Simulation of protein dynamics for mechanistic insight and drug design A thesis submitted to The University of Manchester for the degree of Master of Philosophy in the Faculty of Biology, Medicine and Health 2019 YANG, Zhang School of Health Sciences/Division of Pharmacy List of contents List of figures ............................................................................................................................ 5 List of tables ........................................................................................................................... 14 List of abbreviations ............................................................................................................... 16 Abstract .................................................................................................................................. 18 Declaration ............................................................................................................................. 20 Copyright Statement .............................................................................................................. 21 Acknowledgements ................................................................................................................ 22 Introduction to protein dynamics in drug design .................................................................. 23 In silico design of anti-cancer compounds targeting R-spondin using molecular dynamics and free energy calculations .................................................................................................. 24 1. Background ........................................................................................................................ 24 1.1 Wnt signalling pathway ............................................................................................ 24 1.2 R-spondin signalling and disease ............................................................................. 26 1.3 Structure basis of R-spondin signalling .................................................................... 29 1.4 Protein dynamics...................................................................................................... 31 1.5 Aim of this work ....................................................................................................... 33 2. Computational Theory ...................................................................................................... 35 2.1 Protein-ligand binding site prediction ..................................................................... 35 2.1.1 Shape-based methodology ............................................................................... 36 2.1.2 Energy-based methodology .............................................................................. 37 2.2 Molecular docking .................................................................................................... 38 2.2.1 Molecular docking algorithms .......................................................................... 39 2.2.2 Scoring functions ............................................................................................... 41 2.3 Molecular dynamics ................................................................................................. 43 2.3.1 Molecular mechanics ........................................................................................ 43 2.3.2 Water solvent models ....................................................................................... 45 2.3.3 Periodical boundary conditions ........................................................................ 46 2.3.4 Temperature and pressure regulation .............................................................. 47 2.4 Free energy calculation ............................................................................................ 48 2.4.1 Poisson Boltzmann potential ............................................................................ 48 2.4.2 Generalized Born model ................................................................................... 49 2.4.3 MM-GBSA method ............................................................................................ 50 3. High throughput virtual screening .................................................................................... 51 2 3.1 Compound database refinement ............................................................................. 53 3.2 Binding pocket identification ................................................................................... 55 3.2.1 Closed and Open2 R-spondin binding pockets ................................................. 55 3.2.2 RNF43 binding pockets ..................................................................................... 61 3.2.3 LGR5 binding pockets ........................................................................................ 63 3.3 Molecular docking and protein-ligand interactions ................................................. 66 3.3.1 Methods ............................................................................................................ 66 3.3.2 Closed R-spondin interactions .......................................................................... 67 3.3.3 Open2 R-spondin interactions .......................................................................... 70 3.3.4 RNF43 interactions ............................................................................................ 72 3.3.5 LGR5 interactions .............................................................................................. 76 4 Molecular dynamics simulation ......................................................................................... 79 4.1 Methods ................................................................................................................... 80 4.1.1 Model construction ........................................................................................... 80 4.1.2 Molecular dynamics protocol ........................................................................... 81 4.1.3 Compounds selection and optimization ........................................................... 81 4.1.4 Analysis tools..................................................................................................... 82 4.2 Results: Closed R-spondin ligands ............................................................................ 83 4.2.1 Compound selection ......................................................................................... 83 4.2.2 Ligand Binding stabilities................................................................................... 90 4.2.3 Ligand structure refinement ............................................................................. 94 4.2.4 Special cases .................................................................................................... 100 4.3 Results: Open2 R-spondin ligands .......................................................................... 105 4.3.1 Compound selection ....................................................................................... 105 4.3.2 Ligand Binding stabilities................................................................................. 110 4.3.3 Ligand structure refinement ........................................................................... 113 4.3.4 Special cases .................................................................................................... 118 4.4 Results: RNF43 ligands ........................................................................................... 123 4.4.1 Compound selection ....................................................................................... 123 4.4.2 Ligand Binding stabilities................................................................................. 128 4.4.3 Ligand structure refinement ........................................................................... 137 4.5 Results: LGR5 ligands ............................................................................................. 141 4.5.1 Compound selection ....................................................................................... 141 4.5.2 Ligand Binding stabilities................................................................................. 143 4.5.3 Ligand structure refinement ........................................................................... 149 4.5.4 Special cases .................................................................................................... 155 3 5. Free energy calculation ................................................................................................... 157 5.1 Methods ................................................................................................................. 157 5.2 Binding free energy and energy decomposition .................................................... 159 5.2.1 Closed R-spondin ............................................................................................ 159 5.2.2 Open2 R-spondin ............................................................................................ 162 5.2.3 RNF43 .............................................................................................................. 169 5.2.4 LGR5 ................................................................................................................ 173 6. Discussion .......................................................................................................................