COMPUTATIONAL ANALYSIS OF CYCLOOXYGENASE INHIBITION: ENERGETICS AND DYNAMICS By Christopher Williams Moth Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Chemistry May, 2008 Nashville, Tennessee Approved: Professor Terry P. Lybrand Professor Lawrence J. Marnett Professor Michael R. Waterman Professor Ned A. Porter Professor Alan R. Brash DEDICATION To my father, Brian, who wished he could have been a chemist. To my wife, Valerie, for encouraging me to pursue my dreams. ii ACKNOWLEDGEMENTS There are no adequate words to convey my sincerest thanks to Dr. Terry Lybrand for inviting me into in his lab, and to Dr. Larry Marnett for allowing me to attend his experimental lab meetings. I deeply appreciate the guidance and patience of my committee, as I navigated the murky waters of biomolecular simulation. The interdisciplinary atmosphere at Vanderbilt has provided a wonderful backdrop for all of my studies. Thanks to Al Beth and the Chemical and Physical Biology (CPB) program for funding a flexible starting point for my Ph.D. Studies. I have enjoyed financial support from the National Institute of Health through Chemistry Biology Interface training grant T32 GM65086-02 and N.I.H. Research grants NS33290 (Lybrand) and CA89450 (Marnett). And, thanks to the Vanderbilt Dept. of Chemistry for allowing me to T.A. and grade courses in order to fund the most recent years of my graduate studies. We are incredibly fortunate at the Structural Biology Center to have excellent computing resources, and support infrastructure. Special thanks to Jarrod Smith, Brandon Valentine, Roy Hoffman, Sabuj Pattanayek, and Holland Griffis for all their help keeping our software current, and our hardware working well. Thanks also to Joel Harp for his tireless assistance with my X-ray crystallography questions. For the work in chapter IV, I thank Professor Mihaly Mezei of Mount Sinai School of Medicine for providing his Grand Canonical Monte Carlo code. This work was also made possible, in part, with resources from the Advanced Computing Center for Research and Education at Vanderbilt University. iii Very little of this dissertation would have been completed without the ground- breaking cyclooxygenase simulation work performed by Kristina Furse. Kristina developed the fundamental protocols for simulating the full Cyclooxygenase dimer in a solvated truncated octahedron, the channel_finder utility employed in chapters III and IV, and the steric shielding analysis scripts of chapter IV. iv TABLE OF CONTENTS DEDICATION.....................................................................................................................ii ACKNOWLEDGEMENTS................................................................................................iii LIST OF FIGURES...........................................................................................................vii LIST OF SCHEMES..........................................................................................................ix LIST OF TABLES..............................................................................................................ix Chapter Page I. INTRODUCTION....................................................................................................1 Cyclooxygenase..............................................................................................................1 Mechanism.................................................................................................................4 Active Site..................................................................................................................5 Inhibition....................................................................................................................8 COX-2...........................................................................................................................11 Val-523: The Billion Dollar Residue........................................................................12 Alternate COX-2 Inhibition Strategies.....................................................................16 Indomethacin Amides...............................................................................................19 Dissertation Overview...................................................................................................20 II. ENANTIOSELECTIVE INHIBITION OF CYCLOOXYGENASE-1.................22 Background...................................................................................................................22 Methods.........................................................................................................................26 Results and Discussion..................................................................................................29 Other Proposals.............................................................................................................46 Conclusions...................................................................................................................51 III. THE L472M MUTATION IN COX-2...................................................................53 Background...................................................................................................................53 The Discovery of L472M.........................................................................................53 M472L “Reverse” Mutation in COX-1....................................................................56 Methods.........................................................................................................................57 Structural Analysis of L472M.......................................................................................61 Inspection of Crystal Structures...............................................................................61 Molecular Dynamics Simulation..............................................................................61 Quasi-harmonic Analysis..........................................................................................62 Kinetic Analysis of L472M COX-2-Inhibitor Binding and Inhibition.........................65 Discussion.....................................................................................................................68 v IV. CYCLOOXYGENASE MECHANISTIC INSIGHTS: Second O2 Addition and PGG2 Exit Routes..........................................................72 Introduction...................................................................................................................72 Second O2 Addition..................................................................................................72 PGG2 Exit Routes.....................................................................................................73 Regio- and Stereospecificity of the Second Oxygen Addition......................................76 Results......................................................................................................................76 Discussion.................................................................................................................80 PGG2 Exit Routes in COX............................................................................................83 Channel_finder Results............................................................................................85 Discussion.....................................................................................................................88 Appendix A. NEW AMBER PARAMETERS............................................................................91 α-(R) and α-(S) methyl indomethacin ethanolamide.....................................................91 α-(R) and α-(S) isopropyl indomethacin ethanolamide.................................................92 α-dimethyl indomethacin ethanolamide........................................................................93 Potential Function Parameters.......................................................................................94 REFERENCES..................................................................................................................96 vi LIST OF FIGURES Figure Page I-1. The three-dimensional structure of the COX homodimer...............................2 I-2. Major prostaglandins produced from PGH2....................................................3 I-3. Surface depiction of the arachidonic aAcid binding site in COX...................6 I-4. Examples of COX inhibititors (NSAIDs)........................................................9 I-5. Indomethacin bound to COX-2.....................................................................10 I-6. The COX-2 active site has a larger solvent accessible surface.....................13 I-7. COX-2 selective inhibitor SC-558 bound to the COX-2 active site .............14 I-8. COX-2 selective, diaryl-heterocycle, inhibitors............................................15 I-9. Adding steric bulk converts indomethacin to a COX-2 selective inhibitor...18 II-1. The COX lobby.............................................................................................23 II-2. Conformational strain energy for α-S-methyl and α-R-methyl indomethacin ethanolamides...............................................................................................36 II-3. Conformational strain energy for α-S-isopropyl and α-R-isopropyl