MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Fan Meng Candidate for the Degree DOCTOR OF PHILOSOPHY ______________________________________ Director Dr. Michael W. Crowder ______________________________________ Dr. David L. Tierney ______________________________________ Dr. Carole Dabney-Smith ______________________________________ Dr. Christopher A. Makaroff ______________________________________ Graduate School Representative Dr. Hai-Fei Shi ABSTRACT BIOCHEMICAL CHARACTERIZATION AND ZINC BINDING GROUP (ZBGS) INHIBITION STUDIES ON THE CATALYTIC DOMAINS OF MMP7 (CDMMP7) AND MMP16 (CDMMP16) by Fan Meng Matrix metalloproteinase 7 (MMP7/matrilysin-1) and membrane type matrix metalloproteinase 16 (MMP16/MT3-MMP) have been implicated in the progression of pathological events, such as cancer and inflammatory diseases; therefore, these two MMPs are considered as viable drug targets. In this work, we (a) provide a review of the role(s) of MMPs in biology and of the previous efforts to target MMPs as therapeutics (Chapter 1), (b) describe our efforts at over-expression, purification, and characterization of the catalytic domains of MMP7 (cdMMP7) and MMP16 (cdMMP16) (Chapters 2 and 3), (c) present our efforts at the preparation and initial spectroscopic characterization of Co(II)-substituted analogs of cdMMP7 and cdMMP16 (Chapters 2 and 3), (d) present inhibition data on cdMMP7 and cdMMP16 using zinc binding groups (ZBG) as potential scaffolds for future inhibitors (Chapter 3), and (e) summarize our data in the context of previous results and suggest future directions (Chapter 4). The work described in this dissertation integrates biochemical (kinetic assays, inhibition studies, limited computational methods), spectroscopic (CD, UV-Vis, 1H-NMR, fluorescence, and EXAFS), and analytical (MALDI-TOF mass spectrometry, isothermal calorimetry) methods to provide a detailed structural and mechanistic view of these MMPs. This work is part of overall effort to prepare selective and specific inhibitors against the MMPs, which are expected to be drug candidates in MMP-associated diseases. BIOCHEMICAL CHARACTERIZATION AND ZINC BINDING GROUP (ZBGS) INHIBITION STUDIES ON THE CATALYTIC DOMAINS OF MMP7 (CDMMP7) AND MMP16 (CDMMP16) A DISSERTATION Presented to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry and Biochemistry by Fan Meng The Graduate School Miami University Oxford, Ohio 2016 Dissertation Director: Dr. Michael W. Crowder © Fan Meng 2016 Table of Contents Chapter 1 Introduction: What are matrix metalloproteinases? 1 1.1 Classification of matrix metalloproteinases (MMPs) 2 1.2 Structural features and the catalytic mechanism of MMPs 3 1.2.1. Overall structure of MMPs. 3 1.2.2 Catalytic domains of MMPs. 4 1.2.3 Proposed reaction mechanisms of the MMPs. 6 1.3 Regulation of MMPs 7 1.3.1 Transcriptional regulation. 7 1.3.2 Cell-specific expression of MMPs. 8 1.3.3 Pre-translational regulation of the expression of MMPs 9 1.3.4 Regulation of MMP enzymatic activity 9 1.3.5 Unregulated enzymatic activities of MMPs contribute to multiple pathological processes 11 1.4 Role of MMPs in physiological and pathological conditions 12 1.4.1 Role of MMPs in ECM biology 12 1.4.2 MMPs regulate pathological and physiological events by processing signaling proteins 13 1.4.3 Protective role of MMPs in pathological processes 17 1.5 Review of MMP inhibitors 17 1.5.1 Therapeutic targeting of MMPs 17 1.5.2 Overview of ZBG-based MMPi 19 1.5.3 Challenges and new opportunities for next generation ZBG MMPi 20 1.6 Inhibition studies on MMP7 and MMP16 22 1.7 References 24 1.8 Tables and figures 46 iii Chapter 2 Biochemical and spectroscopic characterization of the catalytic domain of MMP16 (cdMMP16) 60 2.1 Introduction 62 2.2 Materials and methods 65 2.3 Results 72 2.4 Discussion 78 2.5 References 82 2.6 Tables and figures 89 Chapter 3 Biochemical characterization and zinc binding group (ZBGs) inhibition studies on the catalytic domain of MMP7 (cdMMP7) 113 3.1 Introduction 115 3.2 Material and methods 118 3.3 Results 124 3.4 Discussion 128 3.5 Acknowledgements 134 3.6 Reference 135 3.7 Tables and figures 142 Chapter 4 Conclusions of dissertation 161 4.1 Conclusions 162 4.2 References 168 4.3 Table and figure 172 iv List of Tables Table 1.1: Overview of MMP sub-classes, structural elements, and ECM substrates 46 Table 1.2: Overview of cell-specific expression of MMPs under normal physiological conditions 48 Table 1.3: Physiological and pathological events and identified substrates for select MMPs. 49 Table 1.4: Classes of MMP inhibitors (MMPi) 51 Table 2.1: Data fit for EXAFS traces of Zn2-cdMMP16. 89 Table 2.2: Data fit for EXAFS traces of Co2-cdMMP16. 90 Table 2.3: Steady state kinetic constants and metal content of cdMMP16 samples 91 Table 2.4: Kinetic mechanism used to fit stopped-flow fluorescence data and the Dynafit-generated microscopic rate constants. 92 Table 2.5: Secondary structural elements of Zn2-cdMMP16, Co2-cdMMP16, ZnCo-cdMMP16, and metal-free cdMMP16. 93 Table 2.6:Summary of EXAFS fits of Zn2-cdMMP16 and Co2-substituted analogs 94 Table 3.1: Steady-state kinetic constants and metal content for recombinant cdMMP7 analogs 142 Table 3.2: Secondary structural content of Zn2-cdMMP7 and ZnCo-cdMMP7 analogs 143 Table 3.3: Microscopic kinetic constants of the hydrolysis of BML-P131 by Zn2-cdMMP7 144 Table 3.4: Microscopic kinetic constants of as-isolated Zn2-cdMMP7 and Zn2-cdMMP16 from substrate-emission stopped-flow studies 145 Table 3.5: IC50 values of AHA, maltol, TM, and ATM as inhibitors of cdMMP16 and cdMMP7. 146 v Table 3.6: ITC measurements on cdMMP7 and cdMMP16 using ATM and TM as binding groups. 147 Table 4.1: Select inhibitors of MMPs with reported IC50 values 172 vi List of Figures Figure 1.1: Domain structure of represented MMPs. 52 Figure 1.2: Activation of pro-MMPs via cysteine switch 53 Figure 1.3: Overlapped crystal structures of cdMMP7 (green, PDB#1MMP) and cdMMP16 (cyan, PDB#1RMB). 54 Figure 1.4: Proposed reaction mechanisms for MMPs 55 Figure 1.5: Transcriptional, translational, and physiological regulation of MMPs 56 Figure 1.6: Overview of MMPs’ physiological roles, pathological roles and protective roles 57 Figure 1.7: Review of MMPi development and current strategies of therapeutic targeting MMPs 58 Figure 1.8: Overview of experimental approach used in this dissertation. 59 Figure 2.1:Crystal structure of Zn2-cdMMP16 using the coordinates (PDB 1RM8) 95 Figure 2.2: EXAFS of Zn2-cdMMP16 96 Figure 2.3: EXAFS of Co2-cdMMP16 97 Figure 2.4: SDS-PAGE analysis of cdMMP16 purification 98 Figure 2.5: Substrates used in this study 99 Figure 2.6: Relative catalytic activity of Zn2-cdMMP16 in the presence of NaCl 100 Figure 2.7: Circular dichroism of as-isolated Zn2-cdMMP16 (solid), Reconstituted Zn2-cdMMP16 (tight-dot), Co2-cdMMP16 (dash), ZnCo-cdMMP16 (dash dot), and metal-free cdMMP16 (dot) 101 Figure 2.8: Stopped-flow fluorescence traces of 1 M Zn2-cdMMP16 with various concentrations of DNP-Pro-Leu-Ala-Leu-Trp-Ala-Arg-OH 102 Figure 2.9: Stability of cdMMP16 samples using SDS-PAGE 103 Figure 2.10: Relative catalytic activity of metal-free cdMMP16 104 Figure 2.11: MALDI-TOF mass spectra of cdMMP16 in the presence of 2 eq of Co(II) and 5 mM Ca(II) over time 105 vii Figure 2.12: SDS-PAGE of cdMMP16 samples using modified metal incorporation procedure. 106 Figure 2.13: Fluorescence spectra of as-isolated Zn2-cdMMP16 (solid), reconstituted Zn2-cdMMP16 (tight dot) 107 Figure 2.14: Fourier transformed EXAFS data (solid lines) and corresponding best fits (open symbols) for Zn2-cdMMP16 and Co2-cdMMP16 108 Figure 2.15: Optical spectra of Co2-cdMMP16 (dash) and ZnCo-cdMMP16 (solid). Both of samples were diluted in 50 mM Hepes, pH 7.0, containing 5 mM CaCl2 109 1 Figure 2.16: 200 MHz H NMR spectra of Co2-cdMMP16 (top) and ZnCo-cdMMP16 (below). Solvent exchangeable protons are marked with asterisks. 110 Figure 2.17: Fluorescence spectra of 10 M Zn2-cdMMP16 exposed in differential salt concentration 111 1 Figure 2.18: H NMR spectra of reconstituted Zn2-cdMMP16 and as-isolated Zn2-cdMMP16. 112 Figure 3.1: Crystal structure of Zn2-cdMMP7 (PDB #1MMB) 148 Figure 3.2: Structure of substrate 149 Figure 3.3: SDS-PAGE analysis of the two cdMMP7 refolding procedures. 150 Figure 3.4: Circular dichroism spectra of Zn2-cdMMP7 (solid), ZnCo-cdMMP7 (dashed line), and inactive Zn2-cdMMP7 (dotted line) 151 Figure 3.5: Fluorescence emission spectra of ZnCo-cdMMP7 (dash) and as-isolated Zn2-cdMMP7 (solid). 152 Figure 3.6: Fluorescence emission spectra of inactive Zn2-cdMMP7 (dash) and as-isolated, Zn2-cdMMP7 (solid). 153 Figure 3.7: Stopped-flow fluorescence traces of reactions of Zn2-cdMMP7 and with substrate BML-P131 154 Figure 3.8: Stopped-flow fluorescence traces of reactions of A Zn2-cdMMP7 and B Zn2-cdMMP16 with FS-6 fluorescent substrate 155 viii Figure 3.9: Uv-vis spectrum of of the ZnCo-cdMMP7 analog( right top corner) and UV-Vis difference (spectrum of ZnCo analog minus the spectrum of metal-free enzyme) spectrum 156 Figure 3.10: Structures of zinc binding group (ZBG) inhibitors used in this study 157 Figure 3.11: Inhibitory potency of AHA, maltol, TM, and ATM against cdMMP7 and cdMMP16 relative to cdMMP1. 158 Figure 3.12: ITC results of ZBG binding to cdMMP7 and cdMMP16 159 Figure 3.13: Representative binding modes from docking simulations 160 Figure 4.1: Crystal structures of (A) cdMMP1 (yellow, PDB#966C), (B) cdMMP7 (green, PDB#1MMP), (C) cdMMP9(pink, PDB#4H1Q), (D) cdMMP12 (blue,PDB#2POJ), and (E) cdMMP16 (cyan, PDB#1RM8).