Molecular docking of sialic acid and its sialic acid-gadolinium (III ...

M. Khadafi & A. Mutalib Proceeding of The International Seminar on Chemistry 2008 (pp. 333-338)ISBN 978-979-18962-0-7Jatinangor, 30-31 October 2008Molecular docking of sialic acid and its sialic acid-gadolinium (III)poliaminocarboxylate complex conjugate, respectively, towardhemagglutinin of H5N1 viruses of VietnamMahammad Khadafi 1 , Abdul Mutalib 2 *1 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Bandung1,2 Center for Radioisotopes and Radiopharmaceuticals, BATAN, PUSPIPTEK, Serpong*Corresponding author e-mail mutalib@batan.go.id phone number 08121312875AbstractAvian Influenza is one of the infection disease that still become a complex and serious healthproblem. This disease particularly is caused by H5N1 virus which the spikes of virus contain 2 mainproteins that’s call hemagglutinin and neuraminidase. Hemagglutinin is antigenic glycoprotein thatcan be found on the surface of influenza virus which role bind virus to host cell through sialic acidmolecule. The goal of this research is to calculate the binding energy value GdDTPA sialic acidconjugate molecule and determine amino acid residues which give contribution happen hydrogenbonding and hydrophobic interaction in docking process sialic acid and GdDTPA sialic acidconjugate molecule with hemagglutinin as preliminary study for design MRI contrast agentcompound to diagnose avian influenza patient suspect by using MRI contrast agent. Three dimensionstructure of GdDTPA sialic acid conjugate molecule was modelled with MM2 method through Chem3D software. Then docking silmulation was carried out between sialic acid and conjugate moleculewith hemagglutinin active site to determine binding energy value and the amino acid residues onbinding site that can be found hydrogen bonding and hydrophobic interaction by using Chem 3D andArguslab software. From this research, binding energy predicted value of conjugate molecule is -4.26845 kcal/mole with 2 hydrogen bonding interactions on Gln226 and Ser227 and 1 hydrophobicinteraction on Gln226.Keywords: Hemagglutinin, H5N1 virus, sialic acid, GdDTPA, dockingIntroductionOver the past century, emergence of epidemicinfluenza has been serious threat to human health. Theorigin of human influenza viruses is thought to beavian influenza virus because all the subtypes arefound in avian host. Newly adapted avian influenzavirus to human host or reassortant virus could bepandemic because we have no immunity for it, asshown in our history such as 1918 (H1N1), 1957(H2N2) and 1968 (H3N2) pandemics. Recently, thefirst H5 avian influenza virus infected patient wasreported and emergence of new pandemic influenza isalerted (WHO, 2006).Influenza viruses are pleomorphic, envelopedRNA viruses belonging to the family ofOrthomyxoviridae. Protruding from the lipid envelopeare two distinct glycoproteins, the hemagglutinin (HA)and neuraminidase (NA). HA attaches to cell surfacesialic acid receptors, thereby facilitating entry of thevirus into host cells. Since it is the most importantantigenic determinant to which neutralizing antibodiesare directed, HA represents a crucial component ofcurrent vaccines. NA is the second major antigenicdeterminant for neutralizing antibodies. By catalyzingthe cleavage of glycosidic linkages to sialic acid onhost cell and virion surfaces, this glycoproteinprevents aggregation of virions thus facilitating therelease of progeny virus from infected cells. Inhibitionof this important function represents the most effectiveantiviral treatment strategy to date (de Jong & Tihnhien, 2006).Hemagglutinin (HA), the principal antigen on theviral surface, is the primary target for neutralizingantibodies and is responsible for viral binding to hostreceptors, enabling entry into the host cell throughendocytosis and subsequent membrane fusion. Assuch, the HA is an important target for both drug andvaccine development. Although 16 avian andmammalian serotypes of HA are known, only three(H1, H2, and H3) have become adapted to the humanpopulation. HA is a homotrimer; each monomer issynthesized as a single polypeptide (HA0) that iscleaved by host proteases into two subunits (HA1 andHA2). HA binds to receptors containing glycans withterminal sialic acids, where their precise linkagedetermines species preference. A switch in receptorspecificity from sialic acids connected to galactose inα2-3 linkages (avian) to α2-6 linkages (human) is amajor obstacle for influenza A viruses to cross thespecies barrier and to adapt to a new host (Shiya et al.,2005;Suzuki et al., 2000). On H3 and H1 HAframeworks, as few as two amino acid mutations can333

M. Khadafi & A. Mutalib Proceeding of The International Seminar on Chemistry 2008 (pp. 333-338)Jatinangor, 30-31 October 2008switch human and avian receptor specificity (Stevenset al., 2006).Gadolinium ion (Gd 3+ ) is very suitable for MRIcontrast agent compound because of paramagneticproperties and stabilization of its chelat. Gadoliniumcomplex molecules are paramagnetic complex that canbe used widely as MRI contrast agent compound.Some factors which cause Gd complex suitable forthis application are Gd 3+ ion have highestparamagnetic properties because of seven unpairelectron in 4f 7 valence shell, have longest electronicrelaxation time, and can form stable chelate that cancoordinate with two water compound (inner-spherecoordination) (Aime et al., 2002). Some example ofGd 3+ complex compounds that have been accepted inclinical study are Gd-DTPA, Gd-BOPTA, Gd-DOTA(Jacques & Desreux, 2002).Recently, avian influenza viruses have beeninfected human as host cell and cause many suspectedpatient bird flu died in large number. For diagnose thesuspected patient bird flu, some hospital are still usinglaboratory prosedure usually performed byimmunochromatographic or immunofluorescentdetection of influenza virus antigens, or reversetranscriptase polymerase chain reaction (RT-PCR)detection of viral nucleic acids in respiratory specimen(Hien et al., 2004). This method is takes a lot of time(4 to 5 days) to have complete diagnose. So, we studythe stabilization and binding affinity of GdDTPAsialic acid conjugate compound throughcomputational chemistry approach for novel MRIcontrast agent compound to diagnose suspectedhuman bird flu by using MRI contrast agent.of grid resolution and docking method in ArgusLab 4.Docking of ligand sialic acid and conjugate moleculeinto the HA (PDB:2FK0) receptor binding site wasperformed with grid resoution and docking methodresult from validation docking method. Bindingenergy (∆G) result was compared and the bestconformation result of both ligands were analyzed thehydrogen bonding and hydrophobic interaction toidentify spesific contact between ligands and HA.Hardware and Software. Cambridgesoft Chem 3D 8was used for molecular modelling. CambridgesoftChem Draw 8 was used for create gadoliniumcomplex structure. Parameter files GdDTPAmolecular mechanic (MM2) calculation wasperformed on Chem 3D 8. Windows Xp Sp 3 wasused as operating system. Arguslab 4.0.1 was used fordocking simulation and analysis interaction.Results and DiscussionModeling of sialic acid – GdDTPA conjugatemoleculeComplex struture of CHx-A”-Gd-DTPAcompound was drawed with chem draw software tocreate the 2 dimension structure of GdDTPA. The 2dimension structure of GdDTPA compound wasexported to 3 dimension structure by using chem 3Dsoftware. The result compound of GdDTPA structurecomplex can be seen in Fig.1.Materials and MethodsModeling of sialic acid and sialic acid – GdDTPAconjugate molecule.The native sialic acid molecule was modeled byconjugate the 3D structure of CHx-A”-Gd-DTPAusing Chem 3D 8 software. Structure of sialic acid andconjugate molecule GdDTPA – sialic acid were doneminimisation energy job using molecular mechanic(MM2) method. Then, conjugate molecule GdDTPA– sialic acid was modified with replaced the electrondonor atom (N atom) of conjugate molecule to dummyatom (Du atom). Both of structure minimisation resultwere calculate the steric energy summary by usingcompute properties job and done the overlay structurebetween those of structures. The parameter resultfrom minimisation i.e. length bond and angel bondwere calculated the root mean square (RMS) value tofind out the deviation value between both structures.Then, validation docking method was carried out bydocking the native sialic acid into receptor bimdingsite (RBS) of HA by using ArgusLab 4 software.Validation method was done to compare the variationFigure 1 The 3D strucure of GdDTPA complexcompound.The native structure of sialic acid was separated fromthe protein HA co – crystallize structure. Then sialicacid structure was conjugated with GdDTPA structurethrough connecting the Nitrogen atom of imine tailGdDTPA structure to Carbon atom 1 (C-1) of sialicacid structure which have been removed the hydroxyltail (-OH) bind to C-1 atom before. The result ofconjugate structure sialic acid – GdDTPA is showedin Fig.2.334

M. Khadafi & A. Mutalib Proceeding of The International Seminar on Chemistry 2008 (pp. 333-338)Jatinangor, 30-31 October 2008Figure 2 Conjugate structure of sialic acid and sialicacid – GdDTPA compound.Then, conjugate structure and sialic acid nativestructure were done minimisation energy job usingmolecular mechanic MM2 method to make the stableconformation of both structures based onintramolecular interaction among the atomscomposition of both molecules. The result ofminimisation energy was stable conformationstructures of sialic acid and conjugate molecule as weseen in Fig.3.other and also for Oxygen atoms no. 2, 3, 4, 5 andNitrogen atom no. 10. Overall, the conformation ofsialic acid structure from conjugate molecule is closeto sialic acid native structure. This result is agree withcalculation result of RMS (Root Mean Square) lengthbond and angle bond. The RMS of length bond is0.004685 and angle bond is 1.1.9265. This calculationresult show that the RMS value of length bond andangle bond are small. It’s mean that both sialic acidstructures have close conformation structure. So, thereis a little bit deviation between both sialic acidstructures.Figure 4 Overlay structure result between sialic acidnative structure (red) and sialic acidconjugate molecule (green).(a)After that, both sialic acid structures were calculatedthe steric energy summary to know the stablization ofboth compound. The calculations result of stericenergy summary are shown in Tab.1.Tabel 1 The sum of steric energy and itscomposition energy(b)Figure 3 The stable confomation of conjugatemolecule (a) and sialic acid (b) after donethe minimisation energy.Conjugate molecule GdDTPA – sialic acid wasmodified with replacing the electron donor atom(Nitrogen atom) to dummy atom and remove theGdDTPA structure complex. This procedure was doneto know how much the deviation of sialic acidconformation of conjugate molecule to native sialicacid that have been done minimisation energy before.The modified conjugate molecule and sialic acidstructure were done overlay to know the differentconformation of sialic acid in conjugate molecule.From the overlay structure, we see that there is a littledifferent conformation among those structures whichis showed in Fig.4. The atoms Carbon no. 11, 12, 13,14, 15, 16, 17, 18, and 20 show that overlapped eachComposition ofsteric energyConjugatesialic acid(kcal/mole)Nativesialic acid(kcal/mole)Stretch 5.7070 5.6029Bend 24.0232 28.9564Stretch-Bend 0.9994 1.2446Torsion 11.6823 10.7191Non-1,4 VDW -8.7975 -10.77161,4 VDW 15.6697 13.6970Dipole/Dipole -3.8840 -6.2429Total 45.4001 43.2056The result indicate that total steric energy of conjugatemolecule is less stable than native sialic acid, becauseof calculation show that the steric energy total of sialicacid conjugate molecule (45.4001 kcal/mole) is biggerthan sialic acid native structure (43.2056 kcal/mole).Value of steric energy composition for bend energy,non-1,4 VDW energy, and dipole/dipole energy showbig differences. This result impact on the imperfectresult of the overlay structure between both sialic acidmolecules.335

M. Khadafi & A. Mutalib Proceeding of The International Seminar on Chemistry 2008 (pp. 333-338)Jatinangor, 30-31 October 2008Validation docking method in ArgusLab software.Validation docking in ArgusLab was carriedout for validate the docking method and the variationof grid resolution value in docking simulation withArgusLab. This procedure was used to know howdoes the effect of the variation docking method andgrid resolution value in the result of dockingsimulation with ArgusLab. We compare the dockingmethod in ArgusLab between ArgusDock andGADock method and also the variation of gridresolution (0.1 ; 0.15 ; 0.2 ; 0.25 ; 0.3 ; 0.35 ; 0.4 Ǻ).Both docking methods (ArgusDock and GADock)have different approximation. For ArgusDock, thedocking method use structure and ligandapproximation which is docking mechanism directedto limited position only. Meanwhile, GADock useapproximation which ligand is docking directed to allpossible position in receptor (protein) (9). Thisvalidation prosedure base on the different value ofRMSD (Root Mean Square Deviation) between nativesialic acid ligand and copy sialic acid ligand whichdock into the receptor binding site of hemagglutinin.From this validation, we can get the best dockingmethod and value of grid resolution based on thesmallest value of RMSD. In Tab.2, we can see theresult of validation docking method.Tabel 2 The result of validation docking method inArgusLab 4.RMSD value ofGrid resolutiondocking method(Ǻ)GADock ArgusDock0.1 0.963723 4.1462260.15 1.270897 4.2479860.2 0.614733 4.3979560.25 0.867363 4.2468130.3 1.046308 4.4035480.35 0.711158 4.3913810.4 1.024216 4.458099The docking validation method is valid if the RMSDvalue is not more than 2.0000. From this result, wecan conclude that the smallest RMSD value was0.614733. GADock docking method has smallerRMSD value than ArgusDock. So, GADock is muchbetter used in docking prosedure than ArgusDock.The variation value of grid resolution show that 0.2 Ǻgive the smallest RMSD value in GADock dockingmethod. In this case, we use GADock as dockingmethod and 0.2 Ǻ as the value of grid resolution forrunning docking simulation sialic acid native structureand sialic acid conjugate molecule into the receptorbinding site of protein hemagglutinin.Docking simulation and interaction analysis of sialicacid native structure with protein hemagglutininDocking simulation between sialic acid nativestructure and protein hemagglutinin were carried outwith ArgusLab software. The parameter of calculationwas using parameter data from validation dockingresult. In this case, we use protein hemagglutininVietnam (PDB code:2FK0) which is the host sel comefrom human bird flu case. The result of dockingsimulation (in Fig.5a) show that the best pose ofnative sialic acid structure with predicted bindingenergy value (∆G) -3.5443 kcal/mole. This dockingsimulation create 13 poses of native sialic acidstructure and the best pose indicate that sialic acidgive the best conformation which fit into receptorbinding site of protein hemagglutinin. We also analysethe interaction between ligand native sialic acid andreceptor binding site of hemagglutinin protein.From the Fig.5b, we see that there are 4 hydrogenbonding and 6 hydrophobic interactions which can beformed between sialic acid and hemagglutinin.Hydrogen bonding interactions (red line) are involved2 amino acid residues i.e. Glu190 and His 193.Meanwhile, hydrophobic interctions (blue line) areinvolved 3 amino acid residues, that is Leu 194, Gly228, and Ser 227. Many hydrophobic interactions withprotein hemagglutinin this indicate the sialic acidnative structure can’t get into the active site of proteinhemagglutinin. Hydrogen bonding that is formedbetween sialic acid and hemagglutinin have a weakinteraction and long distance. Overall, from the energyand geometry analysis, the interaction between sialicacid native strcuture and protein hemagglutinin showweak interactions.Docking simulation and interaction analysis of sialicacid conjugate structure with protein hemagglutininFor sialic acid conjugate structure, dockingsimulation used the same parameter data from dockingvalidation parameter that have been done before.The different procedure from docking sialic acidnative structure is treatment of ligand. We subjectedligand as rigid ligand For sialic acid conjugatestructure. Beside that, sialic acid native structure istreated as flexible ligand. This was done in order tosialic acid conjugate molecule can’t change itsconformation. So, the docking simulation was runningwith rigid – rigid docking method. We can see inFig.6a, predicted binding energy value (∆G) fromdocking simulation is about -4.26845 kcal/mole. Thisvalue was less than from docking simulation of sialicacid native structure. From energy analysis, it showthat sialic acid conjugate molecule could bind tightlyin hemagglutinin receptor binding site and also couldshift sialic acid native structure.336

M. Khadafi & A. Mutalib Proceeding of The International Seminar on Chemistry 2008 (pp. 333-338)Jatinangor, 30-31 October 2008(a)(b)Figure 5 Result of docking simulation between sialic acid native structure and protein hemagglutinin. (a)Thebest pose of sialic acid with binding energy value, (b) interaction analysis show red line is hydrogenbonding and blue line is hydrophobic interaction.The interaction analysis (Fig.6b) also show that sialicacid conjugate molecule can form 2 hydrogen bondingand 1 hydrophobic interaction with amino acidresidues in hemagglutinin receptor binding site.Hydrogen bonding interactions are involved 2 aminoacid residues Gln 226 and Ser 227 which is Gln 226have strong interaction (2.473235 Å) with sialic acidconjugate molecule. Experiment result from Taiwanresearcher show Gln 226 is a critical amino acidresidue that can make stronger interaction with SA-α-2, 3-Gal than SA-α-2, 6-Gal (Li & Wang, 2006).Hydrophobic interaction only can form 1 interactionwith Gln 226 amino acid. This interaction indicatesialic acid conjugate molecule can get intohemagglutinin active site. Because from hydrophobicinteraction only show fewer interaction than sialic acidnative structure. This interaction analysis can beconcluded that sialic acid conjugate molecule bindtightly than sialic acid native strucutre.Figure 6 Result of docking simulation between sialic acid conjugatemolecule and protein hemagglutinin. (a)Thebest pose of sialic acid with binding energy value, (b) interaction analysis show red line is hydrogenbonding and blue line is hydrophobic interaction.337

M. Khadafi & A. Mutalib Proceeding of The International Seminar on Chemistry 2008 (pp. 333-338)Jatinangor, 30-31 October 2008ConclusionsIn summary, we have determined how sialic acidnative structure and sialic acid conjugate moleculebind with hemagglutinin H5N1 virus using molecularmechanic (MM2) calculation and molecular dockingsimulation. Given the results presented in this report,it indicates that the sialic acid conjugate molecule hasstrong hydrogen bond interactions whereas sialic acidnative structure only shows weak interactions. Most ofthe difference arise from interaction with residue Gln226 amino acid that have strong hydrogwn bondingwith sialic acid conjugate molecule. Fromhydrophobic interactions, sialic acid conjugatemolecule show only a few interaction with amino acidresidues. This show that sialic acid conjugatemolecule can be get into the active site ofhemagglutinin protein. So, we can conclude sialic acid– GdDTPA conjugate molecule can shift the nativestructure of sialic acid and also this conjugatemolecule can be suggested as MRI contrast agentcompound for diagnose suspected human bird fludisease.AcknowledgementsWe thank Dr. Abdul Mutalib for his idea andknowledge, Dr. rer. nat Iwan Hastiawan for hisencourage to finish this research, M. Yusuf, S. Si. forhis knowledge and guidedance, Rustaman, M. Si. forhelpful comment and contunous support, andDepartement of Chemistry Padjadjaran University foreducational support.ReferencesAime, S., Dastru, W., Crich, S. G., Gianolino, E., &Mainero, V. 2002. Biopolimers (Peptida Science).Innovative Magnetic Resonance ImagingDiagnostic Agents Based on Paramagnetic Gd(III)Complexes. 66: 419-428.Hien TT, de Jong M, Farrar J. 2004. Avianinfluenza—a challenge to global health carestructures. N Engl J Med.351:2363 – 2365.Jacques, V. and Desreux, J.F. 2002. Topics In CurentChemistry. New Classes Of MRI Contrast Agent.Springer-Verlag. Berlin.K. Shinya, M. Hatta, S. Yamada, A. Takada, S.Watanabe, P.Halfmann, T. Horimoto, G.Neumann, J.H. Kim, W. Lim, Y. Guan, M. Peiris,M. Kiso, T. Suzuki, Y. Suzuki, Y. Kawaoka. 2005.Characterization of a human H5N1 influenza Avirus isolated in 2003, J. Virol.79:9926–9932Li, M and Bingie Wang. 2006. Computaional Study ofH5N1 Hemagglutinin Binding With SA-a-2,6-Galand SA-a-2,6-Gal. J. Biochem & Biophysical.347:662 – 668.M.D. de Jong and Tran Tinh Hien. 2006. AvianInfluenza A (H5N1) J of Clinical Virology 35:2-13.Stevens, J, Ola Blixt, Terrence M. Tumpey, Jeffery K.Taubenberger, James C. Paulson, Ian A. Wilson.2006. Structure and Receptor Specificity of theHemagglutinin from an H5N1 Influenza Virus.J. Science. 312: 404 – 410.Suzuki, Y., Ito, T., Suzuki, T., Holland Jr., R.E.,Chambers, T.M., Kiso, M., Ishida, H., Kawaoka,Y. 2000. Sialic acid species as a determinant ofthe host range of influenza A viruses. J. Virol.74:11825–11831.The World Health Organization (WHO) Web site.2006. (www.who.int/en/).338