Biochemical Characterization of Human Matrix Metalloproteinases and Their Newly Designed Inhibitors Related to Stroke Qiang Cao

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2010 Biochemical Characterization of Human Matrix Metalloproteinases and Their Newly Designed Inhibitors Related to Stroke Qiang Cao

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COLLEGE OF ARTS AND SCIENCES

BIOCHEMICAL CHARACTERIZATION OF HUMAN MATRIX METALLOPROTEINASES

AND THEIR NEWLY DESIGNED INHIBITORS RELATED TO STROKE

QIANG CAO

A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Degree Awarded: Fall Semester, 2010 The members of the committee approve the dissertation of Qiang Cao defended on July 14, 2010.

Qing-Xiang Amy Sang Professor Directing Dissertation

Yan-Chang Wang University Representative

Hong Li Committee Member

Igor Alabugin Committee Member

Approved:

Joseph B. Schlenoff, Chair, Department of Chemistry and Biochemistry

The Graduate School has verified and approved the above-named committee members.

This dissertation is dedicated to my parents, Shi-De Cao & Mu-Dan Li.

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ACKNOWLEDGEMENTS

There are many people to thank for their support and guidance during the past years. My wife, Juan Zhou, has shown a great deal of patience and personal support through difficult times. Professor Qing-Xiang Amy Sang has provided outstanding scientific guidance and truly developed my interest in cancer research and cardiovascular disease research. A special thanks is given to Drs. Martin Schwartz, Yonghao Jin and Wei Yang for the many discussions about inhibitor interactions the synthesis and modeling of the compounds. I especially thank Mark Dru Roycik for his help on English writing and organizing through the past years and Douglas R. Hurst, Robert Newcomer and Seakoo Lee for their expert guidance throughout my training. I thank the Sang lab members, Chi Ben, Manuel H. Constantino, Suzan Semaan, Dale B. Bosco, Zahraa Khamis, Paul Steward, and other members for their numerous scientific discussions and helps. Last but not least, I would like to thank my family and my friend, Qin Li, for their love and support. Without them, I would not have been able to finish the dissertation on time.

TABLE OF CONTENTS

List of Tables ...... vii List of Figures ...... viii List of Abbreviations ...... x Abstract ...... xii

1. INTRODUCTION ...... 1

1.1 Introduction of stroke and how MMPs are involved ...... 1 1.2 Matrix metalloproteinases and their inhibitors...... 5

2. MATERIALS AND METHODS ...... 25

Materials ...... 25 Total hydrolysis of Quenched Fluorescent Peptide ...... 25 Time Dependence Experiments ...... 26 Estimation of the Michaelis constant (Km) Value...... 26 Competitive inhibition determination ...... 27 app ’ The determination of apparent dissociation constant (Ki or Ki ) 27 MMPI stability in HEPES buffer ...... 28 MMPI stability in EGM-2 MV ...... 28 Human Brain Microvascular Endothelial (hBMEC) Growth ...... 29 Human Mesenchymal Stem Cell (hMSC) Growth...... 29 The assay of inhibition on Endothelial cells migration ...... 29 hBMEC Culture & hMSC Co-Culture ...... 31 MMPI cytotoxicity in EGM-2MV ...... 31 Protease inhibitor preparation ...... 31 The inhibitor treatment on hBMEC, protein extraction preparation 32 Cell sample homogenization using the bead beater ...... 32 TCA/Acetone Precipitation ...... 32 Two-dimensional gel electrophoresis ...... 32 Analysis of Proteins ...... 33 LC- MS/MS ...... 33 Protein Search Algorithm ...... 34

3. RESULTS AND DISCUSSION ...... 35

3.1 Enzyme kinetics ...... 35 3.2 Inhibitor application and characterization in cellular system 67 3.3 Proteomic studies after hBMEC treated with YHJ-6-43 ...... 77

4. CONCLUSION AND FUTURE WORK ...... 86

REFERENCES ...... 88

BIOGRAPHICAL SKETCH ...... 107

LIST OF TABLES

1.1 Selected Variable Residues in the Active Site of MMPs ...... 20 1.2. MMP Inhibitors with a Mercaptosulfide Zinc Binding Group ...... 23 1.3. MMP comparison with deep and shallow P1' substituent ...... 41 3.1 The apparent Ki for YHJ-6-43 and YHJ-6-45 against eight members of MMP family ...... 54 3.2 The apparent Ki of inhibitor modified with fluoride replacement...... 56 3.3 The apparent Ki of inhibitors with Modification on para & meta position at the biphenyl ester ...... 57 3.4 The apparent Ki values of inhibitors with modification for increasing rigidity ...... 59 3.5 The apparent Ki of pyrrolidine-based mercaptosulfonamide inhibitors 60 3.6 The apparent Ki of different types inhibitors ...... 62 3.7 Table of identified proteins by LC MS-MS ...... 81

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LIST OF FIGURES

1.1 The Structure of Blood Brain Barrier ...... 4 1.2 Zinc metalloproteinase classification scheme ...... 6 1.3 The conserved amino acid coordinating with catalytic zinc for the metzicin proteases family ...... 7 1.4 Domain structures of the MMPs...... 8 1.5 Nomenclature of the peptide substrate ...... 11 1.6 The proposed Reaction mechanism for proteolysis by MMPs ...... 12 1.7 The activation network of MMP-9 by MMPs ...... 14 1.8 The activation of proMMP-2 involving MT1-MMP and TIMP-2 ...... 15 1.9 Relationship between enzyme and peptide-based inhibitor structure .. 16 1.10 Common zinc binding groups ...... 18 1.11 Inhibitor zinc binding group ...... 20 1.12 Concept and rationale of novel thiol MMPIs proposed in this study ... 21 1.13 Structures and predicted bindings of mercaptosulfide inhibitor MAG-182 and proposed mercaptosulfonamide inhibitors ...... 23 3.1 Binding and hydrolyzation between enzyme and substrate ...... 35 3.2 Michaelis-Menten hyperbolic curve ...... 38 3.3 The full structure of substrate used in this study ...... 39 3.4 Intensity of cleaved substrate ...... 39 3.5 Km of MMP-2 determination ...... 40 3.6 Km of MMP-9 determination ...... 41 3.7 Time dependence study of YHJ-6-43 against MMP-2 ...... 42 3.8 Time dependence study of YHJ-6-43 against MMP-9 ...... 42 3.9 Time dependence study of YHJ-6-286 against MMP-2 ...... 43 3.10 Time dependence study of YHJ-6-286 against MMP-9 ...... 44 3.11 Enzyme-inhibitor association and dissociation ...... 44 3.12 The three conditions for a reversible, competitive inhibition ...... 45 3.13 Mechanisms of inhibition ...... 50 3.14 Competitive inhibition of YHJ-6-43 ...... 51 app 3.15 The replot of the Ki s ...... 51 3.16 Competitive inhibition of YHJ-6-286 ...... 52 app 3.17 The replot of the Ki s ...... 52 3.18 The structures of YHJ-6-43(trans) Vs YHJ-6-45(Cis) ...... 54 3.19 The structures for inhibitors modified with fluoride replacement ...... 55 3.20 The structures for inhibitors with Modification on para & meta position at the biphenyl ester ...... 57 3.21 The structures of inhibitors with modification for increasing rigidity ... 58 3.22 The structure of pyrrolidine-based mercaptosulfonamide inhibitors ... 60 3.23 The structures of different type of inhibitors ...... 62 3.24 Stability evaluation of YHJ-6-43 in HEPES buffer without TCEP ...... 63 3.25 Stability evaluation of YHJ-6-286 in HEPES buffer without TCEP ..... 64 3.26 Stability evaluation of YHJ-7-52 in HEPES buffer without TCEP ...... 64 3.27 Stability evaluation of YHJ-6-293 in HEPES buffer without TCEP ..... 65 3.28 Stability evaluation of YHJ-7-23 in HEPES buffer without TCEP ...... 65

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3.29 Stability evaluation of YHJ-6-43 in HEPES buffer with TCEP ...... 66 3.30 Stability evaluation of YHJ-6-286 in HEPES buffer with TCEP ...... 66 3.31 Stability of YHJ-6-43 in EGM ...... 69 3.32 Stability of YHJ-6-286 in EGM ...... 69 3.33 Stability of YHJ-6-293 in EGM ...... 70 3.34 Cytotoxicity of YHJ-6-43 in hBMEC culture ...... 71 3.35 Cytotoxicity of YHJ-6-286 in hBMEC culture ...... 71 3.36 Cytotoxicity of YHJ-6-293 in hBMEC culture ...... 72 3.37 Wound-healing assay of YHJ-6-43 in hBMEC culture ...... 73 3.38 Wound-healing assay of YHJ-6-286 in hBMEC culture ...... 73 3.39 Wound-healing assay of YHJ-6-293 in hBMEC culture ...... 74 3.40 Wound-healing assay of ethanol in hBMEC culture ...... 74 3.41 Wound-healing assay of co-cultutre (hBMECs and hMSCs) ...... 76 3.42 Tight junctions and tight junction proteins ...... 78 3.43 The separation results of hBMEC protein extracts ...... 80

LIST OF ABBREVIATIONS rt-PA …………………. recombinant tissue-type plasminogen activator PAR-1 …………………..protease activated receptor MMP .matrix metalloproteinase BBB ………………….blood brain barrier MMPI .MMP inhibitor DD-carboxypeptidase…n-alanyl-n-alanine-cleaving carboxypeptidase MT-MMP .membrane type MMP ECM .extracellular matrix VEGF ……..…………….vascular endothelial growth factor TNF-α…………………….tumor necrosis factor- α IL-1………………………..interleukin 1 AP 1………………………Activator protein 1 EGF………………………Epidermal growth factor TGF-β ……………………Transforming growth factor-β TIMPs …tissue inhibitor of metalloproteinases ZBG………………………..Zinc binding group hBMEC……………………Human brain microvascular endothelial cell Mca …(7-methoxycoumarin-4-yl)acetyl Dpa …N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl FRET …fluorescence resonance energy transfer HEPES ….N-2-hydroxyethylpiperazine-N-2'-ethane sulfonate TCEP ………………………tris(2-carboxyethyl)phosphine TCA………………………...trichloroacetic acid hSMC………………………Human Mesenchymal Stem Cell 2-DE………………………. two dimensional gel electrophorisis IEF………………………….Isoelectric focusing LC……………………………Liquid chromatography QqTOF-MS…...... quadrupole time-of-flight mass spectrometry eIF5A ………………………..Eukaryotic translation initiation factor 5A

ABSTRACT

Matrix metalloproteinases (MMPs), a family of enzymes known for their proteolytic activities on processing extracellular matrix substrates, may play an integral role in blood-brain barrier opening following an ischemic stroke. Several matrix metalloproteinases are proposed to play vital roles in early or late stages of blood brain barrier opening. Matrix metalloproteinase inhibitor (MMPI) has been showing beneficial effect in the treatment of the blood brain opening related to stroke. As old generation matrix metalloproteinase inhibitor failed in oncology clinical trials, our collaborators, Drs. Martin A. Schwartz and Yonghao Jin, have designed and synthesized new biologically friendly mercaptosulfonamide inhibitors. Characterization and selection of effective matrix metalloproteinase inhibitors were performed by evaluating their stability, potency, and selectivity by enzymatic kinetics. According to dissociation constant related to enzyme and inhibitor binding, our data indicates that those inhibitors are capable of inhibiting MMP-2, -9, and membrane-type 1 MMP (MT1-MMP) effectively and selectively. Selected inhibitors were studied with cell wound healing assays in human microvascular endothelial cell model to investigate selected MMPI activities and impact on cell behavior. By blocking MMP activities in cell culture, our inhibitors were able to reduce human brain microvascular endothelial cell wound healing process. Protein expression patterns in cell culture were investigated with proteomics after inhibitor treatment. The reduced expressions of several proteins, which are related to cell division, cell adhesion and cell death, have been discovered. It is also verified the blocking function of our inhibitor in human brain microvascular endothelial cell wound healing assay. Overall, our newly designed matrix metalloproteinase inhibitor efficiently inhibits matrix metalloproteinase which carries intermediate or deep S1’ pocket at protein and cellular level. Upon application of matrix metalloproteinase inhibitor, it has been implicated that blocking of matrix metalloproteinase activities are involved in the decreasing of other cell function modulators. The most potent and specific inhibitors have been

xi selected as promising compounds, which have been further tested in animal models to evaluate their efficacy in the prevention of blood brain barrier opening associated with stroke by our collaborator Dr. Gary A. Rosenberg. This study is the first enzymological and cellular analysis of mercaptosulfonamide inhibitors.

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CHAPTER 1

INTRODUCTION

1.1 Introduction of stroke and how MMPs are involved

Stroke, also called ―brain attack‖, is often referred to as a cerebral accident which leads to the sudden death of some brain cells and brain damage, and is the consequence of diminishing the supply of oxygen and other essential nutrients to the cells in the brain when cerebral blood vessels experience a reduced or disrupted flow of blood. Stroke is the third leading cause of death and the leading cause of severe disability in the United States (Thom et al., 2006; Lloyd-Jones et al., 2009). Stroke can be roughly classified into two major categories, such as ischemic and hemorrhagic strokes. Ischemic stroke accounting for 87 percent of all incidents, is a local blockage of the cerebral artery. The resulting 12% of all cases of hemorrhagic strokes result from a ruptured vessel and causes bleeding into the surrounding brain (Lloyd-Jones et al., 2010). The formation of blood clots in blood vessel may narrow down arteries and start blocking the artery where they occur (Warlow et al., 2001). Theses blood clots are thrombuses, the major cause of ischemic stroke. If a thrombus breaks down, it becomes detached and enters circulation as an embolus, ultimately getting trapped and obstructing a blood vessel. An embolism can also occur after the formation of emboli presenting in the form of blood, fat, or air during surgical procedures (Warlow et al., 2001). Less common causes of ischemic stroke include cerebral venous sinus thrombosis (Stam et al., 2005), carotid dissection and arteritis, infection, and drug abuse, such as the use of cocaine (Hickey et al., 2003). Stroke without an obvious explanation is called "cryptogenic" (Donnan et al., 2008; Guercini et al., 2008). When bleeding, the accumulation of blood may occur within the skull vault and cause hemorrhages such as intracerebral and subarachnoid hemorrhage.

When a diseased blood vessel in the brain bursts, blood is able to leak into brain and causes intracerebral hemorrhage. When a blood vessel outside brain and within skull vault starts to bleed, it causes subarachnoid hemorrhage. Hemorrhagic strokes can also result in tissue injury by bleeding into brain and may cause infarction around the affected area (Donnan et al., 2008; Hickey et al., 2003; Lloyd-Jones et al., 2010). There are many consequences after ischemic stroke onsets. Brain cells start to die if the artery has been blocked for longer than a few minutes. This is the reason that immediate medical treatment for stroke is absolutely critical for stroke patients (Donnan et al., 2008; Hickey et al., 2003; Lloyd-Jones et al., 2010). Because of interruption of oxygen and nutrients to brain, energy dependent processes may fail to function properly, which results into brain cell damage. Free oxygen radials and other reactive oxygen species induced by ischemic stroke may also damage cells (Mannheim et al., 2008). In addition, it is also possible that ischemic stroke result in loss of structural integrity of brain tissue and blood vessels by releasing proteases, such as matrix metalloproteases (MMPs), which may get involved in the breakdown of blood brain barrier (Yang et al., 2007; Mannheim et al., 2008; Ali and Schulz, 2009; Suzuki et al., 2009). Recombinant Tissue-type Plasminogen Activator (rt-PA), a serine protease used as thrombolytic drug, has been the only drug approved by the U.S. Food Drug Administration for ischemic stroke treatment for more than decade. It can activate plasminogen and convert to its active form, plasmin, whose primary function is digestion of fibrin in plasma. Following the digestion of fibrin, the blood flow can be restored because blood clots become easier to be processed by other enzymes (Sheehan et al., 2005). Although clinical studies have demonstrated that rt-PA can reduce the damage from ischemic stroke, there are only a limited number of patients receiving effective rt-PA treatment within first three hours treatment window without apparent hemorrhage complications after the onset of ischemic stroke (Goldstein, 2007). Besides acting as activator of plasminogen and the blood clot dissolver, rt-PA also activates proteases, such as protease activated receptor

(PAR-1) and matrix metalloproteinases (MMPs) which play important roles in degradation of extra cellular matrix and are the major contributors involving in blood brain barrier (BBB) (Figure 1.1) opening, edema and hemorrhage (Goldstein, 2007; Francis et al., 2001). The BBB is a physical barrier between the blood vessels and the central nervous system that controls the entry of molecules and white blood cells from the systemic circulation into the central nervous system and functions to maintain the homeostatic balance of the brain extracellular fluid, to ensure normal brain function (Bock and Haltner, 2006; Hu et al., 2007; Rosenberg et al., 2001). Furthermore, the BBB is formed by cerebral vascular endothelial cells linked together via tight junctions, a basement membrane, pericytes, and astrocytes or glia. Once the opening of BBB occurs, brain cells may get damaged because of the entry of many different toxins. The need to deliver the treatment within three hours of the onset of symptoms, limits the number of treatable patients to less than 5% of stroke patients because disruption of the BBB allows rt-PA to enter brain where it increases the risk of brain edema and hemorrhage. Neuroprotective agents have a variety of beneficial actions, including protecting the BBB and reducing infarct size and cell death. Matrix metalloproteinases (MMPs) are involved in opening the BBB and MMP inhibitors reduce edema and hemorrhage associated with rt-PA treatment (Yang, 2007; Rosenberg et al., 2001 & 2009). The initial opening of the BBB, which is associated with rt-PA toxicity, is due to the activation of MMP-2. There are 25 MMPs, but the gelatinases (MMP-2 and MMP-9), stromelysin-1 (MMP-3) and membrane-type MMP (MMP-14) are the main ones thought to act on the BBB (Gasche, 2001; Sole, et al., 2004; Clark, et al., 1997). 2003). MMP-2 is constitutively expressed in a latent form; early opening of the BBB occurs when MMP-2 is activated by MMP-14 (Hu, et al., 2007; Yamakawa, 2004). Later damage to the neurons and BBB involves the expression of MMP-3 and MMP-9, which are induced in the neuroinflammatory response by cytokines and free radicals (Pfefferkorn and Rosenberg et al., 2003; Rosenberg et al., 2001; Gasche, 2001). Therefore, treatments aimed at expanding the therapeutic window for rt-PA

3 need to block mainly MMP-2, but should have some action against MMP-3 and MMP-9. Animal studies show that if the broad-spectrum MMP inhibitors (MMPIs), BB-1101 (British Biotechnology) are given prior to rt-PA treatment, toxicity is reduced and the therapeutic window is extended (Yang, 2007; Rosenberg et al., 2001 & 2009)

Figure 1.1 The Structure of Blood Brain Barrier. This figure has been modified from (Francis, et al., 2003).

Such agents have the potential for use in clinical studies, and will be tested in preclinical models to identify the optimal agents for use in patients. Inhibitors to MMPs have been developed by a large number of pharmaceutical companies because of the potential for use in the treatment of cancer and arthritis. These agents have been approved for clinical trials, but long-term side-effects, mainly involving painful joints has blocked their use. It has been mentioned that a recent report described a large number of agents that are currently available for clinical use; the authors suggested that brain disorders would be optimal targets for these agents because they could be given for a short period, avoiding long-term side effects (Rosenberg et al., 2001 & 2009).

1.2 Matrix metalloproteinases and their inhibitors

In biological systems, cells are able to produce many different types of enzymes in order to fulfill different roles. Enzymes are able to catalyze hydrolysis reactions by converting proteins, substrates, into different molecules, products, (Garrett and Grisham, 1999). Based on their cleavage locations, those enzymes usually are grouped into exopepetidases, which cleave at the terminal part of proteins, and endopeptidases, which cleave internal peptide bonds of the proteins, respectively (Beynon et al, 1989). Among a great number of endopeptidases, subgroups can be formed based on their active sites, such as serine proteinases, cysteine proteinases, aspartic proteinases, metalloproteinases and others (Stöcker et al., 1995). The zinc metalloproteinase subgroup is a family of zinc-dependent enzyme, which contains a catalytic zinc ion at their active site. There are a large number of enzymes in this zinc metalloproteinase family and are involved in a wide range of biological processes that occur in both physiological and pathological systems. In order to distinguish among them, a classification system has been developed. According to the sequence around the zinc binding motif, most of zinc metalloproteinases may be grouped into zincins, inverzincins, carboxypeptidases

5 and n-alanyl-n-alanine-cleaving carboxypeptidase (DD-carboxypeptidase) as shown in Figure 1.2 (Hooper, 1994).

Zinc metalloproteinases

Zincins Inverzincin Carboxypeptidas s eseses

DD-carboxypeptidase Gluzincin s Metzincins

Astactin Serratia Reprolysin Matrixin

Figure 1.2 Zinc metalloproteinase classification scheme. This figure has been modified from (Hooper, 1994). Matrixin is one of subgroups.

Zincins are zinc metalloproteinases that contain an amino acid sequence of HEXXH (X is any amino acid) and can be further divided into gluzincins, whose third zinc binding ligand is a glutamic acid, and metzincins, which is featured with a long zinc binding sequence HEBXHXBGBXH and followed by a methionine turn (Stöcker et al., 1995). Metzincins contain a highly conserved zinc binding sequence HEBXHXBGBXH (X is any amino acid; B stands for bulky, apolar residue) at the active site (Figure 1.3). Typically, the zinc is coordinated with three amino acid residues followed by a defining methionine-turn in their structures (Bergers and Coussens 2000). Metzincins are further categorized based on the residues following the third ligating histidine and the residues surrounding the methionine in the methionine-turn (Jiang and Bond, 1992).

Figure 1. 3 The conserved amino acid coordinating with catalytic zinc for the metzicin proteases family. This figure was modified from Bergers and Coussens, 2000.

Matrixins is one of the subgroups classified under the heading of metzincins and are commonly known as matrix metalloproteinases because of their proteolytic activities in degrading of multiple proteins in extracellular matrix of tissue and dependence on metal ion for their enzymatic activities (Sang et al., 2006). Collectively, MMPs are able to degrade all of the protein components in extracellular matrix (ECM) which is the network of proteins surrounding cells, release growth factor receptors from the cell surface, the cell membrane or ECM, and active other MMPs (Egeblad and Werb, 2002;. By processing many of bioactive molecules such as cytokines, MMPs are widely involved in cell functions, cell-cell and cell-matrix interactions such as cell adhension, proliferation, migration and invasion (Sang et al., 2006; Park et al, 2010; Zhao et al., 2003). Initially, the enzymatic activity of these enzymes was first observed during the process of tadpole tail metamorphosis (Gross and Lapiere, 1962). By

7 now, there are more than 23 human MMPs that have been identified and reported, in addition to numerous homologues from other species (Kessenbrock, 2010). Based on individual structure, sequence similarity and substrate specificity, MMPs are subdivided into six smaller groups. They are collagenases (MMPs -1, -8, -13, and -18), gelatinases (MMPs -2 and -9), stromelysins (MMPs -3, -10, and -11), matrilysins (MMPs -7 and -26), membrane-type MMPs (MT-MMPs; MMPs -14, -15, -16, -17, -24, and -25) and others (MMPs -12, -19, -20, -21, -23, -27, and -28) (Chen, 2004; Nagase and Woessner, 1999).

Figure 1.4 Domain structures of the MMPs. This figure has been modified from (Brauer, 2006). Four members, MMP-2, -9, -3 and -14, belong to gelatinases, stromelysin and membrane type MMP subgroups, respectively. Besides of conserved domain structure, each of them carrier their own structure features indicated in figure.

MMPs are multi domain enzymes as shown in Figure 1.4 (Brauer, 2006), such as the predomain or signal peptide which directs the secretion of protein from

8 cells, the prodomain, a zinc-binding catalytic domain, and a hinge region with variable lengths followed by a domain with sequence homology to hemopexin which is referred to hemopexin-like domain (overall, 2002; Murphy and Nagase, 2008). The prodomain that contains a conserved unique PRCGV/NPD sequence, maintains the latency of the MMP (proMMP). When removed by proteolysis results in zymogen activation. The prodomain binds to the active site through the coordination between the catalytic zinc and the cysteine. Here, a sulfhydryl serves as the fourth ligand and coordinates with catalytic zinc. The Cys-Zn complex is so-called the ―Cysteine Switch‖ which is essential for maintaining the latency by ligating the catalytic zinc (Van Wart and BIRKEDAL-HANSEN, 1990). In order to activate proMMPs, the interruption of this complex is necessary. Upon the dissociation of the cysteine switch, the thiol group is displaced by a water molecule which is involved in the MMP’s substrate hydrolysis action (Van Wart and Birkedal-Hansen, 1990; Sang et al., 2006). The catalytic domain contains the zinc-binding HEXXHXXGXXH (X: any amino acid) consensus sequence and a conserved methionine, which produces a methionine-turn in the protein chain that provides the base of the active center binding pocket (Jiang and bond, 1992; Murphy and Nagase, 2008). In addition, there are two zinc ions and calcium binding sites at active site which play important roles involved in function and structure stability (Lee, et al., 2007). The catalytic zinc is coordinated with three histidine amino acids and one thiol group in inactive form or a water molecule in active form (Van Wart et al., 1990; Wang et al., 1999; Visse and Nagase, 2003). Besides of zinc ions, one of three calcium ions have been identified and reported to be close to the second zinc binding loop in catalytic domain, together with the second zinc ion, forms zinc-calcium loop which is conserved in the amino acids of all MMPs (Vettakkorumakankav, 1999; Whittaker et al., 1999; Klaus, 2004; Lee et al., 2007). The hinge region acts as a linker region which connects the catalytic domain to the C-terminal hemopexin-like domain (Nagase, 2006). The C-terminal hemopexin-like domain is needed for collagenases to cleave triple helical interstitial collagens. It also

9 contributes to the cell surface activation of proMMPs by MT1-MMP and the binding reaction with TIMPs as well (Neera Borkakoti 2000; Klaus, 2004). As previous studies reported, not all of these domains are essential for every MMP. Some MMPs lack the linker peptide and the hemopexin-like domain. For example, in two of smallest MMPs, MMP-7 (matrilysins) and MMP-26 (endometase), have been characterized by the absence of the C-terminal hemopexin-like domain (Lee, et al., 2007). There are three cysteine-rich repeats within the catalytic domains of gelatinses A (MMP-2) and B (MMP-9). They are fibronectin type II repeats and critical in binding and cleavage of collagen and elastin (Nagase, et al., 2006; Sternlicht and Werb, 2001). The catalytic domains of MMPs all share a sequence similarity and a conserved topology (Bertini, 2003). Based on the topology and secondary structure of MMPs revealed with x-ray crystallization, the active sites of the MMPs have a shallow cleft with a flat non-prime side and a narrow prime side (Sang et al., 2006). There are a few of grooves with various depths around the catalytic zinc at active site and distributed at each side. Those are substrate recognition sites on the proteinases (Gooley et al., 1994; Lovejoy et al., 1994; Bode et al., 1994; Stams et al., 1994; Browner et al., 1995; Lang et al., 2001; Park et al., 2003). They are named following the nomenclature suggested in previous study (Schechter and Berger, 1967). The designation of cleavage site and nomenclature of substrate binding sites are illustrated in Figure 1.5. Towards the N-terminal direction of the scissile peptide bond of a bound peptide substrate, the residues are designated as non-prime sites such as P1, P2, P3, etc. On the C-terminal direction of the scissile peptide bond of a bound peptide substrate, the residues are designated as prime sites as P1’, P2’ and P3’ etc. In accordance with, the numbering at the opposite binding sites of the enzyme are incremented the similar way such as S1, S2 and S3, pockets etc. pockets on left hand side, which is corresponding to the N-terminal of the scissile bond of a peptide substrate, and S1’, S2’ and S3’ etc. pockets on the right hand side, which is corresponding to the C-terminal of the scissile bond of a peptide substrate (Schechter and Berger, 1967, Bode et al., 1999; Park et al., 2003).

S3 …… S2 …… S1.…. S1’…… S2’ …..S3’ (Enzyme) 2+ Zn

P3….… P2 …,… P1……P1’……P2’……P3’ (Substrate)

Cleavage site Figure 1.5 Nomenclature of the peptide substrate. The substrate is cleaved between position P1-P1'.

Among them, S1’ pocket is a hydrophobic and well-defined principal specificity pocket with variable depths in different MMPs, formed and located to the right of the zinc atom (Welch et al.,1996; Bode et al., 1999; Park et al.,2003;Zhang and Kim, 2009). It has been reported that MMPs can be further grouped into three different categories—shallow, intermediate, and deep pockets (Bode et al., 1999; Park et al., 2003). The size of S1’ pocket in this sub-class is limited by one key residue at the active center, this is residue 214. At this position, Arg/Tyr occures crossing the pocket in shallow S1’ pocket such as in the structures of MMP-1 and -7. In the case of MMPs carrying intermediate pocket including MMP-2, -8, -9, and -26, and deep pocket including MMP -12, -13 and -14 are characterized with the presence of Leu 214 (Lang et al., 2001; Morgunova et al., 2002; Rowsell et al., 2002; Park et al., 2003). The differences of S1’ pocket between MMPs have been linked with substrate specificity (Park et al., 2003). It has been concluded that generally MMPs cleave a peptide bond before a residue with a hydrophobic side chain, such as Leu, Ile, Met, Phe, or Tyr, which fits into the S1' pocket. MMPs rarely cleave a peptide bond with a charged residue of the substrate at this position (Folgueras et al., 2004; Amălinei et al., 2007). The proposed reaction mechanism for proteolysis by MMPs has been illustrated in Figure 1.6. When substrate recognized the active site of MMPs, it replaces one of zinc ligands, a water molecule. The scissile amide carbonyl of the substrate coordinates to the active-site zinc ion. The catalytic zinc plays an important role in substrate hydrolysis by acting as a strong electrophile to provide electrophilic force. It assists the water molecule which is hydrogen bonded to the

11 carboxylate group of the catalytic Glu219, to attack substrate by attracting a hydroxide group from this water molecule (Visse and Nagase, 2003; Whittaker et al., 1999). Glu 219 serves to activate this water molecule for attack and to

Figure 1.6 The proposed Reaction mechanism for proteolysis by MMPs. This figure has been modified from (Whittaker et al., 1999).

stabilize the intermediate of substrate hydrolysis reaction (Rob and Judith, 2001; Whittaker et al., 1999). The glutamic acid, so called ―proton shuttle‖, serves as a general base group and extracts a proton from the water molecule. While attacking by water, water donates a proton to glutamic acid, which facilities attack with hydroxide group of the peptide carbonyl group and transfer another proton to

12 the nitrogen on the substrate scissile bond. It results into the substrate cleavage (Rob and Judith, 2001; Whittaker et al., 1999). In order to accomplish their functions, MMPs must be present at the right location with the right amount at the right time and be activated. Thus, they are tightly regulated and controlled at levels of transcription, secretion, activation and inhibition by their activators and inhibitors (Sternlicht and Werb, 2001). It has been indicated that the expression of MMPs can be induced at the gene expression level by many growth factors such as vascular endothelial growth factor (VEGF), hormones, cytokines, UV light and cellular stress etc (Kheradmand et al. 1998; Fini et al. 1998; Sternlicht and Werb, 2001). For instance, cytokines, such as TNF-α and IL-1, and cellular stress, such as UV light, is able to induce the expression of the extracellular stimuli by phosphorylation, c-Jun which is the member of Jun family, which forms a complex with AP-1 and bind to the AP-1 binding sites in the promoter, consequently stimulate the expression of MMPs (Folgueras et al., 2004; Amălinei et al., 2007). The expression of MMPs is also regulated at post-transcriptional level. For example, EGF and phorbol are able to stabilize mRNA transcripts, which encode MMP-1 and MMP-3. On the other hand, TGF-β is capable of destabilizing MMP-13 transcripts (Delany et al.,1995; Vincenti et al.,2001; Overall et al.,1991). Like other proteolytic enzymes, MMPs are generally produced in latent form, that is, inactive proenzymes or zymogens (Hu et al., 2007). Zymogens can be activated by removal or dissociation of the cysteine switch. Various factors are able to fulfill the role involved into the activation of pro-MMPs through their proteolytic activity or ectopic perturbantion on the cysteine-zinc interaction, such as furin-like enzymes, HgCl2, and N-ethylmaleimide (Okumura et al. 1997; Nagase and Woessner, 1999). Tissue plasminogen activator is able to convert plasminogen to plasmin which is involved into the activation of proMMPs such as proMMP-1, proMMP-3, proMMP-7, proMMP-9, proMMP-10 and proMMP-13 (Lijnen 2001; Amălinei et al., 2007). Once proMMPs are activated, they participate into other pro-MMP activation processes (Figure 1.7). In the case of membrane type MMPs (MT-MMPs), MMP-23, and MMP-28, furin-like proteins are able to

13 activate these enzymes intracellularly based on the structure features and similarity (Shiomi, 2003). The activation of MMP-9 is very complex. As demonstrated in figure1.7, following the activation of other MMPs, MMP-9 can be activated by those active MMPs. Plasmin or furin, together, are able to activate all of MMPs. For example, tissue-type plasminogen activator (t-PA) activates proMMP-3 in cell plasma. Following this process, proMMP-9 can be activated by active MMP-3. After proMT1-MMP is activated by furin intracellularly, proMMP-2 can be activated with the help of active MT1-MMP and TIMP-2. And then, active MMP-2 is also able to convert proMMP-9 to active MMP-9 (Philippe et al., 2002; Deryugina et al., 2001).

FIGURE 1.7 The activation network of MMP-9 by MMPs. This figure has been modified from (Philippe et al., 2002).

As previous studies have indicated, the activation of MMP-2 occurs at cell

14 surface and is different with some unique features comparing to the activation processes of other MMPs. It is illustrated in figure 1.8. This process involves several steps. Both of MT-MMP and the tissue inhibitor of matrix metalloproteinases (TIMP-2) are involved in this multiple-step process (Strongin et al., 1995). MT-1-MMP is an excellent activator of MMP-2 among all of the MT-MMPs. In the MT-MMP family, MT4- MMP and human MT2-MMP are the only ones that are unable to activate MMP-2 (Zucker et al., 1998). As shown in figure 1.8, there is a bonding occurring between the N-terminal domain of TIMP-2 and the active site of MT1-MMP. Meanwhile, the C-terminal of TIMP-2 acts as a receptor and binds with the hemopexin domain of proMMP-2. Another MT-1MMP, which is free from binding with TIMP-2 and located near the complex, partially activates proMMP-2 by cleaves the prodomain. There is an autocleavage following this process which releases active MMP-2 (Deryugina et al., 2001; Amălinei et al., 2007).

Figure 1.8 The activation of proMMP-2 involving MT1-MMP and TIMP-2. Cat, catalytic domain; H, hemopexin domain; P, pro domain; N, N-terminal inhibitory domain; C, C-terminal non-inhibitory domain. This figure has been modified from Deryugina et al., 2001.

The regulation of MMP activity becomes very important. Usually, activity of MMPs are well-regulated by their endogenous inhibitors, such as α2-macroglobulin, the tissue inhibitor of matrix metalloproteinases (TIMPs) and other proteases (Nagase and Woodrell, 1999). TIMPs are two-domain structured molecules, having an N-terminal domain and a smaller C-terminal domain, each domain being stabilized by three disulfide bonds (Douglas et al., 1997). TIMPs are believed to inhibit MMPs through a bidentate coordination of the N-terminal of the TIMP to the catalytic zinc ion, leading to the exclusion of water, a necessary factor for catalysis, from the active site (Nagase and Woodrell,1999). Through this bidentate coordination and multiple other interactions between TIMPs and MMPs, TIMPs form strong, noncovalent complexes with MMPs at a 1:1 ratio in a reversible manner and are tight-binding inhibitors of MMPs (Douglas et al., 1997). The imbalance in the related concentrations of enzyme and TIMP has been observed in many degradative diseases such as arthritis, cancer, cardiovascular diseases (Nagase and Hooper, 1996; Woessner et al., 1998). α2-macroglobulin is a major inhibitor of MMPs in tissue fluids. It inhibits most of MMPs by trapping them within the macroglobulin. It plays critical role in the clearance of MMPs in an irreversible way (Sottrup-Jensen and Birkedal-Hansen, 2001). Because their

Figure 1.9 Relationship between enzyme and peptide-based inhibitor structure. This figure is modified from (Whittaker et al., 1999). ZBG: zinc binding group.

endogenous inhibitors are not only acting as MMP inhibitors, but also carrying other biological functions, it causes the limitation on the clinical application of

16 using the TIMPs for potential therapies (Hu et al., 2007). Small molecular weight organic compounds have been developed as Inhibitors of MMPs for decades because of their potential use in the treatment of cancer and arthritis primarily. The principal approach taken for the identification and characterization of synthetic MMP inhibitors is based on sequence information about the cleavage site of substrate. The substrate character of the peptidomimetic part allows the inhibitor to be recognized and to fit into the enzyme active site (Zhang and Kim, 2009). In theory, because the newly developed substrate analogue will bind the enzyme but will not be hydrolyzed, it would be a functional competitive inhibitor. A schematic view of the structural relationship between the inhibitor and enzyme is shown in figure 1.9. In the substrate-based inhibitor design, three classes of compounds have been developed, binding on both sides of zinc binding group (ZBG) by amino acid residues and those in which the amino acid residues are attached on only the left-hand side or only the right-hand side of the ZBG (figure 1.9) (Whittaker et al., 1999). Based on previous studies, it has been found that compounds mimicking the sequence to the right-hand side (prime side) of the active-site and incorporate a hydroxymate ZBG exhibited particularly potent inhibition. In contrast, the corresponding left-hand side inhibitors were reported to possess only modest inhibitory potency (Johnson et al., 1987; Henderson, 1990; Schwartz and Van Wart, 1992). The structures of most sythetic MMP inhibitors (MMPIs) carry two parts, a peptidomimetic backbone and a zinc-binding group (ZBG) (De et al., 1999). For an effective MMP inhibitor, what is important is organic functional groups which serve as ZBG that is capable of chelating the zinc ion at the active site and other functional groups which may interact with enzyme backbone and bind to the enzyme subsites such as S1’, S2’ etc. by forming hydrogen bond or through van der Waals interactions between inhibitor and enzyme (De et al., 1999; Zhang and Kim, 2009). In order to satisfy those requirements, people have designed and synthesized numerous inhibitors with different zinc-binding groups. The most potent inhibitors have been achieved with a hydroxamic acid zinc chelating functionality. Other functional groups effective for MMP inhibition include

17 carboxylate, phosphinate, sulfodiimine, mercaptan, and mercaptosulfide (Hurst, 2004; Schwartz and van Wart 1995; Sang et al. 1999; Sang et al. 2006).

Figure 1.10 Common zinc binding groups. This figure has been modified from (Schwartz, and Van Wart, 1995, Whittaker et al. 1999).

Although numerous inhibitors have been designed and synthesized, there has been only one compound approved for clinical use, which is Periostat™ (CollaGenex Pharmaceuticals, New York, NY) for periodontal inflammation. This compound is a low-dose doxycycline formulation (Pavlaki, 2003). Most of them could not pass oncological clinical trial to be therapeutic agents because of each of them carries different unwanted properies as drug such as toxicity, bioavailability, selectivity etc. (Zhang and Kim, 2009). The lack of MMPIs with sufficient specificities and favorable pharmacological properties is a recognized impediment to the advancement of disease treatment such as stroke treatment. Only a few of MMPIs among numerous hydroxamate inhibitors were reported to

18 be selective for gelatinases (Rossello et al., 2004; Jani et al., 2005; Rossello et al., 2005). These inhibitors may be useful in the biological mechanism studies, but have dim possibilities in clinic application. The Mobashery group has described thiirane-based irreversible MMPIs for gelatinases (Brown et al., 2000; Lim et al., 2004; Ikejiri et al., 2005; Lee et al., 2005). These thiirane MMPIs were reported to be potent, and to selectively inhibit gelatinases A and B (MMP-2 and -9, respectively) in an irreversibly, fashion while sparing the remaining MMPs. However, irreversible inhibitors, especially those with reactive functional groups such as a thiirane, are likely to make poor leading compounds for drug development, given their poor stability and the unpredictable toxicity originating from their inherent chemical reactivity. It is extremely challenging to achieve the inhibitory specificity for MMPs because of the similarity at their active sites (Table 1.1), combined with the intrinsic plasticity and adaptability of the protein environment (Wittaker et al., 1999). The hydroxamate has been indicated to be the most prevalent ZBG used in MMPI design because it has the strongest zinc binding ability (Zhang and Kim, 2009). It has been indicated that hydroxamate is able to bind to the zinc ion in a bidentate fashion, since each oxygen is coordinating to the zinc ion, meanwhile the nitrogen atom may participate in hydrogen bonding (Schwartz and Van Wart, 1992; Whittaker et al., 1999; Browner et al.,1995). Unfortunately, because of all of the side effects resulted from their intrinsic poor bioavailability and their toxicity, none of hydroxamate MMPIs has succeeded in oncological clinical trials (Coussens et al., 2002). In order to overcome the undesired side effects of MMPs, plenty of previous research and design has been focused on modification of the peptidomimetic part. It has been showing disappointing results so far in clinical trials (Skiles et al., 2004). However, research aimed at development of MMPIs, especially those with alternative ZBGs, is still underway because of their great therapeutic potential in stroke, multiple sclerosis, and other cardiovascular diseases (Breuer et al.,2005; Puerta et al.,2006). Future research in MMPI development has to be focused on the selection of biologically friendly ZBGs and on inhibitory specificity for one

MMP at a time. The approach to the problem has been to try to incorporate the thiol into bidentate zinc chelating ligands, which led to the development of the novel 1,2-mercaptosulfide ZBG (figure 1.11) (Schwartz and Van Wart, 1995; Sang et al., 2000; Park et al., 2003; Hurst et al., 2004; Hurst et al., 2005).

Table 1.1 Selected Variable Residues in the Active Site of MMPs (Wittaker et al., 1999). They are very similar at the active sites.

Figure 1.11 Inhibitor zinc binding group

Although thiol ZBGs have the disadvantage of being oxidized to disulfides, it is still an attractive group for incorporating into the design of MMP inhibitors. Previous studies have suggested that although the intrinsic affinity of a

20 monodentate thiol ZBG is less than that of the bidentate groups such as carboxylate or hydroxamate, lower dissolution costs and easier ionization tend to make such inhibitors only slightly less potent than the hydroxamate class of MMP

Figure 1.12 Concept and rationale of novel thiol MMPIs proposed in this study. Figure is indicating the structural differences between the mercaptosulfide and the mercaptosulfonamide inhibitors.

inhibitors (Schwartz and Van Wart, 1992; Whittaker et al., 1999). The advantages of the thiol group as an attractive choice by our collaborators Drs. Drs. Schwartz and Jin at the florida state university are: (i) it is a well known biocompatible metal ligand that has been previously explored in the design of metalloproteinase inhibitors; and (ii) it is especially creditable as possibly therapeutically useful with one successful precedent case, Captopril, a hypertension drug that acts as an

21 inhibitor of angiotension converting enzyme (ACE) (White, 1998), a zinc metalloproteinase similar in many ways to MMPs. Since thiol MMP inhibitors are generally less potent than hydroxamate inhibitors, there have been some attempts to solve this problem, but with limited success in the past (Campbell et al., 1998; Levin et al., 1998). Drs. Schwartz and Jin have successfully approached this problem by introducing an extra zinc binding ligand (Figure 1.12). They first introduced a novel ZBG in MMPI design by incorporating a sulfide, a widely used ligand in host-guest chemistry (Gordon, 1979), into a bidentate thiol ligand. Initial study aimed at identifying the binding characteristics of the mercaptosufide ZBG quickly revealed that compounds with the 1,2-mercaptoethylsulfide moiety gave dramatically better inhibitory activities, comparable to analogous hydroxamate inhibitors (Table 1.2) (Schwartz and Van Wart 1995; Sang et al., 1999; Sang et al., 2000; Jia et al., 2000; Park et al., 2003; Hurst et al., 2004; Hurst et al., 2005). Our two groups have synthesized and biochemically characterized mercaptosulfide MMP inhibitors with attractive profiles for possible application in stroke (Table 1.2), respectively. For example, MAG-243 is an excellent MMP-9 inhibitor, with dissociation constant values in the picomolar range, that spares MMP-1 and MMP-7. Some of these inhibitors have exhibited excellent pharmacological properties in human vascular endothelial and prostate cancer cell cultures, such as stability and non-cytotoxicity (Schwartz and Van Wart 1995; Sang et al., 1999; Sang et al., 2000; Jia et al., 2000; Park et al., 2003; Hurst et al., 2004; Hurst et al., 2005). Most recently, 1,2-mercaptosulfonamides with structural simplicity and synthetic feasibility were explored as a new generation of thiol-based MMPIs with potentially better properties. In our previous works, we disclosed a series of MMPIs with 1,2-mercaptosulfide pharmacophore (Schwartz and Van Wart 1995; Sang et al., 1999; Sang et al., 2000; Park et al., 2003; Hurst et al., 2004; Hurst et al., 2005) and found that they have excellent inhibitory activities and some

Table 1.2 MMP Inhibitors with a Mercaptosulfide Zinc Binding Group. This figure has been modified from (Park et al., 2003).

S1'

Zn2+ O Zn2+ S ' H 1 N Me H Ar S S N S N H S O H O O Ph

Figure 1.13 Structures and predicted bindings of mercaptosulfide inhibitor MAG-182 and proposed mercaptosulphonamide inhibitors.

selectivities. Representive compound MAG-182 has shown excellent inhibitory activities (Table 1.2 and Figure 1.12). Cyclopentane ring incorporated in the inhibitor pharmacophore was envisioned to induce conformational rigidity and increase in vitro potency and pharmacological properties (Schwartz and van Wart, 1995). Based on the structure of MAG-182, we proposed that peptide part of MAG- could be replaced with aryl sulphonamide to derive a new kind of thiol inhibitor with small molecular weight and simple structure, and most specificity, lacking the pepdidomimetic parts (Figure1.13). In this study, I have characterized and evaluated our newly designed and synthezied MMP inhibitors provided by Drs. Schwartz and Jin at enzymatic level

23 by running kinetic studies and in cellular system such as human brain microvascular endothelial cell (hBMEC) related to stroke. Several promising mercaptosofonamide inhibitors have been selected for animal studies. This is the first study on mercaptosulfonamide MMP inhibitors.

CHAPTER TWO

MATERIALS AND METHODS

Materials: All chemicals were purchased from the Fisher Scientific Company except for the following: Human type IV collagen was purchased form Sigma-Aldrich and fluorescent substrate Mca-PLGLDpaAR-NH2 was purchased from Bachem Chemical Company. The synthetic MMP inhibitors were provided by Dr. Martin Schwartz and Dr. Yong-Hao Jin. Matrilysin was provided by Dr. Harold Van Wart’s laboratory, and metalloelastase was provided by Dr. Christine Schiodt’s laboratory. Human fibroblast collagenase (MMP-1) and gelatinase A (72 kDa type IV collagenase, MMP-2) were provided by Dr. Henning Birkedal-Hansen at the National Institutes of Health (Birkedal-Hansen, 1998). Human stromelysin (MMP-3) was provided by Dr. Steve Van Doren from the University of Missouri. Human recombinant matrilysin (MMP-7) was made in Chinese hamster ovary cells and provided by Dr. Harold E. Van Wart from Roche BioSciences, Inc. Gelatinase B (MMP-9) were purified and characterized as described (Sang et al., 1995). Endometase (MMP-26) was expressed in E.coli and extracted, purified and processed for activation in our lab. hBMEC from ScienCell, CA and microvascular endothelial cell medium-2 (EGM-2MV) from Lonza-Clonetics, hMSC from Tulane Center for Gene Therapy and Alpha Minimum Essential Medium (αMEM, Sigma Aldrich). 6-well plates and 24 well-plates were purchased from Fisher Scientific. Invasion chamber was purchased form Becton Dickinson Labware. Ready IPG Strips, pH 3-11, 17 cm long and dithiothreitol were purchased from Promega. Tricine, sodium dodecyl sulfate, urea, and bromophenol blue were purchased from Sigma-Aldrich. CHAPS was purchased from Calbiochem. Total hydrolysis of Quenched Fluorescent Peptide:

A stock endometase (3.8 M) and substrate (Mca-PLGLDpaAR-NH2) mixture was made and incubated at 25 °c for 2 days. It contained 10 L of endometase 25

(3.8 M) and 50 M stock concentration of substrate. It was diluted to 125, 62.5,

31.25 and 15.625 nM with 50 mM HEPES, pH 7.5, 10 mM CaCl2, 0.20 M NaCl, 0.01 % Brij-35 to test the fluorescent intensities. The emergence of fluorescence due to the cleavage of the peptide substrate was monitored using Perkin Elmer LS50B Luminescence Spectrometer (excitation wavelength 328 nM, emission wavelength 393 nM, software FL WinLab v3.0) connected to a temperature controlled water bath. Time Dependence Experiments: Time dependence of inhibitor-enzyme interactions was determined from progress curves in the presence of 1 M of substrate. Inhibitor concentrations near the IC50 values were used. The enzyme (MMP-2, MMP-9) was preincubated with inhibitors (YHJ-6-43, YHJ-6-286) at 25 °c in 50 mM HEPES, pH 7.5, 10 mM

CaCl2, 0.20 M NaCl, 0.01 % Brij-35, 50 M of the reducing agent, tris(2-carboxyethyl)phosphine (TCEP) in 200 L tube, respectively. One assay volume (196 l) of the mixture was taken out at each time point. The reaction was initiated by addition of 4 l of substrate to gve a final concentration of 1 M. The velocities (fluorescent intensity increase / time) of the reactions were collected according to the instruction of the computer program, FLWinlab. All inhibitors were dissolved in dimethylsulfoxide (DMSO), and the substrate (Mca-PLGLDpaAR-NH2) was in 1:1 H2O: DMSO (v/v). As a result, the systems contained 6% DMSO by volume.

Estimation of the Michaelis constant (Km) Value: 10 L of Enzyme (20 nM) was incubated in 186 l 50 mM HEPES, pH 7.5, 10 mM CaCl2, 0.20 M NaCl, 0.01 % Brij-35 buffer for 5 minutes. The reaction was initiated by an addition of 4 L of substrate prepared as mentioned above. As a result, the systems contained 6% DMSO by volume. 8 different substrate concentrations were used to collect the reaction velocity, such as 1000 M, 500 M, 250 M, 12 5 M, 62.5 M, 31.25 M, 15.625 M and 7.8125 M. For each enzyme, the ([S], v) data pairs were fitted to the Michaelis-Menten equation: to

26 estimate the Km and Vmax (maximum reaction rate) values by doing nonlinear curve fitting with Sigmplot 2000.

v = Vmax [S] / (Km + [S]) (Equation 2.1)

Competitive inhibition determination: Enzymatic assays were performed at 25 °C in 50 mM HEPES buffer at pH

7.5 in the presence of 10 mM CaCl2, 0.2 M NaCl, 0.01% Brij-35 and 5 µM of the reducing agent TCEP, with three different substrate concentrations of 1 µM, 5 µM and 10 µM, respectively. The substrate (Mca-PLGLDpaAR-NH2) was in 1:1 H2O: DMSO (dimethylsulfoxide) (v/v). The release of product was monitored by measuring fluorescence (excitation and emission wavelengths of 328 and 393 nm, respectively) with a PerkinElmer luminescence spectrophotometer LS 50B connected to a temperature controlled water bath. All stock solutions of inhibitors were dissolved in DMSO. For inhibition assays, 10 µl of inhibitor stock solution, 176µl of assay buffer, and 10 µl of enzyme stock solution (MMP-9) were mixed and incubated for about 30 min determined from experiments above, prior to initiation of the assay, via addition of 4 µl of substrate 5 pair of (intensity/min) data collected.. Enzyme stock concentration was about 20nM. As a result, the systems contained 6% DMSO by volume. The data was plotted as straight lines using the Henderson equation (referred app to CHAPTER THREE). The slope of each line is the Ki . It is replotted versus the substrate concentration. app ’ The determination of apparent dissociation constant (Ki or Ki ): Enzymatic assays were performed at 25 °C in 50 mM HEPES buffer at pH 7.5 in the presence of 10 mM CaCl2, 0.2 M NaCl, and 0.01% Brij-35 with 1 µM of substrate and 5 µM of the reducing agent TCEP. The substrate

(Mca-PLGLDpaAR-NH2) was in 1:1 H2O: DMSO (v/v), and the systems contained 6% DMSO by volume as well.

The release of product was monitored by measuring fluorescence (excitation and emission wavelengths of 328 and 393 nm, respectively) with a PerkinElmer luminescence spectrophotometer LS 50B connected to a temperature controlled water bath. All stock solutions of inhibitors were dissolved in DMSO. For inhibition assays, 10 µl of inhibitor stock solution, 176µl of assay buffer, and 10µl of enzyme stock solution were mixed and incubated for about 30 min as determined in experiments above, prior to initiation of the assay, via addition of 4 µl of the substrate stock solution. Enzyme concentrations ranged from 0.2 to 7 nM for the app ’ assay. Apparent inhibition constant (Ki or Ki ) values were calculated by fitting the kinetic data to the Morrison equation for tight-binding inhibitors (Park et al., 2000; Morrison, 1969, referred to CHAPTER THREE). MMPI stability in HEPES buffer: Kinetic assays to determine the stability of the MMPIs over various periods of time and in different mediums were carried similarly to those just previously described. The enzyme, in this case MMP-9 with a final concentration of 1 nM, and was incubated with a MMPI at 25 °C in 50 mM HEPES buffer at pH 7.5 in the presence of 10 mM CaCl2, 0.2 M NaCl, and 0.01% Brij-35 with or without 5 µlM of the reducing agent TCEP. All of MMP inhibitors were dissolved and diluted in

HEPES buffer to its relative IC50 (inhibitor concentration at which 50% of enzyme activity is ablated). After 30 min incubation, 4 µl of the fluorescent substrate which was dissolved in a 1:1 solution of DMSO: H2O was added to initiate the assay. Fluorescence readings were taken at several of time points and the relative rate

(υi/ υo) was plotted versus time. MMPI stability in microvascular endothelial cell growth medium (EGM-2 MV): Kinetic assays to determine the stability of the MMPIs over various periods of time and in different mediums were carried similarly to those just previously described. An MMPI (10 µM ) was incubated in EGM-2 MV (5%FBS, 0.04%Hydrocortisone, 0.4%hFGF-B, 0.1%VEGF, 0.1%R3-IGF-1, 0.1%Ascorbic acid, 0.1%hEGF, 0.1%GA-1000) for up to 24 hours at 37°C and 5% CO2 in a cell incubator (Four time points were collected such as 4 hours, 8 hours, 12 hours and

24 hours). 10 µls of MMPI solution were taken at each time point and mixed with MMP-9 for a final concentration of 1 nM. Samples were incubated in 176 µl of 50 mM HEPES buffer at pH 7.5 in the presence of 10 mM CaCl2, 0.2 M NaCl, and 0.01% Brij-35 with 5 µM of the reducing agent TCEP. After 30 min incubation, addition of 4 µl of the fluorescent substrate, dissolved in a 1:1 solution of

DMSO:H2O, initiated the assay which was monitored by fluorescence. The relative rate (υi/ υo) was plotted versus time. Human Brain Microvascular Endothelial (hBMEC) Growth: Passages 6-8 (P6-P8) human brain microvascular endothelial cells (hBMECs,

ScienCell) were plated for a recovery phase of 24 hours at 37°C and 5% CO2 in microvascular endothelial cell growth medium (EGM-2 MV) (5% FBS, 0.04% Hydrocortisone, 0.4% hFGF-B, 0.1% VEGF, 0.1% R3-IGF-1, 0.1% Ascorbic acid, 0.1% hEGF, 0.1% GA-1000). After the recovery phase, hBMECs were seeded into 6-well plates, each well containing 2 mL of EGM-2MV, at a density of ~2,000 cells/cm2. Fresh cell culture medium was added every three days until the cells reached a confluency of approximately ~70% at which time the media was changed to the conditioned forms necessary for observing MMPI cytotoxicity. Human Mesenchymal Stem Cell (hMSC) Growth: Passage 3 human adult bone marrow-derived mesenchymal stem cells (hMSCs, Tulane Center for Gene Therapy) were plated for a recovery phase of 24 hours at 37ºC and 5% CO2 in Alpha Minimum Essential Medium (αMEM, Sigma Aldrich) containing L-glutamine but without ribonucleosides or deoxyribonucleosides, 16.5% FBS, ~2-4 mM L-glutamine, and penicillin and streptomycin as antibiotics. After the recovery phase, hMSCs were seeded into 6-well plates at a density of ~60 cells/cm2 with each well containing 2 mL of cell culture medium. Fresh cell culture medium was added every 3 days until the cells reached a confluence of approximately ~70% at which time the media was changed to the conditioned forms necessary for observing MMPI cytotoxicity. The assay of inhibition on Endothelial cells migration: All experiments for this phase were performed in triplicate, and each iteration consisted of seven wounded cultures utilizing serial concentrations of MMPIs: an

29 untreated control, 1nM MMPI, 10 nM MMPI, 100 nM MMPI, 1µM MMPI, 3 µM MMPI, 10 µM MMPI, 30 µM MMPI and 100 µM. All of inhibitors were diluted with EGM-2 MV (5% FBS, 0.04% Hydrocortisone, 0.4% hFGF-B, 0.1% VEGF, 0.1% R3-IGF-1, 0.1% Ascorbic acid, 0.1% hEGF, 0.1% GA-1000). An 1mL tip was utilized to create a linear wound in a confluent (70~80%) monolayer cell culture (Kinsella et al., 1986; Ettenson et al., 1995). A fine, sterile needle was used to mark a cell-free ―wounded field‖. The wounded culture was kept at 37°C and 5%

CO2 in cell incubator. After 24 hours, wounded cell cultures were washed three times with PBS and stained with 0.1% crystal violet for 15 minutes. Stained cultures were washed with DD H2O three times and left over night to air-dry. Cell cultures were photographed with a Nikon microscope with a 1x objective. The density of cell coverage in the wounded field was quantified utilizing the software ImageJ 1.42 bundled with 32-bit. Statistical method-one way ANOVA was used for subsequent comparative statistical analyses. The impact of MMPI on the migration of hBMECs was concluded based on the results. Since all of inhibitors used for cell culture were dissolved in ethanol, an ethanol control plate was set up based on each concentration of each treatment and treated the same way as other plates. Human brain microvascular endothelial cell Culture & Human mesenchymal stem cell co-Culture: Human brain microvascular endothelial cells were cultured in EGM-2 MV (5% FBS, 0.04% Hydrocortisone, 0.4% hFGF-B, 0.1% VEGF, 0.1% R3-IGF-1, 0.1%

Ascorbic acid, 0.1% hEGF, 0.1% GA-1000) at 37°C and 5% CO2. When both hBMEC and hMSC cultures reached ~70% confluence, cells were sub-cultured into 24-well plates at the following hBMEC to hMSC ratios 50%: 50%, 80%: 20%, 90%: 10%, and 95%: 5%. Upon reaching the desired confluence, a potent synthetic MMP inhibitor (YHJ-6-43) was added to various wells at a concentration of 10 μM. A fine, sterile needle was used to mark a cell-free ―wounded field‖, which was created by gentle scratching with a 1-mL pipette tip. The wounded culture was kept at 37°C and 5% CO2 in a cell incubator. After 24 hours, wounded cell culture were washed three times with PBS and stained with 0.1% crystal violet

30 for 15 minutes. Stained culture were washed with DD H2O for three times and left for over night to air-dry. Cell cultures were photographed with a Nikon microscope with 1x objective. The density of cell coverage in the wounded field was quantified by utilizing the software ImageJ 1.42 bundled with 32-bit. Statistical method-one way ANOVA was used for subsequent comparative statistical analyses. The impact of MMPI on the migration of hBMECs was concluded based on the results. Because all of inhibitors used for cell culture were dissolved in ethanol, an ethanol control plate was set up based on each concentration of each treatment and treated the same way as other plates. MMPI cytotoxicity in EGM-2MV: Upon reaching the desired confluence, hBMECs were treated with fresh EGM-2MV that was conditioned with a logarithmic panel of MMPI concentrations ranging from 1 nM to 100 μM. Specifically, each of the inhibitors were dissolved in an appropriate amount of 100% ethanol to generate an 8 mM stock solutions from which an aliquot was used to produce a series of dilutions with EGM-2MV. The conditioned media was added to the hBMECs and incubated for 24 hours. Cell culture was kept at 37°C and 5% CO2 in a cell incubator. At the end of the treatment period, the media was removed and the cells were trypsinized after being washed 3 times with phosphate-buffered saline. In order to quantify the levels of toxicity these inhibitors had on hBMEC growth and viability, the cells were treated with trypan blue and counted with a hemocytometer. Each of the treatments, and hemocytometer counts, were performed in triplicate and all error bars represent 95% confidence intervals. Protease inhibitor preparation: One tablet of complete mini (Roche, Mannheim, Germany) was dissolved with

2 mL DDH2O and vortexed until the tablet dissolves. Aliquots were stored at -20°C. The inhibitor treatment on hBMEC, protein preparation When hBMEC culture reached about 70% confluence, 10 μM of YHJ-6-43 dissolved in ethanol was added to the cell culture diluted in fresh EGM-2MV. After 24 hours, old cell medium was replaced. Cells were washed with PBS 3 times.

Cells were scratched after application of cell lysis buffer( 7 M urea, 65 mM DTT, 2% CHAPS, 2 M thiourea and protease inhibitors), and collected into an 1.5 mL tube and kept at -85°C for further experiments. Cell sample homogenization using the bead beater: 300 µL of beads was transfered into a 1.5 mL Eppendorf tube containing cells and lysis buffer (Use a cut Eppendorf tube to measure the beads). The Eppendorf tube containing the cells, lysis buffer and beads was set on the bead beater at 4oC. The bead beater was set at speed 6 for 5 seconds for two times and cold in ice for a few seconds between spins. The extracted protein sample was collected in a 2 mL Eppendorf tube. The homogenized sample was sonicated in an ice bath for 10 min. The sample was then centrifuged at 14,000 rpm/4 oC for 10 min. The supernatant, containing the proteins, was then desalted accordingly. TCA/Acetone Precipitation: 100% ice-cold acetone was mixed with 100% trichloroacetic acid (TCA) in a 9:1 ratio. 8 volumes of acetone/TCA solution were added to the sample. It was vortexed and kept at 4°C for about 8 hours or overnight. Then sample was centrifuged at 14,000 rpm for 10 min at 4°C in a microcentrifuge. After the supernatant was removed, 200 µL of lysis buffer containing protease inhibitors was used to reconstitute the precipitate, where the proteins are concentrated. 80% acetone was added into the precipitate, vortexed the mixture, and kept at 4°C for about 4 hours. It was then centrifuged at 14,000 rpm 10 min at 4 °C. Rehydration buffer was applied to reconstitution of the protein pellet. The protein concentration was determined according to the instruction from company (PIERCE Coomassie plus protein assay reagent PROD# 1856210). Two-dimensional gel electrophoresis (2-DE): Rehydration buffer was mixed with sample (100 µg protein, 200 µl total volume) had been agitated for 1 hour at room temperature with a vortex mixer, and then centrifuged. The IPG dry strips (11 cm, pH 3-11) with plastic side peeled off were placed onto samples and were then covered with oil and allowed to rehydrate overnight. The rehydration tray with strips was transferred to the IPGphor instrument (Bio-Rad). The proteins were focused at 250 V for 15

32 minutes, and then 8000 V was maintained for a total of 42,000 Vh per strip. Once completed, the strips were equilibrated for 10 minutes in 2.5 ml of a solution composed of 375 mM Tris-HCl, pH 8.8, 6 M urea, 2% SDS, trace amount of bromophenol blue and 2% dithiothreitol. After this first equilibration, the strips were equilibrated for another 10 minutes in 2.5 ml of a second equilibration buffer composed of 375 mM Tris-HCl, pH 8.8, 6 M urea, 2% SDS, trace amount of bromophenol blue, and 2.5% Iodoacetamide. Equilibrated strips were washed with cathode buffer (0.1 M Tricine, 0.1 M Tris-HCl, pH 8.2, and 0.1% SDS) and placed onto 12% Sodium dodecyl sulfate polyacrylamide gels. The anode buffer consists of 0.2 M Tris-HCl, pH 8.9. Gels were electrophoresed at 200 V until the end of the separation. Gels were stained with sypro ruby (BIO RAD, Hercules, CA) staining following fixing for 1 hour at room temperature. Gels were transferred into destaining solution to be destained for one hour. Gels were washed three times with DD H2O. They were ready for imaging using Typhoon 9410 Scanning Systems (GE Healthcare, Amersham Biosciences), with an excitation wavelength of 457 nm and signals were detected at 610 nm. Analysis of Proteins: After six iterations of 2-DE for each of the three samples, protein spots were manually excised under UV light, combined, incubated with 0.5 µg/mL TPCK-treated Trypsin, which cuts the C-terminal side of Lysine and Argenine amino acids unless next residue is Proline. Proteins contained in picked spots picked were ready for further identification. LC- MS/MS: The enzymatically digested samples were injected onto a capillary trap (LC Packings PepMap) and washed for 5 min with a flow rate of 10 µL/min of 0.1% v/v acetic acid. The samples were loaded onto an LC Packing® C18 Pep Map HPLC column. The elution gradient of the HPLC column started at 3% solvent A, 97% solvent B and finished at 60% solvent A, 40% solvent B for 60 min for protein identification. Solvent A consisted of 0.1% v/v acetic acid, 3% v/v ACN, and

96.9% v/v H2O. Solvent B consisted of 0.1% v/v acetic acid, 96.9% v/v ACN, and

3% v/v H2O. LC-MS/MS analysis was carried out on a hybrid quadrupole-TOF

33 mass spectrometer (QSTAR, Applied Biosystems, and Framingham, MA). The focusing potential and ion spray voltage was set to 275 V and 2600 V, respectively. The information-dependent acquisition (IDA) mode of operation was employed in which a survey scan from m/z 400–1200 was acquired followed by collision induced dissociation of the three most intense ions. Survey and MS/MS spectra for each IDA cycle were accumulated for 1 and 3 s, respectively. Protein Search Algorithm: Tandem mass spectra were extracted by ABI Analyst version 1.1. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.0.01). Mascot was set up to search NCBInr database assuming the digestion enzyme was trypsin. Mascot was searched with a fragment ion mass tolerance of 0.30 Da and a parent ion tolerance of 0.30 Da. Iodoacetamide derivative of Cy, deamidation of Asn and Gln, oxidation of Met, are specified in Mascot as variable modifications. Scaffold 2 (version Scaffold-02-03-01, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications are accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm (Keller, 2002). Protein identifications are accepted if they can be established at greater than 99.0% probability and contain at least 2 identified unique peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003).

CHAPTER THREE

RESULTS AND DISCUSSION

3.1 Enzyme kinetics:

MMPs are proteins that catalyze and speed up the hydrolysis of substrates by lowering the activation energy (ΔG ). According to the Arrhenius equation (equation 3.1), a reduction in ΔG results in an increasing in the rate constant (K) for a given reaction (Garrett and Grisham, 1999).

G k Ae RT (Equation 3.1) (Garrett and Grisham, 1999)

Here, A is a constant for a particular reaction known as the frequency factor, T is the absolute temperature, and R is the gas constant (Copeland, 2000). Enzyme is able to bind and hydrolyze substrate as shown in Figure 3.1.

k1 kcat E+S ES E+P k -1 Figure 3.1 binding and hydrolyzation between enzyme and substrate.

When the reaction reaches equilibrium,

k 1[ES ] k1[E][S] (Equation 3.2)

Introduction of a new constant, Ks, the enzyme-substrate dissociation constant, leads to

[E][S] k 1 K s (Equation 3.3) [ES ] k1

At steady state conditions,

d[ES] k [E][S] (k [ES] k [ES]) 0 (Equation 3.4) dt 1 1 cat

Because the total enzyme concentration ([ET]) is equal to the sum of the free enzyme concentration ([E]) and the substrate bound enzyme concentration ([ES]), it is given that:

[ET ] [E] [ES ](Equation 3.5)

At steady state conditions, the rate of formation of ES is equal to the rate of disappearance of ES, so

k1 ([ ET ] [ES ])[S] (k 1 kcat )[ ES ](Equation 3.6)

Arrange equation 3.6,

([ET ] [ES])[S] k 1 kcat K m (Equation 3.7) [ES] k1

Here, Km is known as the Michaelis constant. Because

d P][ v k [ES](Equation 3.8) dt cat

And

[E ][S] [ES ] T (Equation 3.9 based on Equation 3.7), K m S][

We can get

k [E ][S] V S][ v cat T max (Equation 3.10, the Michaelis-Menten equation) K m S][ K m S][

Vmax is the maximum reaction rate, which represents the limit of the velocity at infinite substrate concentration. At high substrate concentration, it is true that

Vmax kcat [ET ](Equation 3.11)

It is because all of the free enzyme is converted to the enzyme-substrate complex (Garrett, 1999). Based on the Michaelis-Menten equation, we can plot a graph which helps with determine Vmax and Km (Figure 3.2) (Fersht, 2002). Following equation 3.10, when [S] << Km, it become

k v cat [E][S] (Equation 3.12) (Garrett and Grisham, 1999; Copeland, 2000) K m

There are two parameters introduced. One is the turnover number, kcat, which represents the number of catalytic turnover in a given period of time. Another is the Michaelis constant, Km, which is a measure of the affinity between the substrate and enzyme, the lower the value the higher the affinity. Km is also the

37 substrate concentration that gives half of the maximum velocity (Vmax) (Garrett and Grisham, 1999; Copeland, 2000).

Figure 3.2. Michaelis-Menten hyperbolic curve. This figure has been modified from Fersht, 2002).

Total hydrolysis of Quenched Fluorescent Peptide In order to monitor the progress of an enzyme reaction by using fluorescence spectroscopy, a peptide substrate has been used. As shown in Figure 3.3, it contains two parts, a fluorophore and a quencher in close proximity to one another and on opposite sides of the scissile bond. Upon substrate cleavage at scissile bond, they are separated from each other and cause the increase in fluorescence (Copeland. 2000). The substrate used in this study carries the fluorophore (7-methoxycoumarin-4-yl) acetyl (Mca) that has a maximum excitation at 328 nm and emission maximum at 393 nm (Knight, et al.., 1992). Through a short peptide chain, the N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl (Dpa) is attached to this fluorophore with the absorbing group 2,4-dinitrophenyl (Dnp) that shows an overlapping absorption spectrum with the emission of Mca. When the whole substrate sequence is in intact, the quencher-Dnp is significantly 38 quenched the emission of the fluorohphore through fluorescence resonance energy transfer. At the presence of MMPs, it is cleaved at the scissile bond which results into the separation of both of the functional groups. The increased fluorescence intensity is proportional to the rate of products generated form the hydrolysis reaction.

O O O O O H H H N N N N N N N NH H H H 2 O O O NH O N MeO O O 2 NH

NH2+ Cleavage site NH NO 2 2 Figure 3.3. The full structure of substrate used in this study. This figure has been modified from (Knight, et al. 1992).

Tot al hydr ol ysi s

700 y = 4. 5601x + 64. 304 600 500 400 300

( FU) 200 100 0

Fl uor escent i nt ensi t y 0 20 40 60 80 100 120 140 Subst r at e ( nM)

Figure 3.4. Intensity of cleaved substrate. The fluorescence intensity of the standard Mca-Pro-Leu-OH was measured for several concentrations at 25°C. The slope of 4.56 fluorescent units (FU) per nM was used as a conversion factor for the rate of cleavage from FU/min to nM/min of the quenched peptides.

A stock MMP-26 (endometase) and this substrate mixture was prepared and incubated at 25 °c for 2 days. It contained 3.8 M endometase and 50 M the substrate with a complete sequence Mca-PLGLDpaARNH2. The fluorescent intensities at different concentrations of substrate were collected after the substrate was completely hydrolyzed. The slope of the linear regression of intensity on [S] gave the conversion factor as 4.56 Fluorescent unit (FU) / nM (Figure 3.4). Fluorescence was monitored with a Perkin Elmer Luminescence Spectrophotometer LS 50B (FL WinLab v3.0) connected to a temperature controlled water bath.

Estimation of Km Value

The det er minat i on of KM ( MMP-2) 300

270

240

210

180

150 120

React i on r at e 90

0 0 5 10 15 20 25 [ S] µM

Figure 3.5. Km of MMP-2 determination. Initial rates were determined for the hydrolysis of several concentrations of Mca-PLGL-Dpa-AR-NH2 by MMP-2. Data was fit using the Michaelis-Menten equation.

The Km of the efficiently hydrolyzed substrate by MMP-2 was approximated to be greater than 10 µM (Figure 3.5). Because the substrate becomes insoluble once the concentration is greater than 20 µM, this value is an 40 estimated value. The Km of the efficiently hydrolyzed substrate by MMP-9 was approximated to be greater than 10 µM (Figure 3.6). Because the substrate becomes insoluble once the concentration is greater than 20 µM, this value is an estimated value.

The det er minat i on of KM ( MMP-9)

300 270 240 210 180 150 120 React i on r at e 90 60 30 0 0 5 10 15 20 25 [ S] µM

Figure 3.6. Km of MMP-9 determination. Initial rates were determined for the hydrolysis of several concentrations of Mca-PLGL-Dpa-AR-NH2 by MMP-9. Data was fit using the Michaelis-Menten equation.

Time Dependence Experiments Because we are measuring the equilibrium dissociation constant, it is important to ensure that the system is in equilibrium when measurements are made. In order to ensure the characterization of enzyme-inhibitor binding reaction done in equilibrium, time dependant test of inhibitor has been performed to identify how it takes to reach equilibrium for enzyme and inhibitor incubation and binding. Enzymes were incubated with inhibitors (YHJ-6-43, YHJ-6-286) for

41 various periods of time before the initiation of the reaction by the addition of substrate, respectively.

YHJ-6- 43 Ti me denpendence agai nst MMP-2

1 0. 8 0. 6

Vi / Vo 0. 4 0. 2 0 0 20 40 60 80 100 Ti me ( min)

Figure 3.7 Time dependence study of YHJ-6-43 against MMP-2. The equilibrium of binding between enzyme and inhibitor is achieved around 30 minutes.

YHJ-6- 43 Ti me dependence agai nst MMP-9

1 0. 8 0. 6

Vi /0. Vo 4 0. 2 0 0 20 40 60 80 100 Ti me( min)

Figure 3.8 Time dependence study of YHJ-6-43 against MMP-9. Figure is demonstrating that the equilibrium between enzyme and inhibitor was achieved from 20-60 minutes incubation.

Figure 3.7 & 3.8 is showing the reaction of YHJ-6-43 against MMP-2 and MMP-9, respectively. Both curves haven shown concave upward shapes. In between 30-60 minutes, the reactions reached equilibrium status. Around 90 minutes, we see a slightly recovery on enzyme activity. That suggests the equilibrium has been achieved after long time incubation-30 minutes. From the results above, we are able to conclude the interactions between these two enzymes and YHJ-6-43 are slow binding because of taking long time for the interactions reach equilibrium after incubation.

YHJ-6- 286 Ti me dependence agai nst MMP-2 1 0. 8 0. 6

Vi / Vo 0. 4 0. 2 0 0 20 40 60 80 100 Ti me ( min)

Figure 3.9 Time dependence study of YHJ-6-286 against MMP-2. Figure is demonstrating that the equilibrium between enzyme and inhibitor was achieved from 30-60 minutes incubation.

YHJ-6-286 is another inhibitor chosen for evaluation. Figure 3.9 and 3.10 are demonstrating the reactions of YHJ-6-286 against MMP-2 and MMP-9, respectively. Those curve show concave upward shapes. In between 20-60 minutes, the reactions reached equilibrium status. Around 90 minutes, we see slightly recovery on enzyme activity. That suggests the equilibrium is achieved

43 after lone time incubation-within 20-60 minutes incubation. From the results above, we are able to tell the interactions between MMP-2 and MMP-9 and YHJ-6-286 are slow binding because it takes quite long time for the interactions reach equilibrium after incubation.

YHJ-6- 286 Ti me dependnece agai nst MMP-9

1 0. 8 0. 6

Vi / Vo 0. 4 0. 2 0 0 20 40 60 80 100 Ti me ( min)

Figure 3.10 Time dependence study of YHJ-6-286 against MMP-9. Figure is demonstrating that the equilibrium between enzyme and inhibitor was achieved from 20-60 minutes incubation.

app The apparent dissociation constant (Ki ) determination

The equilibrium dissociation constant (Ki) is often used to characterize the potency of a reversible inhibitor and is described by the following equations and Figure. When MMP is mixed with inhibitor, the enzyme-inhibitor complex is formed and is reversible as shown in figure 3.11. A low Ki corresponds to a strong inhibitor.

k3 E+I EI k -3 Figure 3.11 Enzyme-inhibitor association and dissociation

[E][ I] k 3 K i (Equation 3.13) (Copeland, 2000) [EI ] k3

Figure 3.12 The three conditions for a reversible, competitive inhibition based on app the ratio [E]/Ki : classical conditions when the ratio is ≤ 0.01, tight-binding between 0.01 and 100, and titration when ≥ 100. It has been modified form Hurst et al., 2003.

In the case of a classical inhibitor, the assumption is made that the total amount of inhibitor added to the assay is equal to the amount of free inhibitor. In another words, the amount of enzyme-inhibitor complex can be neglected in relation to the total inhibitor concentration ([I]total = [I]free, it is valid when the [E]/Ki ≤ 0.01.). However, for some inhibitors, the so called tight-binding inhibitors, this assumption is no longer valid (Copeland, 2000). In order to determine the Ki of a tight-binding inhibitor, the amount of enzyme- inhibitor complex must be considered (when [E]/Ki > 0.01, [I]total = [I]free + [EI] ) (Bieth, 1995). All of these conditions for reversible, competitive inhibitors have been demonstrate previously described (Bieth, 1995) and demonstrated in Figure 3.12.

The resulting rate equation is arranged in terms of Ki and [E] to This can be written as

1 a [I ] K app [E ](1 a) (Equation 3.14) T i a T

where a is the fraction of total amount of enzyme that is free of inhibitor binding.

[EI ] v a 1 i (Equation 3.15) [ET ] v0

where υi is the initial rate with inhibitor, υo is the initial rate with no inhibitor, and

[E]T is the initial (total) concentration of enzyme. For a competitive tight binding app inhibitor, Ki is influenced by substrate concentration, that is, it is app substrate-dependant. The relation between Ki and Ki for a competitive inhibitor is

app S][ Ki Ki 1 (Equation 3.16) (Cheng and Prusoff, 1973) [K m ]

Solving the equation for total inhibitor concentration,

v ([E ] [I ] K app ) [( E ] [I ] K app )2 [4 E ]K app a i T T i T T i T i v0 [2 ET ] (Equation 3.17)

As mentioned in method, 176 μL of assay buffer, 10 μL of enzyme solution in assay buffer, and 10 μL of inhibitor solution in methanol are incubated for 30

46 minutes, which is determined by time dependence studies, at 25°C to ensure equilibrium conditions have been reached. Following the addition of substrate, the reaction rate is collected or each concentration of inhibitor. By measuring the initial rates with different concentrations of inhibitor and fitting data into Equation app 3.17 or 3.18, we are able to calculate the value of Ki . Based on Equation 16, when the assay is performed under the condition that [S] << KM, the true Ki is app equal to Ki .

The IC50 is another parameter for the inhibition evaluation of a tight-binding, competitive inhibitor can be related to its Ki by the equation

S][ IC50 Ki 1 [5.0 ET ](Equation 3.18) (Barrett, 1995) K m

By definition, when [I] = IC50, vi/v0 = 0.5. After applied into Equation 3.17,

app app 2 IC50 Ki ([ET ] [IC50 ] Ki ) [4 ET ][IC50 ] 0(Equation 3.19)

It is also true,

app 2 app 2 (IC 50 Ki ) ([ET ] [IC 50 ] K i ) [4 ET ][IC 50 ] 0 (Equation 3.20)

Organize and rearrange,

app IC 50 K i [5.0 ET ](Equation 3.21)

app Based on the Equation 3.21, the IC50 is little bit greater than Ki . Although the disadvantage of IC50 is being influence by substrate concentration, it is still a commonly used measure of inhibitor potency (Copeland, 2000). I have investigated more than 47 of mercaptosulfonamides MMP inhibitors against 4 different MMPs. With the help of other lab members (Chi, Manny, Dale,Mark) we evaluated all of inhibitors with 9 different MMPs in our lab.

The competitive mechanism determination of mercaptosulfonamides MMP inhibitors

According to the effects on Km and υmax at the presence of inhibitor in enzyme-substrate system, reversible inhibitors, those reversible inhibitions have grouped into three major mechanisms such as noncompetitive inhibition, uncompetitive inhibition, and competitive inhibition. Noncompetitive inhibitors do not bind the active center of enzymes. They may bind with the free enzyme or the enzyme-substrate complex. Experimentally, there is no change on the value of Km and a decrease on υmax. Comparing with the enzyme-substrate system without inhibitor. If the concentration of substrate is increased, the inhibition can not be overcome (Copeland, 2000). When an uncompetitive inhibitor is introduced into enzyme-substrate system, it causes a proportional decrease on both of υmax and Km since the uncompetitive inhibitor only binds to the enzyme-substrate complex (Copeland, 2000).

In the case of competitive inhibitors, the measured υmax maintains unchanged. The value of Km, however, is increased in the comparison with the enzyme-substrate system without inhibitor. Because the structure of competitive inhibitors is very similar to substrate, they can only bind to the free enzyme. Generally, they bind at the active center, but in rare cases, it may compete with substrate by binding at other sites, which results into the conformational change on the active site. If we increase the concentration of substrate, the inhibition may be overcome. All three mechanisms have been illustrated in Figure 3.13.

In order to identify the mechanism of a tight-binding inhibitor, a linear form of the Morrison Equation (Equation 3.18), which is known as the Henderson equation (Equation 3.22), has been utilized.

[I ] v T K app 0 [E ](Equation 3.22) v i v T 1 i i v0

Here, after we collect reaction rate at several different substrate concentrations, a plot of [I]/(1-υi/υo) versus υo/υi is graphed based on Equation app 3.22. The slope of each curve represents the Ki at each substrate app concentration. The linear form of the replot of Ki versus [S] indicates competitive inhibition. In another words, if the slope of a Henderson plot increases with substrate concentration, this is indicative of a competitive inhibitor. It will not happen to the other two types of inhibitions. It would not affect the slope for noncompetitive inhibition, whereas decreases the slope in the case of uncompetitive inhibition, changing the concentration of substrate (Henderson, 1972). The inhibitors were designed with a zinc coordinating functionality and derived from 1,2-mercaptosulfide MMP inhibitor with the modification on that peptide part of 1,2-mercaptosulfide MMP inhibitor which is replaced with aryl sulphonamide. Since it has been proved that 1,2-mercaptosulfide MMP inhibitor is competitive inhibitor, mercaptosulphonamide inhibitors could be competitive inhibitors as well because of the structure and functionality similarity. However, the binding mechanisms of the mercaptosulfide inhibitors have not been experimentally determined and the possibility for inhibition of enzyme activity by other mechanisms must be considered. To verify a competitive mechanism of inhibition, two inhibitors, YHJ-6-43 and YHJ-6-286, have been assayed with MMP-9 at several substrate concentrations and the data was plotted based on Equation 3.23 as described in methods. As shown in following figures 3.14-3.17, it has been illustrated that along with the increasing of substrate concentration, there is

app an increase on the value of Ki (Figure 3.14 and 3.16). The linear dependence of app Ki versus [S] (Figure 3.15 and 3.17) indicates that our inhibitor is competitive inhibitor.

Figure 3.13 Mechanisms of inhibition. Three different mechanisms have been demonstrated. Figure is modified from Copeland, 2000.

Compet i t i ve I nhi bi t i on YHJ-6- 43 2000

1500

10 uM 1000 5 uM 1 uM ( [ I o/ ( 1- Vi / ) Vo) ( nM) 500

0 0 1 2 3 4 Vo/ Vi Figure 3.14 Competitive inhibition of YHJ-6-43. The inhibitor YHJ-6-43 was assayed for inhibition at substrate concentrations of 1, 5, and 10 µM (triangle, square and diamond, respectively). Data was plotted according to the Henderson equation.

Compet i t i ve i nhi i t i on r epl ot YHJ-6- 43

300

250

200

150

Ki ) 100

Sl ope Sl ent ope ( Appar nM)( 0 2 4 6 8 10 12 - 50 [ S]( uM)

app Figure 3.15 The replot of the Ki s from (Figure 3.14). The linear dependence app between Ki and [S] is indicative of competitive inhibition.

Compet i t i ve I nhi bi t i on YHJ- 6- 286

3000

2500

10 uM 2000 5 uM 1 uM 1500

1000

( [ I o] /500 ( 1- Vi / Vo) ) ( nM)

0 0 1 2 3 4 5 Vo/ Vi

Figure 3.16 Competitive inhibition of YHJ-6-286. The inhibitor YHJ-6-286 was assayed for inhibition at substrate concentrations of 1, 5, and 10 µM (triangle, square and diamond, respectively). Data was plotted according to the Henderson equation.

Compet i t i ve i nhi bi t i on r epl ot of YHJ-6- 286

600 500 400 300 Ki ) 200 100 0 sl ent ope ( Appar nM)( 0 2 4 6 8 10 12 [ S]uM

app Figure 3.17 The replot of the Ki s from (Figure 3.16) that is plotted versus the app substrate concentration. The linear dependence between Ki and [S] is indicative of competitive inhibition.

Inhibitory stereochemistry of mercaptosulfonamide inhibitors As mentioned before, based on the structure of MAG-182, peptide part of MAG- could be replaced with aryl sulphonamide to derive a new kind of thiol inhibitors, 1,2-mercaptosulfonamides, with structural simplicity and synthetic feasibility were explored as a new generation of thiol MMPIs with potentially better properties. The simplest version of this type of inhibitor with a rigid structural skeleton and without peptidomimetic parts that are liable to hydrolysis were prepared and evaluated. Two model (Figure 3.18) mercaptosulfonamides isolated as 1:1 racimic mixtures were assayed to confirm the validity of the design and also study their inhibitory stereochemistry (Table 3.1). They turned out to be very good inhibitors with two attractive features: first, the transisomer consistently had better inhibitory potency than the cis-isomer, different from mercaptosulfide inhibitors; second, they exhibited an certain inhibitory selectivity in that they are potent for MMPs with intermediate and deep pocket (only exception is MMP-3), but significantly weak for MMPs with shallow pocket such as MMP-1 and MMP-7. app As shown in table 3.1, the Ki value of YHJ-6-43 on MMP-1 is 3,400nM which is app app about 29 times lower than the Ki value of YHJ-6-45. YHJ-6-43 has Ki values (18 and 19 nm) on MMP-2 and -9 which is 45 and 51 times higher than which of YHJ-6-45, respectively. These two isomers are showing significant difference on app Ki values. They both demonstrated that they are selectively good inhibitors on MMP-2, -9-12,-13 and MMP-14, whose S1’ sites belong to either intermediate or deep pockets. In the MMPs, the active site can be imagined as consisting of two parts: a catalytic zinc ion with the associated amino acid residues and the surrounding specificity sub sites. The S1’ binding site is a well-define hydrophobic pocket with variable depth and P1’ binding site in MMPs. In regards to the substrate recognition sites, the nature of the P1’ residue is most significant, but the others, specifically the P2’ and P3’ residues, do influence inhibitor potency (Skotnicki, 1999). In order to get more potent or more selective MMP inhibitor, several modifications have been proposed to explore the inhibition potency and selectivity.

O SH H N S O O YHJ-6-43 O SH H N S YHJ-6-45 O O Figure 3.18 The structures of YHJ-6-43(trans) Vs YHJ-6-45(Cis).

Table 3.1 The apparent Ki for YHJ-6-43 and YHJ-6-45 against eight members of MMP family. app Ki (nM) Enzyme YHJ-6-43 YHJ-6-45 MMP-1 3,400 >100K MMP-7 >25K >25K MMP-2 18 810 MMP-9 19 970 MMP-12 7 970 MMP-13 15 200 MMP-14 18 410 MMP-3 >200K >200K

Since YHJ-6-43 has demonstrated good inhibition potency and selectively inhibit MMPs carrying intermediate or deep S1’ pocket. It is an excellent protyotpe for us to derive enhanced inhibitory activities and biological properties. Base on the structure of YHJ-6-43, Dr.s Schwartz and Jin have done a lot of different types of modification such as electrophilic aromatic halogenation, modification on para & meta position at the biphenyl ester, Increasing rigidity by introducing biphenyl group, and pyrrolidine-based mercaptosulphonamide inhibitor. Although it is very

54 difficult to predict the exact binding results because of the complicated environment around the active sites (Sang et al., 2006), we have designed and identified a groups promising MMPIs-pyrrolidine-based mercaptosulphonamide inhibitor.

Fluoride replacement (electrophilic aromatic halogenation)

SH SH H F5 H N N S S O O O O YHJ-6-111 YHJ-6-134

F F SH SH H H N N S S O O O O YHJ-6-133 YHJ-6-147

F F F SH H SH N H S N S O O YHJ-6-141 O O YHJ-6-138 F Figure 3.19 The structures for inhibitors modified with fluoride replacement.

Previous studies have demonstrated that there are a few cases in hydroxamate inhibitors showing excellent inhibition potency-IC50 is in nanomolar range. For example, the best inhibitor groups within all of alanine-based sulfonamide hydroxamates are those containing pentafluorophenylsulfonyl and 3- and 4-substituted phenylsulfonyl P1' groups (Sang et al., 2006). Its IC50 reached 0.09 nM for MMP-2, 6.7 nM for MMP-9. Here, we utilized fluoride functional groups and tried to increase binding affinity since fluoride is an electrophilic group which is able to lower the pKa and help inhibitor to become deprotonated so that providing stronger binding with zinc.

As shown in Figure 3.19, there are several substitutions on benzene ring such as one fluoride, two fluorides and five fluorides at different positions, respectively. Unfortunately, none of them succeeded. According to Table 3.2, they become significantly bad inhibitors. Comparing with YHJ-6-43, they do not carry much inhibition activity. Even worse, the one with five florides, which is YHJ-6-134, is not able to dissolve into HEPES buffer (the buffer used for kinetics.) at effective range. It is very like because of the hydrophobicity has been increased remarkably by introducing additional fluorides. Based on the experimental results, it did not work.

Table 3.2. The apparent Ki of inhibitor modified with floride replacement. Here, insol means insoluble. app Ki (nM) Enzyme YHJ-6-11 YHJ-6-13 YHJ-6-14 YHJ-6-13 YHJ-6-14 YHJ-6-13 YHJ-6-43 1 3 1 4 7 8 MMP-1 >25K N/A N/A N/A N/A N/A 3,400 MMP-7 >50K N/A >25K >12.5K N/A N/A >25K MMP-2 7,200 31K 13K Insol 10K 34K 18 MMP-9 9,600 25K 4,400 Insol 3,500 23K 19 MMP-12 3,500 7,300 2,000 Insol 1,500 2,200 7 MMP-13 3,400 >25K 4,800 >50K 3,400 12K 15 MMP-14 3,000 20K 1,600 >50K 1,300 6,200 18 MMP-3 >200K N/A N/A N/A N/A >25K >200K

Modification on para & meta position at the biphenyl ester

Three other functional groups have been introduced into different positions on biphenyl gourps, such as -CO2MeMethyl Ester, -CONH2 (amide) and –COOH

(Carboxylic) shown in Figure 2.20 and Table 3.3. In genereal, comparing with YHJ-6-43, these modifications improved neither the inhibitory potency, nor selectivity. Surprisingly, YHJ-6-211 shows greatly improved inhibition potency on MMP-12 and MMP-14, with at least 10 folds better than other inhibitors.

Considering the effects on MMP-2 and -9, we do not see any benefitial effect but worse inhibitory potency. What also interesting is with carboxylic group, inhibitors are not only always showing very bad inhibitory potency, but also lose of substrate selectivity. By incorporating those functional groups into inhibitor, we were trying to explore the effects of para and meta position substitution by different organic

O CO2Me O SH SH H H N N S S CO2Me O O O O YHJ-6-205 YHJ-6-176

O CONH O SH 2 SH H H N N S S CONH2 O O O O YHJ-6-181 YHJ-6-211

O CO2H SH O H SH N H S N S CO2H O O O O YHJ-6-182 YHJ-6-207 Figure 3.20 The structures for inhibitors with Modification on para & meta position at the biphenyl ester.

Table 3.3 The apparent Ki of inhibitors with Modification on para & meta position at the biphenyl ester. app Ki (nM) Enzyme YHJ-6-17 YHJ-6-18 YHJ-6-18 YHJ-6-20 YHJ-6-20 YHJ-6-21 YHJ-6-43 6 1 2 5 7 1 MMP-1 >25K 4,000 >50K >200K >200K >25K 3,400 MMP-7 >25K 5,500 >100K >100K N/A >19K >25K MMP-2 74 36 56K 2,800 11K 57 18 MMP-9 46 79 73K 480 >20K 46 19 MMP-12 67 64 50K 310 7,600 4.8 7 MMP-13 29 45 7,000 2,000 >25K N/A 15 MMP-14 460 330 >200K 1,500 >150K 22 18 MMP-3 5,900 1,800 N/A N/A >200K 2,100 >200K

functional groups. Those results are not promising which indicate the interactions between modified functional groups and the environment in S1’ pocket are not strong enough to enhance inhibition potency or selectivity.

Increasing rigidity by introducing biphenyl group

According to previous studies, the incorporation of an extended P1’ group, biphenyl, enhances the inhibitor rigidity and inhibitor selectivity for the deep pocket enzymes over shallow pocket enzymes (Whittaker, 1999). In carboxylic acid derived from D-valine, 4-substitution of the biphenyl ring helped to increase potency over the unsubstituted compound and also helped to improve the pharmacokinetic profile. This compound exhibits selective inhibition of MMP-2 and -3 (O’Brien, 1998).

CO2H

SH H SH N H S N O O YHJ-6-170 S O O YHJ-6-162 Br

SH H N CO2NH2 S O O YHJ-6-153 SH H N S O O YHJ-6-167

CO2Me

SH H N S O O YHJ-6-160 Figure 3.21 The structures of inhibitors with modification for increasing rigidity.

Table 3.4 The apparent Ki values of inhibitors with modification for increasing rigidity. app Ki (nM) Enzyme YHJ-6-17 YHJ-6-15 YHJ-6-16 YHJ-6-16 YHJ-6-16 YHJ-6-43 0 3 0 2 7 MMP-1 ~10K N/A >50K >200K >25K 3,400 MMP-7 N/A >12.5 N/A N/A >50K >25K MMP-2 240 110 150 >100K 7200 18 MMP-9 625 270 200 >100K 9600 19 MMP-12 73 54 140 46K 3500 7 MMP-13 230 90 30 34K 3400 15 MMP-14 120 1100 4400 >200K 3000 18 MMP-3 ~12K ~6000 ~12.5K >200K >200K >200K

As another attempt, we have tested inhibitors without the oxygen between two benzenes, meaning replaced biphenyl ether with biphenyl group. Based on Figure 3.21 and Table 3.4, the apparent Ki values of YHJ-170 are not so good as YHJ-6-43. We do not see the increasing inhibitory potency or selectivity, but worse inhibitory potency. There are four different functional groups utilized for replacement. We were intended to provide and enhance more opportunities and strength of molecular interaction forces such as hydrogen bonds, hydrophobic interactions. For example, with the addition of Br, hydrophobicity of molecular increased, the inhibitory and selectivity is very similar to YHJ-6 170 and worse than YHJ-6-43. In the case of the replacement by carboxyl group, inhibitor lost inhibition activity and selectivity. Although it helped with other types of inhibitors on potency and selectivity, it did not show any promising impact in our inhibitors.

pyrrolidine-based mercaptosulfonamide inhibitor A nitrogen was then introduced into the cyclopentane system to allow for the attachment of additional sidechains that might interact with the non-prime side of the active sites. By incorporate a link atom, N, into the five -membered

O O SH SH H H N N S S - + O O YHJ-6-293 O O YHJ-6-286 Cl H2 N N H2N O

O SH H N S O O YHJ-6-90 N Ph O O

O SH O H O N N S O O YHJ-6-290 N HN O O SH H +NH -Cl N 3 S O O YHJ-7-52 (CH2)3 N

O O O SH H NH N S O O YHJ-7-56 (CH2)3 N

O Figure 3.22 The structure of pyrrolidine-based mercaptosulfonamide inhibitors.

Table 3.5 The apparent Ki of pyrrolidine-based mercaptosulfonamide inhibitors. app Ki (nM) Enzyme YHJ-6-29 YHJ-6-28 YHJ-6-90 YHJ-6-29 YHJ-7-52 YHJ-7-56 YHJ-6-43 3 6 0 MMP-1 ~25K 4,100 >12.5K 2,800 >3,000 >6,000 3,400 MMP-7 >25K >25K >50K >12.5K 4,000 3,000 >25K MMP-2 27 3.9 24 35 2.2 3.8 18 MMP-9 230 15 13 2.4 2.3 1.5 19 MMP-12 38 250 720 2 4.8 2 7 MMP-13 35 50 450 27 2.0 1.7 15 MMP-14 110 11 100 21 2.1 4 18 MMP-3 2,500 460 4,500 37 2,000 810 >200K

60 groups without interruption of ring and make it to be pyrrolidine-based mercaptosulfonamide instead of cyclopentane-based mercaptosulphonamide inhibitor. By converting the inhibitor into a salt form, YHJ-6-293 has been showing very similar inhibition potency as YHJ-6-43. Interestingly, the selectivity has been improved. Beside of sparing MMP-1, -7 and -3, there is about 10 folds difference with preference on MMP2. The introduction of simple urea functionality (YHJ-6-286) or phthalimidoethyl-substituted ureas (YHJ-6-290) at the pyrrolidine nitrogen enhanced the water solubility of these inhibitors, potency and selectivity. The inhibition towards MMP-2 is 4 times better than YHJ-6-43. It is also showing selectivity between MMP-2 and -9 with the preference on MMP-2. It is also give a much better inhibition down to 3.9 nM on MMP-2. The inhibition potency of YHJ-6-290 is more than 7 times better than YHJ-6-43 on MMP-9. Surprisingly, there is a great selectivity enhancement between MMP-2 and -9 with the 15 times difference and preference on MMP-9. In the case of CBZ group substitution, It also carries enhanced more specific inhibition potency. In our study. there are more than 10 different organic functional groups linked to this pyrrolidine-based mercaptosulphonamide inhibitor with promising results on potency and selectivity, respectively. More interestingly, we have explored the potential application of pyrrolidine-based mercaptosulphonamide inhibitors by linking with other long chain which acting as a linker for nanomaterial from our collaborator group.This is the first type of MMP inhibitor reported that links to other organic functional groups without interrupting their inhibition potency comparing its parental compound, YHJ-6-286, (Figure 3.22 and Table 3.5). The linkage makes it possible, monitoring the binding between MMP inhibitor and MMP in more complicated system such as cellular system, which will help us to get further insight of how MMP behave in biological system. Based on all of the results, in comparison with commercial available MMP inhibitor GM6001, which is hydroxamate inhibitor, and the first generation of thiol group in our labs, mercaptosulphonamide inhibitors are excellent MMP inhibitors because of the simple structure, very good inhibitor potency at sub nano molar

61 range which is very close to hydroxamate inhibitors and better selectivity (Figure 2.23).

O SH O H SH N H S N O O S N O O YHJ-6-43 H2N YHJ-6-286 O

O O H HO N NHMe N H O NH GM-6001

SH H O O SH H H H H N H N S NHPmp S NHMe O H H H H O Ph MAG-182 YHJ-73 Figure 3.23 The structures of different type of inhibitors. Mercaptosulfonamide inhibitors (YHJ-6-43& YHJ-6-286); Hydroxamate MMP inhibitor (GM6001); Mercaptosulfide inhibitors (MAG-182& YHJ-73).

Table 3.6 The apparent Ki of different types inhibitors. Mercaptosulfonamide inhibitors (YHJ-6-43& YHJ-6-286); Hydroxamate MMP inhibitor (GM6001) (Galardy et al., 1994); Mercaptosulfide inhibitors ( MAG-182& YHJ-73) (Park et al., 2003). app Ki (nM)

Enzyme YHJ-6-43 YHJ-6-286 GM6001 MAG-182 YHJ-73

MMP-1 3,400 4,100 0.4 49 >12K

MMP-7 >25K >25K N/A 40 1,000

MMP-2 18 3.9 0.5 1.1 20

MMP-9 19 15 0.2 0.57 8.6

MMPI stability in enzyme kinetic buffer

Stability of inhibitors in enzyme kinetic buffer has been evaluated. As shown in Figure 3.24, without reducing reagent, tris(2-carboxyethyl)phosphine (TCEP), inhibition activity of YHJ-6-43 lasts about 6 hours starting at 80 % to 20% of in inhibition on enzyme activity. In Figure 3.25, without reducing reagent, TCEP, YHJ-6-286 maintains its inhibition activity for about 6 hours starting at 60% to 20% of inhibition on enzyme activity. In Figure 3.26, without reducing reagent, TCEP, YHJ-7-52 is able to maintain its inhibition activity for about 6 hours starting at 60% to 20% of inhibition on enzyme activity. As demonstrated in Figure 3.27, without reducing reagent, TCEP, YHJ-6-293 is able to maintain its inhibition activity for about 8 hours starting at 40% to 20% of inhibition on enzyme activity. In Figure 3.28, without reducing reagent, TCEP, YHJ-7-23 is able to maintain its inhibition activity for about 8 hours starting at 40% to 20% of inhibition on enzyme activity. Without reducing reagent, mercaptosulfonamide inhibitor is able to maintain more than 20% of inhibition activity for about 6 hours.

Figure 3.24 Stability evaluation of YHJ-6-43 in HEPES buffer without TCEP.

Figure 3.25 Stability evaluation of YHJ-6-286 in HEPES buffer without TCEP.

Figure 3.26. Stability evaluation of YHJ-7-52 in HEPES buffer without TCEP.

Figure 3.27. Stability evaluation of YHJ-6-293 in HEPES buffer without TCEP.

Figure 3.28. Stability evaluation of YHJ-7-23 in HEPES buffer without TCEP.

As we mentioned in chapter one, although thiol group is a very attractive zinc binding group, it also carries disadvantages. One of them is that thiol group can be oxidized when exposed to air. That means, when exposed to air, the inhibition activity of inhibitors is going to be eliminated gradually. As we have demonstrated, mercaptosulfonamide inhibitor can maintain their inhibition activity more 20% for 6 hours, which may be caused by oxidation of thiol group. That leads us to think

65 what if we have reducing reagent in HEPES buffer, how long can mercaptosofonamide inhibitor last?

Figure 3.29 Stability evaluation of YHJ-6-43 in HEPES buffer with TCEP.

Figure 3.30 Stability evaluation of YHJ-6-286 in HEPES buffer with TCEP.

With reducing reagent, TCEP, indicated in Figure 3.29, YHJ-6-43 is able to maintain its inhibition activity starting at 70% to 60% of inhibition on enzyme activity for 24 hours. As shown in Figure 3.30, with reducing reagent, TCEP,

YHJ-6-286 is able to maintain its inhibition activity starting at 65% to 40% of inhibition on enzyme activity for 24 hours. As shown in Figure 3.31, with reducing reagent, TCEP, YHJ-6-90 is able to maintain its inhibition activity starting at 50% to 40% of inhibition on enzyme activity for 24 hours. As shown in Figure 3.32, with reducing reagent, TCEP, YHJ-6-176 is able to maintain its inhibition activity starting at 45% to 40% of inhibition on enzyme activity for 24 hours. When TCEP, the reducing reagent present, YHJ-7-23, as shown in Figure 3.33, is able to maintain its inhibition activity starting at 60% to 30% of inhibition on enzyme activity for 24 hours.

3.2 Inhibitor application and characterization in cellular system

Enzymatic activities of MMPs are very important in pathological and physiological processes, such as the opening of BBB related to stroke (Asahi et al., 2001). Within those four major MMPs involved into stroke, MMP-2 and -9 are the two most powerful and critical proteases in the progress of the BBB opening. Specifically, the expression of MMP-2 has been shown to increase in early stages of reperfusion in the foot processes of astrocytes near blood vessels, where it can participate in the initial opening of the BBB (Rosenberg et al., 2001). MMP-9 on the other hand, which is absent under physiological conditions, is expressed at 24 hours in blood vessels and infiltrating neutrophils, reaching a maximum at 48 hours in focal cerebral ischemia with delayed reperfusion during an ischemic stroke, suggesting its participation in the late stages of BBB disruption and opening, neuronal cell death, and hemorrhage after incident (Rosenberg et al., 2001). Furthermore, both gelatinases may also participate in a hemorrhagic stroke when an artery in the brain bursts as both interfere with cell-matrix interactions and are capable of triggering caspase-mediated cytotoxicity in brain endothelial cells (Cunningham et al., 2005). Many studies in animal models indicate that the reduction of the incidence of toxicity associated with the use of t-PA may be a consequence from the application of matrix metalloproteinase

67 inhibitors (MMPIs), especially inhibitors specific for MMP-2 and -9 (Rosenberg et al.,2003; Lo et al.,2004). In order to evaluate our MMP inhibitors, hBMEC culture have been chosen to fulfill the job. Wound healing assay of hBMEC culture provides a relevant model to illustrate the proteolytic functions of MMPs. MMPs participate in regulation of wound repair processes by involving into different physiological mechanism. For example, as mentioned in previous studies, MMPs are involved into activation and processing of cytokines and chemokines which are important elements in inflammation by cleaving and release those molecules from the cell surface chemokines (Parks et al., 2004; Yong et al., 2005, Clark-Lewis et al., 1995; Zlotnik et al., 2006 . MMPs contribute to re-epithelization by cleaving and processing components of cell-cell junction and cell matrixin the epithelium (Atkinson et al., 2007). As well, MMPs participate into the ECM remodeling related to wound repair by directly proteolytic degatdation of proteins such as collagens (Singer and Clark, 1999, Sawicki et al., 2005). Wound healing model is a good model which provides a platform for us to study the impact of selected MMP inhibitors on MMPs-related cell behavior since MMPs are key regulators of tissue repair. In order to evaluate our inhibitors in cellular system,

MMPI Stability in Cell Culture Medium

Before observing the effects of these compounds in pseudo-physiological settings, it was necessary to assess the biological compatibility of these compounds. As a preliminary assessment of MMPI stability, YHJ-6-43, YHJ-6-286 and YHJ-6-293 were subjected to in vitro analyses aimed towards providing a rough estimate as to how long these compounds will successfully inhibit MMP activity. By observing a change in the fluorescence of a synthetic substrate, indicating greater MMP activity, in endothelial growth medium (EGM), stability of YHJ-6-43, YHJ-6-286 and YHJ-6- 293 was good (Figure 3.31, 3.32 and 3.33). Starting with a concentration of 10 μM, both compounds exhibited significant

68 inhibitory potential up to 8 hours after initiating incubation, with YHJ-6-43 still inhibiting ≥80% of MMP-9 activity 24 hours after initiation. YHJ-6-286, though to a

YHJ-6- 43 st abi l i t y i n EGM

100 80 60 40 act i vi t y 20 orli Nomral zed Enzyme 0 0 5 10 15 20 25 30 Ti me ( hour )

Figure 3.31 Stability of YHJ-6-43 in EGM. 10 μM of inhibitor shows 80% inhibition activity against MMP-9 even after 24 hours.

YHJ-6- 286 st abi l i t y i n EGM

60 50 40 30

act i20 vi t y 10 orli Nomral zed Enzyme 0 0 5 10 15 20 25 30 Ti me ( hour )

Figure 3.32 Stability of YHJ-6-286 in EGM. 10 μM of inhibitor shows 50% inhibition activity against MMP-9 even after 24 hours.

Stabi l i t y of YHJ-6- 293 i n EGM

60 50 40 30

act i20 vi t y 10 o a i mal Nor zed Enzyme 0 0 5 10 15 20 25 30 Ti me ( hour )

Figure 3.33 Stability of YHJ-6-293 in EGM. 10 μM of inhibitor shows 40% inhibition activity against MMP-9 even after 24 hours.

lesser extent, still possessed some inhibitory potential, blocking ~50% of MMP-9 activity at the same time point. YHJ-6-293, which was showing similar stability with YHJ-6-286, was able to block ~50% of MMP-9 activity after 24 hours.

MMPI Cytotoxicity and Wound Healing

As both compounds proved potent for multiple biologically significant MMPs, in addition to their considerable ex vivo stability, the next steps were to evaluate any toxicity the compounds may elicit to a line of human brain microvascular endothelial cells (hBMECs) and observe any effects they may impose during a physiological process, such as wound healing. For both YHJ-6-43, YHJ-6-286 and YHJ-6-293 concentrations in excess of 30 μM proved cytotoxic to the hBMECs, killing ≥30% of the culture, while minimal death was observed at concentrations ≤30-μM and essentially no death at concentrations ≤10-μM (Figure 3.34, 3.35,andFigure 3.36).

Figure 3.34 Cytotoxicity of YHJ-6-43 in hBMEC culture. (*: Comparing with control, it is significant different p<0.05).

Figure 3.35 Cytotoxicity of YHJ-6-286 in hBMEC culture.

Figure 3.36 Cytotoxicity of YHJ-6-293 in hBMEC culture.

Wound healing assay

A wound healing cell model (hBMEC culture) has been used to investigate the impact of selected MMP inhibitors in hBMC culture. Because of ptoteolytic activities of MMPs, they are greatly involved into the BBB opening related to stroke. By blocking the proteolytic activities of MMPs, the repression of wound healing repair was expected by application of MMP inhibitors. In this study, YHJ-6-286 dramatically reduced wound recovery at concentrations of 100 and 30 μM, it has shown more than 50% of cell recovery inhibited (Figure 3.38). In the case of YHJ-6-43, it is significantly (P ≤ 0.05) hampering the recovery of hBMECs by more than 50% at 100 μM and ~50% at concentrations of 10, 3, and 1μM (Figure 3.37). At 100, 30 and 10 μM, YHJ-6-293 gives almost identical repression on wound healing process, but with an interesting results which promoting cell growth at low concentrations (100 nM, 10 nM) (Figure 3.39). In order to eliminate the effects of inhibitor solvent, ethanol, we have also tested the impact of ethanol in hBMECs wound healing assay. As

Figure 3.37 Wound-healing assay of YHJ-6-43 in hBMEC culture. Comparing with control without MMP inhibitor treatment, wound healing process has been inhibited partially and significantly at 100, 30 and 10 μM by ~50% or greater. (*: Comparing with control, it is significant different p<0.05).

Figure 3.38 Wound-healing assay of YHJ-6-286 in hBMEC culture. Comparing with control without MMP inhibitor treatment, wound healing process has been inhibited partially and significantly at 100 and 10 μM by ~50% or greater. (*: Comparing with control, it is significant different p<0.05).

Figure 3.39 Wound-healing assay of YHJ-6-293 in hBMEC culture. (*: Comparing with control, it is significant different p<0.05).

Figure 3.40 Wound-healing assay of ethanol in hBMEC culture. (*: Comparing with control, it is significant different p<0.05).

74 shown in figure 40, comparing with control without MMP inhibitor treatment, although there was a reduction when the system carried 2.5% ethanol, the others tested did not show significant impact on hBMEC wounded culture recovery progress. These synthetic inhibitors at low micromolar concentrations (10 μM) reduced the wound recovery process of human brain microvascular endothelial cells (hBMECs). The mechanisms by which these inhibitors prevent recovery remain unknown. We speculate that MMPs cleave ECM proteins and that some of these fragments may promote cell survival and growth. MMPs, or other metalloproteinases, may also release ECM bound growth factors or activate pro-growth factors on the cell surface or in cell culture media. Thus, inhibition of metalloenzyme activity would thereby mitigate the growth promoting effects of metalloenzymes. In order to further improve the repression of hBMEC wound healing process is resulted from the inhibition of MMP’s activities. We have utilized a co-culture of hMSC and hBMEC model to perform further evaluation. Human adult bone- marrow derived mesenchymal stem cells (hMSCs) are human adult stem cells that reside within the bone marrow compartment. Because of their therapeutic potential in regenerative medicine, orthopedic and ischemic myocardial diseases, hMSCs have attracted lots of attention for clinical use recently (Young, et al., 1998; Sato, et al.,2005; Shi, et al.,2007). It also has been reported that hMSCs are able to secret a series of growth factors and neurotrophic factors and differentiate into a variety of cell types such as smooth muscle cells and endothelial cells, resulting in increased vascularity and improved cardiac function, in addition to accelerating dermal repair in acute and chronic wounds (Badillo, et al., 2007; Volk, et al., 2007; Wu, et al., 2007; Yoshikawa, et al., 2008) . Furthermore,in the tumor microenviroment, mesenchymal stem cells is able to differentiate into vascular endothelial cells and help with angiogenesis during tumor progression, which may be a imprtant pathway of promoting tumor growth (Zhang, et al., 2010). Another work has been done in stroke related animal model has indicated that intravenous infusion of hMSCs is able to promote vascular

75 endothelial growth factor (VEGF) secretion, VEGF receptor 2 (VEGFR2) expression and angiogenesis in the ischemic boundary zone after stroke (Chen, 2003). It is also addressed that VEGF treatment significantly enhanced production of matrix metalloproteinase (MMP)-1, -3, and -9 by human smooth muscle cells (Wang, 1998). It is reseanable to hypothesize that hMSCs is capable of helping with HBMEC wound healing process.

Figure 3.41 Wound-healing assay of co-cultutre (hBMECs and hMSCs) with and without inhibitor treatment: wound healing of the co-culture was increased in the absence of YHJ-6-43 in comparison to its addition. (*: Comparing with control hBMECs, it is significant different p<0.05).

In this study, we revealed that hBMECs co-cultured with 5% or 10% of hMSCs exhibit better wound healing results, yet, higher percentages of hMSCs do not appear to promote recovery (figure 3.41). At 10% and 5% of hSMC in hBMEC culture, There were promoting on wound healing process with 10% and 6% higher recovery than control, respectively. It is accordance to what have been reported that hMSCs are able to help with wound healing process. With application of inhibitor, we see not only the reacovery promoted by 10% and 5% of hSMC, but also indicating reducing the healing process in HBMEC culture. When it is 10% and 5% of hMSC in HBMEC culture, YHJ-6-43 was able to

76 repress about 20% and 10% recovery comparing with the culture without inhibitor treatment, respectively. The mechanisms by which these inhibitors inhibit co-culture wound recover are unknown. Because hMSCs are able to release a series of growth factors and may be able to differentiate into endothelial cells, it enhanced the wound healing process in hBMEC culture. Meanwile, MMPs still played important roles here. By inhibiting their proteolytic activities, our inhibitors reduced the recovery process in co-culture wound healing model.

3.3 Proteomic studies after hBMEC treated with YHJ-6-43

One of important structure properties in BBB is the tight junction, which is also known as a zonula occludens. The tight junction is one of junctions that join brain endothelial cells together closely and help to stabilize surrounding tissue structure and regulate the exchanging of substance between outside and inside of brain environments. It is the main structure that responsible for the barrier properties. (Reese and Karnovsky, 1967; Van Deurs and Koehler, 1979; Wolburg and Lippold , 2002; Yang et al., 2007). The tight junction has been studied for decades. It has been identified that many proteins are involved into the formation and function of tight junction. Those proteins are listed in figure 3.42. Several tight junction proteins have been indentified in brain endothelial cell tight junction. MMPs are required for numerous developmental disease related processes because of their potent ability to degrade various types of proteins such as structural proteins of ECM to participate into the remodeling of ECM. In addition to their ECM substrates, MMPs also are able to regulate cell behavior such as cell-cell adhension and cell-matrix interaction by cleaving and releasing cell surface molecules, other nonmatrix proteins around cells (Sternlicht et al., 2000; Stocker et al., 1995). They are greatly involved into the opening of BBB related to stroke. Previous studies have indicated that MMPs are able to degrade tight junction proteins such as occludin and claudins which are the major structure components of BBB (Yang et al., 2007). The first reported tight junctional transmembrane

77 molecule was occludin (Furuse et al., 1993; Ando-Akatsuka et al., 1996; Wolburg and Lippold , 2002). It has been indicated that occludin is needed to regulate blood brain barrier properties in cells (Wolburg and Lippold , 2002). For example, the phosphorylated occluding is able to regulate tight junction permeability (Hirase et al., 2001). It is also responsible for sealing of tight junctions (Lacaz-Vieira et al., 1999). The claudins are involved into maintain BBB properties. (Morita et al., 1999; Tsukita and Furuse, 1999). It has been approved the the opening of BBB is prevented at the presence of MMP inhibitor, BB1101. Meanwhile, the degradations of tight junction proteins. Occludins and claudins (-5) were prevented (Yang et al., 2007). The detailed mechanism remain unclear that how MMPs are involved into these processes and the impact of MMP activities on the degradation of other proteins.

Figure 3.42 Tight junctions and tight junction proteins. Figure has been modified from Wolburg and Lippold , 2002. It is indicating the major components of tight junction.

Understanding these processes will lead us into a more rational approach toward the treatment of MMPs mediated diseases. In this first study on the mercaptosulfornamide inhibitors, we would like to get insight views on how our

78 mercaptosulfornamide inhibitor affects the degradation or expression of proteins by inhibiting the activity of MMPs. The proteins expressed in hBMEC culture with/ without MMP inhibitor treatment have been separated with two dimensional gel electrophorisis (2-DE). Proteins interested have been identified with LC-MS/MS. 2-DE, the combination of Isoelectric focusing (IEF) with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), is a powerful analytical technique that has been commonly used to separate large scale of complex mixture of proteins extracted from cells, tissues, or other biological samples. This analytical technique separates proteins in two steps based on two independent properties of proteins. Proteins in mixture are separated by two different properties. Isoelectric focusing is the first-dimension. It is capable of separating proteins according to their isoelectric points (PI). Sample was applied in a gel with a pH gradient. If PI of a protein is higher than the pH in the region where protein locates, it will migrate towards the cathode till it reaches the place in which region the pH is the same as its PI, that is, the overall charge of this protein is zero. Because it carries net charge and can not be affected by electrical attraction anymore. The second dimension is SDS-PAGE gel electrophoresis which is a method used to separate proteins according to the sizes of proteins. Since different proteins with similar molecular weights may migrate differently due to their differences in secondary, tertiary or quaternary structure, we used SDS, an anionic detergent to denature proteins by reducing and linearizing proteins to their primary structure. Proteins are coated with uniform negative charges. In electrophoresis, an electric current is used to move the protein molecules across a polyacrylamide gel. The gel matrix is formed by the polymerization of the monomer molecule acryrylamide, which is crosslinked by N,N'-methylene-bis-acrylamide. The polymization was initiated by addition of free radicals generated by ammonium persulfate (APS) and catalyzed by N,N,N',N'-tetramethylethylene diamine (TEMED). The polyacrylamide gel is a

79 cross-linked matrix that acts as a sieve to help catch the molecules as they are transported by the electric current in electrophoresis. The negatively charged

Ethanol control 2

14 13 14 13 4 7 6 7 6 4 3 3 1 1

A: hBMEC Control B: EOH treated hBMECControl

14 13

7 6 4 3 1

C: YHJ-6-43 treated hBMEC

Figure 3.43 The separation results of hBMEC protein extracts. A: hBMEC control;B: hBMEC treated by ethanol only; C: hBMEC treated by YHJ-6-43 (10uM).

protein molecules are attracted to the positive end by the electric current. During traveling through the gel network, they encounter resistance from this polyacrylamide network. Smaller molecules are able to go through the network faster than the larger proteins. After a certain period of time, by stopping the electric current, all of proteins are separated and stay in different positions in the gel.

hBMEC culturue have been prepared. Upon reaching about 70~80% confluency, MMP inhibitor, YHJ-6-43, was applied. Since inhibitor was dissolved into ethanol, we have set up an ethanol control to help us with distinguish the impacts caused by inhibitor and inhibitor. Proteins were properly extracted and desalted following our protocol. Proteins in mixture have been successfully separated by 2-DE. The pH rang is 3-11 and distributed starting at lower to higher along with the gel from left to right. Proteins with similar PI have been further separated in the gel. Based on different molecular weight, they are distributed into gel. Proteins with smaller molecular weight stay at the lower pat of gel. In another words, proteins with higher molecular weight locate at the higher portion in the gel. The results have been shown in Figure 3.43 (A-C). A software, progenesis, has been used to compared gels among treatments. By evaluating with progenesis, we were able to identify the proteins changed after inhibitor treatment. We have picked 20 spots showing changes for protein identification.

Table 3.7. Table of identified proteins with LC MS-MS. Spot Protein name Mw/pI PI # 1 Galectin-1 14.7 5.34 3 Isoform 2 of Eukaryotic translation initiation 20.2 6.52 factor 5A-1 4* Peptidyl-prolyl cis-trans isomerase B 23.7 9.42 6* Glutathione S-transferase P 23.3 5.43 7 triosephosphate isomerase 1 isoform 2 30.8 5.65 13 Calreticulin 48.1 4.29 14* Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 37.3 5.6

Liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-QqTOF-MS) is an analytical technique which combines the physical

81 separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. It is a powerful technique used for many applications which has very high sensitivity and specificity. It is feasible to be used in the study of proteomics where components of a complex mixture must be detected and identified (Campbell et al., 1998). Selected spots have been identified and listed in table 3.7.

Proteins identified:

Galectin-1:

Galectin-1， a member of galectin family which has been characterized with

ß-galactoside-binding, is the first protein identified in this family (Van den Brule et. al., 1979; He and Baum, 2006). There are around 14 members reported by now (Chobot et. al., 2002; La et. al., 2003). It has bee indicated that galectins are found in multiple locations in cells such as the cytosol, nucleus and membrane compartments, meaning they are wildly involved into many biological processes (La et. al., 2003). Galectin-1, has been identified as one of substrates of MMPs and is expressed in many types of cells, healthy and malignant cell lines (Prudova et. al., 2010; He and Baum, 2006). General, it is secreted into the ECM and play critical roles in effecting of cell behavior (such as cell adhesion, cell proliferation and cell death) (He and Baum, 2006).It has also been reported that this protein is involved into cancer invasion and metastasis, wound healing, angiogenesis, and immune functions (He and Baum, 2006; Wu et. al., 2009). The overexpression of Galectin-1 is strongly linked to the increase of invasiveness of cancer cells. it promotes the invasion of cells by up-regulating the expressing of MMP-2 and -9 (Wu et. al.,2009). Both of them are not clear that how galectins work as multifunction proteins and how they are secreted from cells.

Isoform 2 of Eukaryotic translation initiation factor 5A-1:

Eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein and expressed in all of eukaryotes (Hopkins et al., 2008). It, containing the posttranslationally synthesized amino acid hypusine, makes this proteins unique (Park et al., 1981; Cooper et al.,1982 ; Hopkins et al., 2008). Several studies have been done and proved that this protein plays critical role for normal function of mammalian cells and participates into regulation of cell behavior ( cell proliferation, and apoptosis) (Tome and Gerner, 1997; Caraglia et al., 2003). For example, previous study has indicated that the wound healing processes of human heptocellular carcinoma cell lines have been significantly suppressed by applying an inhibitor (N1-guanyl-1,7-diamino heptane ), which is for the first step of elF5A hypusination that converts this protein into its active form (Lee et al.,.) How eiF5A plays the roles in cells remain unkown (Hopkins et al., 2008).

Peptidyl-prolyl cis-trans isomerase B:

The Peptidyl-prolyl cis-trans isomerase B, also refers to cyclophilin B or PPIB, is a regulator that permits cells to function normally by modulating a range of cellular functions such as the folding, transport of proteins (Shaw, 2007; Thorpe et al., 2001). After expressed and secreted, it locates at the chondrocyte cell surface and processed by MMPs (MMP-1,-2,-3,-9 and -13) (De Ceuninck et al., 2003).Cyclophilin B has been demonstrated to affect gene expression, cell proliferation and cell growth related to STAT5 and prolactin by protein-protein interaction (Rycyzyn and Clevenger, 2002). It has also been indicated that have shown that the expression of cyclophilin B is associated with malignant progression and regulation of genes implicated in the pathogenesis of breast cancer (Fang et al., 2009).

Glutathione S-transferase P:

Glutathione S-transferases a large family of enzymes including GST isoform A, GST isoform M (GSTM), and GST isoform P (GSTP). They are in charging of

83 catalyzing the conjugation reaction between electrophilic compounds and reduced glutathione, whose level in cell are greatly related to production of reactive oxygen species. It is involved into cellular proliferation, apoptosis interrupting the production of reactive oxygen species (Yin et al., 2001; Conklin et al., 2009)

Triosephosphate isomerase 1 isoform 2:

Triosephosphate isomerase 1 is a central and concerved glycolytic enzyme that is important for energy production in the cytoplasm of neurons and play an important role in glycolysis, gluconeogenesis, fatty acid synthesis and the pentose shunt, which are essential for cell growth and maintenance (Cheng et al., 1990). The hypoxia-indcible factor may initiate the expression of triosephosphate isomerase 1(Bernhard et al., 2004). When cells are at the proliferative state, the expression of triosephosphate isomerase 1 may also be stimulated by cell culture serum (Boye, 1991). The deficiency of Triosephosphate isomerase 1 results into a progressive, sever neurological disorder-triosephosphate isomerase deficiency (Ralser et al., 2008).

Calreticulin:

Calreticulin has been first isolated decads ago by Ostwald and MacLennan (Ostwald et al.,1974; Johnson et al.,2001). Calreticulin is a highly conserved major ca2+ binding protein of the endoplasmic reticulum (ER). It has been indicated that it localizes on the surface of multiple cell types such as platelets, fibroblasts, apoptotic cells, and endothelial cells (Nanney et al., 2008). Previous studies have reported that calreticulin is involved into wound environment as a stress or injury-related protein (Conway et al., 1995; Boraldi et al., 2007; Hopf et al., 2007; Malda et al., 2007). It may be playing critical roles in wound healing based on its biological activities which are largely related to cellular adhesion, migration, and phagocytosis. For example, Calreticulin binds to the cytoplasmic

84 tail of alpha integrins, acting as an escort through the ER, and maintains appropriate calcium balance for integrin signaling and adhesion-dependent functions of matrix proteins with significant impact on regulating cell shape, motility, and spreading (Coppolino et al., 1997& 2000; Kwon et al., 2000). In addition, the integrin binding basement membrane protein, laminin, interacts with Calreticulin to mediate migration (McDonnell et al., 1996; White et al., 1995). The express patern of Calreticulin also can affect matrix protein expression, such as fibronectin, and cell adhesion and spreading related to integrin function (Rauch et al., 2000; Szabo et al., 2007; Nanney et al., 2008). The regulation role of calreticulin on the activities of MMP has been described previously based on the studies on the calreticulin deficient cell culture. It has been ddemonstrated that there is a significant decrease in the MMP-9 and increase in the MMP-2 activity and expression in the calreticulin deficient cells (Wu et al., 2007).

Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 :

Guanine nucleotide-binding proteins, G-proteins, are a family of proteins involved into signal transduction outside of cell and changes inside the cell. They are able to communicate with many other signaling proteins or factors (Neves et al., 2002). In summary, by working with proteomics of hBMEC culture sample treated with mercaptosulphonamide MMP inhibitor, we were able to identify several proteins which are shown decreasing of concentration upon the presence of YHJ-6-43. Several of proteins are directly related to the regulation of MMP activities such as Galectin-1, Calreticulin and Peptidyl-prolyl cis-trans isomerase B. They are all participate into modulation of cell behavior (proliferation, growth, death or adhension). It also provides evidence that MMPI may be able to suppress wound healing process by inhibiting MMP activities and affecting the expression level of cell function modulators listed above. The mechanism about how MMP inhibitor participates in those processes is not clear.

CHAPTER FOUR

CONCLUSION AND FUTURE WORK

In summary, the modification at the non-prime site of inhibitor by incorporate a link atom, N, into the five carbon ring and make it to be pyrrolidine-based mercaptosulfonamide instead of cyclopentane-based mercaptosulfonamide such as YHJ-6-43, makes good inhibitors that show good inhibition potency and selectively inhibit MMPs carrying intermediate or deep S1’pocket. The demonstrated importance of the non-prime side in MMP inhibitory potency and the elucidation of different inhibition profiles in term of S1’ pocket at active center of MMPs. We have identified proteins down-regulated which are related to cell function modulation by proteomic studies after the application of MMP inhibitor from hBMEC culture. Seven out of 20 samples have been demonstrated that they are related to cell behavior such as cell proliferation, cell death, cell growth, and apoptosis, respectively. These results are in agreement with what we observed from hBMEC wound healing assay, that is, cells recovery was dramatically suppressed by mercaptosulfonamide inhibitors. Based on the result from wound healing assay of co-culture model of hMSC and hBMEC, it is very clear that our inhibitor has been inhibiting MMP’s activity at cellular level efficiently. This is the first study on mercaptosulfonamide inhibitors. We have characterized the newly designed and synthesized mercaptosulfonamide MMP inhibitors are reversible, slow-tight binding inhibitors which are relatively more stable and bio-friendly. They have demonstrated excellent MMP inhibition features at protein level and are very effective in stroke related cellular system. Those results may facilitate the design of more potent, selective MMP inhibitors. This may then lead to the development of new pharmaceuticals, which inhibit undesired pathological processes, such as the BBB opening related to stroke and cancer metastasis, in particular, while minimizing the side effects from the treatment. Based on our studies, selected potent MMP inhibitors have been sent

86 to our collaborators for animal studies. Further investigations may identify investigational new drug candidates for the treatment of stroke.

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BIOGRAPHICAL SKETCH

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional:

Florida State University Ph.D. 2003-2010 Biochemistry Florida A&M University M.S. 2000-2003 Environmental Chemistry Tianjin Normal University B.S. 1993-1997 Chemistry

Positions and Employment: 2003-present, Teaching Assistant, Department of Chemistry and Biochemistry, FSU 2000-2003, Research Assistant in Environmental Science institution, Florida A&M University 1997-1999, Chemistry Instructor, the Tianjin 43rd High school.

Research Experience:

Research project for M.S. training at Florida A&M University is on ―Toxicity of naphthalene photodegradation products to native bacteria‖

Research projects at Florida State University are on matrix metalloproteinase biochemistry, enzyme inhibition kinetics, and cell biology of human endothelial and prostate cancer cells.

Research Publications:

Q.-X. Sang, Y. Jin, R.G. Newcomer, S.C. Monroe, X. Fang, D.R. Hurst, S. Lee, Q. Cao, and M.A. Schwartz (2006) Matrix Metalloproteinase Inhibitors as Prospective Agents for the Prevention and Treatment of Cardiovascular and Neoplastic Diseases. Current Topics in Medicinal Chemistry. 6, 289-316. Invited review.

Park H.I., Lee S., Ullah A., Cao Q., Sang Q.X..(2010) Effects of detergents on catalytic activity of human endometase/matrilysin 2, a putative cancer biomarker. Anal Biochem. 396(2):262-8. Epub 2009 Oct 8.

Newcomer, R.G.; Roycik, M.D.; Cao, Q.; and Sang, Q.X. (2010) Matrix metalloproteinase inhibition in cerebral and cardiovascular diseases. Curr. Top. Med. Chem. Invited Review, Submitted.

Wang-Yong Yang, Qiang Cao, Catalina Galvis, Qing-Xiang A. Sang, Igor V. Alabugin (2010). Intracellular DNA damage by lysine-acetylene conjugates. J. Nucleic Acid Research, accepated for publication.

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Xu Wang, Qiang Cao, Qing-Xiang A. Sang, Mark R. Emmett, Alan G. Marshall. (2010) Systematic phosphoprotein analysis in the ARCaP cells by optimized enrichment method and FTICR mass spectrometry. Manuscript in preparation.

Qiang Cao, Fu-li Yu, Qing-Xiang Amy Sang. (2010) Inhibition of Human Endometase/Matrilysin-2 by TIMP-2 and Comparison with Matrilysin and Metalloelastase. Anal Biochem. Manuscript in preparation.

Qiang Cao, Yong-hao Jin, M.H. Constantino, M.A. Schwartz, Qing-Xiang Amy Sang. (2010) Design, synthesis, and characterization of 1,2-mercapto aryl sulphonamide inhibitors of matrix metalloproteinases. J. Bio. Chem. Manuscript in preparation.

Qiang Cao, Donghong Min, Yonghao Jin, Marty Schwartz, Qing-Xiang Amy Sang, Wei Yang. (2010) Quantitative Prediction of the Stereo-isomeric effects of the binding of A Mercaptosulfonamide inhibitor on matrix metalloproteinase-9 (MMP-9): A double blind test on the QM/MM OSRW algorithm. Manuscript in preparation.

Conference Abstracts and Presentations:

Y. Jin, Q. Cao, Q.-X. Sang and M.A. Schwartz (2006) Zinc Metalloproteinase Inhibitors with 1, 2-Dihydroxy benzene and 3-Hydroxy-4-pyrone as Zinc Binding Groups. 231st American Chemical Society National Meeting & Exposition, March 26-30, 2006, Atlanta, Georgia. Poster Presentation. Y. Jin, Q. Cao, C. Ben, Q.-X. Sang, and M.A. Schwartz. Design and Syntheses of Novel MMP Inhibitors Containing a Mercaptosulphonamide Zinc-bind Group. Invited Talk. American Chemical Society, Florida Section Meeting, Orlando, Florida. May 10, 2007. Talk number 46. M.D. Roycik, Q. Cao, C.S. Yun, Y. Jin, S.M. Hira, M.A. Schwartz, G.F. Strouse, and Q.-X. Sang (2008) Role of Human Mesenchymal Stem Cells and Matrix Metalloproteinases in Vascular Wound Healing. Poster number 26, Page 30. First Southeast Stem Cell Consortium, University of Georgia, Athens, GA. October 23-24, 2008. Invited talk. Cao, Q.; Jin, Y.; Schwartz, M.A.; Ben, C.; Roycik, M.D.; and Sang, Q.X. (2009) Targeting metalloproteinases involved in stroke-associated blood brain barrier permeability. American Chemical Society, the 85th Annual Florida Meeting and Exposition (FAME), Orlando, Florida. May 14th, 2009. Poster Presentation. Qiang Cao, Mark D. Roycik, Yonghao Jin, Dale B. Bosco, Martin A. Schwartz, and Qing-Xiang Amy Sang (2009). Investigation on co-culture model of HMSC and HBMEC treated with novel synthetic MMPI. 2nd Annual Southeast Stem Cell Consortium, University of Georgia, Athens, Georgia. October 15th-16th, 2009. Poster Presentation.

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Sang, Q.X.; Jin, Y.; Lee, S.; Cao, Q.; Ben, C.; Roycik, M.D.; Qi, B.; and Schwartz, M.A. (2009) Metizincin metalloproteases and their function in cancer, stroke, and inflammatory diseases. American Chemical Society, the 85th Annual Florida Meeting and Exposition (FAME), Orlando, Florida. May 15th, 2009. Invited talk. Roycik, M.D.; Cao, Q.; Jin, Y.; Hira, S.M.; Breshike, C.; Strouse, G.F.; Schwartz, M.A.; and Sang, Q.X. (2009) Investigation of matrix metalloproteinases in human mesenchymal stem cells towards mechanisms for cardiac repair. American Chemical Society, the 85th Annual Florida Meeting and Exposition (FAME), Orlando, Florida. May 15th, 2009. Poster Presentation. Sang, Q.X.; Cao, Q.; Roycik, M.D.; Jin, Y.; Ben, C.; and Schwartz, M.A. (2009) Human metalloproteinases and their inhibitors in microvascular wound healing, blood-brain permeability, and adult mesenchymal stem cell differentiation. Atherosclerosis Gordon Research Conference, Tilton School, Tilton, New Hampshire. June 21st-26th, 2009. Poster Presentation. Sang, Q.X.; Cao, Q.; Roycik, M.D.; Jin, Y.; Ben, C.; and Schwartz, M.A. (2009) Role of human matrix metalloproteinases and mesenchymal stem cells in microvascular endothelial wound healing and 3-D blood-brain barrier permeability. Gordon Research Conference on Biomaterials: Biocompatability/Tissue Engineering, Holderness School, Holderness, New Hampshire. July 19th-24th, 2009. Poster Presentation. Sang, Q.X.; Roycik, M.D.; Cao, Q.; Jin, Y.; Ben, C.; and Schwartz, M.A. (2009) Role of human matrix metalloproteinases and their inhibitors in mesenchymal stem cell differentiation and vascular endothelial and smooth muscle cell wound healing. National Institutes of Health, NHLBI Symposium on Cardiovascular Regenerative Medicine, Bethesda, Maryland. October 14th-15th, 2009. Poster Presentation. Roycik, M.D.; Cao, Q.; Jin, Y.; Bosco, D.B.; Schwartz, M.A.; and Sang, Q.X. (2009) Exploring roles of matrix metalloproteinases in human mesenchymal stem cell differentiation with novel synthetic inhibitors. 2nd Annual Southeast Stem Cell Consortium, University of Georgia, Athens, Georgia. October 15th-16th, 2009. Invited Talk. Roycik, M.D.; Cao, Q.; Jin, Y.; Ben, C.; Constantino, M.H.; Bosco, D.B.; Schwartz, M.A.; and Sang, Q.X. (2010) Matrix metalloproteinase inhibitors in vascular wound healing and mesenchymal stem cell differentiation. American Chemical Society, the 86th Annual Florida Meeting and Exposition (FAME), Palm Harbor, Florida. May 13th-15th, 2010. Invited Talk. M.H. Constantino, Y. Jin, M.A. Schwartz, Q. Cao, and Q.-X. Sang (2010). Novel mercaptosulfonamide metalloproteinase inhibitors targeting cerebral and cardiovascular diseases and cancer. American Chemical Society, the 86th Annual Florida Meeting and Exposition (FAME), Innisbrook Resort and Gulf Club, Tampa, Florida. May 13-15, 2010. Poster presentation.

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