PERSPECTIVE Challenges in the determination of the binding modes of non-standard ligands in X-ray crystal complexes Alpeshkumar K. Malde • Alan E. Mark Received: 8 October 2010 / Accepted: 25 October 2010 / Published online: 4 November 2010 Ó Springer Science+Business Media B.V. 2010 Abstract Despite its central role in structure based drug design the determination of the binding mode (position, orientation and conformation in addition to protonation and tautomeric states) of small heteromolecular ligands in pro- tein:ligand complexes based on medium resolution X-ray diffraction data is highly challenging. In this perspective we demonstrate how a combination of molecular dynamics simulations and free energy (FE) calculations can be used to correct and identify thermodynamically stable binding modes of ligands in X-ray crystal complexes. The conse- quences of inappropriate ligand structure, force field and the absence of electrostatics during X-ray refinement are high- lighted. The implications of such uncertainties and errors for the validation of virtual screening and fragment-based drug design based on high throughput X-ray crystallography are discussed with possible solutions and guidelines. Keywords X-ray crystallography Á Ligand design Á Molecular dynamics simulations Á Free energy calculations Á Binding mode Abbreviations aaRSs Aminoacyl-tRNA synthetases ATB Automated Topology Builder CDK Cyclin Depdendent Kinase CNS Crystallography and NMR System CR6 1-deoxy-1-acetylamino-b-D-gluco-2- heptulopyranosonamide FE Free Energy GLG a-D-glucopyranosyl-2-carboxamide GPb Glycogen Phosphorylase b HIV-1 Human Immunodeficiency Virus-1 JG-365 Ac-Ser-Leu-Asn-Phe-W[CH(OH)CH 2 N]-Pro- Ile-Val-OMe L-Ser L-Serine LIGA (4-{4H-indeno[1,2-c]pyrazol-3-yl}pyridine) LIGB (4-{1H,4H-indeno[1,2-c]pyrazol-3- yl}pyridine) MD Molecular Dynamics MM Molecular Mechanics Pab-NTD N-terminal editing domain of Pyrococcus abyssi threonyl-tRNA synthetase PDB Protein Data Bank PDE4B Phosphodiesterase 4B PNMT Phenylethanolamine N-methyltransferase QM Quantum Mechanics ROL Rolipram SC558 1-phenylsulfonamide-3-trifluoromethyl-5-p- bromophenylpyrazole SKF 1,2,3,4-tetrahydro-isoquinoline-7- sulphonicacidamide TetR Tet repressor protein Introduction X-ray crystallography is an indispensable tool in structural biology and rational drug design. However, while the Electronic supplementary material The online version of this article (doi:10.1007/s10822-010-9397-6) contains supplementary material, which is available to authorized users. A. K. Malde Á A. E. Mark (&) School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, QLD 4072, Australia e-mail: a.e.mark@uq.edu.au A. E. Mark Institute for Molecular Bioscience, University of Queensland, St. Lucia QLD 4072, Australia 123 J Comput Aided Mol Des (2011) 25:1–12 DOI 10.1007/s10822-010-9397-6