© 2015 Indian Journal of Pharmaceutical Sciences | Published by Wolters Kluwer - Medknow 439

*Address for correspondence E-mail: sirsendu@nitrkl.ac.in

Molecular Docking and Interactions of Pueraria Tuberosa with Vascular Endothelial Growth Factor ReceptorsS. ASTHANA, T. AGARWAL, S. SINGOTHU, A. SAMAL1, I. BANERJEE, K. PAL, K. PRAMANIK AND S. S. RAY*Department of Biotechnology and Medical Engineering, National Institute of Technology, 1Super Speciality Hospital, Rourkela-769 008, India

Asthana, et al.: Molecular Interactions of Pueraria Tuberosa with VEGFR’s

Pueraria tuberosa is known for its therapeutic potentials in cardiovascular disorders, but its effect in angiogenesis has not been studied so far. In this study, a computational approach has been applied to elucidate the role of the phytochemicals in inhibition of angiogenesis through modulation of vascular endothelial growth factor receptors: Vascular endothelial growth factor receptor‑1 and vascular endothelial growth factor receptor‑2, major factors responsible for angiogenesis. Metabolite structures retrieved from PubChem and KNApSAcK – 3D databases, were docked using AutoDock4.2 tool. Hydrogen bond and molecular docking, absorption, distribution, metabolism and excretion and toxicity predictions were carried out using UCSF Chimera, LigPlot+ and PreADMET server, respectively. From the docking analysis, it was observed that puerarone and tuberostan had significant binding affinity for the intracellular kinase domain of vascular endothelial growth factor receptors‑1 and vascular endothelial growth factor receptor‑2 respectively. It is important to mention that both the phytochemicals shared similar interaction profile as that of standard inhibitors of vascular endothelial growth factor receptors. Also, both puerarone and tuberostan interacted with Lys861/Lys868 (adenosine 5'‑triphosphate binding site of vascular endothelial growth factor receptors‑1/vascular endothelial growth factor receptors‑2), thus providing a clue that they may enforce their inhibitory effect by blocking the adenosine 5'‑triphosphate binding domain of vascular endothelial growth factor receptors. Moreover, these molecules exhibited good drug‑likeness, absorption, distribution, metabolism and excretion properties without any carcinogenic and toxic effects. The interaction pattern of the puerarone and tuberostan may provide a hint for a novel drug design for vascular endothelial growth factor tyrosine kinase receptors with better specificity to treat angiogenic disorders.

Key words: Angiogenesis, VEGFR1, VEGFR2, phytochemicals, molecular docking, ADME and Tox

Angiogenesis is a complex and intricate processwherein the preexisting blood vessels give rise tothe new ones[1,2]. These blood vessels distributethe nutrients; carry out gas exchange and transportwaste, thus maintaining a balanced and healthyenvironment throughout the tissue[3]. Optimallyregulated angiogenesis is essential for proper growthand development of tissue. On the other hand, aderegulated angiogenesis leads to various diseasessuch as cancer, obesity, diabetes, arthritis, uterinebleedings,Alzheimer’s, osteoporosis, stroke andatherosclerosis.Thereexistvariouschemicalmediatorslike vascular endothelial growth factors (VEGF),fibroblast growth factor (FGF), epidermal growthfactor (EGF), angiostatin and interferon, whichregulate the angiogenic process[4].Amongst all suchmediators, VEGF is the most prominent inducer

of angiogenesis. VEGF is essential in migrating,proliferating and forming of capillary like structuresby endothelial cells.VEGF executes its actions bybinding to its receptors (VEGFR1 andVEGFR2)on the endothelial cells, thus activating the cascadeofmany downstream signaling pathways essentialfor angiogenesis[5]. Chemicals that interrupt thissignaling cascade act as angiogenesis inhibitors.Angiogenic inhibitors have potential therapeuticutility in cancer, age relatedmacular degeneration

Research Paper

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Accepted 03 August 2015Revised 24 January 2015Received 16 March 2014

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and other diseases where vascular proliferationleads to pathogenesis. Recently, a number of plantderived natural compounds or phytochemicals havebeen reported possesing a potential antiangiogenicactivity[6,7]. Schindleret al. andBagliet al. reportedthat theflavonoids like genistein and luteolin have aprofound inhibitory effect onVEGF secretion in thecancercells, thus inhibiting the tumorgrowth[8,9].Also,curcumin has been reported for its antiangiogenicpotentials through down-regulation of VEGFexpression and inhibitionofVEGFRs[10].

Pueraria tuberosa or IndianKudzu iswidely knownin traditional medicines to have manymedicinalbenefits. It has exhibited cardiotonic, aphrodisiac,antihyperglycemic, antilipidemic and galactogogicactivities[11-14].TubersofPueraria tuberosa are rich indaidzin,puerarin,puerarone,genistein,puetuberosanol,tuberostan, tuberosin, andpuerarin4’,6’-diacetate[15-17].Though it hasmuch health beneficial effect but itseffect on angiogenesis not been studiedmuch.

In the current investigation,wehave tried to evaluatethe effect of various phytochemicals derived fromaforesaid plant source on theVascular EndothelialGrowth FactorReceptors (VEGFRs) using in silicoapproach.Thephytochemicalswere screened for theirtoxic andmutagenic effects. Further, the nontoxicand nonmutagenic phytochemicalswere dockedontokinasedomainofVEGFR1andVEGFR2.

MATERIALS AND METHODS

Protein structure retrieval and active site predictions:The three-dimensional structuralmodelsof intracellularkinase domain ofVEGFR1 andVEGFR2 proteinswerededucted fromRCSBProteinDataBank (http://www.rcsb.org).Cleaning and optimizing the proteinmodel geometrywas carried out by the removal ofmiscellaneous ligands and other hetero-atoms suchas water and ions using theArgus Lab Software.Further, the proteinmodelswere used for the activesite prediction usingCASTpCalculations (ComputedAtlasofSurfaceTopographyofproteins) (http://stsfw.bioengr.uic.edu/castp/calculation.php).Amongst allpredicted sites, the site having the catalytic aminoacidswas selected for docking.The clue about theamino acids involved in protein catalysiswas gainedfrom theUniProtServer (http://www.uniprot.org/).

Substrate selection:The three-dimensional PDB structures of thephytochemicals present in the aforesaid plants andthe standard FDA approved drugmolecules wereretrieved fromKNApSAcK-3D (http://knapsack3d.sakura.ne.jp/) andPubChem (http://pubchem.ncbi.nlm.nih.gov/) databases using PRODRGServer (http://davapc1.bioch.dundee.ac.uk/prodrg/). Further, energyminimizationand ligandoptimizationwerecarriedoutusingArgus lab software.

Molecular property predictions:The selected phytochemicals were examined fortheirmolecular properties, absorption, distribution,metabolism and excretion (ADME) and toxicityusing PreADMET (http://preadmet.bmdrc.org/).Thephytochemicals,which passed the selection criteriawereused for thedocking analysis.

Molecular docking:The selected phytochemicalswere docked into theactive sites of theproteinmodels usingAutoDock4.2software tool. In brief, polar hydrogen andKollmancharges were added to the protein models anddockingwas donebyLamarckianGeneticAlgorithmusing a standard protocol on the basis a populationsizeof 150 randomlyplaced individuals; amaximumnumber of 2.5×107 energy evaluations, amutationrate of 0.02 and a crossover rate of 0.8[18]. Twentyindependent docking runs were carried out andresultswere clustered according to the 1.0ǺRMSDcriteria. The grid maps representing the proteinswere calculated using auto grid and grid sizewas set to 60×60×60 points with grid spacing of0.375Ǻ.Thereafter, the interaction pattern in theprotein-ligand complexwas visualized usingUCSFchimera[19] andLigPlot+[20].

RESULTS AND DISCUSSIONS

VEGFR1 and VEGFR2 are the transmembranetyrosine kinase receptorswith all the three domains:Extracellular domain, a transmembrane region, andan intracellular domain.AlthoughVEGFR1 wasearlier consideredas anegative regulatorofVEGFR2,but recently it was also found to accumulate theangioblast cells in the blood vessels. On the otherhand,VEGFR2plays a primary role in angiogenesis,vascularpermeability anddifferentiationof the abovementioned angioblast cells[21].

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Usually, theFDAapproved antiangiogenic drugs aretargeted at the intracellular domain of theVEGFRs,including the adenosine5'-triphosphate (ATP)bindingdomain.ATPbindingdomaingains a special attentionwhile considering receptor tyrosine kinases asATPhydrolysis plays a crucial role in their activation.Thus, inhibitionof thisdomainmaypotentially inhibitthe signaling pathways. Keeping this perspectiveinmind,we evaluated the inhibitory potentials ofphytochemicalsbyblocking theATPbindingdomainofVEGFRs.Recently, interactionbetween juxtamembranedomain and catalytic domainofVEGFR2has gaineda significant attention. Juxtamembrane domainconnects the transmembranedomainwith thecatalyticintracellular domain in receptor tyrosine kinases[22].Solowiejet al. have reported that thepresenceof theJuxtamembranedomainofVEGFR2has a substantialeffect on inhibitor binding to the catalytic domainofVEGFR2. It was observed that the presence ofjuxtamembrane domain enhanced the affinity ofAxitinib for the catalytic domain ofVEGFR2[23].However, absenceof an intactX-Raycrystallographicstructure for the same, limits it use for the presentin silico investigation. Thus, three-dimensionalstructures of catalytic domain ofVEGFR1 (3HNG)andVEGFR2 (3VHE)were taken for the analysisfromRSCBPDB (Table 1). For in silico analysis ofthe ligand-protein interaction, theknowledgeof activesiteof theprotein is essential because ligandsusuallytarget the active sites.The active site of proteinswaspredictedusingCASTpcalculations.

The three-dimensional structure of phytochemicalswas isolated fromKNApSacK-3D and PubChemdatabase. Screening of the phytochemicals wascarried out using PreADMET server andwas basedon theirmolecular properties includingdrug likeness,carcinogenicity andmutagenicity.The ligandswhichpassed the selection criteriawere further subjectedto docking analysis. 18 out of 140moleculeswerefound tobenontoxicandwereused fordocking studyusingAutoDock4.2. Nontoxic 18molecules weredockedwith the intracellular domain ofVEGFRs,within which puerarone and tuberostan gave bestbindingwithVEGFR1 andVEGFR2. Further studywas continuedwith these twomolecules.The originand chemical structures of these twomolecules ismentioned inTable 2 andfig. 1.

Puerarone and tuberostan, bothwere found to followLipinski’s rule of fivewhich permit to assess the

pharmacokinetics of the drug, including absorption,distribution, metabolism and excretion (ADME).Furthermore, they were recognized as nontoxic,nonmutagenicandnoncarcinogenic innature (Table3).

Puerarone (KNApSAcK_3D ID C00009871)displayed best affinity towards the intracellulardomain ofVEGFR1with a binding energy of -9.91kcal/mol (Ki: 0.182 µM). On the other hand,tuberostan (KNApSAcK_3D IDC00010049) showedhighest affinity towards the intracellular domain ofVEGFR2withabindingenergyof -9.32kcal/Mol (Ki:0.148 µM). When compared with all the FDAapproved drugs asmentioned in theTable 4, itwasfound that puerarone showed better binding affinitywithVEGFR1 intracellular domain except pazopaniband vatalanibwhere it showed comparable results.Similarly, tuberostan showedbetter results thanall thedrugs except pazopanib and axitinibwhere it showedalmost equal results. InTable 4, values in bold fontrepresent best affinity of the testmoleculeswith theintracellulardomainofVEGFR1andVEGFR2.

For an effective binding of a ligand to a proteinreceptor, the affinityof the ligand towards theproteinaswell as the stabilityof theprotein ligand complex,are some of the important benchmarks.The above

TABLE 1: TARGETS TO CHECK THE EFFECT ON ANGIOGENESISProteins Notations PDB ID Organism ReferencesVascular endothelial growth factor receptor 1

VEGFR1 3HNG Homo Sapiens

[24]

Vascular endothelial growth factor receptor 2

VEGFR2 3VHE Homo Sapiens

[25]

TABLE 2: NAME AND THE ORIGIN OF THE SELECTED PHYTOCHEMICALSKNApSAcK ID Name of the

PhytochemicalOrigin Ref

C00009871 Puerarone Pueraria tuberosa (vidarikanda) [26]C00010049 Tuberostan Pueraria tuberosa (vidarikanda) [27]

Fig 1: Chemical structures of potential anti-VEGFR phytochemicals from Pueraria tuberosa: (a) puerarone; (b) tuberostan.

ba

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mentionedbindingenergies alongwith the interactionprofile (hydrogenbondsandhydrophobic interactions)provide an important indication for the affinity andstability of ligandwith the protein. The hydrogenbonds and hydrophobic interactions formed betweenproteins andphytochemicalswere illustrated throughLigPlot+ (Table 5). Interaction profileswere figuredout only for the best possible orientation of theselected ligandmolecule. For LigPlot+ analysis, anillustrative figure was generated for each ligand,describingbonded aswell as nonbonded interactionsbetween the ligands andprotein.

The hydrophobic amino acids of the intracellulardomainofVEGFR1,whichengage in thehydrophobicinteractions with puerarone, are Leu833,Val841,

Ala859, Lys861, Leu882,Val892,Val907,Val909,Tyr911, Cys912, Gly915, Leu1029, Cys1039, andPhe1041.Within the commercial inhibitors, vatalanibbound to theVEGFR1 intracellular domainmorestrongly than the others. Interestingly, the interactionprofile of vatalanib interacted hydrophobicallywithLeu833,Ala859, Lys861, Leu882,Val907,Val909,Tyr911,Leu1029,Cys1039, andPhe1041, and formedhydrogenbondswithGlu878whichwassimilar to thatofpuerarone. It is important tomention thatAsp1040residue ofVEGFR1was involved in hydrogen bondformationwith pazopanib, axitinib, vatalanib andsorafenib.Lys861 forms amajor amino acid residueofATPbinding sites in intracellulardomainVEGFR1and interestingly, both, puerarone andmost of theinhibitors interactedwith it.Thisprovidesus the cluethat pueraronemay block theATP binding site ofVEGFR1, thus enforcing their inhibitory effect.

Equivalently, tuberostanwas found to interactmoreeffectively with the intracellular kinase domainof VEGFR2 through eleven hydrophobic aminoacids, including Leu833, Lue840,Val848, Lys868,Glu885, Leu889,Val899,Val916, Phe918, Cys919,Lys920,Gly922,Cys1045,Asp1046, and Phe1047.Among the inhibitors, axitinib showed the highestaffinity for intracellular domain ofVEGFR2.Both

TABLE 3: MOLECULAR PROPERTIES OF THE SELECTED PHYTOCHEMICALS − PUERARONE AND TUBEROSTANProperties Puerarone TuberostanMolecular weight (g/mol) 336.33804 348.34874Log P 5.09 5.87HB donor 2 0HB acceptors 5 5Ames test Nonmutagen NonmutagenCarcinogenicity Noncarcinogen NoncarcinogenHuman intestinal absorption (%) 93.881 95.894HB: Hydrogen bond

TABLE 4: BINDING INTERACTIONS OF PHYTOCHEMICALS ALONG WITH INHIBITORS WITH VEGFRS−DOCKING AND CHIMERA ANALYSISPDB ID Ligands Minimum binding energy (kcal/mol) Ki (µM) Hydrogen Bonds Interacting amino acids

VEGFR13HNG Test molecule

Puerarone −9.91 0.182 0 -Tuberostan −8.44 0.646 0 -

Standard inhibitorsSunitinib −7.44 7.0 3 Cys912 (2), Cys1039Pazopanib −10.19 0.034 3 Lys861, His1020, Asp1040Axitinib −9.86 0.059 3 Cys1018, Asp1040 (2)Vandetanib −8.54 0.5493 0 -Vatalanib −12.18 0.0018 3 Glu878, Cys912, Asp1040Sorafenib −9.44 0.120 3 Cys912 (2), Asp1040

VEGFR23VHE Test molecule

Puerarone −7.91 1.58 1 Cys1045Tuberostan −9.32 0.148 0 -

Standard inhibitorsSunitinib −6.26 51.98 4 Asp814, Lys868, Ala 881, Leu1049Pazopanib −9.58 0.095 1 Asp1046Axitinib −9.94 0.052 1 Lys868Vandetanib −7.36 4.01 0 -Vatalanib −8.78 0.367 1 Asp1046Sorafenib −7.7 2.26 1 Lys868

Values in bold face indicate the test molecule with a better minimum binding energy. VEGFRs: Vascular endothelial growth factor receptors

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tuberostan and axitinib were found to share theirhydrophobic interactions with Leu840, Val848,Lys868,Glu885, Leu889,Val899,Val916, Phe918,Cys919, Gly922, Cys1045, and Phe1047.Also,tuberostan formed hydrophobic interaction withAsp1046,whichon the contrary,was involved in thehydrogenbondingwith axitinib.Similar toVEGFR1,Lys868 forms a crucial residue forATP binding inthe case ofVEGFR2whichwas recognized to showhydrophobicbondingwith tuberostan andcommercialinhibitors. Both the proteins and all the inhibitorsshowed hydrophobic interaction at theATP bindingsite, but tuberostan, additionally, showedmaximumhydrophobic interactionandhighestbindingaffinityasper theLigPlot+ andChimera analysis.Thus,makingit a better candidate for inhibition of intracellular

domain ofVEGFR2.The interaction profile of thephytochemicalswith theVEGFRsusingAutodock4.0,Chimera andLigPlot+, canbeviewed infig. 2.

In this complete study in silico was done to explorethe therapeutic potentials of the phytochemicalsderived fromPueraria tuberosa.Theoverall analysissuggested that puerarone and tuberostan could showa high affinity towards the critical proteinsVEGFR1and VEGFR2, respectively. The study providesa hint for the design of novel drug leads againstVEGFRs for the cureofmultiple angiogenicdiseases;exploiting the pharmacological aspects of thesecompounds.Further, in vitro and in vivo validationofthe estimatedmechanism aswell as the therapeuticpotentials of the selected phytochemicals is required.

TABLE 5: MOLECULAR INTERACTIONS OF THE SELECTED PHYTOCHEMICALS AND STANDARD INHIBITORS WITH VEGFRS − LIGPLOT+ ANALYSISPDB ID Ligands Hydrogen Bonds Hydrophobic Interactions

VEGFR13HNG Test molecules

Puerarone Glu878, Glu910 Leu833, Val841, Ala859, Lys861, Leu882, Val892, Val907, Val909, Tyr911, Cys912, Gly915, Leu1029, Cys1039, Phe1041

Tuberostan - Leu833, Val841, Ala859, Lys861, Glu878, Ile881, Leu882, Ile885, Val892, Val907, Val909, Cys1018, Leu1029, Ile1038, Cys1039, Asp1040

Standard inhibitorsSunitinib Cys912 (2), Cys1039 Leu833, Val841, Ala859, Glu878, Leu882, Val907, Val909, Glu910,

Tyr911, Gly915, Asn916, Leu1029, Asp1040, Phe1041Pazopanib Asp1040 Leu833, Val841, Lys861, Glu878, Val892, Val909, Tyr911, Cys912,

His1020, Leu1029, Cys1039, Phe1041Axitinib Asp1040 (3) Asp807, Lys861, Glu878, Ile881, Leu882, Vaal892, Cys1018, Ile1019,

His1020, Arg1021, Ile1038, Cys1039Vandetanib - Val841, Glu878, Ile881, Leu882, Ile885, Val891, Val892, Val909,

Leu1013, Leu1029, Ile1038, Cys1039, Asp1040, Phe1041Vatalanib Glu878, Cys912, Asp1040 (2) Leu833, Ala859, Lys861, Leu882, Val891, Val907, Val909, Glu910,

Tyr911, Leu1013, Leu1029, Ile1038, Cys1039, Phe1041Sorafenib Cys912, Asp1040 Leu833, Val841, Ala859, Lys861, Glu878, Ile881, Leu882, Val892,

Val909, Glu910, Tyr911, Gly915, Leu1013, Cy1018, Cys1039VEGFR2

3VHE Test moleculesPuerarone Glu885, Cys1045 Leu840, Val848, Ala866, Lys868, Lue889, Val916, Gly922, Asn923,

Lue1035, Asp1046, Phe1047Tuberostan - Lue840, Val848, Lys868, Glu885, Leu889, Val899, Val916, Phe918,

Cys919, Lys920, Gly922, Cys1045, Asp1046, Phe1047Standard inhibitors

Sunitinib Asp814, Lys868, Ala 881, Leu1049 Cys817, Leu882, Ser884, Glu885, Ile888, Asp1046, Gly1048Pazopanib Asp1046 Val848, Ala866, Val867, Lys868, Glu885, Ile888, Leu889, Val899,

Val914, Val916, Cys919, Gly922, Leu1035, Cys1045, Phe1047Axitinib Asp1046 Leu840, Val848, Ala866, Lys868, Glu885, Leu889, Val899, Val916,

Phe918, Cys919, Gly922, Leu1035, Cys1045, Phe1047Vandetanib Lys868, Glu885, Ile888, Leu889, Val899, Val916, Leu1035, Cys1045,

Asp1046, Phe1047, Gly1048Vatalanib Val848, Lys868, Glu885, Leu889, Val899, Val914, Val916, Leu1035,

Cys1045, Asp1046, Phe1047Sorafenib Lys868 Leu840, Val848, Ala866, Glu885, Leu889, Val899, Val916, Glu917,

Cys919, Phe918, Leu1035, Cys1045, Asp1046, Phe1047(2), Gly1048Presence of bold faced amino acid indicates the interaction of the phytochemical at ATP binding site of the VEGFRs. VEGFRs: Vascular endothelial growth factor receptors

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Financial support and sponsorship:Nil.

Conflicts of interest:There are no conflicts of interest.

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