Diterpene Derivative) Ligands with Selected Venom Toxins: Evidences from Docking Studies Kadiyala Gopi, Mrunalini Sarma, Arnold Emerson and Muthuvelan
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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.