig 1b). HO two compounds; a derivative of Andrographolide from Andrographis lineate which has an Fig. 1.(b) Bio- active compound isolated ability to neutralize the venom (data unpublished) and Andrographolide from Andrographis Toxins from Andrographis lineata. paniculata for our molecular activity studies with selected venomous toxins like Disintegrin, Six toxins selected were; Disintegrins, Aggretin, Echicetin, Acutolysin C, Denmotoxin and Aggretin, Echicetin, Acutolysin, Irditoxin & Haditoxin. These toxins; were selected based on Haditoxin. All the crystal structure of the toxins was downloaded from the Protein Data Bank their importance, activity & function and more importantly on the availability of their 3D (PDB) http://www.rcsb.org/pdb/home/home.do. structure in the PDB. Currently, the computer aided molecular docking studies present a very advantageous and reasonable way of finding the efficacy of these compounds. Further, this kind Disintegrins (1J2L) are a family of proteins that act as fibrinogen receptor antagonists [13]; they of docking will provide with an overview on how the compounds react and bind with the are hemorraghic toxins commonly found in Coratlid and Viperid venoms [14]. Disintegrins components of the snake venom, before testing them in lab. So, in this present study we have basically block integrin binding with its ligands [15], and cell matrix inhibitions [16]. performed docking using AutoDock 4.2 to find out the binding efficiency of our two com- Aggretin (3BX4) is a novel hemotoxin isolated from Calloselasma rhodostoma [17], it pounds (ligands) against the above addressed six toxins, and the results are reported and functions as a platelet activating protein [18], which can be blocked by monoclonal antibodies. discussed. Once computationally proven it will be an easier job to develop it as a drug and also Structurally Aggretin is a heterodimeric protein of 29 KDa in weight [19] and belong to the to synthesize a much better ligand. family of C. Type Lectins [20].

MATERIALS AND METHODS Echicetin (1OZ7) is a toxin isolated from Indian saw-scaled viper [21] and is commonly found Bioactive Compound in various snake venoms. It is a heterodimeric C-type lectin which is known to inhibit platelet Ligand 1 aggregation [22]. Acutolysin C is a hemorrhagic toxin, a zinc finger metalloproteinase [23]. It Andrographis paniculata was collected from area around Vellore district, Tamil Nadu, India. is isolated from the venom of Agkistrodon acutus and has a mass of 22KDa [24]. Denmotoxin is a three fingered neurotoxin found in Mangrove catsnake [25], which exhibits a bird specific *Corresponding author. neurotoxicity [26]. Haditoxin is a neurotoxin from venom of Ophiophagus Hannah, with Muthuvelan. B, antagonist effect for muscle and neuronal acetylcholine receptors [27]. Professor Docking School of Bio Sciences and Technology, Preparation of protein files and grid box VIT University, Vellore – 632014 Protein coordinate files were prepared, as the first step in docking; the input format for protein Tamil Nadu, India files in AutoDock was pdb. The file initially loaded was not a charged file and the Kollman charges were added to it, along with polar hydrogens to form hydrogen bonds with the ligand.

Journal of Pharmacy Research Vol.4.Issue 4. April 2011 1069-1072 Kadiyala Gopi et al. / Journal of Pharmacy Research 2011,4(4),1069-1072 Once the protein files were ready, a grid box is set to cover the protein, in which the number if points in X, Y, Z are to be adjusted along with the spacing between the grid points. The default was 0.375 Å, which is a quarter of the length of carbon – carbon single bond. After all the coordinates were set the file was saved as a grid parameter file (gpf), which is run in AutoGrid.

Preparation of ligand Similar to protein file, the ligand file is also in the pdb format and is saved as a charged file. The number of torsions for the ligands are fixed, i.e the bonds that can be rotated (single bonds) [28]. Running AutoGrid and AutoDock Once the protein and ligand files are prepared the grid parameter file is run in AutoDock. AutoDock calculates the interaction energy of each point in the three dimensional. Separate energies are calculated for each atom of the ligand including the hydrogen bond energies [29]. After running AutoGrid, a docking parameter file is saved, the actual docking simulation is done by AutoDock, in which interaction energy is calculated for each orientation and the ligand then ultimately finds the most favorable conformation with the best binding energy. The number of runs for each docking experiment was set to 100, to find the most suitable binding energy, and the number of evals was kept to 25,000. The RMSD tolerances were set between 0.5 Å to 1.0 Å. After the AutoDock is run, to display the results a dlg file is opened and all the different 100 run results are analyzed.

RESULTS Fig. 3 Comparison of interaction of Aggretin with both ligands (A) Docked conformation of In order to study and compare the efficiency of the two ligands in neutralizing the toxins of Aggretin with Ligand 1. (B) Docked conformation of Aggretin with Ligand 2. (C) Interacting snake venom, we docked the toxins and ligands using the AutoDock 4.2 program. AutoDock residues of Aggretin, with Ligand 1. (D) Interacting residues of Aggretin with Ligand 2. (E) 3 computed the binding energy and inhibition constant for these dockings to interpret the results D structure of fitting of Ligand 1 with Aggretin. (F) 3 D structure of fitting of Ligand 2 with of the bindings. For each dock 100 docking runs were initiated with 25,000 evlas and 150 Aggretin. population size and RMSD tolerances was set within the limit of 0.5 Å to 1.0 Å. The best binding energy was selected from amongst the 100 runs and reported along with its respective value of inhibition constant in table 1. The docked energy of binding for these ligands was in the range of -11.92 to -4.75 kcal/mol. All ligands bound to the binding site of the toxins, except for Acutolysin. The interacting residues of the toxin and ligands are shown (Fig 2a-d to Fig 7a-d), blue color molecule representing the ligand and the green color representing the toxin. The three dimensional conformation of the interaction of ligands and toxin is also presented (Fig 2e-f to Fig 7e-f). Table 1. Binding energy, number of clusters and Inhibition constant of dockings of the six toxins with both the Ligand 1, 2 Ligand 1 Ligand 2 Toxins PDB No. of Binding Inhibition No. of Binding Inhibition Code clusters Energy Constant clusters Energy Constant Kcal/mol µM Kcal/mol µM Disintegrin 1J2L 2 -5.34 122.48 3 -5 215.11 Aggretin 3BX4 2 -5.78 58.41 2 -4.88 266.27 Echicetin 1OZ7 2 -9.32 148.22 2 -6.29 24.54 Acutolysin 1QUA 8 -4.9 256.07 5 30.24 0 Irditoxin 2H7Z 2 -11.92 1.82 3 -4.75 332.04 Haditoxin 3HH7 5 -6.44 19.07 5 -4.98 224.54

Fig. 4 Comparison of interaction of Echicetin with both ligands (A) Docked conformation of Echicetin with Ligand 1. (B) Docked conformation of Echicetin with Ligand 2. (C) Interacting residues of Echicetin with Ligand 1. (D) Interacting residues of Echicetin with Ligand 2. (E) 3 D structure of fitting of Ligand 1 with Echicetin. (F) 3 D structure of fitting of Ligand 2 with Echicetin.

Fig. 5 Comparison of interaction of Acutolysin with both ligands (A) Docked conformation of Fig. 2 Comparison of interaction of Disintegrin with both ligands (A) Docked conformation Acutolysin with Ligand 1. (B) Docked conformation of Acutolysin with Ligand 2. (C) Interact- of Disintegrin with Ligand 1 (B) Docked conformation of Disintegrin with Ligand 2. (C) ing residues of Acutolysin with Ligand 1. (D) Interacting residues of Acutolysin with Ligand 2. Interacting residues of Disintegrin, with Ligand 1. (D) Interacting residues of Disintegrin (E) 3 D structure of fitting of Ligand 1 with Acutolysin. (F) 3 D structure of fitting of Ligand 2 with Ligand 2. (E) 3 D structure of fitting of Ligand 1 with Disintegrin. (F) 3 D structure of with Acutolysin. fitting of Ligand 2 with Disintegrin. Journal of Pharmacy Research Vol.4.Issue 4. April 2011 1069-1072 Kadiyala Gopi et al. / Journal of Pharmacy Research 2011,4(4),1069-1072

Fig. 6 Comparison of interaction of Irditoxin with both ligands (A) Docked conformation of Fig. 7 Comparison of interaction of Haditoxin with both ligands (A) Docked conformation Irditoxin with Ligand 1. (B) Docked conformation of Irditoxin with Ligand 2. (C) Interacting of Haditoxin with Ligand 1. (B) Docked conformation of Haditoxin with Ligand 2. (C) Inter- residues of Irditoxin with Ligand 1. (D) Interacting residues of Irditoxin with Ligand 2. (E) 3 acting residues of Haditoxin with Ligand 1. (D) Interacting residues of Haditoxin with Ligand D structure of fitting of Ligand 1 with Irditoxin. (F) 3 D structure of fitting of Ligand 2 with 2. (E) 3 D structure of fitting of Ligand 1 with Haditoxin. (F) 3 D structure of fitting of Ligand Irditoxin. 2 with Haditoxin. From table 1 it is seen that docking of Irditoxin with ligand 1 has the minimum binding energy of -11.92 Kcal/mol amongst all the twelve dockings. However, the same toxin has Andrographolide is a diterpene which has been studied in detail for its various properties maximum binding energy with the ligand 2; -4.75 Kcal/mol. Also, the runs for docking with [31-33]. Various docking studies on Andrographolide also have been performed, specifically ligand 1 fall into two clusters, compared to ligand 2 which has three energy clusters. From Fig for HIV [34, 35]. For the first time we are reporting the use of Andrographolide (ligand 1) and 6c & Fig 6d, it is seen that while Ligand 1 has interactions with two amino acid residues of its derivative (ligand 2) for acting against snake venom, because the source plant extracts were the toxin, ALA & GLY, ligand 2 is interacting majorly with water molecules and one amino used to treat snake bites in rural India and other part of the globe from the time immemorial. acid residue ASN. The difference in the interacting residues may be attributed to this vast Overall our study reveals that both the bio active compounds are effective against toxins from difference in the binding energy; however, this can be confirmed only with further studies. different snake venom, thus making it a more versatile treatment for snake bites as opposed to Next, Echicetin is the toxin having second minimum binding energy of -9.32 Kcal/mol with venom specific antibodies. ligand 1 whereas it has a moderately higher binding energy in case of docking with ligand 2. But converse to docking of Irditoxin, here, Ligand 1 is interacting only with a single water In principle the main aim of the study was to check the effectiveness of the ligands in molecule of Echictein (Fig 4c) while Ligand 2 is interacting with ARG residue of the toxin neutralizing the effect of these snake venom toxins. The docking method provided a compara- along with the same water molecule (Fig 4d). Again, the reason for such difference is yet to be tive view of the action of both the bio active compounds. The data provided by AutoDock studied and understood. Meanwhile, both the dockings have same number of energy clusters. provides a simple way of quantifying the results. It was observed that both the ligands show It is seen from the table that like both the above toxins; Irditoxin & Echicetin; Haditoxin again activity against these toxins. has minimum binding energy of -6.44 Kcal/mol with ligand 1 compared to -4.98 Kcal/mol of ligand 2. Here in both the dockings the interacting residue is TYR and even the water The isolation of all the six toxins has been reported earlier in the literature [13, 18, 21, 23, 25, molecules almost same (Fig 7c, 7d), along with identical energy clusters. Disintegrin and 27]. As mentioned in material and methods, toxins Disintegrin, Aggretin, Echicetin and Aggretin are seen to have almost similar energies on docking with both the toxins. However, Acutolysin are hemotoxins. Disintegrins inhibit integrin functions thus blocking the cell Disintegrin has a slightly less binding energy with ligand 1; -5.78 Kcal/mol than binding signaling pathways. Studies have been conducted to find monoclonal antibodies to block the energy of Aggretin with ligand 1; -5.34 Kcal/mol. Disintegrin docking with ligand 2 has -5 action of disintegrins [36, 37], similarly antibodies to Aggretin have also been produced [17, Kcal/mol of binding energy and Aggretin has -4.88 Kcal.mol. Further from Fig 2c & 2d it is 20]. Echicetin in cross linking with GPIb acts as a platelet activator [38] but the antibodies seen that interacting residues of i.e. ASP, GLY are same in docking with both the ligands. against these do not show any effect [39]. So antibodies that have been produced are basically Along with Disintegrin, interacting residues of Aggretin are also almost same in both the to stop the specific function but none of these are reported to be used as treatment for snake cases; SER, LEU, PHE & LYS. However, in docking with ligand 1 PRO residue is involved, bites. Docking of these metalloproteniases with ligands have been studied [40], but dockings which is not seen in docking with ligand 2. The energy clusters of 2, for Aggretin dockings are for snake venom have not been performed yet with these toxins. same with both ligands. While in case of Disintegrin docking with ligand 1 has 2 energy clusters while with ligand 2 are 3. Acutolysin is the only toxin amongst all that does not show Irditoxin and Haditoxin belong to the class of three finger neurtotoxins which are nicotinic binding with ligand 1. It displays a very high positive binding energy of 30.24 Kcal/mol, acetylcholine receptors [41]. Homology modeling and docking studies have been performed to showing that ligand 2 is unable to inhibit Acutolysin. But on the other hand, it is seen that understand their mechanism of action [42]. All the toxins have been studied in detail, but there it binds with ligand 2, although with binding energy which is the highest amongst all the is no report for studying them as components of snake venom and finding treatment for the cases, -4.9 Kcal/mol. Along with these results, it is observed in table 1 that, the inhibition same. In our study we have used them as components of snake venom which have to be constant is directly proportional to the binding energy in docking with both the ligands. In neutralized. docking with ligand 1 Irditioxin which has the least binding energy has the least inhibition constant of 1.82 uM, and Acutolysin which has the highest binding energy has the highest Overall from the result it is understood that both the bio active compounds (ligand 1, 2) have inhibition constant of 256.07 uM. Similarly in docking with ligand 2, Echicetin has the significant binding activity against the proposed toxins. However, ligand 1 has better binding minimum binding energy and the minimum inhibition constant of 24.54 uM (except Acutolysin in all the cases than ligand 2. In terms of binding energy, it was seen that ligand 1 has a lesser which does not show binding with ligand 2), and Irditoxin which has the highest binding binding energy in all the cases. With the toxin Acutolysin it was seen that in the 100 runs there energy has the highest inhibition constant 332.04 uM. The reasons behind this are still to be was no structural conformation that gave a negative binding energy with ligand 2, but with understood. ligand 1 it was observed that the binding was appropriate and gave a negative binding energy. Comparing the other toxins it was seen that the binding energy of toxins with ligand 1 was DISCUSSION & CONCLUSION always less than that with ligand 1. Although in some cases it was just a small difference, like As mentioned in the introduction, the treatment for snake bites is still in progress [16, 30]. Disintegrin, Aggretin and Haditoxin, the interacting amino acids were almost same in all the Because, the use of antibodies is widely done but it is not a completely effective treatment, three cases. For the other three toxins Echictein, Acutolysin and Irditoxin there was a since these antibodies are mostly monoclonal and effective only against specific snake venoms reasonable difference in the binding energies with both the ligands, and there were completely [5] and also it causes significant side effects in most of the cases. So, more flexible and widely different interacting amino acid residues. So it can be assumed that the reason for difference in applicable treatments need to be searched preferably from the plant sources as pointed out the dockings with both the ligands may be due to the structural differences and their interaction earlier. In this context, in our study we have attempted to find bio active compounds which with the different residues. From Fig 1-6e, 1-6f it is evident that there is a difference in the have broad spectrum activity, i.e activity against components of several snake venoms. binding cavity of the ligands for Echictein, Acutolysin and Irditoxin.

Journal of Pharmacy Research Vol.4.Issue 4. April 2011 1069-1072 Kadiyala Gopi et al. / Journal of Pharmacy Research 2011,4(4),1069-1072 From the result it is evident that even a small structural change is enhancing the binding 22. Navdaev A, Clemetson JM, Polgár J, Kehrel BE, Glauner M, Magnenat E, Wells TNC, Clemetson efficiency. So there is a possibility of using such bioactive compounds as lead molecule to KJ (2001) Aggretin, a Heterodimeric C-type Lectin from Calloselasma rhodostoma (Malayan Pit Viper), Stimulates Platelets by Binding to a2ß1 Integrin and Glycoprotein Ib, Activating Syk and develop target based derivatives after performing the modeling and simulation studies in Phospholipase C?2, but Does Not Involve the Glycoprotein VI/Fc Receptor ? Chain Collagen respect to drug development. The binding energy can be further reduced to upto -70 Kcal/mol Receptor. J. Biological Chemistry 76: 20882-20889. to give a better and stronger binding. Over the years the ligand- protein docking studies have 23. Jasti J, Paramasivam M, Srinivasan A, Singh TP (2004) Crystal structure of echicetin from Echis carinatus (Indian saw-scaled viper) at 2.4 resolution. J. Mol. Bio 335: 167-176. presented a successful use for developing new compounds [41] for pharmaceutical industries. 24. Navdaev A, Dörmann D, Clemetson JM, Clemetson KJ (2001) Echicetin, a GPIb-binding snake If methods such as computer aided drug designing, virtual screening [42] are followed; C-type lectin from Echis carinatus, also contains a binding site for IgMKresponsible for platelet pharmaceutical companies will be leading in finding safe cure not only for snake envenomation agglutination in plasma and inducing signal transduction. Blood 97: 2333–2341. but for any other diseases. 25. 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Journal of Pharmacy Research Vol.4.Issue 4. April 2011 1069-1072