How to Calculate Binding Constants for Drug Discovery Studies

María J.R. Yunta^1,

¹Departamento de Química Orgánica I, Facultad de Química, Universidad Complutense, Madrid, Spain

Cite this paper:
María J.R. Yunta. How to Calculate Binding Constants for Drug Discovery Studies. American Journal of Modeling and Optimization. 2013; 1(3):61-70. doi: 10.12691/ajmo-1-3-7

Abstract

Free energy calculations to predict binding ability of molecules to receptors are a main subject for drug design. A wide range of theoretical methods has been applied to the calculation of binding constants. This mini-review seeks to identify such methods to better achieve correct predictions for drug candidates.

Keywords:
molecular modeling binding constant computer aided drug design

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References:

[1]	Guvench, o., MacKerell Jr. A.D. Computational evaluation of protein - small molecule binding. CurrOpinStruct Biol., 2009, 19, 56-61.
[2]	Wang, W.; Donini, O.; Reyes, C. M.; Kollman, P. A. Biomolecular simulations. Recent developments in force fields, simulations of enzyme catalysis, protein-ligand, protein-protein and protein-nucleic acid noncovalent interactions.Annu. ReV. Biophys. Biomol.Struct. 2001, 30, 211-243.
[3]	Wan, S.; Coveney, P. V.; Flower, D. R. Peptide recognition by the T cell receptor: comparison of binding free energies from thermodynamic integration, Poisson-Boltzmann and linear interaction energy approximations. Philos. Trans. R. Soc. A 2005,363, 2037-2053.
[4]	Kovalskyy, D.; Dubyna, V.; Mark, A. E.; Korenelyuk, A. A molecular dynamics study of the structural stability of HIV-1 protease under physiological conditions: The role of Na+ ions in stabilizing the active site. Proteins: Struct, Funct. Bioinf. 2005, 58, 450-458.
[5]	Bountis, T. (Ed.), Proton Transfer in Hydrogen-Bonded Systems, NATO ASI Series B: Physics, vol. 291, Plenum Press, New York, 1992, pp. 1-355.
[6]	Fornabaio, M., Cozzini, P., Mozzarelli, A., Abraham, D. J., Kellogg, G. E. Simple, Intuitive Calculations of Free Energy of Binding for Protein-Ligand Complexes. 2. Computational Titration and pH Effects in Molecular Models of Neuraminidase-Inhibitor Complexes. J. Med. Chem. 2003, 46, 4487-4500.
[7]	Miranda, C., Escartí, F., Lamarque, L., Yunta, M. J. R., Navarro, P., GarcíaEspaña, E., Jimeno, M. L. New 1H-pyrazole-containing polyamine receptors able to complex L-glutamate in water at physiological pH values.J. Am. Chem. Soc, 2004, 126, 823-833.
[8]	Kellogg, G. E., Fornabaio, M., Spyrakis, F., Lodola, A., Cozzini, P., Mozzarelli, A., Abraham, D. J. Getting it right: modeling of pH, solvent and “nearly” everything else in virtual screening of biological targets. J. Molec. Graph. Mod., 2004, 22, 479-486.
[9]	Dong, F., Olsen, B., Baker, N. A. Computational Methods for Biomolecular Electrostatics. Meth. Cell Biol. 2008, 84, 843-870.
[10]	Miteva, M.A., Tufféry, P., Villoutreix, B.O. PCE: web tools to compute protein continuum electrostatics. Nuc. Acids. Res., 2005, 33, w 372-w 375.
[11]	Varekova, R. S., Geidl, S., Ionescu, C-M., Skrehota, O., Bouchal, T., Sehnal, D., Abagyan, R., Koca, J. Predicting p Ka values from EEM atomic charges. J. Cheminform. 2013, 5:18.
[12]	Witham, S., Talley, K., Wang, L., Zhang, Z., Sarkar, S., Gao, D., Yang, W., Alexov, E. Developing hybrid approaches to predict p Ka values of ionizable groups. Proteins, 2011, 79, 3389-3399.
[13]	Yikilmaz, E., Rodgers, D. W., Miller, A-F. The Crucial Importance of Chemistry in the Structure-Function Link: Manipulating Hydrogen Bonding in Iron-Containing Superoxide Dismutase. Biochemistry 2006, 45, 1151-1161.
[14]	Nielsen, J. E., McCammon, J. A. On the evaluation and optimization ofprotein X-ray structures for pKa calculations, Protein Sci. 2003, 12, 313-326.
[15]	Chipot, C., Porhorille, A. (Eds.) Free energy calculations. Theory and applications in chemistry and biology series. 2007.
[16]	Reddy, M. R., Erion, M.D., Agarwal, A. Free energy calculations: useand limitations in predicting binding affinities, Rev. Comput. Chem. 2000, 16, 217-304.
[17]	Straatsma, T. P. Free energy by molecular simulation, 2007, in Reviews in Computational chemistry. Vol 9 (Eds. K. B. Lipkowitz and D. B. Boyd), John Wiley & Sons, Inc., Hoboken, NJ, USA, pp 81-127.
[18]	Wu, K. W., Chen, P.C., Wang, J., Sun, Y. C. Computation of relative binding free energy for an inhibitor and its analogs binding with Erk kinase using thermodynamic integration MD simulation. J. Comput. Aided Mol. Des. 2012, 26, 1159-69.
[19]	Åqvist, J., Luzhkov, V. B., Brandsal, B. O. Ligand binding affinities from MD simulations. Acc. Chem. Res. 2002, 35, 358-365.
[20]	Jorgensen, W. L., Tirado-Rives, J. Potential Energy Functions for Atomic-Level Simulations of Water and Organic and Biomolecular Systems. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 6665-6670.
[21]	Ode, H.; Matsuyama, S.; Hata, M.; Hoshino, T.; Kakizawa, J.; Sugiura,W. J. Med. Chem. 2007, 50, 1768-1777.
[22]	Stoica, I., Sadiq, S. K., Coveney, P. V. Rapid and Accurate Prediction of Binding Free Energies for Saquinavir-Bound HIV-1 Proteases. J. Am. Chem. Soc. 2008, 130, 2639-2648.
[23]	Kongsted, J., Ryde, U. An improved method to predict the entropy term with the MM/PBSA approach.J. Comput. Aid. Molec. Design, 2009, 23, 63-71.
[24]	Gohlke, H., Case, D. Converging Free Energy Estimates: MM-PB (GB) SA Studies onthe Protein-Protein Complex Ras-Raf. J.Comput. Chem. 2004, 25,238-250.
[25]	Page, C.S., Bates, P.A. Can MM-PBSA Calculations Predict the Specificities of Protein Kinase Inhibitors? J. Comput. Chem., 2006, 27, 1990-2007.
[26]	Pearlman, D.A. Evaluating the molecular mechanics Poisson-Boltzmann surface areafree energy method using a congeneric series of ligands to p38 MAP kinase. J. Med. Chem. 2005, 48, 7796-7807.
[27]	Onufriev, A., Bashford, D., Case, D.A. Modification of the generalized Bornmodel suitable for macromolecules. J. Phys. Chem. B, 2000,104, 3712-3720.
[28]	Rizzo, R. C., Aynechi, T., Case, D.A., Kuntz, I. D. Estimation of absolute free energies of hydration using continuum methods: accuracy of partial charge models and optimization of non-polar contributions. J. Chem. Theory Comput. 2006, 2, 128-139.
[29]	Archontis, G.; Simonson, T., Karplus, M. Binding free energies and free energycomponents from molecular dynamics and Poisson-Boltzmann calculations. Application to amino acid recognition by aspartyl-tRNAsynthetase. J. Mol. Biol .2001, 306, 307-327.
[30]	Archontis, G., Simonson, T. Proton Binding to Proteins: A Free-Energy Component Analysis Using a Dielectric Continuum Model. Biophys. J., 2005, 88, 3888-3904.
[31]	Schutz, C. N., Warshel, A. What are the dielectric constants of proteins and how tovalidate electrostatic models? Proteins-Structure, Function and Genetics, 2001, 44, 400-417.
[32]	Simonson, T. Electrostatics and dynamics of proteins. Reports of Progress in Physics, 2003, 66, 737-787.
[33]	Tamamis, P., Morikis, D.,Floudas, C. A., Archontis, G. Species specificity of thecomplement inhibitor compstatin investigated by all-atom molecular dynamicssimulations. Proteins: Structure, Function & Bioinformatics, 2010, 78, 2655-2667.
[34]	Hayes, J. M., Archontis, G. MM-GB (PB) SA Calculations of Protein-Ligand Binding Free Energies, Molecular Dynamics - Studies of Synthetic and Biological Macromolecules, Wang, L. (Ed.), 2012, In Tech, Ch. 9; pp 171-190.
[35]	Yang, C-Y., Sun, H., Chen, J., Nikolovska-Coleska, Z., Wang, S. Importance of ligandre organization free energy in protein-ligand binding affinity prediction. J. Am. Chem. Soc. 2009, 131, 13709-13721.
[36]	Kollman, P. A., Massova, I., Reyes, C., Kuhn, B., Huo, S., Chong, L., Lee, M., Lee, T.,Duan,Y., Wang W., Donini, O., Cieplak, P., Srinivasan, J., Case, D. A., Cheatham, T.E. III, Calculating structures and free energies of complex molecules: combiningmolecular mechanics and continuum models. Acc. Chem. Res., 2000, 33,889-897.
[37]	Case, D.A., Cheathham, T. E., III, Darden, T., Gohlke, H., Luo, R., Merz, K. M., Jr., Onufriev, A., Simmerling, C., Wang, B., Woods, R.The Amber biomolecular simulation programs. J. Comput. Chem., 2005, 26, 1668-1688.
[38]	Rocchia, W., Alexov, E., Honig, B. Extending the applicability of the nonlinear Poisson-Boltzmann equation: Multiple dielectric constants and multivalentions. J. Phys. Chem. B, 2001, 105, 6507-6514.
[39]	Du, J., Sun. H., Xi, L., Li, J., Yang, Y., Liu, H., Yao, X. Molecular modeling study of Checkpoint Kinase 1 inhibitors by multiple docking strategies and Prime/MMGBSA. J. Comput. Chem., 2011, 32, 2800-2808.
[40]	Hou, T., Wang, J., Li, Y., Wang, W. Assessing the performance of the MM/PBSA and MM/GBSA methods. 1. The accuracy of binding free energy calculations basedon molecular dynamics simulations. J. Chem. Inform. Model, 2011, 51, 69-82.
[41]	Singh, N., Warshel, A. Absolute binding free energy calculations: On the accuracy of computational scoring of protein-ligand interactions. Proteins: Structure, Function& Bioinformatics, 2010, 78, 1705-1723.
[42]	Gilson, M. K., Zhou, H-X. Calculation of protein-ligand binding affinities. Ann. Rev. Biophys. Biomol. Struct., 2007, 36, 21-42.
[43]	Kuhn, B., Gerber, P., Schulz-Gasch, T., Stahl, M. Validation and use of the MMPBSAapproach for drug discovery. J. Med. Chem., 2005, 48, 4040-4048.
[44]	Hayes, J.M., Skamnaki, V.T., Archontis, G., Lamprakis, C.,Sarrou, J., Bischler, N., Skaltsounis, A. L., Zographos, S.E. ,Oikonomakos, N. G. Kinetics, in silicodocking, molecular dynamics, and MM-GBSA binding studies on prototypeindirubins, KT 5720, and staurosporine as phosphorylase kinase ATP-binding site inhibitors: The role of water molecules examined. Proteins: Structure Function &Bioinformatics, 2011, 79, 703-719.
[45]	Rastelli, G., Del Rio, A., Degliesposti, G., Sgobba, M. Fast and accuratepredictions of binding free energies using MM-PBSA and MM-GBSA. J. Comput. Chem. 2009, 31, 797-810.
[46]	Hu, R., Barbault, F., Maurel, F., Delamar, M., Zhang, R. Molecular dynamicssimulations of 2-amino-6-arylsulphonylbenzonitriles analogues as HIV inhibitors: interaction modes and binding free energies. Chem. Biol. Drug.Des., 2010, 76, 518-526.
[47]	Balius, T. E., Rizzo, R.C. Quantitative prediction of fold resistance forinhibitors of EGFR. Biochemistry, 2009,48, 8435-8448.
[48]	Kollman, P.A., Massova, I., Reyes, C., Kuhn, B., Huo, S. H., Chong, L., Lee. M., Lee, T., Duan, Y., Wanh, W., Donini, O., Cieplak, P., Srinivasan, J., Case, D.A., Cheatham, T. E. III. Calculating structures and free energies of complex molecules: Combining molecular mechanics and continuum models. Acc. Chem. Res., 2000, 33, 889-897.
[49]	Lee, M.S., Olson, M.A. Calculation of absolute protein-ligand bindingaffinity using path and endpoint approaches. Biophys. J. 2006, 90, 864-877.
[50]	Baris, I., Tuncel, A., Ozber, N., Keskin, O., Kavakli, I. H. Investigation of theInteraction between the Large and Small Subunits of Potato ADP-GlucosePyrophosphorylase. PLoSComput.Biol., 2009,5, e 1000546.
[51]	Chen, Q., Cui, W., Ji, M. Studies of chirality effect of 4-(phenylamino)-pyrrolo2, 1-f 1, 2, 4 triazine on p38alpha by molecular dynamics simulationsand free energy calculations. J.Comput. Aided. Mol. Des. 2009, 23, 737-745.
[52]	Li, W. H., Tang, Y., Hoshino, T., Neya, S. Molecular modeling of humancytochrome P450 2W1 and its interactions with substrates. J. Mol. Graph. Model, 2009, 28, 170-176.
[53]	Yang, C. Y., Sun, H. Y., Chen, J. Y., Nikolovska-Coleska, Z., Wang, S. M. Importance of Ligand Reorganization Free Energy in Protein-Ligand Binding-Affinity Prediction. J. Am. Chem. Soc. 2009, 131, 13709-13721.
[54]	Xie, L., Evangelidis, T., Xie, L., Bourne, P.E. Drug Discovery Using Chemical Systems Biology: Weak Inhibition of Multiple Kinases May Contribute to the Anti-Cancer Effect of Nelfinavir. PLoSComput. Biol., 2011, 7, e 1002037.
[55]	Feig, M., Onufriev, A., Lee, M.S., Im, W., Case, D.A., Brooks, C. L. III. Performancecomparison of generalized born and Poisson methods in the calculation ofelectrostatic solvation energies for protein structures. J. Comput. Chem., 2004, 25, 265-284.
[56]	Onufriev, A., Bashford, D., Case, D.A. Exploring protein native states andlarge-scale conformational changes with a modified generalized born model. Proteins, 2004, 55, 383-394.
[57]	Weiser, J., Shenkin, P.S., Still, W. C. Approximate atomic surfaces from linearcombinations of pairwise overlaps (LCPO). J. Comput. Chem. 1999, 20, 217-230.
[58]	Gouda, H., Kuntz, I.D., Case, D.A., Kollman, P.A. Free energy calculations for theophylline binding to an RNA aptamer: Comparison of MM-PBSA and thermodynamic integration methods. Biopolymers, 2003, 68, 16-34.
[59]	Lee, M.R., Duan, Y., Kollman, P.A. Use of MM-PB/SA in estimating the freeenergies of proteins: Application to native, intermediates, and unfolded villinheadpiece. Proteins 2000, 39, 309-316.
[60]	Ahmed, M., Sadek, M.M., Serrya, R.A., Kafafy, A-H.N., Abouzid, K. A., Wang, F. Assessment of new anti-HER2 ligands using combined docking, QM/MM scoring and MD simulation. J. Mol. Graph. Mod. 2013, 40, 91-98.
[61]	Grant, J. A., Pickup, B. T., Nicholls, A. A smooth permittivity function for Poisson-Boltzmann solvation methods, J. Comput. Chem. 2001, 22, 608-640.
[62]	Kollman, P.A., Massova, I., Reyes, C., Kuhn, B.,Huo, S., Chong, L., Lee, M., Lee, T.,Duan, Y., Wang, W., Donini, O., Cieplak, P., Srinivasan, J., Case, D.A., Cheatham, T.E. III. Calculating structuresand free energies of complex molecules: combining molecularmechanics and continuum models, Acc. Chem. Res. 2000, 33, 889-897.
[63]	Fogolari, F., Brigo, A., Molinari, H. Protocol for MM/PBSA moleculardynamics simulations of proteins.Biophys. J. 2003, 85 159-166.
[64]	Gohlke, H., Kiel, C., Case, D.A. Insights into protein-protein bindingby binding free energy calculation and free energy decomposition forthe Ras-Raf and Ras-RaIGDS complexes, J. Mol. Biol. 2003, 330, 891-913.
[65]	Simonson, T., Georgios, A., Karplus, M. Free Energy SimulationsCome of Age: Protein-Ligand Recognition. Acc. Chem. Res. 2002, 35,430-437.
[66]	Steinbrecher, T.,Hrenn, A., Dormann, K., Merfort, I.,Labahn,A. Bornyl (3,4,5-trihydroxy)-cinnamatean optimized human neutrophilelastase inhibitor designed by free energy calculations. Bioorg. Med. Chem. 2008, 16, 2385-2390.
[67]	Lu, N., Kofke, D. A., Woolf, T. B. Improving the Efficiency andReliability of Free Energy Perturbation Calculations Using OverlapSampling Methods. J. Comput. Chem. 2004, 25, 28-39.
[68]	Jorgensen, W. L., Thomas, L. T. Perspective on Free-EnergyPerturbation Calculations for Chemical Equilibria. J. Chem. TheoryComput. 2008, 4, 869-876.
[69]	Zhou, Huan-Xiang; Gilson, Michael K. Theory of free energy and entropy in noncovalentbinding. Chem. Rev. 2009109, 4092-4107.
[70]	Swanson, Jessica M J., Henchman, Richard H., Andrew McCammon, J. Revisiting free energy calculations: a theoretical connection to mm/pbsa and direct calculation of the association free energy. Biophys J. 2004, 86, 67-74.
[71]	Khavrutskii, I.V., Wallqvist, A. Computing relative free energies of solvation usingsingle reference thermodynamic integration augmented with hamiltonian replica exchange. J. Chem. Theor.Comput. 2010, 6, 3427-3441.
[72]	Brown, S. P., Muchmore, S.W. Rapid estimation of relative protein-ligand bindingaffinities using a high-throughput version of mm-pbsa. J. Chem. Inf. Model. 2007, 47, 1493-1503.
[73]	Michel, J.,Verdonk, M.L., Essex, J. W. Protein-Ligand Binding Affinity Predictions by Implicit Solvent Simulations: A Tool for LeadOptimization?J. Med. Chem. 2006, 49, 7427-7439.
[74]	For reviews, see: Free Energy Calculations in Rational Drug Design; Reddy, M. R., Erion, M.D., Eds.; Kluwer/Plenum Press: New York, 2001.
[75]	Cross, J. B., Thompson, D.C. Rai, B. K., Baber, J.C., Fan, K. Y., Hu, Y., Humblet, C.C omparison of Several Molecular Docking Programs: Pose Prediction and Virtual Screening Accuracy. J. Chem. Inf. Model. 2009, 49, 1455-1474.
[76]	Friesner, R.A., Murphy, R. B., Repasky, M. P., Frye, L.L., Greenwood, J. R., Halgren, T. A., Sanschagrin, P. C.. Mainz, D.T. Extraprecision glide: docking and scoring incorporating a model of hydrophobicenclosure for protein-ligand complexes. J. Med. Chem. 2006, 49, 6177-6196.
[77]	Amorim, H.L., Caceres, R.A., Netz, P.A. Linear interaction energy (LIE) method in lead discovery and optimization. Curr. Drug Targets, 2008, 9, 1100-1105.
[78]	Gutierrez-de-Terán, H., Åqvist, J. Linear interaction energy: method and applications in drug design. Methods Mol. Biol. 2012, 819, 305-323.
[79]	Zhou, R.H., Friesner, R.A., Ghosh, A., Rizzo, R.C., Jorgensen, W.L., Levy, R.M. New linear interaction method for binding affinity calculations using a continuum solvent model. J. Phys. Chem. B, 2001, 105, 10338-10397.
[80]	Huang, D., Catflisch, A. Efficient evaluation of binding free energy using continuum electrostatics solvation. J. Med. Chem., 2004, 47, 5791-5797.
[81]	Carlsson, J., Ander, M., Nervall, M., Åqvist, J. Continuum solvation models in the linear interaction energy method. J. Phys. Chem. B, 2006, 110, 12034-12041.
[82]	Alonso, H., Blinznyuk, A. A., Gready, J. E. Combining docking and molecular dynamic simulations in drug design. Med. Res. Rev. 2006, 26, 531-568.
[83]	Amaro, R.E., Baron, R., McCammon, J.A. An improved relaxed complex scheme for receptor flexibility in computer-aided drug design. J. Comput. Aided Mol. Des. 2008, 22, 693-705.
[84]	Lin, J. H., Perryman, A. L., Schames, J. R., McCammon, J, A. The relaxed complex method: accommodating receptor flexibility for drug design with an improved scoring scheme. Biopolymers, 2003, 68, 47-62.
[85]	Chodera, J.D., Mobley, D.L., Shirts, M. R., Dixon, R.W., Branson K., Pande V. S. Alchemical free energy methods for drug discovery: progress and challenges. Curr. Opin. Struct. Biol., 2011, 21, 150-160.
[86]	Kitchen, D. B., Decornez, H., Furr, J. R., Bajurath, J. Docking and scoring in virtual screening for drug discovery: methods and applications. Nat. Rev. Drug Discov. 2004, 3, 935-949.
[87]	Taylor, R.D., Jewsbury, P. J., Essex, J. W. A review of protein-small molecule docking methods. J. Comput. Aided Mol. Des. 2002, 16, 151-166.
[88]	Huang, S-Y., Zou, X. Advances and challenges in protein-ligand docking. Int. J. Mol. Sci. 2010, 11, 3016-3034.
[89]	Lee, W., Park, H., Lee, S. Molecular dynamics free energy simulation study to rationalize the relative activities of PPAR δ agonists. Bull. Kor. Chem. Soc., 2008, 29, 363-371.
[90]	Lu, N., Woolf, T. B. Chapter 6: Understanding and improving free energycalculations in molecular simulations: Error analysis and reduction methods. In: Free Energy Calculations: Theory & Applications in Chemistry & Biology (Springer Seriesin Chemical Physics Vol.86), Chipot, C., Pohorille, A. (Eds), Springer-Verlag, 2007, Berlin-Heidelberg.
[91]	Schwab, F., van Gunsteren, W.F., Zagrovi, B. Mechanism for the transport of ammonia within carbamoyl phosphate synthetase determined by molecular dynamics simulations. Biochemistry 2008, 47, 2935-2944.
[92]	Åqvist, J., Luzhkov, V.B., Brandsdal, B.O. Ligand binding affinities from MDsimulations. Acc. Chem. Res., 2002, 35, 358-365.
[93]	Gohlke, H., Kiel, C., Case, D.A. Insight into protein-protein binding by binding freeenergy calculation and free energy decomposition for the Ras-Raf and Ras-RalGDScomplexes. Journal of Molecular Biology, 2003, 330, 891-913.
[94]	Olsson, T.S.G., Williams, M.A., Pitt, W.R., Ladbury, J. E. Thethermodynamics of protein-ligand interaction and solvation:insights for ligand design. J. Mol. Biol., 2008, 384, 1002-1017.
[95]	Ma, X.H., Wang, C.X., Li, C.H., Chen, W.Z. A fast empirical approach to binding free energy calculations based on protein interface information. Prot. Eng.2002, 15, 677-681.
[96]	Gilson, M.K., Zhou, H.-X. Calculation of protein-ligand bindingaffinities.Annu. Rev. Biophys. Biomol. Struct.2007, 36, 21-42.
[97]	Exner, O. Entropy-enthalpy compensation and anticompensation: solvation and ligand binding. Chem. Commun. 2000, 1655-1656.
[98]	Sharp, K. Entropy-enthalpy compensation: fact or artifact? Protein Sci. 2001, 10, 661-667.
[99]	GallicchioE, Levy, R. M. Recent Theoretical and Computational Advances for Modeling Protein-Ligand Binding Affinities.Adv Protein ChemStruct Biol. 2011.85, 27-80.
[100]	Ford, D.M. Enthalpy-entropy compensation is not a generalfeature of weak association. J. Am. Chem. Soc. 2005, 127, 16167-16170.
[101]	Sharp, K. Entropy-enthalpy compensation: fact or artifact? Prot. Sci. 2001, 10, 661-667.
[102]	Houk, K. N., Leach, A.G., Kim, S. P., Zhang, X. Bindingaffinities of host-guest, protein-ligand and protein-transitionstate complexes. Angew. Chem., Int. Ed. 2003, 42, 4872-4897.
[103]	Whitesides, G. M., Krishnamurthy, V. M. Designing ligands tobind proteins. Q. Rev. Biophys. 2005, 38, 385-395
[104]	Williams, D. H., Stephens, E., O’Brien, D. P., Zhou, M. Understandingnoncovalent interactions: ligand binding energy andcatalytic efficiency from ligand-induced reductions in motionwithin receptors and enzymes. Angew. Chem., Int. Ed. 2004, 43, 6596-6616.
[105]	Muley, L., Baum, B., Smolinski, M., Freindorf, M., Heine, A., Klebe, G., Hangauer, D. Enhancement of hydrophobic interactions and hydrogen bond strength by cooperativity: synthesis, modeling and MD-simulations of a congeneric series of thrombin inhibitors. J. Med. Chem. 2010, 53, 2126-2135.
[106]	Baum, B., Muley, L., Smolinski, M., Heine, A., Hangauer, D., Klebe G. Non-additivity of functional group contributions in protein-ligand binding: a comprehensive study by crystallography and isothermal titration calorimetry. J. Mol. Biol. 2010, 397, 1042-1054.
[107]	Scatena, L. F., Brown, M. G., Richmond, G. L. Water athydrophobic surfaces: weak hydrogen bonding and strong orientationeffects. Science 2001, 292, 908-912.
[108]	Scatena, L. F., Brown, M. G., Richmond, G. L. Water at hydrophobic surfaces: weak hydrogen bonding and strong orientation effects. Science, 2001, 292, 908-912.
[109]	Dill, K. A., Truskett, T. M., Vlachy, V., Hribar-Lee, B. Modelingwater, the hydrophobic effect, and ion solvation. Annu. Rev. Biophys. Biomol. Struct. 2005, 34, 173-199.
[110]	Meyer, E. A., Castellano, R. K., Diederich, F. Interactions witharomatic rings in chemical and biological recognition.Angew. Chem., Int. Ed. 2003, 42, 1210-1250.
[111]	Snyder, P. W., Mecinovic, J., Moustakes, D.T., Thomas, S.W. III, Harder, M., Meck, E. T., Lockett, M. R., Héroux, A., Sherman, W., Whitesides, G. M. Mechanism of the hydrophobic effect in the biomolecular recognition of arylsulphonamides by carbonic anhydrase. PNAS, 2011, 108, 17889-17894.
[112]	García-Sosa, A. T. Hydration properties of ligands and drugs in protein binding sites: tightly-bound, bridging water molecules and their effects and consequences on molecular design strategies. J. Chem. Inf. Mod. 2013, 53, 1388-1405.
[113]	Michel, J., Tirado-Rives, J., Jorgensen, W. L. Prediction of the Water Content in Protein Binding Sites. J. Phys. Chem. B, 2009, 113, 13337-13346.
[114]	Garcia-Sosa, A. T., Mancera, R. L., Dean, P.M. WaterScore: anovel method for distinguishing between bound and displaceablewater molecules in the crystal structure of the binding site ofprotein-ligand complexes. J. Mol. Model. 2003, 9, 172-182.
[115]	Amadasi, A., Surface, J. A., Spyrakis, F., Cozzini, P., Mozzarelli, A., Kellogg, G. E. Robust classification of “relevant” watermolecules in putative protein binding sites. J. Med. Chem. 2008, 51, 1063-1067.
[116]	Boehm, H.-J., Boehringer, M., Bur, D., Gmuender, H., Huber,W., Klaus, W., Kostrewa, D., Kuehne, H., Luebbers, T., Meunier-Keller, N., Mueller, F. Novel inhibitors of DNA gyrase: 3Dstructure based biased needle screening, hit validation by biophysicalmethods, and 3D guided optimization. A promising alternativeto random screening. J. Med. Chem. 2000, 43, 2664-2674.
[117]	Garcia-Sosa, A. T., Firth-Clark, S., Mancera, R. L. Includingtightly-bound water molecules in de novo drug design. Exemplificationthrough the in silico generation of poly (ADP-ribose)-polymerase ligands. J. Chem. Inf. Model. 2005, 45, 624-633.
[118]	Vijayan, R., Sahai, M.A., Czajkowski, T., Biggin, P. C. A comparative analysis of the role of water in the binding pockets of ionotropic glutamate receptors. Phys. Chem. Chem. Phys. 2010, 12, 14057-14066.
[119]	http://www.desertsci.com/ (accessed September 3, 2013).
[120]	Dodson, G. G., Lane, D. P., Verma, C. S. Molecular simulations of protein dynamics: New windows onmechanisms in biology. EMBO Reports 2008, 9,144-150.
[121]	Okazaki, K. I., Takada, S. Dynamic energy landscape view of coupled binding and proteinconformational change: Induced-fit versus population-shift mechanisms. Proc. Natl. Acad. Sci. USA. 2008, 105, 11182-11187.
[122]	Wong, C.F. Flexible ligand-flexible protein docking in protein kinase systems. Biochim. Biophys. Acta-Proteins and Proteomics 2008, 1784, 244-251.
[123]	Nabuurs, S. B., Wagener, M., De Vlieg, J. A flexible approach to induced fit docking. J. Med. Chem .2007, 50, 6507-6518.
[124]	Koska, J., Spassov, V. Z., Maynard, A. J., Yan, L., Austin, N., Flook, P. K., Venkatachalam, C M. Fully automatedmolecular mechanics based induced fit protein-ligand docking method. J. Chem. Inf. Mod.2008, 48, 1965-1973.
[125]	Sánchez-Moreno, M., Gómez-Contreras, F., Navarro, P., Marín, C., Olmo, F., Yunta, M.J.R., Sanz, A.M., Rosales, M. J., Cano, C., Campayo, L.Phthalazine Derivatives Containing Imidazole Rings Behave as Fe-SOD Inhibitors and Show Remarkable Anti-T. cruzi Activity in Immunodeficient-Mouse Mode of Infection. J. Med. Chem., 2012, 55, 9900-9913.
[126]	Cornell, W. D., Cieplak, P., Bayly, C.I., Gould, I. R., Merz, K. M. Jr., Ferguson, D. M., Spellmeyer, D.C., Fox, T., Caldwell, J. W., Kollman, P.A. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc 1995; 117, 5179-5197.
[127]	HyperChem(TM) Professional 8.0, Hypercube, Inc., 1115 NW 4th Street, Gainesville, Florida 32601, USA.
[128]	Miranda, C., Escartí, F., Lamarque, L., Yunta, M. J. R., Navarro, P., García-España, E., Jimeno, M. L. New 1H-pyrazole-containing polyamine receptors able to complex L-glutamate in water at physiological pH valuesJ. Am. Chem. Soc. 2004, 126, 823-833 and references cited therein.
[129]	Reviriego, F., Navarro, P., García-España, E., Albelda, M. T., Frias, J.C., Domènech, A., Yunta, M. J. R., Costa, R., Ortí, E.Diazatetraester 1H-pyrazolecrowns as fluorescent chemosensors for AMPH, METH, MDMA (ecstasy), and dopamine. Org. Let .2008; 10, 5099-5102.
[130]	Moro, S., Deflorian, F., Bacilieri, M., Spalluto, G. Ligand-based homology modelling as attractive tool to inspect GPCR structural plasticity.Curr. Pharm. Des. 2006, 12, 2175-2185.
[131]	Colotta, V., Catarzi, D., Varano, F., Capelli, F.,Lenzi, O., Filacchioni, G., Martini, C., Trincavelli, L., Ciampi, O., Pugliese, A.M., Pedata, F.,Schiesaro, A., Morizzo, E., Moro, S. New 2-Arylpyrazolo 3, 4-cquinoline Derivatives as Potent and Selective Human A3 Adenosine Receptor Antagonists. Synthesis, Pharmacological Evaluation, and Ligand-Receptor Modeling Studies. J. Med. Chem. 2007, 50, 4061-4074.