Université Pierre et Marie Curie Ecole doctoral de Chimie Moléculaire de Paris Centre Institut Parisien de Chimie Moléculaire/ Equipe MACO On the Role of Protons in the Reactivation of Acetylcholinesterase: Quantum and Molecular Mechanics Studies Présentée par : Thomas Driant Pour obtenir le grade de Docteur de l’université Pierre et Marie Curie Soutenance publique prévue le 22 septembre 2017 Devant un jury composé de : Madame Isabelle NAVIZET Professeur Rapporteur Monsieur Sam de VISSER Professeur Rapporteur Madame Shina Caroline Professeur Examinateur Lynn KAMERLIN Monsieur Florian NACHON Ingénieur d'études et Examinateur de fabrications HDR Monsieur Philippe KAROYAN Professeur Examinateur Monsieur Matthieu SOLLOGOUB Professeur Examinateur Monsieur Etienne DERAT Maître de Conférences HDR Directeur de thèse Acknowledgments My PhD Project was carried out at the Institut Parisien de Chimie Moléculaire, directed by Doctor Corinne Aubert. I worked as a member of the MACO team directed by Professor Louis Fensterbank. Firstly, I would like to address sincere thanks to Professor Isabelle Navizet, Professor Sam de Visser, Professor Shina Caroline Lynn Kamerlin, Professor Philippe Karoyan, Docteur Florian Nachon, and Professeur Matthieu Sollogoub for accepting to review my thesis and be part of my Jury. I am especially grateful to Doctor Etienne Derat for trusting me with this project. Looking back at my progress and maturation as a researcher I am proud of what I achieved and have you to thank for it. You have always been available for me to help me, guide me, teach me, and discuss the project. I am honoured by the confidence you put in me and I hope to have risen to the opportunity you have given me three years ago. I am also grateful to Professor Shina Caroline Lynn Kamerlin for hosting me in her team in Uppsala University for three months. I thank you for the substantial contribution you have made to my scientific growth throughout our collaboration. I also thank all the members of both the MACO team and the team of the Kamerlin lab, you are wonderful colleagues and I deeply enjoyed working with all of you. Finally, I would like to thank my friends, my family and Anne for their love and support during those three years. 3 Table of Contents ACKNOWLEDGMENTS ............................................................................................ 3 TABLE OF CONTENTS .............................................................................................. 4 GENERAL INTRODUCTION .................................................................................... 11 I. Biological significance of acetylcholinesterase ........................................................ 11 I.1. Role as a mediator of signal transmission .................................................................... 11 I.2. Target of nerve agents.................................................................................................. 13 II. Scope of this work .................................................................................................. 15 CHAPTER 1: INTRODUCTION ON ACETYLCHOLINESTERASE: STRUCTURE, FUNCTION, AND MECHANISM .............................................................................. 19 I. Structure of acetylcholinesterase ........................................................................... 19 I.1. Active site ..................................................................................................................... 23 I.2. Enzyme channel of AChE .............................................................................................. 29 II. Activity of acetylcholinesterase .............................................................................. 38 II.1. Regular catalytic activity ............................................................................................... 38 II.2. Inhibition by organophosphates .................................................................................. 40 II.3. Reactivation of acetylcholinesterase............................................................................ 46 III. Molecular simulations of AChE ............................................................................... 54 III.1. Regular catalytic activity ............................................................................................... 54 III.2. Inhibition by nerve agents ............................................................................................ 66 III.3. Reactivation by oximes ................................................................................................. 76 IV. Conclusions on acetylcholinesterase literature ....................................................... 84 CHAPTER 2: INTRODUCTION TO THE COMPUTATIONAL METHODS ..................... 87 I. Quantum mechanics .............................................................................................. 87 I.1. From molecular orbital method to Hartree-Fock theory ............................................. 88 I.2. Density functional theory ............................................................................................. 93 I.3. Valence bond theory .................................................................................................... 97 II. Molecular mechanics ............................................................................................. 99 II.1. Classical force fields ...................................................................................................... 99 II.2. Polarizable force fields ............................................................................................... 101 II.3. Molecular dynamics ................................................................................................... 103 II.4. Constant pH Molecular Dynamics .............................................................................. 104 4 II.5. Empirical Valence Bond .............................................................................................. 105 III. QM/MM ................................................................................................................ 87 III.1. Additive or Subtractive ............................................................................................... 109 III.2. Boundary scheme ....................................................................................................... 111 III.3. Interactions between QM and MM ............................................................................ 113 III.4. How to set up QM/MM simulations .......................................................................... 114 IV. Conclusion ............................................................................................................ 118 CHAPTER 3: TO DIVIDE AND CONQUER .............................................................. 121 I. Introduction .......................................................................................................... 121 II. Minimal model...................................................................................................... 124 III. The role of Glu334 ................................................................................................. 128 IV. The oxyanionic hole .............................................................................................. 131 V. The aromatic rings in the active site of AChE ......................................................... 136 V.1. Benzene ...................................................................................................................... 137 V.2. Phenol ......................................................................................................................... 144 V.3. Indole .......................................................................................................................... 150 VI. Discussion ............................................................................................................. 156 VII. Conclusions ........................................................................................................... 160 CHAPTER 4: QM/MM SIMULATIONS OF ACETYLCHOLINESTERASE REACTIVATION BY 2-PAM ............................................................................................................ 163 I. Computational details ........................................................................................... 163 II. Destabilization of the frontier orbitals of the systems ............................................ 169 III. Orientation of 2-PAM ............................................................................................ 173 IV. Study of the protonation state of Glu334 .............................................................. 173 V. VX-inhibited AChE reactivation by 2-PAM .............................................................. 175 V.1. Reactivation of AChE with an unprotonated Glu202 ................................................. 176 V.2. Reactivation of AChE with a protonated Glu202 ....................................................... 179 V.3. Reactivation with an unprotonated His447 and a protonated Glu202 ..................... 181 V.4. QM model confirmation ............................................................................................. 182 V.5. Alternate approach of 2-PAM towards the phosphorus ............................................ 184 VI. Deprotonation of 2-PAM in the active site of AChE ...............................................