ABSTRACTObjective: The aim of this study was to study the interactions of Aloin and microdontin with antimalarial drug targets.Methods: The ADMET properties of Aloin and microdontin were analyzed using LigandScout and Osiris Molecular Property Predictor tools. Theprotein-ligand docking was performed in AutoDock software. AutoDock result was analyzed using PyMol and LigPlot+ software.Results: ADMET analysis suggests no major side effects for both Aloin and microdontin. Docking results show that Aloin had the highest significancewith Plasmodium falciparum calcium-dependent protein kinase (PfCDPK2) with a free binding energy of â€“8.01 Kcal/Mol, Ki value of 1.35 ÂµM and6 hydrogen bonds. Microdontin had the highest significance toward Glutaredoxin-1 with â€“8.04 Kcal/Mol, Ki value of 1.28 ÂµM and 3 hydrogen bonds.Conclusion: Based on the observed results for the studied drug targets, the proposed mechanism of action of Aloin is suggestively concluded asPfCDPK2 and for microdontin as glutaredoxin-1.Keywords: Anti-malaria, Aloin, Microdontin, Autodock, Ligandscout

Ragunathan, Adhithya

Ravi, Lokesh

English

Innovare Academic Sciences: E-Journals

Vol 9, Issue 2, 2016Online - 2455-3891 Print - 0974-2441POTENTIAL DRUG TARGETS FOR ALOIN AND MICRODONTIN: AN IN SILICO ANALYSISLOKESH RAVI*, ADHITHYA RAGUNATHANDivision of Biomolecules and Genetics, School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, India. Email: lokesh.ravi@vit.ac.inReceived: 17 December 2015, Revised and Accepted: 04 March 2016ABSTRACTObjective: The aim of this study was to study the interactions of Aloin and microdontin with antimalarial drug targets.Methods: The ADMET properties of Aloin and microdontin were analyzed using LigandScout and Osiris Molecular Property Predictor tools. The protein-ligand docking was performed in AutoDock software. AutoDock result was analyzed using PyMol and LigPlot+ software.Results: ADMET analysis suggests no major side effects for both Aloin and microdontin. Docking results show that Aloin had the highest significance with Plasmodium falciparum calcium-dependent protein kinase (PfCDPK2) with a free binding energy of –8.01 Kcal/Mol, Ki value of 1.35 μM and 6 hydrogen bonds. Microdontin had the highest significance toward Glutaredoxin-1 with –8.04 Kcal/Mol, Ki value of 1.28 μM and 3 hydrogen bonds.Conclusion: Based on the observed results for the studied drug targets, the proposed mechanism of action of Aloin is suggestively concluded as PfCDPK2 and for microdontin as glutaredoxin-1.Keywords: Anti-malaria, Aloin, Microdontin, Autodock, Ligandscout.INTRODUCTIONAloe vera predominantly distributed in the tropical and subtropical regions throughout the world is a succulent xerophyte belonging to the family Liliaceae. Though A. vera plants are indigenous to Africa now, their existence could be found all over the world [1]. This genus Aloe contains more than 600 species [2]. Their water storage tissue is large, and so they are capable of adapting to areas with low water availability [1]. A. vera consists of many biologically important compounds. Aloe percrassa is, traditionally, used in Ethiopia to treat Malaria; it is also used to treat wounds and gastrointestinal problems; it is locally known as Ere-Senay in Ethiopia. Recently, Gereziher et al. evaluated the antimalarial potency of anthrone derivatives and leaf latex extracted from Aloe percrassa. Chloroquine-resistant Plasmodium berghei was used to infect the test mice. It was reported that anti-malarial activity of Aloin and microdontin were lower than that of the leaf latex and concluded that the components of leaf latex had synergistic activity [2]. Several anti-malarial studies are being carried out all over the globe using Aloe and other medicinal, to compensate the current demand for the effective anti-malarial drug. The mortality rates of malaria have decreased by 47% in the global level and by 54% in the WHO African region; yet, 5,84,000 people died due to malaria in 2013 out of which 78% were children under the age of 5 [3].Here, in this study, Aloin and microdontin were docked with some of the known Plasmodium proteins to find the potential drug targets using AutoDock. The studied proteins were; P. falciparum lactate dehydrogenase (PfLDH) with PDB ID: 2 × 8L. It plays a key catalytic role in the reduction of pyruvate [4]. Plasmepsin-2 with PDB ID: 1LF4. It is an asparatic protease, with an important role in hemoglobin degradation pathway [5]. Thioredoxin-2 with PDB ID: 3UL3, it is a redox protein that guards the plasmodium cells against high fluxes of reactive oxygen species [6]. Glutaredoxin-1 with PDB ID: 4MZC, it is also a redox protein, working against reactive oxygen species [7]. P. falciparum protein kinase-5 with PDB ID: 1OB3. Is an important protein kinase in P. falciparum and is believed to be essential for regulation of nuclear division [8]. Falcipain-2 with PDB ID: 2GHU. It is a cysteine protease, with key function in degradation of hemoglobin [9]. P. falciparum phosphoethanolanime methyltransferase with PDB ID: 3UJ9. It is a methyltransferase enzyme, with a key function in membrane biogenesis in Plasmodium. This protein is unique for Plasmodium and hence is an ideal drug target [10]. P. falciparum ADP-Ribosylation factor-1 with PDB ID: 3LRP. It is an important vesicular trafficking protein [11]. P. falciparum gamete antigen-27 with PDB ID: 1N81. It is a crucial protein involved in the development of the gamete in P. falciparum [12].METHODSAll protein molecules were downloaded from RCSB Protein Data Bank website. Structure with the following PDB ID were used in this study; 3UL3, 1LF4, 3UJ9, 1OB3, 2X8L, 1N81, 4MVF, 3LRP, 4MZC, 2GHU, 3K7Y. The structure of the Aloin was downloaded from PubChem website, Aloin 313325. The structure of microdontin was sketched using ChemSketch. Properties of the ligand molecules were analyzed using LigandScout.Protein-ligand docking was performed using AutoDock. 4.2.1 [13-15], using Generic Algorithm parameters. Binding sites of each protein molecule was predicted using MetaPocket online tool [16,17]. The docking results from AutoDock were saved in pdb file format and were analyzed using PyMol [18,19] and LigPlus software [20,21] to view the docking positions and bonding.Osiris property explorer an online molecular properties prediction tool was used to analyze the biological properties of Aloin and micrdontin. LigandScout [22,23] was used to analyze the chemical nature and structural arrangement of the ligand molecules.RESULTSLigandScout analysis of properties of Aloin and microdontin are listed in Table 1. The pictorial representation of H-bond acceptors and H-bond donors are given in Fig. 1. Osiris toxicity analysis suggested no major side effects, signifying their drug ability. A drug score of with yellow color code is considerable as an acceptable drug molecule as given in Table 2.Auto dock results of Ligand-Protein docking for Aloin and 10 chosen drug target proteins are listed in Table 3, in the order of their binding Research ArticleAsian J Pharm Clin Res, Vol 9, Issue 2, 2016, 194-196 Ravi and Ragunathan 195energy requirement. Similarly, Auto dock results of Ligand-Protein docking for Aloin and 10 chosen drug target proteins are listed in Table 4, in the order of their binding energy requirement.Aloin showed the highest affinity toward P. falciparum calcium-dependent protein kinase (PfCDPK-2) with binding energy of –8.01 Kcal/Mol and with IC50 value of 1.35 uM. The PyMol analysis of Aloin and PfCDPK-2 complex is given in Fig. 2. Aloin formed 6 hydrogen bonds with 4 amino acid residues, Ile-212 (2.0Å), Cys-149 (1.9Å, 2.5Å and 2.1Å), Gly-151 (2.2Å) and Glu-153 (2.1Å).Microdontin showed the highest affinity toward Glutaredoxin-1 with a binding energy of –8.04 Kcal/Mol and IC50 value of 1.28 uM. LigPlot + analysis of microdontin and glutaredoxin-1 is given in Fig. 3. microdontin formed 3 hydrogen with two amino acid residues, Lys-82 (2.96Å) and Leu-69 (2.90Å and 2.76Å).DISCUSSIONLigand scout and osiris toxicity analysis revealed the physiochemical properties of the ligand molecules. Both Aloin and microdontin Fig. 1: H-bond acceptor and donor sites in Aloin and microdontin, (HBA: Hydrogen bond acceptor, HBD: Hydrogen bond donor, AR: Aromatic ring)Fig. 2: PyMol view of Aloin and Plasmodium falciparum calcium-dependent protein kinases-2 complexTable 3: AutoDock results of Aloin with chosen target proteinsTarget proteinBinding energyInhibition constantNumber of H-bondsPfCDPK2 –8.01 1.35μM 6Thioredoxin-2 –7.49 3.22 μM 7Glutaredoxin-1 –7.11 6.19 μM 6PfPMT –7.00 7.35 μM 6PfPK5 –6.76 11.09 μM 6Plasmepsin-2 –6.18 29.36 μM 8PfLDH –5.97 42.01 μM 4Falcipain-2 –5.91 46.50 μM 6PfARF1 –5.58 81.87 μM 6Pfg27 –5.23 146.48 μM 5PfCDPK-2: Plasmodium falciparum calcium-dependent protein kinases, PfPMT: Plasmodium falciparum phosphoethanolamine, PfLDH: Plasmodium falciparum lactate dehydrogenase, PfPK-5: Plasmodium falciparum protein kinase 5Table 4: AutoDock results of microdontin with chosen target proteinsTarget proteinBinding energyInhibition constantNumber of H-bondsGlutaredoxin-1 –8.04 1.28 μM 3Plasmepsin-2 –7.92 1.57 μM 4Thioredoxin-2 –7.70 2.25 μM 5PfPK5 –7.50 3.17 μM 5PfLDH –7.27 4.69 μM 7PfPMT –6.47 18.13 μM 1PfCDPK2 –6.42 19.61 μM 4Falcipain-2 –6.00 39.92 μM 5PfARF1 –4.78 312.08 μM 2Pfg27 –3.32 3.7 mM 3PfCDPK-2: Plasmodium falciparum calcium-dependent protein kinases, PfPMT: Plasmodium falciparum phosphoethanolamine, PfLDH: Plasmodium falciparum lactate dehydrogenase, PfPK-5: Plasmodium falciparum protein kinase 5Table 2: Osiris molecular property analyzer drug score for Aloin and microdontinParameters Aloin MicrodontinScore for cLogP 0.997 0.983Score for LogS 0.89 0.608Score for Mol.Wt 0.727 0.316Score for drug likeness 0.089 0.338No risk for mutagenicity 1 1No risk for tumorigenicity 1 1No risk of irritating effects 1 1No risk reproductive effects 1 1Total drug score 0.444 (yellow) 0.351 (yellow)Table 1: Ligand scout analysis of Aloin and microdontinParameters Aloin MicrodontinFormula C21H24O9 C30H32O11Mol. weight 420.41 Da 568.57 DacLogP –1.536 0.354TPSA 171.07 197.37HBA 9 10HBD 8 8HBA: Hydrogen bond acceptor, HBD: Hydrogen bond donor, AR: Aromatic ring, TPSA: Topological polar surface areaAsian J Pharm Clin Res, Vol 9, Issue 2, 2016, 194-196 Ravi and Ragunathan 196contains a high number of H-Bond Acceptors and H-Bond Donors. Osiris toxicity analysis displayed that, both Aloin and microdontin possess no major side effects and has significant properties for being used as drug. The report by Gerziher et al., 2014, demonstrated that Aloin and microdontin did not display any adverse side effects and toxicity, with a maximum of 2 g/kg body weight of pure compound for up to 14 days in mice models. Though the anti-malarial activity of Aloin and microdontin have been proven in-vitro and in-vivo (Gerziher et al., 2014), the mechanism by which these compounds exerts its action is still unclear. It was also reported by Gerziher et al., 2014, that Aloin and microdontin displayed reduced activity individually when compared to crude latex.Auto dock results along with visual confirmation have led to the suggestion of possible target proteins of the test molecules. Aloin showed the highest significance of inhibition toward PfCDPK-2 (RCSB-PDB ID: 4MVF). Aloin demonstrated 1.35 μM inhibition constant with -8.01 Kcal/Mol binding energy. Since Aloin is rich in hydroxyl group, it has formed 6 hydrogen bonds with PfCDPK-2. This suggests PfCDPK-2 could be the potential target protein for Aloin to exert its antimalarial activity. CDPKs are one of the most common drug targets used for malaria an d other protozoan diseases since CDPKs does not exist in mammals. Microdontin on the other hand demonstrated highest significant inhibition toward glutaredoxin-1 (RCSB-PDB ID: 4MZC). It is a well-known drug target andGrx-1 is a key redox protein present in Plasmodium. Microdontin showed 1.28 μM inhibition constant with –8.04 Kcal/Mol free binding energy and formed 3 hydrogen bonds.CONCLUSIONThe objective of this study was achieved by suggesting the potential target protein for Aloin and microdontin for its anti-malarial activity. PfCDPK-2 has been suggestively concluded as the possible drug target for Aloin. CDPK family proteins are a novel drug targets for plasmodium diseases since CDPKs does not exist in mammals.Glutaredoxin-1 has been suggestively concluded as the potential drug target for microdontin. Since Grx-1 plays key role in the protection of plasmodium cells against Reactive-Oxygen-Species, inhibiting these proteins would lead to the destruction of plasmodium cells by reactive oxygen species. Since Gerziher et al., 2014 has reported that Aloin and microdontin showed reduced anti-malarial activity when administered individually, it could be suggested that, these two molecules work synergistically together by inhibiting PfCDPK-2 and Grx-1, respectively.REFERENCES1. Kani ka P, Dinesh KP. Medicinal importance, pharmacological activities, and analytical aspects of Aloin: A concise report. J Acute Dis 2013;2(4):262-3.2. Gerziher G, Daniel B, Kaleab A. Isolation, characterization and in vivo antimalarial evaluation of anthrones from the leaf latex of Aloe percrassa Todaro. J Nat Rem 2014;14(2):119-26.3. World Health Organization. World Maralaria Report 2014. United Kingdom Houses of Parliament on 9 December 2014.4. Maulana T, Hari P. Tea leaves extracted as anti-malaria based on molecular docking plants. Proc Environ Sci 2013;17:188-94.5. Silva AM, Lee AY, Gulnik SV, Maier P, Collins J, Bhat TN, et al. Structure and inhibition of plasmepsin II, a hemoglobin-degrading enzyme from Plasmodium falciparum. Proc Natl Acad Sci U S A 1996;93(19):10034-9.6. Sharma A, Sharma A, Dixit S, Sharma A. Structural insights into thioredoxin-2: A component of malaria parasite protein secretion machinery. Sci Rep 2011;1:179.7. Yogavel M, Tripathi T, Gupta A, Banday MM, Rahlfs S, Becker K, et al. Atomic resolution crystal structure of glutaredoxin 1 from Plasmodium falciparum and comparison with other glutaredoxins. Acta Crystallogr D Biol Crystallogr 2014;70:91-100.8. Holton S, Merckx A, Burgess D, Doerig C, Noble M, Endicott J. 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Pharmacophore-based discovery of FXR-agonists. Part II: Identification of bioactive triterpenes from Ganoderma lucidum. Bioorg Med Chem 2011;19:6779-91. Fig. 3: LigPlot + analysis of microdontin and glutaredoxin-1 complex

POTENTIAL DRUG TARGETS FOR ALOIN AND MICRODONTIN: AN IN-SILICO ANALYSIS

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POTENTIAL DRUG TARGETS FOR ALOIN AND MICRODONTIN: AN IN-SILICO ANALYSIS

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