Research Journal of Recent Sciences _________________________________________________ ISSN 2277-2502 Vol. 3(ISC-2013), 408-414 (2014) Res. J. Recent. Sci. International Science Congress Association 408 Molecular Modeling and Docking Studies of Neu5Ac2en analogues against Cholera toxin Jino Blessy John and Jeya Sundara Sharmila Department of Bioinformatics, Karunya Univesity, Karunya Nagar, Coimabtore-641114, Tamil Nadu, INDIAAvailable online at: www.isca.in, www.isca.me Received 30th November 2013, revised 17th January 2014, accepted 9th March 2014 AbstractNeu5Ac2en (2-deoxy-2, 3-didehydro-N-acetylneuraminic acid) analogues were modified in two different positions C-4 and C-9 were investigated using molecular modeling and molecular docking techniques. Cholera toxin is an protein complex made up of AB5 subunits secreted by the pathogenic organism Vibrio cholerae. In present days these organism shows resistance towards antibiotics. In our present study, Cholera toxin 3D protein structure was optimized and minimized using maestro v9.2. Twelve synthetic Neu5Ac2en analogues were modeled using ACD/ChemSketch and optimized in LigPrep which is a tool in Schrödinger suite. Active site of cholera toxin protein was analyzed using PDBsum database. Molecular docking of Neu5Ac2en analogues into the active site of cholera toxin protein were carried out using Glide v5.7. All the 12 analogues of Neu5Ac2en show good binding affinity towards the cholera toxin with least docking (XPG) energy score and also these analogues have good pharmacological properties. Neu5Ac2en analogues blocks the binding site residues of cholera toxin directly through intermolecular hydrogen bonding. Keywords: Molecular modeling, molecular docking, Neu5Ac2en analogues, Cholera toxin. Introduction Sialic acid or N-acetylneuraminic acid (Neu5Ac or NeuNAc) is the derivatives of neuraminic acid with 9 carbon monosaccharides found in the terminal components of glycoconjugates found in the cell surface of mammalian and blood cells of some non-mammalian and they involved in cellular and molecular interactions1-3. Sialic acid residues mask the bacterial cell surface from the host immune system and some of the bacterial pathogens are Campylobacter jejuni, Escherichia coli, Haemophilus influenza, Haemophilus ducreyi, Neisseria gonorrhoeae, Neisseria meningitides and Streptococcus agalactiae10. Sialic acids are the natural ligands for both Hemagglutinin (HA) and Neuraminidase (NA)11. It is difficult to explain the general role of sialic acid because it involve directly or indirectly in various cellular events. Due to their negative charge Neu5Ac functions are divided into two groups. First, sialic acid act as the masking agent which protects recognition sites such as proteins, macromolecules and receptor molecules. Second, sialic acid is the ligand for large number of molecules such as hormones, lectins, antibodies, and inorganic cations12. 2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid (Neu5Ac2en) is the derivative of unsaturated sialic acid. Neu5Ac2en is a sialosyl cation transition-state analogue and it is a strong inhibitor core template. The structurally modified Neu5Ac2en analogues give more effective inhibitors13. Also Neu5Ac2en and N-acyl derivatives inhibit Newcastle disease virus (NDV), simian virus 5, and Sendai virus sialidases14. Cholera toxin (CT) is a protein complex produced by the bacterium Vibrio cholera15. CT is made up of six protein subunits one A subunit with enzymatic activity and five B subunit with receptor binding. Vibrio cholera sticks to the human small intestinal epithelium cells and cause diarrhea due to the production of cholera toxin. In 1970s ganglioside (GM1) was recognized as the receptor for CT16. However, antibiotics are commonly administered as part of the treatment regimen. In present days, the organismbecomes resistance towards the multiple antibiotics17. The active site of cholera toxin is found in the single B-subunit and also a single solvent mediated hydrogen bond from the amino acid residue Gly 33 to the neighboring subunit. The majority of the interaction between the receptor and the cholera toxin is due involvement of two terminal sugar of GM1 such asgalactose and sialic acid18. The binding site of cholera toxin can be blocked by inhibition using sialic acid analogues19. In our present work, molecular modeling of Neu5Ac2en and its derivatives have substituted in two different positions C-4 and C-9. The Neu5Ac2en analogues were modeled using chemical drawing software chemsketch. Absorption, Distribution, Metabolism and Excretion (ADME) properties were done using QikProp to know the pharmacological activity of the compound as the drug molecule. The 3D crystal structure of cholera toxin is available in the protein data bank (PDB). Docking was done to study the binding mode of Neu5Ac2en analogues into the binding pocket of cholera toxin. Furthermore we describe the effective interaction between the Neu5Ac2en analogues and the target protein cholera toxin. Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 408-414 (2014) Res. J. Recent. Sci. International Science Congress Association 409 Material and Methods Molecular Modeling of Neu5Ac2en analogues and their modifications in two different positions C-4 and C-9 were collected from the literature shown in (table-1). The Neu5Ac2en derivatives are designed as described by Suzuki, et al.,20. Molecular Docking: Ligand preparation: The two dimensional chemical structures of the molecule are converted into three dimensional structures using a tool LigPrep in Schrödinger suite21. LigPrep produces different conformation of structure from each input with different ring conformations, ionization states, tautomers and stereochemistries. Ligands are minimized based upon the force field OPLS (Optimized Potentials for Liquid Simulations)22. The pharmacological properties of the ligands were calculated using QikProp software. In our study all the 12 compounds are biological active compounds. Protein preparation: The 3D crystal structure of cholera toxin protein PDB id is 3CHB was downloaded from the protein data bank (PDB). The structural features and active site residues of the choler toxin protein 3CHB were analyzed using PDBsum database. Protein preparation wizard Maestro v9.2 is used to fix the atomic representations of protein and its optimization23. Minimization was carried out using molecular mechanics force field OPLS22. A receptor grid was generated in the binding pocket of cholera toxin protein using Glide v5.724. Glide Docking: Two molecules bound to each other to form a stable complex to predict the preferred orientation of the both molecule is called docking25. Docking was carried out using the software Glide v5.724. In the grid box of protein the prepared and optimized ligands were flexibly docked using Monte carlo based simulation algorithm (MCSA) based minimization. In our study two subsequent docking procedure were used they are standard precision (SP) and extra precision (XP)26. 32 poses were generated for each ligand during XP docking the conformation of each ligand with best pose was retaining after post docking. The molecules were ranked based on XPG score with least binding energy and glide score. Table-1 Neu5Ac2en Analogues and their derivatives Neu5Ac2en derivatives Substituent Neu5Ac2en R1=OH R2=OH 4-O-amidinomethyl-Neu5Ac2enOCHC(=NH)NH OH 4-O-carbamoylmethyl-Neu5Ac2en OCHC(=O)NH OH 4-O-thiocarbamoylmethyl-Neu5Ac2en OCHC(=S)NH OH 4-O-cyanomethyl-Neu5Ac2en OCHCN OH 9-acetamido-4-O-amidinomethyl-Neu5Ac2en OCHC(=NH)NH NHAc 9-acetamido-4-O-carbamoylmethyl-Neu5Ac2en OCHC(=O)NH NHAc 9-acetamido-4-O-thiocarbamoylmethyl-Neu5Ac2en OCHC(=S)NH NHAc 9-acetamido-4-O-cyanomethyl-Neu5Ac2en OCHCN NHAc 9-azido-4-O-amidinomethyl-Neu5Ac2en OCHC(=NH)NH N 9-azido-4-O-carbamoylmethyl-Neu5Ac2en OCHC(=O)NH N 9-azido-4-O-methoxy-iminomethyl- Neu5Ac2en OCHC(=NH)OCH N Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 408-414 (2014) Res. J. Recent. Sci. International Science Congress Association 410 Results and Discussion Structural analysis of 3CHB: The three dimensional structure of cholera toxin protein 3CHB in complex with GAL-NGA- GAL-GLU-SIA were downloaded from PDB. The protein consists of 103 residues and comprises of 2 helices and 6 strands. The residues such as GLU11, TYR12, HIS13, LYS34, GLU51, GLN56, HIS57, ILE58, GLN61, TRP88, ASN90 and LYS91 were localized to be in van der Waal contact with GAL-NGA-GAL-GLU-SIA constitute as the active site residues. 3CHB residues GLU11, TYR12, HIS13, GLY33, LYS34, GLU51, GLN56, HIE57, ILE58, GLN61, TRP88, ASN90 and LYS91 were involved in hydrophobic interactions with GAL-NGA-GAL-GLU-SIA (figure 1). The above analysis were done using PDBsum database27. Cholera toxin- Neu5Ac2en analogues docking complex: Cholera toxin-Neu5Ac2en analogues docking complex was shown in (table-2 and 3). The good interaction between these analogues is due to various interactions such as hydrogen bond, hydrophobic, hydrophilic, electrostatic and steric interactions. Neu5Ac2en analogues such as Neu5Ac2en show least glide score of -8.08 and glide energy of -39.92 Kcal/mol with seven intermolecular hydrogen bond, 4-O-carbamoylmethyl-Neu5Ac2en -7.46 and -40.40 Kcal/mol with four intermolecular hydrogen bond, 4-O-cyanomethyl-Neu5Ac2en -7.37 and -43.97 Kcal/mol with seven intermolecular hydrogen bond, 4-O-thiocarbamoylmethyl-Neu5Ac2en -7.35 and -41.25 Kcal/mol with four intermolecular hydrogen bond, 9-azido-4-Ocarbamoylmethyl-Neu5Ac2en -7.15 and -45.88 Kcal/mol with seven intermolecular hydrogen bond, 9-acetamido-4-Ocyanomethyl-Neu5Ac2en -7.09 and -41.01 with seven intermolecular hydrogen bond, 4-O-amidinomethyl-Neu5Ac2en -6.84 and -37.99 Kcal/mol with seven intermolecular hydrogen bond, 9-acetamido-4-O-carbamoylmethyl-Neu5Ac2en -6.75 and -36.36 Kcal/mol with five intermolecular hydrogen bond, 9-acetamido-4-O-amidinomethyl- Neu5Ac2en -6.71 and -38.41 with five intermolecular hydrogen bond, 9-azido-4-Oamidinomethyl-Neu5Ac2en -6.53 and -39.78 Kcal/mol with four intermolecular hydrogen bond, 9-acetamido-4-O-thiocarbamoylmethyl-Neu5Ac2en -6.52 and -37.11 Kcal/mol with seven intermolecular hydrogen bond and 9-azido-4-O-methoxy-iminomethyl-Neu5Ac2en -6.18 and -46.29 Kcal/mol with five intermolecular hydrogen bond. Figure-1 Graphical representation of interaction of ligands GAL-NGA-GAL-GLU-SIA with active site of 3CHBTable-2 Glide docking score and glide energy of cholera toxin-Neu5Ac2en analogues Neu5Ac2en derivative Glide energy (Kcal/mol) Glide score Neu5Ac2en -39.92 -8.08 4-O-carbamoylmethyl-Neu5Ac2en -40.40 -7.46 4-O-cyanomethyl-Neu5Ac2en -43.97 -7.37 4-O-thiocarbamoylmethyl-Neu5Ac2en -41.25 -7.35 9-azido-4-O-carbamoylmethyl-Neu5Ac2en -45.88 -7.15 9-acetamido-4-O-cyanomethyl-Neu5Ac2en -41.01 -7.09 4-O-amidinomethyl-Neu5Ac2en -37.99 -6.84 9-acetamido-4-O-carbamoylmethyl-Neu5Ac2en -36.36 -6.75 9-acetamido-4-O-amidinomethyl-Neu5Ac2en -38.41 -6.71 9-azido-4-O-amidinomethyl-Neu5Ac2en -39.78 -6.53 9-acetamido-4-O-thiocarbamoylmethyl-Neu5Ac2en -37.11 -6.52 9-azido-4-O-methoxy-iminomethyl- Neu5Ac2en -46.29 -6.18 Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 408-414 (2014) Res. J. Recent. Sci. International Science Congress Association 411 Table-3 Intermolecular hydrogen bond interaction between the cholera toxin-Neu5Ac2en analogues Neu5Ac2en derivative Ligand atom Protein Distance ( Å ) Residue Atom Neu5Ac2enO38 H33 H37 O O H30 H32 LYS:91 GLN:56 GLN:56 GLN:61 ASN:90 HIE:13 HIE:13 HZ1 O O HE22 HD22 O O 1.774 1.684 2.121 2.477 1.817 1.859 1.914 4-O-carbamoylmethyl-Neu5Ac2en H35 O19 O23 H34 ASN:90 TRP:88 HIE:13 GLU:51 OD1 HE1 H OE1 1.976 1.972 1.993 1.732 4-O-cyanomethyl-Neu5Ac2en H33 O O42 H34 H35 O15 O19 GLU:51 LYS:91 ASN:90 GLN:56 GLN:56 GLN:61 TRP:88 OE1 HZ1 HD22 O O HE22 HE1 2.023 1.860 1.561 2.376 2.096 2.431 2.187 4-O-thiocarbamoylmethyl-Neu5Ac2en O19 H34 H36 H37 GLN:61 ILE:96 ILE:96 ALA:97 HE21 O O O 1.877 2.016 1.837 1.625 9-azido-4-O-carbamoylmethyl-Neu5Ac2en N25 H36 O O O46 H45 H44 HIE:13 HIE:13 ASN:90 ASN:90 LYS:91 GLN:56 GLN:61 HE2 O HD22 HD22 HZ1 O OE1 2.055 2.034 2.207 2.248 1.696 1.974 1.997 9-acetamido-4-O-cyanomethyl-Neu5Ac2en H39 O14 O O48 O15 H38 H36 GLN:56 TRP:88 ASN:90 LYS:91 HIE:13 HIE:13 HIE:13 O HE1 HD22 HZ2 H O O 2.027 2.397 2.041 1.916 2.045 2.064 2.031 4-O-amidinomethyl-Neu5Ac2en O46 H42 O H35 H36 O15 O13 LYS:91 GLN:56 ASN:90 HIE:13 HIE:13 TRP:88 HIE:13 HZ1 O HD22 O O HE1 H 1.788 1.910 1.818 2.473 1.812 2.166 1.832 9-acetamido-4-O-carbamoylmethyl-Neu5Ac2en H39 H38 O51 O O26 GLN:61 GLN:56 LYS:91 ASN:90 HIE:13 OE1 O HZ1 HD22 H 1.939 1.784 1.952 1.877 2.044 9-acetamido-4-O-amidinomethyl-Neu5Ac2en O19 H51 H49 H39 H37 GLN:61 GLU:11 GLU:11 ALA:97 ALA:97 HE21 OE2 OE2 O O 2.097 1.875 1.980 2.164 1.981 Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 408-414 (2014) Res. J. Recent. Sci. International Science Congress Association 412 Neu5Ac2en derivative Ligand atom Protein Distance ( Å ) Residue Atom 9-azido-4-O-amidinomethyl-Neu5Ac2en O14 H37 O O47 TRP:88 HIE:13 ASN:90 LYS:91 HE1 O HD22 HZ1 2.008 2.022 1.691 1.704 9-acetamido-4-O-thiocarbamoylmethyl- Neu5Ac2en O22 H39 H38 O O51 H49 H50 GLN:61 GLN:56 GLN:56 LYS:91 ASN:90 ASN:90 HIE:13 HE22 O O HZ1 HD22 OD1 O 2.270 1.978 1.878 1.771 1.893 2.042 2.069 9-azido-4-O-methoxy-iminomethyl- Neu5Ac2en O18 N25 H37 H45 O49 GLN:61 LYS:91 GLN:56 GLU:11 HIE:13 HE21 HZ2 O O H 2.033 1.942 1.864 1.924 1.986 Figure-2 Neu5Ac2en docked in cholera toxin binding pocket Figure-3 4-O-carbamoylmethyl- Neu5Ac2en docked in cholera toxin binding pocket Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 408-414 (2014) Res. J. Recent. Sci. International Science Congress Association 413 Table-4 QikProp Pharmacopore Prediction Ligands Name Molecular Weight H-bond donors H-bond acceptors QPlogPo/w Neu5Ac2en 293.273 6 13 -2.452 4-O-carbamoylmethyl- Neu5Ac2en 350.325 7 15 -3.652 4-O-cyanomethyl-Neu5Ac2en 332.310 5 14 -2.651 4-O-thiocarbamoylmethyl-Neu5Ac2en 366.385 7 15 -2.224 9-azido-4-O-carbamoylmethyl-Neu5Ac2en 375.338 6 16 -3.980 9-acetamido-4-O-cyanomethyl-Neu5Ac2en 373.362 5 15 -2.831 4-O-amidinomethyl-Neu5Ac2en 349.340 8 14 -2.792 9-acetamido- 4-O-carbamoylmethyl-Neu5Ac2en 391.377 7 16 -3.952 9-acetamido- 4-O-amidinomethyl-Neu5Ac2en 390.392 8 15 -3.187 9-azido-4-O-amidinomethyl-Neu5Ac2en 374.353 7 15 -3.229 9-acetamido-4-O-thiocarbamoylmethyl- Neu5Ac2en 407.438 7 15 -2.388 9-azido-4-O-methoxy-iminomethyl- Neu5Ac2en 389.364 5 15 -2.225 QikProp pharmacokinetic prediction: QikProp is used to predict the molecular properties of the known drug. Pharmacokinetic properties like molecular weight, hydrogen bond donor, hydrogen bond acceptor, oral absorption, QPlogPo/w are calculated based on Lipinski’s rule. Conclusion Docking study revealed that the Neu5Ac2en analogues with substitution in two different position C-4 and C-9 have good interaction in the binding site of cholera toxin protein. Among the 12 analogues 6 shows least glide (docking) score for the compounds Neu5Ac2en, 4-O-carbamoylmethyl-Neu5Ac2en, 4-O-cyanomethyl-Neu5Ac2en, 4-O-thiocarbamoylmethyl-Neu5Ac2en, 9-azido-4-O-carbamoylmethyl-Neu5Ac2en, 9acetamido-4-O-cyanomethyl-Neu5Ac2en such as -8.08, -7.46, -7.37, -7.35, -7.15 and -7.09 respectively and minimum glide energy such as -39.92 Kcal/mol, -40.40 Kcal/mol, -43,97 Kcal/mol, -41.25 Kcal/mol, -41.01 Kcal/mol and -41.01 Kcal/mol respectively. Neu5Ac2en analogues blocked the active site residues LYS:91, GLN:56, GLN:61, ASN:90, HIE:13, ALA:97, GLU:11, TRP:88 and ILE:96 directly through intermolecular hydrogen bonding. Neu5Ac2en analogues have better pharmaceutical properties thus it could be used as futuristic drug for cholera. Acknowledgement The authors acknowledge the support given by DST-SERB Ref No. (SR/FT/LS-157/2009), Govt. of India, New Delhi and for research activities in the Department of Bioinformatics, Karunya University. Reference 1.Schauer R., Achievements and challenges of sialic acid research, Glycoconj.J., 17, 485–499 (2000)2.Varki A.,Biological roles of oligosaccharides: all of the theories are correct, Glycobiology.,3, 97–130 (1993)3.Vimr E. R, Kalivoda K.A, Deszo E.L. and Steenbergen S.M., Diversity of microbial sialic acid metabolism, Microbiol. Mol. Biol., 68,132–153 (2004) 4. Avril T., Wagner E.R., Willison H.J. and Crocker P.R., Sialic acid-binding immunoglobulin-like lectin 7 mediates selective recognition of sialylated glycans expressed on Campylobacter jejuni lipooligosaccharides, Infect. Immun., 74, 4133–4141 (2006)5.Vimr E., Steenbergen S. and Cieslewicz M., Biosynthesis of the polysialic acid capsule in Escherichia coli K1, J. Ind. Microbiol., 15, 352–360 (1995)6.Bauer S.H., Mansson M., Hood D.W., Richards J.C., Moxon E.R. and Schweda E.K.,A rapid and sensitive procedure for determination of 5--acetyl neuraminic acid in lipopolysaccharides of Haemophilus influenzae: a survey of 24 non-typeable H. influenzae strains, Carbohydr. Res., 335, 251–260 (2001)7.Schilling B., Goon S, Samuels N.M., Gaucher S.P., Leary J.A., Bertozzi C.R. and Gibson B.W., Biosynthesis of sialylated lipooligosaccharides in Haemophilus ducreyi is dependent on exogenous sialic acid and not mannosamine. Incorporation studies using -acylmannosamine analogues, -glycolylneuraminic acid, and 13C-labeled acetylneuraminic acid, Biochemistry., 40, 12666–12677 (2001) Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 408-414 (2014) Res. J. Recent. Sci. International Science Congress Association 414 8.Ram S., Sharma A.K., Simpson S.D., Gulati S., McQuillen D.P., Pangburn M.K. and Rice P.A., A novel sialic acid binding site on factor H mediates serum resistance of sialylated Neisseria gonorrhoeae, J. Exp. Med., 187, 743–752 (1998)9. Hammerschmidt S., Hilse R., van Putten J.P., Gerardy-Schahn R., Unkmeir A. and Frosch M., Modulation of cell surface sialic acid expression in Neisseria meningitidis via a transposable genetic element, EMBO J., 15, 192–198 (1998)10.Lewis A.L., Hensler M.E., Varki A. and Nizet V.,The group B streptococcal sialic acid O-acetyltransferase is encoded by neuD, a conserved component of bacterial sialic acid biosynthetic gene clusters, J. Biol. Chem., 281, 11186–11192 (2006)11.Varki N.M. and Varki A., Diversity in cell surface sialic acid presentations: implications for biology and disease, Lab Invest.,87, 851–857 (2007)12. Janas T. and Janas T., Polysialic acids: structure and properties. In Polysaccharides — Structural Diversity and Functional Versatility, Marcel Dekker., 707-727 (2005)13.Fgedi and Per., The organic chemistry of sugars., Washington, DC: Taylor and Francis,. 822–823 (2006)14.Meindl P., Bodo G., Palese P., Schulman J. and Tuppy H., Inhibition of neuraminidase activity by derivatives of 2-deoxy-2,3-dehydro-Nacetylneuraminic acid, Virology., 58, 457–63 (1974)15.Ryan K.J. and Ray C.G., Sherris Medical Microbiology., McGraw Hill. 375 (2004) ISBN 0-8385-8529-9. 16. Holmgren J., Lo¨nnroth I. and Svennerholm L., Fixation and inactivation of cholera toxin by GM1 ganglioside, Scand J Infect Dis.,, 77–78 (1973) 17. Kitaoko M, Miyata S.T., Unterweger D. and Pukatzki S., Antibiotic resistance mechanisms of Vibrio cholera, J Med Microbiol., 60, 397-407 (2011)18. Ethan A., Merritt, Sarfaty S., Focco VAN DEN Akker, Cecile L’HOIR, Joseph A., Martial and Wim G.J. Hol., Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide, Protein Science., 3, 166-175 (1993)19. Sharmila, D.J.S. and veluraja R., Monosialogangliosides and Their Interaction with Cholera Toxin– Investigation by Molecular Modeling and Molecular Mechanics, Journal of Biomolecular Structure and Dynamics., 21, 591-613 (2004)20. Suzuki T., Ikeda K, Koyama N., Hosokawa C., Kogure T., Takahashi T., Jwa Hidari K.I-P, Miyamoto D., Tanaka K. and Suzuki Y., Inhibition of human parainfluenza virus type 1 sialidase by analogs of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, Glycoconjugate Journal., 18, 331-337 (2001)21.Chen I.J. and Foloppe N.J., Drug-like bioactive structures and conformational coverage with the LigPrep/ ConfGen suite: comparison to programs MOE and catalyst, J Chem Inf Model., 50, 822-39 (2010)22. Kaminski G.A., Friesner R.A., Tirado-Rives J. and Jorgensen W.L., Evaluation and reparametrization of the OPLS- AA force field for protein via comparison with accurate quantum chemical calculations on peptides, J Phys ChemB.,105, 6474-6477 (2001)23.Maestro 9.0, versuib 70110, Schrodinger, New York (2009)24.Friesner R.A., Banks J.L., Murphy R.B., Halgren T.A., Klicic J.J. and Mainz D.T., et al., Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy, J Med Chem., 47, 1739 -1749 (2004)25.Lengauer T. and Rarey M., Computational methods for biomolecular docking, Curr Opin Struct Biol., , 402-406 1996)26. Friesner R.A., Murphy R.B., Repasky M.P., Frye L.L., Greenwood J.R. and Halgren T.A., et al., Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes, J Med Chem., 49, 6177-6196 (2006) 27.Merritt E.A., Sarfaty S., van den Akker F., L'Hoir C., Martial J.A. and Hol W.G., Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide, Protein Sci., 3, 166-175 (1994)