University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2018-04-10 Targeting Drug Resistance In HCV NS3/4A Protease: Mechanisms And Inhibitor Design Strategies Ashley N. Matthew University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Biochemistry Commons, Biophysics Commons, and the Structural Biology Commons Repository Citation Matthew AN. (2018). Targeting Drug Resistance In HCV NS3/4A Protease: Mechanisms And Inhibitor Design Strategies. GSBS Dissertations and Theses. https://doi.org/10.13028/M23X3H. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/969 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. TARGETING DRUG RESISTANCE IN HCV NS3/4A PROTEASE: MECHANISMS AND INHIBITOR DESIGN STRATEGIES A Dissertation Presented By ASHLEY NICOLE MATTHEW Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY APRIL 10th, 2018 MD/PHD PROGRAM ii TARGETING DRUG RESISTANCE IN HCV NS3/4A PROTEASE: MECHANISMS AND INHIBITOR DESIGN STRATEGIES A Dissertation Presented By ASHLEY NICOLE MATTHEW This work was undertaken in the Graduate School of Biomedical Sciences MD/PHD PROGRAM The signatures of the Dissertation Defense Committee signify completion and approval as to style and content of the Dissertation _______________________________________________ Celia A. Schiffer, Ph.D., Thesis Advisor _______________________________________________ Paul Thompson, Ph.D., Member of Committee _______________________________________________ William Royer, Ph.D., Member of Committee _______________________________________________ Kendall Knight, Ph.D., Member of Committee _______________________________________________ Stefan Sarafianos, Ph.D., Member of Committee The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee _______________________________________________ Daniel Bolon, Ph.D, Chair of Committee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the School. _______________________________________________ Kendall Knight, Ph.D. Dean of the Graduate School of Biomedical Sciences APRIL 10th, 2018 iii TABLE OF CONTENTS Page # ABSTRACT ......................................................................................................... vii DEDICATION ..................................................................................................... viii ACKNOWLEDGEMENTS ..................................................................................... x LIST OF TABLES ............................................................................................... xiv LIST OF FIGURES AND SCHEMES .................................................................. xv LIST OF THIRD PARTY COPYRIGHTED MATERIAL .................................... xviii List of Symbols, Abbreviation or Nomenclature ........................................... xix Chapter I ............................................................................................................... 1 Introduction .......................................................................................................... 1 1.1 Hepatitis C Virus Epidemiology ........................................................................... 2 1.2 Hepatitis C Viral Proteins and Life Cycle ............................................................ 3 1.3 Direct-Acting Antivirals against Hepatitis C Virus ................................................ 6 1.4 NS3 Bifunctional Protein and Protease Architecture ........................................... 9 1.5 Small Molecule Inhibition of HCV NS3/4A Protease ......................................... 12 1.6 Development of Pan-genotypic Protease Inhibitors .......................................... 17 1.7 Substrate Recognition and Protease Inhibitor Resistance ................................ 21 1.8 Mechanisms of Resistance for Single Substitutions ......................................... 26 1.9 Emergence of double substitution variants ....................................................... 31 1.10 Scope of Thesis .............................................................................................. 32 Chapter II ............................................................................................................ 34 Clinical signature variant of HCV NS3/4A protease uses a new mechanism to confer resistance........................................................................................... 34 Preface ..................................................................................................................... 365 2.1 Abstract ................................................................................................................ 36 2.2 Introduction ......................................................................................................... 37 2.3 Results ................................................................................................................. 43 2.3.1 Inhibitors are highly susceptible to the Y56H/D168A NS3/4A variant ............ 43 2.3.2 Double mutant cycle analysis reveals interdependency of Tyr56 and Asp168 for most PIs ............................................................................................................. 50 2.3.3 Crystal structures of protease-inhibitor complexes ........................................ 53 2.3.4 Danoprevir resistance to the Y56H/D168A variant is solely due to the D168A substitution .............................................................................................................. 55 2.3.5 Resistance mechanism of grazoprevir and analogs against single and double mutants ................................................................................................................... 60 2.3.6 Altered dynamics correlate with structural mechanisms of resistance ........... 62 2.4 Discussion ........................................................................................................... 69 iv 2.5 Methods ............................................................................................................... 74 2.5.1 Inhibitor Synthesis .......................................................................................... 74 2.5.2 Expression and Purification of NS3/4A Constructs ........................................ 74 2.5.3 Determination of the Inner Filter Effect .......................................................... 75 2.5.4 Determination of Michaelis–Menten (Km) Constant ....................................... 76 2.5.5 Enzyme Inhibition Assays .............................................................................. 76 2.5.6 Cell-Based Drug Susceptibility Assays .......................................................... 77 2.5.7 Crystallization and structure determination .................................................... 77 2.5.8 System Preparation for Molecular Dynamics Simulations ............................. 78 2.5.9 Molecular Dynamics Simulations ................................................................... 79 2.5.10 Correlated Motions ....................................................................................... 81 2.6 Acknowledgements ............................................................................................ 82 Chapter III ........................................................................................................... 83 Hepatitis C Virus NS3/4A Protease Inhibitors Incorporating Flexible P2 Quinoxalines Target Drug Resistant Viral Variants ....................................... 83 Preface ....................................................................................................................... 84 3.1 Abstract ................................................................................................................ 85 3.2 Introduction ......................................................................................................... 85 3.3 Chemistry ............................................................................................................. 92 3.4 Results and Discussion ...................................................................................... 98 3.4.1 Modifications of P1’ and P4 Capping Groups .............................................. 102 3.4.2 SAR Exploration of P2 Quinoxaline ............................................................. 104 3.4.3 Effects of P2 Substituent Size and Flexibility ............................................... 118 3.5 Conclusions ....................................................................................................... 122 3.6 Methods ............................................................................................................. 123 3.6.1 General ........................................................................................................ 123 3.6.2 Typical procedures for the synthesis of protease inhibitors using