Development of Neutral Phosphotyrosine Mimetics As a Protein Tyrosine Phosphatase Inhibitor and Studies on Its Inhibition Mechanism

DEVELOPMENT OF NEUTRAL PHOSPHOTYROSINE MIMETICS AS A PROTEIN TYROSINE PHOSPHATASE INHIBITOR AND STUDIES ON ITS INHIBITION MECHANISM

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Junguk Park

*****

The Ohio State University 2005

Dissertation Committee: Approved by Professor Dehua Pei, Advisor

Professor Ross Dalbey ______Professor Sean Taylor Dehua Pei Graduate Program in Chemistry

ABSTRACT

Reversible phosphorylation of proteins on tyrosyl residues is one of the most important processes in various cellular signaling pathways. A proper level of phosphorylation is controlled by the opposing actions of protein tyrosine kinases (PTKs), and protein tyrosine phosphatases (PTPs).

Thus far, a great deal of efforts was focused on development of the specific PTP inhibitor not only because it would be a useful tool for studying the physiological role of this enzyme, but also because inhibitors against PTPs could provide potential therapeutic agents. Almost all PTP inhibitors developed so far contain a negatively-charged nonhydrolyzable pY mimetic as the inhibitor core structure, although it may impede their membrane permeability. This dissertation describes the development of neutral pY mimetics as PTP inhibitors and their inhibition mechanisms.

First, we have shown that peptidylaldehydes act as reversible, slow-binding inhibitors with modest potency against classical PTPs and DSPs (VH1 and VHR). The mechanism of inhibition was investigated by 1H-13C HSQC spectroscopy using Cinn-

GEE specifically labeled with 13C at the aldehyde carbon and the wild type/mutant

PTP1B. It was revealed that the aldehyde group of the inhibitor formed an imine/enamine adduct with the guanidine group of Arg-221 in the PTP1B active site, resulting in loss of ii its activity. Based on this information, we tested peptidylaldehydes aginst SH2 domains, which also contain Arg in their pY binding site. Biochemical as well as spectroscopic data showed that peptidylaldehydes also bind to SH2 domains with the same inhibition mechanism. These results strongly suggested that these aldehydes should provide a general, neutral core structure for the further development of potent, specific, and membrane permeable inhibitors against PTPs and SH2 domains.

iii

Dedicated to my parents and family

iv ACKNOWLEDGMENTS

I would like to thank my advisor, Professor Dehua Pei, for intellectual support and encouragement through my graduate studies. In addition, I truly admire and appreciate his enthusiasm for science. I also would like to thank the professors in

Johnston Laboratory, Drs Ross Dalbey, Ming-Daw Tsai and Sean Taylor for their kind suggestions and support.

I also thank to my great seniors and lab mates, Drs Peng Wang, Kiet Nguyen,

Mike Sweeney, Hua Fu and Ms. Anne-Sophie Wavreille for their helpful scientific discussion. Their assiduous scientific passions not only stimulate me to work hard in the lab, but also make “Pei Group” as an enjoyable place to work. I am also thankful for my friends, Drs. Taejin Yoon and Minyong Chen for their unforgettable friendship. And I want to thank to Drs. Chunhua Yuan, In-Ja Byeon and Charles Cottrell for grateful discussion as well as excellent technical assistance for HSQC experiments.

Lastly, I truthfully thank to E. J. for her tremendous support as a colleague, a friend and my wife.

v VITA

1972...... Born – Seoul, South Korea

1997...... B.S. Chemistry, Chung-Ang University Seoul, Korea

1999...... M.S. Chemistry, Chung-Ang University Seoul, Korea

2000 – 2003...... Graduate Teaching and Research Assistant The Ohio State University.

2004...... Evans Fellow Department of Chemistry, The Ohio State University.

2005 – present...... Graduate Research Assistant The Ohio State University

PUBLICATIONS

Research Publications

1. M. C. Sweeney, A-S Wavreille, J. Park, J.P. Butchar, S. Tridandapani, and D. Pei, Decoding Protein-Protein Interactions through Combinatorial Chemistry: Sequence Specificity of SHP-1, SHP-2 and SHIP SH2 Domains. Biochemistry, 2005, 44, 14932-14947

2. J. Park and D. Pei, trans-β-Nitrostyrene Derivatives as Slow-Binding Inhibitors of Protein Tyrosine Phosphatases. Biochemistry, 2004, 43, 15014-15021

3. J. Park, H. Fu, and D. Pei, Peptidyl Aldehydes as Slow-Binding Inhibitors of Dual Specificity Phosphatases. Bioorg. Med. Chem. Lett., 2004, 14, 685-687

vi 4. J. Zhu, E. Dizin, X. Hu, A-S Wavreille, J. Park, and D. Pei, S-Ribosylhomo cysteinase (LuxS) is a Mononuclear Iron Protein. Biochemistry, 2003, 42, 4717- 4726

5. J. Park, H. Fu, and D. Pei, Peptidyl Aldehydes as Reversible Covalent Inhibitors of Src Homology 2 Domains. Biochemistry, 2003, 42, 5159–5167

6. H. Fu, J. Park, and D. Pei, Peptidyl Aldehydes as Reversible Covalent Inhibitors of Protein Tyrosine Phosphatases. Biochemistry, 2002, 41, 10700-10709

7. S-W Ham, J. Park, S. Lee, and J. S. Yoo, Selective Inactivation of Protein Tyrosine Phosphatase PTP1B by Sulfone Analogs of Naphthoquinone. Bioorg. Med. Chem. Lett., 1999, 9, 185-186

8. S-W Ham, J. Park, S. Lee, W. Kim, K. Kang, and K. H. Choi, Naphthoquinone Analogs as Inactivators of cdc25 Phosphatase. Bioorg. Med. Chem. Lett., 1998, 8, 2507-2510

9. S-W Ham, J. S. Yoo, J. Park, and H. Cho, Kinetic Analysis of Inactivation of the cdc25 Phosphatase by Menadione. Bull. Korean Chem. Soc., 1998, 19, 29-31

FIELDS OF STUDY

Major Field: Chemistry

vii TABLE OF CONTENTS

P a g e

Abstract……………………………………………………………………………… ii Dedication...…………………………………………………………………………. iv Acknowledgments…………………………………………………………………... v Vita …………………………………………………………………………………. vi List of Tables………………………………………………………………………... xi List of Figures……………………………………………………………………….. xii List of Abbreviations………………………………………………………………... xvi

Chapters:

1. General Introduction……………………………………………………….... 1 1.1 Protein Tyrosine Phosphatases (PTPs)…………………………….... 1 1.2 Classification of the PTPs…………………………………………... 2 1.3 Catalytic Mechanism of the PTPs…………………………………... 4 1.4 PTP Inhibitors………………………………………………………. 7 1.5 Development of the pY analogs…………………………………….. 8

2. Peptidylaldehydes as Reversible Covalent Inhibitors of PTPs…………….... 13 2.1 Introduction…………………………………………………………. 13 2.2 Experimental Procedures…………………………………………… 14 2.2.1 Materials………………………………………………………... 14 2.2.2 Synthesis of the Peptidylaldehyde Inhibitors…………………... 14 2.2.3 Protein Purification……………………………………………... 20 2.2.4 Site Directed Mutagenesis of PTP1B…………………………... 21 2.2.5 PTP Inhibition Assay………………………………………….... 22 2.2.6 Inactivation of Inhibitor Cinn-GEE by Cysteamine……………. 23 2.2.7 NMR Spectroscopy of [13C]Cinn-GEE and [13C]Cinn-GEE-PTP1B Complex...... ………….... 23 2.3 Results………………………………………………………………. 24 2.3.1 Inhibition of PTPs by Simple Aldehydes……………………...... 24

viii 2.3.2 Slow-Binding Inhibition of PTPs by Cinn-GEE………………... 25 2.3.3 Cinn-GEE Is Active-Site Directed…………………………….... 26 2.3.4 Mechanism of Inhibition………………………………………... 27 2.4 Discussion…………………………………………………………... 30

3. Peptidylaldehydes as Slow-Binding Inhibitors of DSPs……………………. 49 3.1 Introduction…………………………………………………………. 49 3.2 Experimental Procedures…………………………………………… 50 3.2.1 Purification of GST-VH1……………………………………….. 50 3.2.2 DSP Inhibition Assay…………………………………………… 51 3.2.3 Competition between Cinn-GEE and pNPP for binding to VH1.. 52 3.2.4 NMR Spectroscopy of [13C]Cinn-GEE and [13C]Cinn-GEE-VH1 Complex………………………………….. 52 3.3 Results and Discussion………………………………………………. 53 3.3.1 Inhibition of VH1 by Cinn-GEE………………………………… 53 3.3.2 Mechanism of Inhibition………………………………………… 54

4. Peptidylaldehydes as Reversible Covalent Inhibitors of SH2 Domains……... 60 4.1 Introduction………………………………………………………….. 60 4.2 Experimental Procedures……………………………………………. 62 4.2.1 Materials………………………………………………………… 62 4.2.2 Synthesis of Peptidylaldehyde Inhibitors……………………….. 62 4.2.3 Activation of SHP-1 by peptidylaldehydes……………………... 66 4.2.4 SH2 Domain-Inhibitor Binding Assay………………………….. 67 4.2.5 NMR Spectroscopy of [13C]Cinn-GEE and [13C]Cinn-GEE-PTP1B Complex………………………………. 68 4.2.6 Mass Spectrometry……………………………………………… 69 4.2.7 Synthesis of Peptidyl Cinnamaldehyde Library and Screening ... 70 4.3 Results………………………………………………………………. 71 4.3.1 Stimulation of SHP-1 Activity by Peptidylaldehydes…………... 71 4.3.2 Direct Binding between Cinn-GEE and SH2 Domains…………. 74 4.3.3 Cinn-GEE is Active-Site Directed………………………………. 75 4.3.4 Cinn-GEE/SH2 Domain Interaction is Reversible……………… 75 4.3.5 Mechanism of Cinn-GEE/SH2 Domain Interaction…………….. 76 4.3.6 Screening of Cinn-XXXX-NH2 Library………………………... 78 4.4 Discussion…………………………………………………………… 78

5. trans-β-Nitrostyrene (TBNS) Derivatives as Slow-Binding ix Inhibitors of PTPs...... 94 5.1 Introduction………………………………………………………...... 94 5.2 Experimental Procedures……………………………………………. 95 5.2.1 Materials………………………………………………………… 95 5.2.2 Site Directed Mutagenesis of PTP1B…………………………… 95 5.2.3 Synthesis of TBNS Derivatives…………………………………. 96 5.2.4 PTP Assay………………………………………………………. 98 5.2.5 Substrate Protection…………………………………………….. 98 5.2.6 Competition between TBNS and Iodoacetic Acid……………… 99 5.2.7 NMR Spectroscopy of [α-13C]TBNS and [α-13C]TBNS-PTP1B Complex………………………………… 99 5.3 Results………………………………………………………………. 99 5.3.1 Inhibition of PTPs by TBNS and Its Derivatives……………….. 99 5.3.2 Slow-Binding Inhibition of PTPs by TBNS…………………….. 102 5.3.3 TBNS is Active-Site Directed…………………………………... 103 5.3.4 Electronic Absorption Spectroscopy of E•I* Complex…………. 104 5.3.5 1H-13C HSQC NMR Spectroscopy of E•I* Complex…………… 104 5.3.6 Effect of Active-Site Mutations on E•I* Complex……………… 106 5.4 Discussion………………………………………………….………... 107

6. Determination of the Substrate Specificity of Methionine Aminopeptidases by Combinatorial Peptide Library Screening……………………………...... 121 6.1 Introduction………………………………………………………….. 121 6.2 Experimental Procedures……………………………………………. 122 6.2.1 Materials………………………………………………………… 122 6.2.2 Synthesis of Dde-linker on the TentaGel……………………….. 123 6.2.3 Synthesis of the Methionine (M) Libraray……………………… 124 6.2.4 Library Screening……………………………………………….. 125 6.2.5 Partial Edman Degradation, Cleavage and Sequencing of Peptides…………………………………………. 127 6.3 Results and Discussion……………………………………………… 128 6.3.1 M Library Design and Synthesis………………………………... 128 6.3.2 Cleavage and Sequencing of Peptides…………………………... 131

Appendix…………………………………………………………………………….. 132 Bibliography…………………………………………………………………………. 133

x LIST OF TABLES

Table Page

2.1 Inhibitory activity of aldehydes against various PTPs………………………. 35

5.1 Inhibition constants of TBNS derivatives against PTP1B and SHP-1(ΔSH2)………………………………………………………………... 111

6.1 Peptides selected from E. coli. MetAP screening…………………………… 136

xi LIST OF FIGURES

Figure Page

1.1 Protein tyrosine phosphorylation…………………………………………. 10

1.2 Schematic representation of the catalytic mechanism of the PTP………… 11

1.3 Structures of the non-hydrolyzing phosphotyrosine analogs……………… 12

2.1 Mechanism of catalysis and inactivation by α-haloacetophenone

derivatives...... 36

2.2 Synthesis of N-[4-(3-Oxo-1-propenyl)benzoyl]-Gly-Glu-Glu-NH2………. 37

13 2.3 Synthesis of N-[4-(3-[ C]Oxo-1-propenyl)benzoyl]-Gly-Glu-Glu-NH2…. 38

13 2.4 Synthesis of N-[4-(3-Oxo-1-[ C]propenyl)benzoyl]-Gly-Glu-Glu-NH2…. 39

2.5 Structures of PTP inhibitors……………………………………………….. 40

2.6 Plot of remaining PTP activity against 4-carboxy-cinnamaldehyde

Concentration……………………………………………………………… 41

2.7 Slow-binding inhibition of PTP1B by Cinn-GEE…………………………… 42

2.8 Competition between Cinn-GEE and substrate for binding to PTP1B………. 43

2.9 HSQC spectra of 13C-labeled Cinn-GEE in the presence and absence

of PTP1B…………………………………………………………………….. 44

2.10 HSQC spectra of 13C-labeled Cinn-GEE in the presence of C215A PTP1B… 45 xii 2.11 HSQC spectra of 13C-labeled Cinn-GEE (compounds 14 and 17)

in the presence and absence of PTP1B……………………………………… 46

2.12 Inactivation of Cinn-GEE by cysteamine……………………………………. 47

2.13 Proposed mechanism of inhibition of PTP by cinnamaldehyde derivatives… 48

3.1 Structures of peptidyl cinnamaldehydes used in Chapter 3…………………. 56

3.2 Slow-binding inhibition of VH1 by Cinn-GEE……………………………... 57

3.3 Competition between Cinn-GEE and pNPP for binding to VH1……………. 58

3.4 Inhibition of VH1 by peptidyl aldehydes……………………………………. 59

4.1 Synthesis of N-{2-[4-(3-Oxo-1-propenyl)phenoxy]acetyl}-Ala-Arg-Leu-

NH2…………………………………………………………………………... 83

4.2 Structures of SH2 inhibitors…………………………………………………. 84

4.3 Activation of SHP-1 by peptidyl aldehydes…………………………………. 85

4.4 BIAcore analysis of the binding of GST-SH2(N) to immobilized Cinn-GEE. 86

4.5 Competition of Cinn-GEE and peptide IYpYANLI for

binding to GST-SH2(N)……………………………………………………… 87

4.6 Time-dependent dissociation of the Cinn-GEE-SH2 domain complex……… 88

4.7 HSQC spectra of 13C-labeled Cinn-GEE in the presence and absence of SH2

Protein……………………………………………………………………….. 89

4.8 Deconvoluted spectra from LC-ESI MS analyses of the

xiii [13C]Cinn-GEE-MBP-SH2(N) complex…………………………………….. 90

4.9 Comparison of the stimulatory effect of Cinn-GEE on wild-type and

R30K SHP-1…………………………………………………………………. 91

4.10 Proposed mechanism of inhibition of SH2 domains by peptidyl

cinnamaldehyde derivatives……………………………………………….... 92

4.11 Synthesis of peptidyl cinnamaldehyde library……………………………… 93

5.1 Structures of TBNS derivatized PTP inhibitors…………………………….. 112

5.2 Absorption spectra of TBNS……………………………………………….... 113

5.3 Slow-binding inhibition of PTP1B by TBNS……………………………….. 114

5.4 Competition for PTP binding between TBNS and pNPP or IDA…………... 115

5.5 Effect of PTP1B on the absorption spectra of TBNS……………………….. 116

5.6 HSQC spectra of [α-13C]TBNS in the absence and presence of PTP1B or

free thiol……………………………………………………………………... 117

5.7 HSQC spectra of [α-13C]TBNS in the presence of various PTP1B mutants... 118

5.8 Proposed mechanism for the slow-binding inhibition of PTPs by TBNS

derivatives………………………………………………………………...... 119

5.9 2-D SDS-PAGE analysis of freshly prepared PTP1B………………………. 120

6.1 Synthesis of Dde linker on the TentaGel S NH2 (90 μm)…………………... 133

6.2 Scheme of the M library screening for MetAP…………………………….... 134

xiv 6.3 MS spectrum of the Partial Edman Degradation Product for peptide

sequencing…………………………………………………………………… 135

xv LIST OF ABBREVIATIONS

Abu Aminobutyric acid

Ac acetyl aq aqueous

β β-Alanine

BCIP 5-Bromo-4-chloro-3-indoyl phosphate

BOC t-butyloxycarbonyl

BSA Bovine serum albumin

Bz benzoyl

°C degrees Celsius calcd calculated

Cys, C Cysteine

CDC Cell division cycle

δ chemical shift in parts per million downfield from tetramethylsilane d doublet (spectra); day(s)

DCM Dichloromethane

Dde 1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl

xvi DIPEA N,N-Diisopropylethylamine

DMAP 4-dimethylamino pyridine

DMF N,N-Dimethylformamide

DMSO dimethylsulfoxide

DNA Deoxyribonucleic acid dNTPs Deoxyribonucleic acid triphosphates

DSP Dual specificity phosphatase

EDC 1-Ethyl-3-(3-dimethyl aminopropyl)-carbodiimide

EDTA N,N,N’,N’-Ethylendiamine tetraaceticacid equiv. equivalent

ESI-MS Electrospray ionization-mass spectrometry

Fmoc 9-Fluorenylmethoxycarbonyl

γ gamma g gram(s)

GSH Glutathione

GST Glutathione S-transferase h hour(s)

HBTU N-[1H-Benzotriazol-1-yl)(dimethylamino)-methylene]-N-

Methylmethanaminium hexa fluorophosphates N-oxide

xvii HEPES N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid

HPLC High performance (or pressure) liquid chromatography

HOBt 1-Hydroxybenzotriazole

HSQC Heteronuclear sigle quantum correlation (or coherence)

IPTG Isopropyl-β-D-thiogalacytoside

J coupling constant in Hz (NMR)

L liter(s) m milli; multiplet (NMR)

MBP Maltose binding protein

μ micro

M moles per liter

MeOH Methanol

MHz megahertz min minute(s) mol mole(s)

MS mass spectrometry; molecular sieves

MT Mutant m/z mass to charge ratio (MS)

Nic-OSu Nicotinic acid O-succimimide

xviii Nle Norleucine

NMM N-Methylmorpholine

NMR nuclear magnetic reasonance p para

PAGE Polyacrylamide gel electrophoresis

PCR Polymerase chain reaction

PITC Phenylisothiocyanate

PNPP p-Nitrophenylphosphate ppm parts per million

PTP Protein tyrosine phosphatase pY Phosphotyrosine q quartet (NMR) s singlet (NMR); second(s)

SA-AP Streptavidin-alkaline phosphatase conjugate

SDS Sodium dodecylsulfate

SPR Surface plasmon resonance spectroscopy t tertiary (tert) t triplet (NMR)

TCEP Tris(2-carboxyethyl)phosphine

xix TEA Triethylamine

TFA Trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

TMS trimethylsilyl

Tris-buffer Tris(hydroxymethyl)aminomethane-buffer

WT Wild type

Standard one letter codes are used for deoxynucleotides occurring in PCR primers, and standard one and three letter codes are used for 20 natural amino acids

CHAPTER 1

GENERAL INTRODUCTION

1.1 Protein Tyrosine Phosphatase (PTP)

Protein phosphorylation is one of the most important processes to transmit cellular signals in vivo, and it is estimated that one third of the mammalian proteins are phosphorylated [1]. In fact, most of them are phosphorylated on serine/threonine residues in fact, and compared to serine/threonine phosphorylation, smaller extent of tyrosine phosphorylation (~ 0.05 %) is occurred in cells [2]; however it has been shown that tyrosine phosphorylation is also essential in controlling various cellular events, such as cellular growth, differentiation, metabolism and cell cycle, etc [3]. Thus far, ≥ 800 phosphotyrosine (pY), ≥ 3,500 phosphoserine (pS) and phosphothreonine (pT) motifs are experimentally identified from different eukaryotic species.

Protein tyrosine phosphorylation can alter conformation, catalytic activity or localization of the proteins, as well as mediate protein-protein interactions during the signal transduction pathway (Figure 1.1) [3]. Thus, abnormal protein tyrosine phosphorylation can disrupt cellular signaling, resulting in many human diseases, such as cancer, diabetes, and inflammatory diseases [4, 5]. Two enzyme families - protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) - act together to

1 maintain the proper level of protein phosphorylation in vivo [6]. PTKs catalyze the reaction of adding the phosphate group on the tyrosine residue, while PTPs catalyze removing of the phosphate group from the phosphotyrosine residue. Although more studies has been focused on PTKs than on PTPs and the exact roles of the PTPs are still unclear, recent studies has made it clear that PTPs as well as PTKs play the significant roles in controlling the level of tyrosine phosphorylation, and in regulating various physiological processes [7, 8].

1.2 Classification of the PTPs

It has been shown that ~ 100 PTP genes are encoded in human genome [7], and it is almost identical to the number of PTKs (~ 90 PTKs) encoded in human genome [9], probably suggesting they could have comparable substrate specificities [7]. Based on the amino acids sequences of the catalytic domains, PTPs can be divided into four different groups [7, 10, 11]. The major group is class I cysteine-based PTPs, which contain the common signature motif (C(X)5R(S/T)) in their active site. Class I PTPs can be further divided into two subgroups based on the degree of homology between catalytic domains.

Among class I PTPs, 38 PTPs can be separated as classical PTPs [12], which are strictly phosphotyrosine specific, and others are grouped as VH-1 like PTPs, which are also called dual-specificity protein phosphatase (DSP) [13, 14]. Unlike classical PTPs, DSPs can hydrolyze phosphoserine and phosphothreonine as well as phosphotyrosine.

Comparing X-ray crystal structures of classical PTPs and DSPs [15, 16], overall architectures including the signature motif around the active sites are conserved. One critical difference between two subfamilies exists in the depth of the active-site pocket. In

2 the dual-specificity phosphatase VHR, the depth of the active-site pocket is ~ 6 Å deep, while the active-site pocket of the classical phosphotyrosine phosphatase PTP1B is ~ 9 Å deep. The difference of the active-site depth has been proposed to make the classical

PTPs be highly specific to phosphotyrosine, because the depth of the pocket is too deep for pS/pT to reach the active-site cysteine residue that is located at the bottom of the active-site pocket and critical for PTP catalysis (discussed below) [17]. Furthermore, classical PTPs are subdivided into two classes, receptor-like PTPs and nonreceptor-like

PTPs, based on the location of the proteins [6], and VH-1 like PTPs are more diverse, thus it is separated into several groups depending on its structure and cellular functions

[7].

Class II cysteine-based PTPs is the low-molecular weight (18 KDa) phosphatase

(LMPTP) [18]. LMPTPs are widely distributed and well conserved from bacteria to human, while their in vivo functions are not yet well-defined [7]. Including prokaryotic

LMPTPs, class II enzymes are also phosphotyrosine specific; however LMPTPs rarely share sequence homology with other PTPs except the signature motif [7, 18].

Cdc25 phosphatases represent class III cysteine-based PTPs [19]. In human, there are three Cdc25 homologues, Cdc25A, B and C, and they play important roles in cell cycle progression by dephosphorylating and activating cyclin-dependent kinases [20-24].

Based on substrate specificity, Cdc25 can be classified to DSPs, and it also contains the signature motif in the active site; however the catalytic domain structure of Cdc25 is unique among the cysteine-based PTPs [24]. The folding pattern of Cdc25 would rather be more closely related to that of a bacterial rhodanese from Azotobacter vinelandii, which transfer of sulfane sulfur from thiosulfate to cyanide [7, 10, 25].

3 The last class of the protein phosphatases is aspartic acid-based PTPs, which were newly identified [26-28]. It contains the Eya proteins, which show dual specificity, and play a significant role in eye development. Exact catalytic mechanism of the aspartic acid-based PTPs is still needed to be investigated, and structural information is also required to clearly understand this family of the enzymes.

1.3 Catalytic mechanism of the PTPs

As previously mentioned, all of the cysteine-based PTPs share the common signature motif, C(X)5RS/T. One more residue which is significant in enzyme catalysis is the conserved aspartic acid [11, 15, 29]. Although there are other considerably important residues during the catalysis [11, 12], we will focus on the roles of the signature motif and the conserved aspartic acid, and it would be helpful to understand the catalytic mechanism of the PTPs.

Arg221 (numbering in PTP1B)

In order to be dephosphorylated, the pY should bind into the enzyme-active pocket first. The arginine residue in the signature motif directly involves in binding of the pY, and it is highly conserved among all the PTPs. Structural studies showed that, once the pY comes into the active site, a guanidinium group of the arginine binds to two oxygen atoms of the phosphate group, and provides a strong ionic interaction for the binding of the pY. In fact, mutation of the Arg to Ala significantly decreases the substrate binding affinity (~ 100 folds) [30]. In addition, this Arg to Ala mutant showed ~ 104 folds lower kcat, suggesting Arg221 also involves in enzyme catalysis [31, 32].

Cys215

Dephosphorylation by the PTPs occurs via the two step reaction (Figure 1.2) [33-

35]. First, the phosphate group is attacked by catalytic cysteine, resulting in the formation of the phospho-enzyme intermediate (E-P). Next, the enzyme is reactivated by hydrolyzing the phosphate group by the water molecule in the active site. It has been shown that the catalytic cysteine residue is essential for catalysis [34], and highly conserved among all the cysteine-based PTPs. Structural studies showed that the cysteine residue is located at the bottom of the active-site pocket, and it is properly directed toward the incoming phosphate group for the nucleophilic substitution reaction[17]. One of the interesting features of the catalytic cysteine is that it has lower pKa value (pKa ~

4.5) than normal cysteine residues (pKa ~ 8.5), thus it exists as a thiolate ion, which could be better nucleophile than a thiol [36, 37]. Based on the X-ray crystal structures of the PTPs [29, 38, 39], this atypical pKa of the cysteine is accomplished by stabilizing the thiolate ion through the ionic interactions between Cys215 and Arg225 as well as hydrogen bonding network of the amide N-H in the enzyme active site. In addition, the

α-helix motif that is located next to the pY binding loop also contribute in lowering the pKa of Cys215. In fact, disruption of the hydrogen bonding network by site directed mutagenesis of the active-site residue increases the pKa value of the catalytic cysteine, resulting in lowering the enzyme activity [37].

Almost all of the PTPs contain the cysteine residue in their active site; however, some of the PTPs contain the aspartic acid rather than the cysteine (see above).

Unfortunately, the catalytic mechanism of the aspartic acid-based PTPs is still unclear.

5 However, interestingly, recent reports showed that the Cys to Asp mutant of PTP1B maintains some extent of the activity [40]. Therefore, it might be possibly expected that the aspartic acid-based PTPs share the common catalytic mechanism with the cysteine- based PTPs except the identity of the nucleophile in their active site.

Serine/Threonine222

There are two major roles of the serine/threonine222 were reported. One is that the serine/threonine assists the catalytic cysteine to position toward the incoming phosphate group [16, 29, 38]. The other is that it stabilizes the thiolate ion in hydrolysis of the E-P [41]. Therefore, mutation of the serine/threonine affects not only formation of the E-P (~ 10 folds decrease), but also hydrolysis of the E-P (~ 100 folds decrease) [10,

11].

Asp181

Asp181 is located out of the active site in the free enzymes, which is called

“WPD (Trp-Pro-Asp) loop” and is very flexible during catalysis, but it comes close to the active site when the substrate binds to the enzyme [15, 29]. It plays a critical role in both the formation and the hydrolysis of the E-P, but not in the substrate binding [42, 43]. In the first step, it acts as a general acid to provide the leaving phenolate ion with a proton, making it better leaving group. In the next step, it takes a proton from the water molecule in the active site to make it better nucleophile, resulting in acceleration of the E-P hydrolysis. Because of that, the Asp to Ala mutant PTP has ~ 105-folds lower activity

6 than wild type, while the binding affinity of the mutants remains similar. Therefore, the

Asp mutant is commonly used as a PTP substrate trapping mutant [44].

1.4 PTP inhibitors

As previously mentioned, PTPs involve in various important signaling pathways and malfunctions of the PTPs were observed in many human diseases [45]. Therefore one can expect that well developed PTP inhibitors could be the leads for novel drug discovery as well as useful tools for studying cellular signal transduction pathways [11, 46].

However, development of the PTP inhibitors might be more challenging than PTK inhibitors, as it is difficult to find a specific inhibitor toward a certain PTP. Because PTPs share the highly conserved active site, it might be difficult to get a specific inhibitor. In addition, the biological studies suggest that a lot of PTPs regulate more than one signaling pathway [11]; therefore inhibition of the PTP may cause undesired side effects in vivo. Therefore, more careful approaches are needed to develop the PTP inhibitor as a new therapeutic reagent.

For instance, a great deal of effort was focused on development of the PTP1B inhibitor for last ten years [5, 11, 46]. PTP1B is one of the well studied PTPs [47], and it belongs to the cysteine-based class I PTPs. It was first purified as a catalytic domain of

37 KDa [48, 49], however cDNA of the PTP1B revealed that it has C-terminal extension including the hydrophobic tail that interact with the endoplasmic reticulum (ER) membrane [50-53]. Like other class I PTPs, it contains the signature motif in the active site, and structural and biochemical studies showed that it shares the common catalytic mechanism of the PTPs [38]. One of the physiological roles of PTP1B is that it act as a

7 negative regulator in insulin signaling, thus inhibition of PTP1B could be an attractive way to cure type 2 diabetes by stimulating the insulin signaling [54]. Nonetheless, PTP1B may not be a good drug target by using a PTP inhibitor because it also involves in various other signaling pathways [11]. However, animal studies [54, 55] showed somewhat promising results. It was shown that increased insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) phosphorylation was observed in PTP1B-/- mice, resulting in enhancing sensitivity to insulin in skeletal muscle and liver. In addition, these mice were resistant to high-fat diet induced obesity. Most surprising observation is that PTP-/- mice showed normal and healthy behavior, proposing that the drug against PTP1B action might not have any undesired side effect in vivo [11].

As discussed previously, PTP inhibitors are also useful tools for studying the cellular signaling pathway. For this purpose, in many cases, one may need to inhibit all of the PTPs by using a non-specific PTP inhibitor[56]. In fact, a lot of non-specific PTP inhibitors and the inhibitor cocktails that are commercially available have been used in the various cellular and molecular biology studies [57]. Therefore, development of the general PTP inhibitor is also significant, while specificity is absolutely required with a view to discovering a drug.

1.5 Development of the pY analogs

Because of the reasons discussed above, a great deal of effort has been focused on the development of the PTP inhibitors [11, 46]. In general, most of the PTP inhibitors contain the non-hydrolyzable pY analog as a core structure, which is essential for the binding of the inhibitor indeed [15]. A number of non-hydrolyzable pY analogs were

8 found so far, and a majority of them in fact have at least one anionic charge (Figure 1.3)

[58-73]. An anionic pY analog could be more potent than the neutral one because it can mimic the ionic interaction between the phosphate group and the arginine residue in the active site [30]. However, it also impedes the cellular membrane permeability of the inhibitor, so its practical application is still in doubt. Therefore, the potent and neutral pY-analog inhibitors are needed to overcome this problem.

Although the pY analog is essential for the binding, the small pY analog molecule itself is not very potent and selective inhibitor. In order to improve the pY analog inhibitor, one approach is adding optimal peptide sequence to the pY analog. The interaction between the peptide and the protein surface could make the inhibitor more potent as well as more selective. For instance, one of the common pY analogs, phosphonodifluoromethyl phenylalanine (F2Pmp) has 2.5 mM as KI against PTP1B [62].

To improve its potency, the known PTP1B substrate sequence (DADE(F2Pmp)L-NH2) was added, and, as a result, KI was decreased to ~ 200 nM against PTP1B [74, 75]. It strongly suggests that derivatization of the pY mimetics with the proper peptide (or other moieties) is also important for developing the PTP inhibitor.

In summary, ~ 100 PTPs are encoded in human genome, and they involve in a variety of cellular signaling pathways. However, the exact roles of the PTPs are still unclear; in addition, malfunction of the PTPs were found in many human diseases.

Therefore, as a tool of the molecular and cellular biology studies as well as a lead for drug discovery, development of the potent and selective PTP inhibitor is required.

ATP ADP = OH OPO 3 PTK

N N H H O PTP O

= Y HOPO 3 H2O pY

Altered Conformational Change of Binding to SH2 Catalytic Activity Change Localization or PTB Domains

Signal Transmission

Figure 1.1. Protein tyrosine phosphorylation. PTK, preotein tyrosine kinase; PTP, protein tyrosine phosphatase; pY, phosphotyrosine; SH2, Src Homology 2; PTB, phosphotyrosine binding.

Figure 1.2. Schematic representation of the catalytic mechanism of the PTP [47](using PTP1B numbering).

CO2H

O CO2H

O CO2H F P OH F OH H2N

CO2H

α,α-difluorobenzyl phosphonic acid O-malonyltyrosine Cinnamic acid

OH O HO NH OH O HO N NH O N H OH COOH O O

2-(oxalylamino)-benzoic acid 5-carboxy-2-naphtholic acid Salicyclic acid derivative

Figure 1.3. Structures of the non-hydrolyzing phosphotyrosine analogs [11]

CHAPTER 2

PEPTIDYLALDEHYDES AS REVERSIBLE COVALENT INHIBITORS OF

PROTEIN TYROSINE PHOSPHATASES

2.1 Introduction

As discussed in Chapter 1, the neutral pY analog is one of the most important aspects of the PTP inhibitor development. Thus, for last several years, our laboratory has been undertaking an approach to designing PTP inhibitors by covalently modifying its conserved active-site residues with the neutral pY mimetics. In fact, we have previously demonstrated that α-haloacetophenone derivatives act as potent, time-dependent inactivators of PTPs by alkylating the active-site cysteine (Figure 2.1) [76, 77]. In addition, we have showed that α-haloacetophenone is membrane permeable, resulting in increasing phosphoprotein level in α-haloacetophenone treated B-cell. Furthermore, these inhibitors showed moderate selectivity toward PTPs over DSPs. However, one drawback of the α-haloacetophenone inhibitor is a specificity problem as shown in other irreversible inhibitors, which indicates that it could possibly interact with any cysteine of the proteins and permanently damages the undesired proteins. Therefore, our research has been focused on the reversible and neutral pY analog development for recent a few years,

13 and here, we report that simple aldehydes act as slow-binding, reversible inhibitors of

PTPs by forming an imine/enamine adduct with the active-site arginine.

2.2 Experimental Procedures

2.2.1 Materials

All of the chemicals for general solid phase peptide synthesis were purchased from Advanced ChemTech (Louisville, KY), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Oligonucleotides were purchased from Integrated

DNA Technologies (Coralville, IA). SP Sepharose® Fast Flow resin was from Pharmacia

(Piscataway, NJ). Pfu Turbo® DNA polymerase (Stratagene. La Jolla, CA) was used to amplify the nucleotides and GeneAmp® PCR System 2400 (Applied Biosystems, CA) was used to perform the polymerase chain reactions (PCR). Lambda 20 UV/Vis spectroscopy (Perkin Elmer, MA) was used for measuring UV absorption. NMR experiments were performed on a Bruker (Billercia, MA) DMX-600 NMR spectrometer.

2.2.2 Synthesis of the Peptidylaldehydes Inhibitors

Peptidylaldehydes inhibitors were chemically synthesized by Dr. Hua Fu following the synthetic schemes shown in Figure 2.2, 2.3, and 2.4.

Synthesis of Methyl 4-[2-(1,3-Dioxolane-2-yl)-ethenyl]benzoate (6) This

compound was prepared by modification of a literature procedure [78].

Methyl 4-formylbenzoate (0.82 g, 5.0 mmol) and

tris(methoxyethoxyethyl)amine (TMA) (1.62 g, 5.0 mmol) were dissolved

14 in 30 mL of dichloromethane under argon at room temperature. A saturated aqueous

K2CO3 solution (30 mL) and (1,3-dioxolane-2-yl-methyl)triphenylphosphonium bromide

(2.15 g, 5 mmol) were added, and the reaction mixture was refluxed (~ 40 oC) for 16 h with vigorous stirring. The reaction product was extracted into dichloromethane (3 × 20 mL), washed with water (20 mL), and dried over MgSO4. Compound 6 was obtained as a

67:33 mixture of Z and E isomers after chromatography on a silica gel column (6:1

1 hexane/ethyl acetate) (yield: 1.16 g, 99.1%). H NMR (250 MHz, CDCl3) δ 7.97-8.10 (m,

2H, Ar), 7.52-7.76 (m, 2H, Ar), 6.83-6.93 (m, 1H, ArCH=CHCH), 6.36 (dd, 0.33H,

ArCH=CHCH E, J = 5.8 Hz, 16.1 Hz), 5.81 (dd, 0.67H, ArCH=CHCH Z, J = 7.4 Hz,

11.8 Hz), 5.45 (d, 0.67H, ArCH=CHCH Z, J = 7.4 Hz), 5.39 (d, 0.33H, ArCH=CHCH E,

13 J = 5.9 Hz), 3.88-4.18 (m, 7H, CH3, OCH2CH2O); C NMR (62.5 MHz, CDCl3) δ

166.63, 141.31, 134.39, 133.47, 131.39, 130.58, 130.13, 129.83, 127.64, 104.06, 99.98,

65.76, 65.61, 52.31.

Synthesis of Methyl 4-(3-Oxo-1-propenyl)benzoate (9) Compound 6 (0.12 g,

O H 0.5 mmol) was added to 5 mL of 90% aqueous TFA and the solution was

stirred for 1 h. After removal of solvents by rotary evaporation, the residue

was dried over P O in vacuo to a white solid as pure E isomer (95 mg, O OMe 2 5

1 quantitative yield). H NMR (250 MHz, CDCl3) δ 9.74 (d, 1H, CHO, J = 7.5 Hz), 8.09 (d,

2H, Ar, J = 8.4 Hz), 7.87 (d, 2H, Ar, J = 8.4 Hz), 7.77 (d, 1H, ArCH=CHCH, J = 16.1

13 Hz), 6.87 (dd, 1H, ArCH=CHCH, J = 7.5 Hz, 13.3 Hz), 3.91 (s, 3H, OCH3); C NMR

(62.5 MHz, CDCl3) δ 194.12, 166.61, 151.62, 132.86, 131.44, 129.47, 52.59.

15 Synthesis of 4-(3-Oxo-1-propenyl)benzoic acid (3) Compound 9 (48 mg) was

O H added to 3 mL of an aqueous NaOH solution (40 mg, 1 mmol) and the

solution was stirred overnight at room temperature. After removal of

O OH methanol by rotary evaporation, the remaining solution was diluted with 5 mL of H2O and acidified to pH 2 with HCl. The solution was extracted with ethyl acetate

(3 × 10 mL) and the combined organic phase was dried over MgSO4. Evaporation of solvent afforded 41 mg of a light yellow solid (92.4% yield). 1H NMR (250 MHz,

CDCl3) δ 10.46 (s, 1H, COOH), 9.78 (d, 1H, CHO, J = 7.6 Hz), 8.14 (d, 2H, Ar, J = 8.3

Hz), 7.88 (d, 2H, Ar, J = 8.3 Hz,), 7.78 (d, 1H, ArCH=CHCH, J = 16.1 Hz), 6.89 (dd, 1H,

13 ArCH=CHCH, J = 7.5 Hz, 16.0 Hz); C NMR (62.5 MHz, CDCl3) δ 194.15, 164.15,

151.24, 134.52, 131.07, 129.45.

Synthesis of Ethyl 4-[2-(1,3-dioxolane-2-yl)-ethenyl]benzoyl carbonate (7) An

aqueous NaOH solution (0.8 g dissolved in 8 mL of H2O) was added to O O H compound 6 (1.16 g, 5.0 mmol) dissolved in 8 mL of methanol. The

O O O O solution was stirred for 16 h at room temperature, followed by solvent evaporation. The solid residue was dried over P2O5 in vacuo to produce the corresponding sodium salt, which was stable during storage in a –5 oC freezer. Ethyl chloroformate (0.26 g, 24 mmol) was added to a 15-mL suspension of the salt (0.14 g) in

CH2Cl2 under argon and the mixture was stirred for 2 h at room temperature and filtered.

The filtrate was evaporated under reduced pressure to obtain 0.12 g of an oil (isomer Z :

1 E = 62 : 38) (86% yield). H NMR (250 MHz, CDCl3) δ 8.02-8.08 (m, 2H, Ar), 7.60-7.72

(m, 2H, Ar), 6.86-6.92 (m, 1H, ArCH=CHCH), 6.42 (dd, 0.38H, ArCH=CHCH E, J = 5.8 16 Hz, 16.1 Hz), 5.86 (dd, 0.62H, ArCH=CHCH Z, J = 7.4 Hz, 11.8 Hz), 5.46 (d, 0.62H,

ArCH=CHCH Z, J = 7.4 Hz), 5.41 (d, 0.38H, ArCH=CHCH E, J = 5.9 Hz), 4.29-4.42 (m,

13 2H. CH2CH3), 3.91-3.94 (m, 4H, OCH2CH2O), 1.29-1.38 (m, 3H, CH2CH3); C NMR

(62.5 MHz, CDCl3) δ 161.80, 149.93, 149.30, 143.33, 143.09, 134.00, 132.24, 131.57,

131.28, 131.11, 130.99, 130.39, 128.19, 127.85, 127.57, 103.85, 99.85, 66.71, 65.79,

65.63, 14.15.

Synthesis of N-[4-(3-Oxo-1-propenyl)benzoyl]-Gly-Glu-Glu-NH2 (5) The

tripeptide Gly-Glu-Glu was synthesized on 0.18 g of Rink resin (0.11

mmol) using standard Fmoc/HBTU/HOBt solid-phase peptide

chemistry. Next, 61.3 mg (0.21 mmol) of anhydride 7 in 2.5 mL

CH2Cl2 and 100 µL of N-methylmorpholine were added to the resin suspended in 2.5 mL of anhydrous DMF. The mixture was shaken for 8 h at room temperature. Ninhydrin tests indicated complete acylation of the N-terminal amine. The solvents were drained and the resin was washed with DMF (5 × 5 mL) and CH3OH (3 × 5 mL). Deprotection of side- chain as well as aldehyde protecting groups and cleavage from the resin were carried out with a cocktail containing 4 mL of 90% TFA in water, 0.1 mL of anisole, and 0.15 mL of thioanisole for 3 h at room temperature. TFA, H2O and other volatile chemicals were removed under a gentle flow of nitrogen, and the residue was triturated five times with diethyl ether. Compound 5 was obtained as a brownish solid in the pure E isomer form

1 (yield 48 mg). H NMR (250 MHz, D2O): 9.62 (d, CHO, J = 7.4 Hz), 7.79-7.86 (m, Ar,

ArCH=CHCH), 6.87 (dd, ArCH=CHCH, J = 7.5 Hz, 16.0 Hz), 4.11-4.42 (m, α-CH of

Glu), 3.84 (s, α-CH2 of Gly), 2.37-2.42 (m, 2 CH2CH2COOH), 1.96-2.12 (m, 2 17 + + CH2CH2COOH). HRESI-MS: C22H26O9N4Na ([M+Na] ), calcd. 513.1592, found

513.1576.

Synthesis of Methyl 4-(3-[13C]Oxo-1-propenyl)benzoate (10)

13 O H (Triphenylphosphoranylidene)-[1- C]acetaldehyde (11) was synthesized * H as previously described [79]. The crude aldehyde 11 (0.85 g, 2.8 mmol)

O OMe after crystallization was dissolved in 25 mL of benzene and mixed with methyl 4-formyl benzoate (0.7 g, 4.3 mmol). The solution was stirred for 6 h at room temperature and the solvent was removed by rotary evaporation. Chromatography on a silica gel column (hexane/ethyl acetate = 6:1) afforded 0.17 g of a white solid (26% yield

1 13 3 1 for two steps). H NMR (250 MHz, CDCl3): δ 9.75 (dd, 1H, CHO, JH-H = 7.6 Hz, JC-H

3 3 = 173.1 Hz), 8.09 (d, 2H, Ar, JH-H = 6.7 Hz), 7.64 (d, 2H, Ar, JH-H = 8.4 Hz), 7.50 (d,

3 3 1H, ArCH=CHCH, JH-H = 16.0 Hz, JC-H = 33.5 Hz), 6.78 (ddd, 1H, ArCH=CHCH E,

3 2 13 JH-H = 7.5 Hz, 16.0Hz, JC-H = 0.85 Hz), 3.95 (s, 3H, OCH3); C NMR (62.5 MHz,

CDCl3) δ 193.65.

Synthesis of Methyl 4-[2-(1,3-Dioxolane-2-[13C]yl)-ethenyl]benzoate (12) A

O O solution of ethylene glycol (0.11 g, 1.78 mmol) in benzene (5 mL) was

H added into a round-bottomed flask containing compound 10 (0.17 g, 0.89

O OMe mmol), p-toluenesulfonic acid monohydrate (3 mg), MgSO4 (0.22 g, 1.78 mmol), and benzene (25 mL). The reaction mixture was heated to reflux for 8 h. After cooling, solid NaHCO3 (5.4 mg, 0.064 mmol) was added to the mixture to neutralize the p-toluenesulfonic acid and the mixture was stirred for 30 min. The reaction mixture was 18 then filtered through a pad of anhydrous NaHCO3 and the filter cake was washed with

CH2Cl2 (3 × 10 mL). The filtrate was concentrated and purified by flash column chromatography on a silica gel column (6:1 hexane/ethyl acetate) to obtain 0.14 g of a

1 3 white solid (67% yield). H NMR (250 MHz, CDCl3) δ 7.99 (d, 2H, Ar, JH-H = 6.6 Hz),

3 3 3 7.63 (d, 2H, Ar, JH-H = 6.6 Hz,), 6.87 (dd, 1H, ArCH=CHCH, JH-H = 16.0 Hz, JC-H = 6.7

3 3 Hz), 6.37 (dd, 1H, ArCH=CHCH E, JH-H = 5.8 Hz, 16.0 Hz), 5.39 (dd, 1H, JH-H = 5.9

2 13 Hz, JC-H = 167.43 Hz), 3.88-4.04 (m, 7H, OCH3, OCH2CH2O); C NMR (62.5 MHz,

CDCl3) δ 104.08.

Synthesis of Ethyl 4-[2-(1,3-dioxolane-2-[13C]yl)-ethenyl]benzoyl carbonate

1 (13) This was prepared as described for 7. H NMR (250 MHz, CDCl3) δ 8.03 (d, 2H, Ar,

O O 3 3 JH-H = 8.0 Hz), 7.51 (d, 2H, Ar, JH-H = 8.05 Hz), 6.82 (dd, 1H, H 3 3 ArCH=CHCH, JH-H = 15.8 Hz, JC-H = 7.45 Hz), 6.31 (dd, 1H, O O O O 3 3 2 ArCH=CHCH E, JH-H = 5.6 Hz, 15.9Hz), 5.46 (dd, 1H, JH-H = 5.6 Hz, JC-

H = 168.1 Hz), 4.40 (m, 2H, OCH2CH3), 3.96-4.09 (m, 4H, OCH2CH2O), 1.44 (t, 3H,

13 OCH2CH3); C NMR (62.5 MHz, CDCl3) δ 103.52.

13 Synthesis of N-[4-(3-[ C]Oxo-1-propenyl)benzoyl]-Gly-Glu-Glu-NH2 (14)

This was prepared from anhydride 13 in a similar manner to 5. 13C NMR (62.5 MHz,

CDCl3) δ 194.43.

Synthesis of Methyl 4-[13C]cyanobenzoate (15) This compound was synthesized

19 from [13C]KCN according to a literature procedure

1 (Scheme 3) [80]. H NMR (250 MHz, CDCl3) δ 8.13 (d,

2H, Ar, J = 8.2 Hz), 7.80 (d, 2H, Ar, J = 8.2Hz), 3.95 (s, 14 15 13 3H, OCH3); C NMR (62.5 MHz, CDCl3) δ 118.35.

Synthesis of Methyl 4-[13C]formyl benzoate (16) Nickel/aluminum alloy (1:1

w/w, 80 mg) was added to methyl 4-[13C]cyanobenzoate 15 (640 mg, 3.95

mmol) in aqueous formic acid [10 mL, 3:1 formic acid/water (v/v)]. The

suspension was refluxed for 1 h, cooled, and filtered, and the residual alloy was rinsed with ethanol (2 mL), chloroform (4 mL), and diethyl ether (5 mL). The filtrate was extracted with chloroform (2 × 20 mL). The organic phase was washed with water (15 mL) and then a saturated sodium bicarbonate solution (15 mL). The water and sodium bicarbonate phases were back-extracted with chloroform (10 mL) and then diethyl ether (10 mL). The combined organic phase was dried over anhydrous magnesium sulfate and evaporated in vacuo to yield 0.53 g of a semi-solid (81% yield),

1 which was used without further purification. H NMR (250 MHz, CDCl3) δ 10.08 (d, 1H,

*CHO, J = 162.5 Hz), 8.18 (d, 2H, Ar, J = 8.3 Hz), 7.96 (d, 2H, Ar, J = 8.23 Hz), 3.94 (s,

13 3H, OCH3); C NMR (62.5 MHz, CDCl3) δ 191.88.

13 Synthesis of N-[4-(3-Oxo-1-[ C]propenyl)benzoyl]-Gly-Glu-Glu-NH2 (17)

This was prepared from aldehyde 16 in a similar manner to 5. 13C NMR

(62.5 MHz, CDCl3) δ 152.16.

20 2.2.3 Protein Purification

The catalytic domain of SHP-1, SHP-1(∆SH2), and VHR were purified by Dr.

Gulnur Arabaci from a recombinant Escherichia coli (E. coli) strain as previously described [81]. The catalytic domain of PTP1B (residues 1–321) was sub-cloned into pET-22(b) plasmid by Dr. Kirk Beebe using pBS-PTP1B (supplied by Dr. Zhong-In

Zhang) as a template.

To purify PTP1B, E. coli cells carrying pET-22(b)-PTP1B were grown in LB media (4 L) supplemented with 75 mg/mL ampicilline, 10 g/L trypton, 5 g/L yeast extract and 10 g/L NaCl at 37 °C to an OD600 of 0.6. The cells were induced by the addition of 300 μM isopropyl β-D-thiogalactoside (IPTG) and continued grown at 30 °C for an additional 4 h. The cells were harvested by centrifugation at 5,000 r.p.m. at 4 °C for 10 min, and resuspended in 100 mL of the lysis buffer (30 mM HEPES, pH 7.2, 4 mM EDTA, 10 mM NaCl, 10 mM β-mercaptoethanol, 200 μM PMSF, 2 mg trypsin inhibitor, 5 mg chicken egg white lysozyme, and 500 mg prostamine sulfate). The resuspended cells were lysed twice by a Thermo French Press at 500 psi. The lysate was centrifuged at 12,000 r.p.m. for 30 min at 4 °C, and the supernatant was loaded onto the

SP Sepharose column (2.5 cm × 10 cm), which was pre-equilibrated with the low salt buffer (30 mM HEPES, pH 7.2, 10 mM NaCl, 1mM EDTA, and 1mM β- mercaptoethanol). After loading the protein, the column was washed with 500 mL of the low salt buffer, and the protein was eluted by using linear ionic gradient (10 mM – 500 mM NaCl). The protein fractions were pooled and concentrated to ~ 2 mL at 4 °C by using an Amicon stirred cell equipped with YM-3 cellulose membrane. The concentrated

21 protein was stored at – 80 °C with 30 % glycerol for the general purpose. For NMR experiment, the protein was stored in the absence of glycerol. The concentration of the protein was measured by Bradford assay using bovine serum albumin (2 mg/mL) as a standard.

2.2.4 Site Directed Mutagenesis of PTP1B

Site-directed mutagenesis was carried out with pET-22(b)-PTP1B plasmid as template using the QuickChange mutagenesis kit (Stratagene, CA). The DNA primers

(purchased from Integrated DNA Technologies, Inc., IW) used here were as follows:

C215A, 5’-GTTGTGGTGCACGCCAGTGCAGGCATC-3’;

R225A, 5’-GCAGGCATCGGCGCGTCTGGAACCTTC-3’.

The identity of all DNA constructs was confirmed by DNA sequencing. Expression in

Escherichia coli and purification of recombinant PTP1B mutants were performed as described in wild type PTP1B purification.

2.2.5 PTP Inhibition Assays

Stock solutions of inhibitors were prepared in dimethyl sulfoxide (DMSO) and their concentrations were calculated from the known inhibitor masses and solvent volumes. A typical reaction (total volume 1 mL) contained 50 mM HEPES (pH 7.4), 1 mM EDTA, 1 mM tris(carboxyethyl)phosphine (TCEP), 50 mM NaCl, 5% (v/v) DMSO,

0.1–0.2 µM PTP, and 0–2000 µM inhibitor. After incubation of the enzyme with the inhibitor for 1 h at room temperature, the reaction was initiated by the addition of 1.0 mM p-nitrophenyl phosphate (pNPP). The reaction progress was monitored at 405 nm on a

22 Perkin-Ellmer UV-Vis spectrophotometer. The IC50 values were determined by plotting the remaining activity as a function of inhibitor concentration, and the KI* values were obtained by fitting the data to the Michaelis-Menten equation. To determine the KI value, the reaction was initiated by addition of enzyme (0.2 µM) into the above reaction mixture, which also contained 1.0 mM pNPP. The reaction was monitored continuously on the UV-Vis spectrophotometer and the initial reaction rates (<15 s) were fitted to the

Michaelis-Menten equation.

To determine the rate constant k6, the enzyme (1.0 µM) was incubated with 50–

100 µM inhibitor in 100 µL of the above reaction buffer for 3 h at room temperature. The mixture was rapidly diluted into 900 µL of the same reaction buffer containing 1.0 mM pNPP (without inhibitor). Reactivation of PTP activity was monitored at 405 nm and the progress curves were fit to the equation

-k6t Abs405 = vs[t – (1 – e )/k6] where vs is the final steady-state velocity. The rate constant k5 was calculated using the equation KI* = KIk6/(k5 + k6).

2.2.6 Inactivation of Inhibitor Cinn-GEE by Cysteamine

Cinn-GEE (1 mM) was incubated with 100 mM cysteamine hydrochloride (which had been neutralized by the addition of 1.0 equivalent of NaOH) in 100 µL of a 97:3

DMSO/H2O mixture for 24 h. The treated inhibitor was then used in inhibition assays as described above. As a control, inhibition assay also performed with the same amount of cysteamine hydrochloride/NaOH solution.

23 2.2.7 NMR Spectroscopy of [13C]Cinn-GEE and [13C]Cinn-GEE-PTP1B Complex

All NMR experiments were performed on a Bruker DMX-600 NMR spectrometer equipped with a triple-resonance and 3-axes gradient probe at 300 K. The 2D 1H-13C heteronuclear single-quantum coherence spectra (HSQC) were acquired using a constant- time sensitivity-enhanced method [82, 83]. All samples were dissolved in a buffer containing 5 mM Hepes (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 2 mM β-

1 mercaptoethanol (94:6 H2O:D2O). The spectral widths were 9615 Hz in the H dimension and 19621 Hz in the 13C dimension, with carrier frequencies at 4.7 and 150 (for 13C at the aldehyde position) or 100 ppm (for 13C at the benzylic position), respectively.

2.3 Results

2.3.1 Inhibition of PTPs by Simple Aldehydes

Peptidyl aldehydes represent an important class of inhibitors for cysteine proteases which, like PTPs, also utilize an active-site cysteine as the catalytic nucleophile

[84]. These aldehydes bind to the proteases through the formation of a covalent but reversible hemithioacetal adduct with the active-site cysteine. Calpeptin, a potent dipeptide aldehyde inhibitor of calpain, has recently been reported to show modest cross reactivity toward several PTPs [85, 86]. These observations led us to hypothesize that aryl substituted aldehydes might serve as selective PTP inhibitors. We therefore tested several aryl aldehydes (Figure 2.5) against tyrosine phosphatases PTP1B and SHP-1, and a dual-specificity phosphatase VHR. 4-Carboxycinnamaldehyde (3) was the most active against PTPs among the three aldehydes tested, with IC50 values of 230 and 970 µM against SHP-1 and PTP1B, respectively (Figure 2.6 and Table 2.1). It showed no

24 significant inhibition against VHR up to 2 mM. Benzaldehyde was the least active inhibitor against all PTPs, presumably because its carbonyl group cannot readily reach the nucleophile(s) in the deep active-site pocket. All of the inhibitors were less active against VHR, which has a wider, shallower active site pocket as compared to PTPs [16].

Due to limited solubility, the IC50 values for some enzyme/inhibitor pairs could not be determined.

2.3.2 Slow-Binding Inhibition of PTPs by Cinn-GEE

The simple aldehydes presumably interact with only the active-site pocket of

PTPs, limiting their inhibitory potency. One approach to improving their potency is to modify the aldehydes with functional groups that can interact with the protein surfaces near the active site. These additional interactions would also confer selectivity for a particular PTP on the inhibitor. Moran et al. have shown that attachment of a tripeptide,

Gly-Glu-Glu-NH2 (GEE), to the para position of cinnamic acid results in a potent inhibitor (compound 4) for PTP1B (KI = 79 nM), although cinnamic acid alone shows only weak activity [73]. As a proof of principle, we attached the tripeptide GEE to the para position of cinnamaldehyde to obtain N-[4-(3-oxo-1-propenyl)benzoyl]-Gly-Glu-

Glu-NH2 (Cinn-GEE) (compound 5 in Figure 2.5). Cinn-GEE was synthesized from commercially available materials as detailed under Experimental Procedures (Figure 2.2).

Cinn-GEE exhibited time-dependent inhibition toward PTP1B, SHP-1, and VHR.

It resulted in biphasic curves when the hydrolysis of pNPP by PTPs was monitored in a continuous fashion, indicative of slow-binding inhibition (Figure 2.7.A) [87]. The inhibition kinetics can be described by the following equation,

25 KI k5 E + I E•I E•I* k6

where KI is the equilibrium inhibition constant for the formation of the initial complex,

E•I, and k5 and k6 are the forward and reverse rate constants for the slow conversion of the initial E•I complex into a tight complex E•I*, respectively. The overall potency of the inhibitor is described by the overall equilibrium constant, KI* = KIk6/(k5 + k6). The KI* values of Cinn-GEE were 5.4, 10.7, and 288 µM against PTP1B, SHP-1, and VHR, respectively (Table 2.1). In order to distinguish the observed slow-binding behavior from time-dependent inactivation, PTP1B was pre-incubated with excess Cinn-GEE to form the E•I* complex, which was then rapidly diluted into an assay solution containing pNPP as substrate. The reaction progress as a function of time is shown in Figure 2.7.B. The slow reactivation of the enzyme with time is consistent with the slow-binding mechanism. Curve fitting indicates that the rate for reactivation of PTP1B (k6) is 0.56

-1 min . Based on the KI value of 550 µM for Cinn-GEE binding to PTP1B, the rate for the conversion of noncovalent E•I complex to the covalent E–I complex was determined as

56 min-1. 4-Carboxycinnamaldehyde exhibited similar slow-binding behavior towards the

PTPs , although the KI, k5, and k6 values could not be reliably determined due to the weak binding affinity. The tripeptide (GEE-NH2) alone did not inhibit any of the enzymes.

2.3.3 Cinn-GEE Is Active-Site Directed

Competition with substrate was determined by measuring kobs, the pseudo-first- order rate constant for onset of inhibition, from progress curves obtained at constant

26 inhibitor but varying pNPP concentrations. Although the data are somewhat scattered due to technical difficulties in attaining final steady states in a reasonable time frame, there is clearly an inverse correlation between kobs and pNPP concentration, as expected from competitive binding of Cinn-GEE and pNPP to the same active site (Figure 2.8). In another experiment , PTP1B was first treated with a slight molar excess of p-hydroxyl α- bromoacetophenone, which inactivates PTPs by selectively alkylating the catalytic cysteine [76], and then examined for binding to Cinn-GEE by NMR spectroscopy (see below). Alkylation of the active-site cysteine abolished the ability of PTP1B to bind

Cinn-GEE. These data strongly suggest that Cinn-GEE is active-sited directed.

2.3.4 Mechanism of Inhibition

We initially hypothesized that the E•I complex was the noncovalent complex of

PTP1B and Cinn-GEE, whereas the E•I* represented a covalent adduct formed between the inhibitor aldehyde and the thiol group of the enzyme active-site cysteine. To test this hypothesis, we synthesized Cinn-GEE labeled with 13C at the aldehyde carbonyl carbon

(Figure 2.3 compound 14) and analyzed both the inhibitor and the enzyme-inhibitor complex by 1H–13C HSQC NMR spectroscopy. When dissolved in the PTP assay buffer

(pH 7.4), [13C]Cinn-GEE alone showed a single cross peak at δ 9.64 (1H) and δ 201 (13C) in the NMR spectrum (Figure 2.9.A). This suggests that the free inhibitor existed predominantly in the aldehyde form in the aqueous solution. Upon the addition of increasing concentrations of PTP1B (with preincubation), the signal of the free inhibitor decreased, with concomitant appearance of two major peaks at δ 7.80/130 and

δ 7.65/132, respectively, and a minor peak at δ 7.76/137 (Figure 2.9.B). Finally, when 27 PTP1B was added in excess, the free inhibitor signal was completely converted into the above three peaks (Figure 2.9.C). No other signals were observed that could be attributed to either the free inhibitor or the enzyme-inhibitor complex. The same three peaks were also observed in the 1-D 13C NMR spectrum of the PTP1B/Cinn-GEE complex. These unexpected results (chemical shift values and the appearance of multiple peaks) are inconsistent with the formation of a hemithioacetal. On the basis of these observations, we proposed that the inhibitor aldehyde could react with the guanidine group of the catalytic arginine residue (Arg-221 in PTP1B) to form an imine or enamine adduct.

To establish the role of Arg-221 in inhibitor binding, we performed the same

HSQC experiment with C215A and R221A mutant forms of PTP1B. The identity of both mutants was established by sequencing their coding regions as well as the absence of significant catalytic activity [30, 88, 89]. Consistent with our current mechanistic proposal, the C215A mutant retained its ability to bind [13C]Cinn-GEE and produced exactly the same three cross peaks in the HSQC spectrum as the wild-type enzyme

(Figure 2.10). The result with the R221A mutant is more complex. While the three cross peaks observed with wild-type and C215A PTP1B were clearly absent, we also failed to observe any signal corresponding to the free inhibitor at δ9.64/201. Careful inspection of the entire spectrum (δ0–10 for 1H and δ0–230 for 13C) revealed a single cross peak of weaker-than-expected intensity at δ7.2/121. We did notice that upon the addition of Cinn-

GEE, a significant fraction of the R221A protein precipitated in the NMR tube, likely causing the weak absorption signal. Our tentative explanation is that the R221A mutant is still capable of binding to Cinn-GEE at high concentrations through noncovalent interactions (500 µM PTP1B and 200 µM Cinn-GEE were used in the NMR experiment). 28 The δ7.2/121 peak may represent the equilibrium signal from two or more rapidly inter- converting species. Thus, the data clearly rule out Cys-215 and are highly suggestive of

Arg-221 as the nucleophile responsible for reaction with Cinn-GEE.

To gain further insight into the nature of the Cinn-GEE/Arg adduct (imine vs enamine), we synthesized Cinn-GEE with 13C labeling at the benzylic position (Scheme

2.4 compound 17) and examined its complex with wild-type PTP1B by HSQC experiments. The free inhibitor showed a single cross peak at δ7.85/157, consistent with an sp2 carbon in conjugation with an electron-withdrawing aldehyde (Figure 2.11.A).

Addition of excess PTP1B resulted in disappearance of the free inhibitor signal and appearance of two new cross peaks at δ4.08/47 and δ4.12/47 positions (Figure 2.11.B).

Unfortunately, this region of the spectrum is crowded by many signals of buffer materials. To ascertain that the peaks at δ4.08/47 and δ4.12/47 are bona fide signals of the PTP1B/inhibitor complex, a control experiment was performed under exactly the same conditions as in Figure 2.11.B, except that compound 14 was employed. We again observed the three cross peaks at δ~7.7/~135 (as in Figure 2.9), but no signal at either

δ7.85/157 or δ4.1/47 region (Figure 2.11.C). The chemical shift values of δ∼4.1/47 indicate that the benzylic carbon is sp3 hybridized in the PTP1B/inhibitor complex, suggesting an enamine structure as the enzyme-inhibitor adduct (see Discussion).

There is a slight possibility that the observed inhibitory activity of Cinn-GEE may be due to contamination by a small amount of the more potent cinnamic acid 4 (which could be formed through air oxidation of Cinn-GEE). This notion was tested by treating the inhibitor with excess cysteamine, which reacts with aldehydes to form a stable five- membered thiazolidine ring but should have no effect on the cinnamic acid inhibitor. As 29 expected, prior treatment of Cinn-GEE with excess cysteamine abolished its inhibitory activity against PTP1B (Figure 2.12). In addition, the cinnamic acid 4 was reported as a simple competitive inhibitor [73], whereas the observed inhibition in this work exhibited slow-binding characteristics, consistent with the formation of a reversible enzyme- inhibitor adduct. Taken together, these data rule out the possibility that the observed inhibition was caused by cinnamic acid contamination.

2.4 Discussion

We have been pursuing a different approach to PTP inhibitor design. Our strategy is to covalently modify the conserved active-site residues of PTPs using hydrophobic core structures [76]. Because the catalytic cysteine is conserved among all PTPs and is exceptionally acidic (e.g., pKa = 4.7 in Yersinia PTP) [37], we reasoned that a peptidyl aldehyde might selectively inhibit PTPs by forming a hemithioacetal adduct with the cysteine thiolate. Since aryl aldehydes are only marginally soluble in water, they should have relatively low desolvation energy, a property that should make them more membrane permeable as well as promote binding to an enzyme active site. In this work, we show that certain aryl-substituted aldehydes indeed act as reversible inhibitors of

PTPs. As one might expect from their small sizes, the simple aldehydes do not have high affinity to the PTP active site (IC50 values in the high µM to mM range). However, attachment of a tripeptide GEE to the para position of cinnamaldehyde resulted in an inhibitor of substantially improved affinity to both PTP1B (KI* = 5.4 µM) and SHP-1

(KI* = 10.7 µM). This suggests that by attaching a properly designed PTP recognition

30 motif or by screening a combinatorial library, one should be able to obtain highly potent and specific inhibitors against a PTP of interest.

Cinn-GEE behaves as a slow-binding inhibitor. Since simple aldehydes (e.g., 4- carboxycinnamaldehyde) exhibit similar slow-binding inhibition, the E•I to E•I* conversion is likely due to structural changes within the active site. Initially, it appeared that the slow-binding behavior could be explained by the time-dependent formation of a reversible hemithioacetal adduct between the aldehyde and the active-site cysteine. Such a mechanism is commonly observed for inhibition of cysteine proteases by peptidyl aldehydes [37, 90]. Surprisingly, 1H–13C HSQC experiments with 13C-labeled Cinn-GEE

(at the aldehyde carbon) and PTP1B showed three cross peaks at δ 7.80/130, δ 7.65/132, and δ 7.76/137 positions (Figure 2.9). These results are inconsistent with the formation of a hemithioacetal, as a hemithioacetal would show no more than two cross peaks at δ ~5.0

(for the aldehyde-derived 1H) and δ ~85 (13C) [91-93]. The hemithioacetal mechanism is further ruled out by the observation that mutation of the catalytic Cys-215 to alanine had no effect on the HSQC spectrum. The data also rule out the possibility of a free aldehyde

(δ 9.64/201) or the hydrate form (which should have a single cross peak at δ ~5.2/~95) as the bound inhibitor form in the PTP active site.

We propose that the bound inhibitor forms an enamine adduct with the guanidine group of Arg-221 (Figure 2.13). Arg-221 is a universally conserved residue present in the

PTP signature motif, HCxxGxxR(S/T). It is critical for pY substrate binding and transition-state stabilization by forming a pair of hydrogen bonds between the phosphate non-bridging oxygen atoms and the Nε and Nη of Arg-221 [15]. Binding of the cinnamaldehyde derivatives likely places the carbonyl group at a similar position as the 31 phosphate of a substrate. The close proximity between the aldehyde and the guanidinium group presumably drives the deprotonation of the guanidinium group and the formation of a conjugated imine intermediate. Conjugate addition at the benzylic position by a yet unidentified nucleophile produces the enamine structure as the E•I* complex (Figure

2.13). This mechanistic proposal is supported by both experiments specifically designed to test the model. First, mutation of Arg-221 to alanine abolished the enamine structure, as evidenced by the absence of the three HSQC cross peaks observed in wild-type and

C215A proteins. Second, HSQC experiment with 13C-labeled Cinn-GEE showed that the chemical shift values for the benzylic proton/carbon decreased from 7.85/157 ppm to

~4.1/47 ppm upon binding to PTP1B. The latter values indicate that the benzylic carbon must be sp3 hybridized in the E•I* complex, as predicted by our model. The identity of the nucleophile is not yet well established. It may be either a protein side chain (e.g., the second amino group of Arg-221 or Asp-181 which is the general acid/base during catalysis) or a buffer species (e.g., β-mercaptoethanol or a water molecule). The high field position of the absorption (δ4.1/47) is most consistent with a thiol being the nucleophile, as the 13C chemical shift values for the benzylic carbon should be well above

60 and 50 ppm when it is directly attached to an oxygen and nitrogen, respectively. The only cysteine near the PTP1B active site is Cys-215, which can be ruled out since its mutation does not affect the enamine structure. Therefore, we propose that the unknown nucleophile is most likely β-mercaptoethanol in the buffer, which was used to maintain

PTP1B activity.

Our proposed mechanism also provides a sensible explanation for all of the other experimental observations. Since the formation of enamine is readily reversible, it 32 explains the reversible nature of the inhibitors. Because formation of the imine intermediate requires the expulsion of a water molecule from a relatively deep active site, the reaction is expected to be slow, as is observed. The unknown nucleophile may add from either side of the double bond and the resulting enamine double bond may assume either cis or trans configuration. This can account for the formation of multiple products in the HSQC spectra. Finally, the observed chemical shift values (δ ~132) for the aldehyde-derived carbon atom suggest that this carbon atom is sp2 hybridized in the E•I* complex. Since the 13C resonance of a typical imine is at δ 150–160, the observed δ values are more consistent with an enamine structure, as predicted by the model. Because we have failed to observe the imine intermediate under any conditions, we propose that conjugate addition by the unknown nucleophile is a rapid step.

In retrospect, the lack of reaction between the aldehyde group and the active-site cysteine of PTPs is not entirely surprising. A nucleophile must approach from above or below the carbonyl plane in order to have a successful addition reaction. Peptidyl aldehyde inhibitors of cysteine proteases are so designed that the aldehyde carbonyl occupies the same position as the amide carbonyl of a scissile bond. Thus, in the E•I complex, the cysteine thiol is properly positioned for nucleophilic attack on the aldehyde.

In PTPs, the thiolate is situated at the bottom of the active-site pocket [15]. This position is ideally suited for a back attack on an incoming phosphate group, with a co-linear S–P–

OPh bond angle in the transition state. However, when cinnamaldehyde binds to the PTP active site, the thiolate is likely face the edge of the carbonyl plane. This geometric restriction presumably prevents a successful nucleophilic attack. On the other hand, Arg-

221 side chain is on the wall of the active-site pocket [15]. This position permits a side 33 attack on the carbonyl plane. Burridge and co-workers have found that calpeptin has modest inhibitory activity against several PTPs but did not examine its mechanism of inhibition [85, 86]. Very recently, Urbanek et al. reported a series of 9,10- phenanthrenediones as fairly potent inhibitors of PTP CD45 but not against other PTPs

[94]. On the basis of the slow-binding behavior of the 1,2-diketones, these investigators suggested the formation of a hemithioketal adduct between the inhibitor and the active- site cysteine. In light of the findings in this work, it will be of interest to determine whether calpeptin and the 1,2-diketones also inhibit PTP by covalently modifying the active-site arginine. Note that 1,2-diketo compounds (e.g., phenylglyoxal and 1,2- cyclohexanedione) are commonly used to selectively modify arginine residues in proteins.

In summary, we have demonstrated that certain aryl-substituted aldehydes act as slow-binding inhibitors of PTPs. The time-dependent inhibition is due to the formation of a reversible adduct between the inhibitor and the conserved active-site arginine. These aryl aldehydes and ketones should provide a general core structure that can be further developed into highly potent and specific inhibitors against PTPs.

Inhibitor Inhibition Constant (IC50 or KI*, µM)

PTP1B SHP-1 VHR

1 NA NA NA

2 15% (at 200 µM) NA NA

3 970a 230a >2000a

4 0.079b

5 5.42 ± 0.94c 10.7 ± 1.4c 288 ± 112c

Table 2.1. Inhibitory Activity of Aldehydes against Various PTPs a b c IC50 values; KI value from Moran et al. (20); KI* values; NA, no significant activity.

Figure 2.1. Mechanism of catalysis and inactivation by α-haloacetophenone derivatives − − − [76]. Pi, pyrophosphate; X , Br or Cl ; R, H, CH3 or CH2CO2H

CHO O O 1) NaOH O TMA/K2CO3 H 2) EtOCOCl + HO Br- + reflux PPh3 O OMe

O OMe Z/E = 67/33 6

O O O O

NH2뺾EE H H TFA 5

Z/E < 5/95 O OOO ONH뺾EE

Z/E =62/38 7 8

Figure 2.2. Synthesis of N-[4-(3-Oxo-1-propenyl)benzoyl]-Gly-Glu-Glu-NH2.

OH CHO O O * *

H HOCH2CH2OH + Ph3P=CH뺺 *HO H PTSA 11 reflux O OMe

O OMe 10 O OMe 12

O O O O OH * * * 1) NaOH NH 뺾EE 2) EtOCOCl H 2 H TFA

O O O O NH뺾EE O NH뺾뻅 EE H 2 13 14

13 Figure 2.3. Synthesis of N-[4-(3-[ C]Oxo-1-propenyl)benzoyl]-Gly-Glu-Glu-NH2

OH Br N *CN O * H * O Ni/Al

Cu*CN HCO2H/H2O

O OMe O OMe O OMe ONHEEH뺾뻅2 15 16 17

13 Figure 2.4. Synthesis of N-[4-(3-Oxo-1-[ C]propenyl)benzoyl]-Gly-Glu-Glu-NH2

O OH O H H

1 2 OOH 3 O OH OH

OOH O OH

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

OOH O OH 4 5

Figure 2.5. Structures of PTP inhibitors.

1.0 VHR 0.8

0.6

0.4 PTP1B Rel. Activity Rel.

0.2 SHP-1

0 0 500 1000 1500 2000 [I] (킡 )

Figure 2.6. Plot of remaining PTP activity against 4-carboxy-cinnamaldehyde concentration. All of the activities are relative to those in the absence of inhibitor.

41 A

0.25 B 0.20

0.15 405 A 0.10

0.05

0.0 0 50 100 150 200 250 300 Time (s)

Figure 2.7. Slow-binding inhibition of PTP1B by Cinn-GEE. (A) Hydrolysis of pNPP (1.0 mM) by PTP1B (0.2 µM) in the presence of indicated amounts of Cinn-GEE. The reactions were initiated by the addition of enzyme as the last component. (B) Hydrolysis of pNPP by reactivated PTP1B. The enzyme (1.0 µM) was preincubated with Cinn-GEE (75 µM) for 3 h before being diluted 10-fold into the reaction buffer containing 1.0 mM pNPP.

0.14

0.12 ) -1 0.10 (min

obs 0.08 k

0.06

0.04 0.0 0.5 1.0 1.5 2.0 2.5 3.0 [pNPP] (mM)

Figure 2.8. Competition between Cinn-GEE and substrate for binding to PTP1B. Reaction time courses were initiated by the addition of PTP1B (0.3 µM) to assay mixtures containing fixed inhibitor concentration (40 µM) and varying pNPP concentration. The kobs values were obtained from fitting individual times courses to –kobst equation: [P] = vst + [(vi – vs)/kobs](1 – e ). The solid line through data is a best fit to the data according to equation: kobs = kmax/(1 + [pNPP]/KM).

Figure 2.9. HSQC spectra of 13C-labeled Cinn-GEE (compound 14) in the presence and absence of PTP1B. (A) 200 µM Cinn-GEE only; (B) 200 µM Cinn-GEE and ~200 µM PTP1B; and (C) 200 µM Cinn-GEE and ~300 µM PTP1B. PTP1B concentration was based on the Bradford assay using bovine serum albumin as standard.

Figure 2.10. HSQC spectra of 13C-labeled Cinn-GEE (compound 14; 200 µM) in the presence of C215A PTP1B (500 µM).

Figure 2.11. HSQC spectra of 13C-labeled Cinn-GEE (compounds 14 and 17) in the presence and absence of PTP1B. (A) 200 µM inhibitor 17 only; (B) 200 µM inhibitor 17 and 500 µM PTP1B; and (C) 200 µM inhibitor 14 and 500 µM PTP1B.

1 cysteamine cysteamine + Cinn-GEE 0.8 Cinn-GEE

0.6 405 A 0.4

0.2

0 0 50 100 150 200 250 300 Time (s)

Figure 2.12. Inactivation of Cinn-GEE by cysteamine. PTP1B (0.2 µM) was preincubated with Cinn-GEE alone (20 µM), cysteamine alone (200 µM), or Cinn-GEE (20 µM) and cysteamine (200 µM) before being added to the reaction mixture containing 1.0 mM pNPP (pH 7.4).

S S S O H slow S fast H2NN fast H2NN H2N N H2NN H2O RSH KI H O k5 H N H NH NH2 + NH2 k6 SR

EI R R R E뷞 E*뷞

Figure 2.13. Proposed mechanism of inhibition of PTP by cinnamaldehyde derivatives. RSH, β-mercaptoethanol.

CHAPTER 3

PEPTYDYL ALDEHYDES AS SLOW-BINDING INHIBITORS OF DUAL

SPECIFICITY PHOSPHATASES

3.1 Introduction

In chapter 2, peptidylaldehydes were shown to modify the active-site arginine

(Arg 221 in PTP1B) of protein tyrosine phosphatases with covalent and reversible manner, resulting in inhibition of the enzymes. As mentioned in chapter 1, there are three types of protein phosphatases. One is potein tyrosine phosphatase (PTPs) that catalyzes the hydrolysis of phosphotyrosine (pY) residues in proteins (e.g. PTP1B and SHP1 ), whereas protein serine/threonine phosphatases (e.g. PP1 and PP2A) remove the phosphoryl group from phosphoseryl and phosphothreonyl residues [7, 10]. In addition, a third class of dual-specificity phosphatases (DSPs; e.g. VH1 and cdc25) are capable of hydrolyzing both phosphoseryl/ phosphothreonyl and phosphotyrosyl residues. Like

PTPs, DSPs have been shown to be involved in important cellular events, such as intracellular signaling. For examples, VH1 (Vaccinia open reading frame H1) phosphatase, [14] which was the first DSP identified, can reverse Stat-1 activation in vaccinia virus-infected cells. [95] Another DSP, human VHR (VH1 related) phosphatase,

[16, 96] was shown to play a critical role in intracellular signaling mediated by mitogen-

49 activated protein (MAP) kinase [97, 98]. Despite significant recent progresses, the precise cellular roles of the DSPs are still largely unknown. Therefore, specific inhibitors against

DSPs may provide useful probes for studying their physiological functions and potential therapeutic agents. However, unfortunately, there have been relatively few reports [99-

103] on DSP inhibitors.

Because DSPs share the same signature motif (HCX5RS/T), [104] active-site structure, [97] and catalytic mechanism as PTPs, [36] we reasoned that the peptidyl aldehydes might also inhibit DSPs by modifying the arginine in the signature motif. Here, we show that this indeed the case, suggesting cinnamaldehyde as a general, neutral protein phosphatase inhibitor.

3.2 Experimental Procedures

3.2.1 Purification of GST-VH1

E. coli BL21(DE3) cells carrying pGEX-VH1 plasmid, which was kindly provided by Dr. Kuan-liang Guan, were grown in LB media supplemented with 75 mg/mL ampicilline, 10 g/L trypton, 5 g/L yeast extract and 10 g/L NaCl at 37 °C to an

OD600 of 0.6. The cells were induced by the addition of 300 μM isopropyl β-D- thiogalactoside (IPTG) and continued grown at 30 °C for an additional 4 h. The cells were harvested by centrifugation at 5,000 r.p.m. at 4 °C for 10 min, and resuspended in

100 mL of the lysis buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM EDTA, 10 mM β-mercaptoethanol, 200 μM PMSF, 20 mg/L of trypsin inhibitor, and 0.5% prostamine sulfate). The cells were strirred for 20 min at 4 °C and sonicated with 5 × 10 s pulses. The crude lysate was centrifuged at 12,000 r.p.m for 30 min at 4 °C, and the clear 50 supernatant was loaded onto the GST affinity (Pharmacia, NJ) column (2.5 cm × 2.5 cm), which was pre-equilibrated with the GST-binding/wash buffer (20 mM HEPES, pH 7.4,

100 mM NaCl, 1mM EDTA, and 10mM β-mercaptoethanol). The column was washed with 200 mL GST-binding/wash buffer, and the protein was eluted by the GST-elution buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 1mM EDTA, 10mM β-mercaptoethanol and 10 mM reduced glutathione). The protein fractions were pooled and concentrated to

~ 1 mL at 4 °C by using an Amicon stirred cell equipped with YM-3 cellulose membrane, and the buffer was exchanged to GST-binding/wash buffer at the same time. The concentrated protein was stored at – 80 °C with 30 % glycerol for the general purpose.

For NMR experiment, the protein was stored in the absence of glycerol. The concentration of the protein was measured by Bradford assay using bovine serum albumin (2 mg/mL) as a standard.

3.2.2 DSP Inhibition Assay

A typical reaction (total volume 1.0 mL) contained 50 mM imidazole (pH = 7.0),

1 mM EDTA, 0.05% (v/v) β-mercaptoethanol, 5% DMSO, 0.1–0.2 μM VH1 (or 0.5–1

μM VHR), and 0–2700 μM inhibitor. After incubation of the enzyme with the inhibitor for 1h at room temperature, the reaction was initiated by the addition of 5 mM pNPP. The reaction progress was monitored at 405 nm on a Perkin-Ellmer UV/Vis spectrophotometer.

To determine the rate constant k6, VH1 (0.2 µM) was incubated with 200–300 µM inhibitor in 100 µL of the above reaction buffer for 1.5 h at room temperature. The

51 mixture was rapidly diluted into 900 µL of the same reaction buffer containing 1.0 mM pNPP (without inhibitor). Reactivation of PTP activity was monitored at 405 nm for 15 min and the progress curves were fit to the equation

-k6t Abs405 = vs[t – (1 – e )/k6] where vs is the final steady-state velocity. The rate constant k5 was calculated using the equation KI* = KIk6/(k5 + k6).

3.2.3 Competition between Cinn-GEE and pNPP for binding to VH1

Enzymatic reactions were initiated be the addition of VH1 (0.2 μM) to the assay buffer containing a fixed amount of Cinn-GEE (100 μM) and varying pNPP concentrations (1.2–30 mM). The kobs values were obtained by fitting individual time

-kobst courses to the equation: [p] = vs + [(vi – vs)/kobs](1 – e ). The data were fit according to the equation: kobs = kmax/(1 + [pNPP]/KM).

3.2.4 NMR Spectroscopy of [13C]Cinn-GEE and [13C]Cinn-GEE-VH1 Complex

NMR experiments were performed on a Bruker DMX-600 NMR spectrometer equipped with a triple-resonance and 3-axes gradient probe at 300 K. The 2D 1H-13C heteronuclear single-quantum coherence spectra (HSQC) were acquired using a constant- time sensitivity-enhanced method. (36, 37) [13C]Cinn-GEE (400 μM) was added to the buffer (10% D2O, 15 mM HEPES, pH 7.4, 100 mM NaCl, 1mM EDTA, and 1mM

TCEP) containing 240 μM VH1. The reaction was incubated for 30 min at room

52 temperature, followed by centrifugation to remove ant precipitation. Clear [13C]Cinn-

GEE-VH1 complex solution (~ 500 μL) was taken and used for NMR experiment.

3.3 Results and Discussion

3.3.1 Inhibition of VH1 by Cinn-GEE

We first tested p-carboxycinnamaldehyde (compoubd 1 in Figure 3.1) against

DSPs VH1 and VHR.11 While compound 3 showed only ~40% inhibition of VHR at 2.5 mM, it was more potent against VH1 with an IC50 value of ~1 mM, which is comparable

7 to that against PTP1B (IC50 = 970 μM). Next, the peptidyl aldehyde Cinn-GEE was tested against the enzymes. Cinn-GEE behaved as a slow-binding inhibitor toward VH1, as evidenced by the biphasic reaction progress curves (Figure 3.2.A) and the time- dependent reactivation of inhibited enzyme upon dilution (Figure 3.2.B). The inhibition kinetics can be described by the equation as mentioned Chapter 2:

KI k5 E + I E•I E•I* k6

,where KI is the equilibrium constant for the formation of the initial E•I complex, whereas k5 and k6 are the forward and reverse rate constants for the interconversion between the

E•I complex and the final E•I* complex. The overall equilibrium constant (KI*) is described by KI* = KIk6/((k5 + k6) and represents the overall potency of the inhibitor. The

KI and KI* values of Cinn-GEE against VH1 were xx and 18 ± 3 μM, respectively.

Fitting of the reactivation curve in Figure 3.2.B against the equation [87]

-k6t Abs405 = vs[t – (1 – e )/k6] 53

-1 produced the reactivation rate constant (k6 = 0.68 min ). The forward rate constant for conversion of E•I to E•I* was calculated using the equation KI* = KIk6/((k5 + k6) (k5 = xx

-1 min ). Cinn-GEE also inhibited VHR, but with much lower potency. The KI* value for

VHR was 290 ± 110 μM (chapter 2) whereas the KI, k5, or k6 values could not be accurately determined, due to the lower potency.

Comparison of the potency of compounds 3 and 5 (Figure 3.1) against VH1 revealed that the addition of tripeptide Gly-Glu-Glu improved the affinity by >50-fold.

To determine whether other tripeptides could produce the same effect, we tested several other peptidyl cinnamaldehydes (Figure 3.1 compounds 18-20), which had previously been selected from a combinatorial library against PTP1B, against VH1. Despite their structural similarities to Cin-GEE, these compounds showed much lower potency against

VH1 than Cinn-GEE (Figure 3.4). These data indicate that specific peptide structures (or other structures) and a proper linkage between the peptide and the cinnamaldehyde are both necessary to generate potent peptidyl cinnamaldehyde inhibitors of VH1.

3.3.2 Mechanism of Inhibition

To determine whether Cinn-GEE is active site-directed, hydrolysis reactions were carried out at a fixed Cinn-GEE concentration and varying substrate concentrations [1.2–

30 mM p-nitrophenyl phosphate (pNPP)]. An inverse correlation was observed between the kobs, the pseudo-first-order rate constant for onset of inhibition, and pNPP concentration (Figure 3.3). This result indicates that Cinn-GEE binds to the active site of

VH1. The mechanism of inhibition was further investigated using 13C-labeled Cinn-GEE

54 and 1H-13C heteronuclear single-quantum correlation spectroscopy as previously described for PTP1B (chapter 2). Unfortunately, these experiments failed to provide any mechanistic insight, due to difficulty in preparing highly concentrated VH1/Cinn-GEE samples for the NMR experiments. Attempts to capture the covalent adduct through reduction with sodium cyanoborohydride followed by detection by mass spectrometry also failed, as was the case with PTP1B. However, on the basis of the similarities in their active-site structures and the inhibition kinetics, we propose that Cinn-GEE inhibits DSPs via a mechanism similar to that of PTPs (Figure 2.13), by covalently modifying the conserved active-site arginine (Arg-116 in VH1). The failure to be reduced by sodium cyanoborohydride is consistent with the formation of an enamine adduct, as previously observed for PTPs (chapter 2)

In summary, we have demonstrated that Cinn-GEE is a slow-binding inhibitor of moderate potency against DSPs. Together with our earlier reports, our results suggest that cinnamaldehyde could be used as a general pY mimetic for inhibition of PTPs (e.g. classical PTPs and DSPs). More potent and specific inhibitors against these pY-binding proteins may be developed by designing and screening combinatorial libraries of peptidyl cinnamaldehydes.

O H O H

O H

O OH O OH

O O H H N NH2 N NH O N N O N N 2 H H H H O O O O O OH O 3 O OH HO 518 O H O H

O HO O OH O O H H N N O O N NH2 H H O N O O N NH2 H O O OH 19 O OH 20

Figure 3.1. Structures of peptidyl cinnamaldehydes used in Chapter 3.

A 0.25 0 μΜ 20 μΜ 0.20 30 μΜ 60 μΜ 100 μΜ 0.15 200 μΜ

405 300 μΜ 400 μΜ

Abs 0.10

0.05

0.00 0200400600800 Time (s) B 0.10

0.08

0.06 405

Abs 0.04

0.02

0.00 0 200 400 600 800 Time (s)

Figure 3.2. Slow-binding inhibition of VH1 by Cinn-GEE. (A) Hydrolysis of pNPP (5 mM) by VH1 (0.1 μM) in the presence of indicated amounts of Cinn-GEE. The reactions were initiated by the addition of VH1 as the last component. (B) Hydrolysis of pNPP by reactivated VH1. VH1 (0.2 μM) was preincubated with Cinn-GEE (260 μM) for 3 h before being diluted 10-fold into a reaction solution containing 5 mM pNPP.

0.25

0.20 ) -1 0.15 (min

obs 0.10 k

0.05

0.00 0 5 10 15 20 25 30 [pNPP] (mM)

Figure 3.3. Competition between Cinn-GEE and pNPP for binding to VH1. Reactions were initiated be the addition of VH1 (0.2 μM) to solutions containing a fixed amount of Cinn-GEE (100 μM) and varying pNPP concentrations (1.2–30 mM). The kobs values were obtained by fitting individual time courses to the equation: [p] = vs + [(vi – -kobst 12 vs)/kobs](1 – e ). The solid line is a best fit to the data according to the equation: kobs = kmax/(1 + [pNPP]/KM).

Figure 3.4. Inhibition of VH1 by peptidyl aldehydes 2–5. VH1 (0.2 mM) was preincubated with each inhibitor (40 mM) in the reaction buffer (50 mM imidazol, pH 7.0, 1mM EDTA, 0.05% (v/v) β-mercaptoethanol, 5% DMSO) for 1 h at room temperature and the reaction was then initiated by the addition of 5 mM pNPP (final concentration) and monitored on a UV–Vis spectrophotometer.

CHAPTER 4

PEPTIDYLALDEHYDES AS REVERSIBLE COVALENT INHIBITORS OF SRC

HOMOLOGY 2 DOMAINS

4.1 Introduction

Protein-protein interaction is an important aspect of cellular processes. Many of these interactions are mediated by small modular domains, which recognize peptide motifs in their partner proteins. The Src homology 2 (SH2) domain was one of the first such modular domains identified [105]. Since then, dozens of other modules have now been discovered (e.g., SH3, PH, PDZ, FHA, and PTB domains) [106]. SH2 domains contain ~100 amino acids and are often found in signaling proteins. A large number of

SH2 domains have already been identified and the human genome encode at least 95 SH2 domains [107]. Their function is to bind specific phosphotyrosine (pY)-containing proteins and promote protein-protein interactions. The structural basis for SH2 domain- pY interaction has been very well studied. SH2 domains bind to their partner proteins by recognition of linear pY-containing sequence motifs [108, 109]. A key interaction, which is common to all SH2 domains, is the insertion of the pY side chain into a deep pocket in the SH2 domain, where an invariant arginine residue (Arg βB5) forms a bidentate interaction with the pY phosphate group [110-113]. Additional binding energy is

60 provided by interactions between amino acids adjacent to pY, particularly the three residues immediately C-terminal to pY, and the less conserved surface of the SH2 domain. This latter interaction also governs the selectivity of a given SH2 domain in binding to a specific pY partner.

The blockade of SH2 domain-dependent protein-protein interactions has emerged as a new strategy for treating a plethora of diseases related to cell signaling, such as cancer, osteoporosis, allergy, asthma, and inflammation [114]. A variety of inhibitors have been developed against the SH2 domains of Src kinase and adapter protein Grb2

(reviewed in [114, 115]). A Src SH2 domain inhibitor (AP22408) has been shown to reduce the bone resorptive activity of osteoclasts in rat [116]. In addition to their therapeutic values, SH2 domain inhibitors also provide useful probes for elucidating the cellular function of the SH2 domain-containing protein.

Since the natural ligands of SH2 domains are pY peptides, there are two challenges in designing SH2 domain inhibitors. The first is to find a proper small- molecule replacement for the peptide backbone, whereas the second is to design pY isosteres that are capable of interacting with the highly positively charged pY binding pocket and yet have the desired pharmacological properties (e.g., membrane permeability). The first challenge has largely been overcome, as evidenced by the availability of many potent, nonpeptidic SH2 inhibitors [115]. On the other hand, finding the proper pY isostere has been much more difficult. All of the reported SH2 inhibitors contain negatively charged phosphonates [59, 117-119] or carboxylates [71, 120-123] as pY isosteres, which adversely affect their membrane permeability.

61 In the previous chapters, we described that peptidyl aldehydes act as reversible and covalent inhibitors of the PTPs. Now, we report here that peptidyl aldehydes also can act as covalent inhibitors of SH2 domains derived from protein tyrosine phosphatase

SHP-1 [6]. This finding should open the door to a new family of neutral pY mimetics, which have much better membrane permeability and can potentially overcome the second challenge in designing SH2 domain inhibitors.

4.2 Experimental Procedures

4.2.1 Materials

SHP-1 and its catalytic domain, SHP-1(∆SH2), were purified from a recombinant

Escherichia coli strain as previously described [81]. The N- and C-terminal SH2 domains, either as their maltose-binding protein (MBP) or glutathione-S-transferase

(GST) fusion proteins or as C-terminal histidine tagged proteins, were expressed and purified as previously described [124]. All of the peptide synthesis reagents were purchased from Advanced ChemTech (Louisville, KY). The Rink resin had a loading capacity of 0.7 mmol/g. All other chemicals were obtained from Aldrich or Sigma.

4.2.2 Synthesis of Peptidylaldehyde Inhibitors

Peptidylaldehydes inhibitors were chemically synthesized by Dr. Hua Fu, and the synthetic scheme for compound 21 is described in Figure 4.1.

62 Synthesis of t-Butyl 2-(4-Formylphenoxy)acetate (23) NaH (0.26 g, 11 mmol)

CHO was added to a solution of 4-hydroxybenzaldehyde (1.22 g, 10 mmol) in

O 20 mL of DMF at room temperature and the resulting mixture was O OtBu t stirred for 30 min under argon. BrCH2COO Bu (1.95 g, 10 mmol) was then added to the above solution, and the reaction was continued for 1 h at 80 oC. After cooling, the reaction mixture was partitioned between ethyl acetate and water. The organic layer was sequentially washed with 1 M aqueous NaOH and water, dried over anhydrous MgSO4, and evaporated to an oil, which was subsequently purified by silica gel chromatography (4:1 hexane/ethyl acetate) (yield: 1.56 g or 66%). 1H NMR (250

MHz, CDCl3): 9.89 (s, 1H, CHO), 7.88 (d, 2H, J = 13.2 Hz, Ar), 7.00 (d, 2H, J = 13.2 Hz,

13 Ar), 4.59 (s, 2H, OCH2CO), 1.51 (s, 9H, tBu); C NMR 190.74, 167.15, 162.77, 131.93,

130.56, 114.85, 82.91, 65.51, 27.79.

Synthesis of t-Butyl 2-{4-[2-(1,3-Dioxolane-2-yl)-ethenyl]phenoxy}acetate (24)

O O Compound 23 (1.56 g, 6.6 mmol) and tris[2-(2-

H methoxyethoxy)ethyl]amine (TDA-1) (2.14 g, 6.6 mmol) were dissolved

O O OtBu in dichloromethane (40 mL) under argon at room temperature. A saturated aqueous K2CO3 solution (40 mL) and (1,3-dioxolane-2-yl-methyl)triphenylphosphonium bromide (2.85 g, 6.6 mmol) were added, and the reaction mixture was refluxed at 40 oC for 18 h with vigorous stirring. The reaction mixture were extracted with dichloromethane (2 x 20 mL), washed with water (15 mL), and dried over MgSO4.

Compound 24 was obtained as a mixture of E and Z isomers (yield: 1.77 g, 87.6%). 1H

NMR (250 MHz, CDCl3): 7.26-7.36 (m, 2H, Ar), 6.83-6.89 (m, 2H, Ar), 6.72-6.76 (2d,

63 1H, ArCH E and Z), 6.01 (dd, 0.41 H, ArCHCHCH E, J = 5.7 Hz, 15.8 Hz), 5.65 (dd,

0.59 H, ArCHCHCH Z, J = 7.3 Hz, 11.8 Hz), 5.52 (d, 0.59 H, ArCHCHCH Z, J = 7.3

Hz), 5.41 (d, 0.41 H, ArCHCHCH E, J = 5.7 Hz), 4.51 (s, 2H, OCH2CO), 3.92-4.08 (m,

4H, OCH2CH2O), 1.49 (s, 9H, tBu).

Synthesis of Ethyl 2-{4-[2-(1,3-Dioxolane-2-yl)-ethenyl]phenoxy}acetyl

Carbonate (25) An aqueous NaOH solution (0.93 g dissolved in 6 mL of water) was

O O added to compound 24 (1.77 g, 5.8 mmol) dissolved in 10 mL of

H methanol. The solution was refluxed for 8 h until the ester is completely O O O O OEt hydrolyzed, and the solvent was removed by rotary evaporation. The residue was dried over P2O5 in vacuo to afford a white solid. The solid was suspended in

25 mL of dichloromethane and ethyl chloroformate (3.78 g) was added. The mixture was stirred for 40 min at room temperature and filtered. The filtrate was evaporated under reduced pressure to produce 7 as a 4:1 mixture of Z and E isomers (yield: 1.35 g, 72.4%).

1 H NMR (250 MHz, CDCl3) δ 7.32-7.36 (m, 2H, Ar), 6.89-6.92 (m, 2H, Ar), 6.72-6.77

(2d, 1H, ArCH E and Z), 6.06 (dd, 0.21 H, ArCHCHCH E, J = 5.6 Hz, 15.7 Hz), 5.64

(dd, 0.79 H, ArCHCHCH Z, J = 7.4 Hz, 11.8 Hz), 5.48 (d, 0.79 H, ArCHCHCH Z, J =

7.4 Hz), 5.38 (d, 0.21 H, ArCHCHCH E, J = 5.6 Hz), 4.76 (2s, 2H, OCH2CO Z and E),

4.32-4.41 (m, 2H, OCH2CH3), 3.81-4.08 (m, 4H, OCH2CH2O), 1.30-1.41 (t, 3H,

OCH2CH3).

Synthesis of N-{2-[4-(3-Oxo-1-propenyl)phenoxy]acetyl}-Ala-Arg-Leu-NH2

(21) The tripeptide Ala-Arg-Leu was synthesized on 0.15 g of Rink resin

(0.10 mmol) using standard Fmoc/HBTU/HOBt solid-phase chemistry.

Next, 64 mg (0.20 mmol) of anhydride 7 in 2.5 mL of dichloromethane and 100 µL of N-methylmorpholine were added to the resin suspended in 2.5 mL of anhydrous DMF. The mixture was shaken for 8 h at room temperature. Ninhydrin tests indicated complete acylation of the N-terminal amine. The solvents were drained and the resin was washed with DMF (5 x 5 mL) and methanol (3 x 5 mL). Deprotection of side chains as well as aldehyde protecting group and cleavage from the resin were carried out with a cocktail containing 4 mL of 90% TFA in water, 0.1 mL of anisole, and 0.15 mL of thioanisole for 3 h at room temperature. After removing the volatile chemicals by a gentle flow of nitrogen, the residue was triturated five times with diethyl ether. Compound 21 was obtained as a brownish solid in the pure E isomer form. HPLC analysis showed a single major peak of at least 80% purity (monitored at 291 nm, which is the λmax of the

+ cinnamaldehyde group). HRESI-MS: C26H40N7O6 , calcd 546.3035, found 546.3122.

Synthesis of N-[4-(3-Oxo-1-propenyl)benzoyl]-Gly-Glu-Glu-NH2 (5), N-[4-(3-

13 [ C]Oxo-1-propenyl)benzoyl]-Gly-Glu-Glu-NH2 (14), 4-(3-Oxo-1-propenyl)benzoic

Acid (3), and Ethyl 4-[2-(1,3-Dioxolane-2-yl)-ethenyl]benzoyl Carbonate (7) These compounds were synthesized as described in chapter 2.

65 Synthesis of N-[4-(3-Oxo-1-propenyl)benzoyl]-Gly-Glu-Glu-βAla-βAla-βAla-

ε (N -biotinyl)Lys-NH2 (22). The unbiotinylated precursor of this compound was

synthesized from anhydride 9 on the solid phase in a manner

similar to compound 21. The fully deprotected compound (1.8

mg, 1.6 µmol) containing a single amino group on the lysyl residue was dissolved in 1 mL of 30 mM Na2HPO4 (pH 9.2) and N- hydroxysuccinimidobiotin (0.5 µmol) in 10 µL of DMF was added. The mixture was incubated at room temperature for 2.5 h. The use of substoichiometric amount of the biotinylating agent was to avoid binding to the streptavidin sensorchip (see below) by free biotin. HPLC analysis showed an approximately 50:50 mixture of biotinylated and unbiotinylated peptides, which was essentially free of other species when monitored at

+ 291 nm. ESI-MS: C47H68N11O15S , calcd 1058.4601, found 1058.4.

4.2.3 Activation of SHP-1 by Peptidylaldehydes.

Phosphatase activity of SHP-1 was measured with p-nitrophenyl phosphate

(pNPP) as substrate. The assay reaction (total volume of 50 µL) contained 50 mM Hepes, pH 7.4, 50 mM NaCl, 1 mM EDTA, 1 mM tris(carboxyethyl)phosphine, 10 mM pNPP,

0–2000 µM peptidyl aldehyde (or other effectors), and 0.5 µg of SHP-1. The reaction was initiated by the addition of SHP-1 as the last component and allowed to proceed at room temperature (~ 23 ˚C) for 30 min. The reaction was then quenched with 950 µL of 1

M NaOH, and the absorbance at 405 nm was measured on a Perkin-Elmer UV-vis spectrophotometer. The SHP-1 reactivity reported was relative to that of SHP-1 in the absence of any SH2 inhibitor. The time-course experiment was carried out under similar 66 conditions except that the reaction (total volume of 1.0 mL) contained 45 µM Cinn-GEE and 10 µg of SHP-1.

4.2.4 SH2 Domain-Inhibitor Binding Assays.

Surface plasma resonance measurements were carried out on a Pharmacia

BIAcore 3000 instrument. A biosensor chip containing immobilized streptavidin was conditioned with 1 M NaCl in 50 mM NaOH according to manufacturer’s instructions.

The biotinylated Cinn-GEE (compound 22) was immobilized onto the biosensor chip by flowing a 0.8 µM solution of 22 for 8 s (flow rate = 15 µL/min). Increasing concentrations of GST-SH2(N) protein (0–15 µM) in HBS-EP buffer (10 mM Hepes, pH

7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% polysorbate 20) was flowed over the sensorchip for 10 min at a flow rate of 15 µL/min. A blank flow cell (no compound 22) was used as the control to correct for any signal due to the solvent bulk and/or nonspecific binding interactions. In between two runs, the chip surface was regenerated by flowing 0.05% SDS in HBS-EP buffer for 10 s at a flow rate of 30 µL/min. Data analysis was carried out by plotting the equilibrium response units (RUeq), obtained by subtracting the response of the blank flow cell from that of the Cinn-GEE immobilized flow cell, against the SH2 protein concentration. The dissociation constant (KD) was obtained by fitting the data to the equation

RUeq = RUmax • [SH2]/(KD + [SH2])

67 where RUeq is the measured response unit at a certain SH2 concentration and RUmax is the maximum response possible. Binding of C-SH2 domain was carried out similarly, except that higher SH2 protein concentrations were used (5–50 µM) and the C-SH2 domain contained a C-terminal histidine tag.

For competition experiments, a biotinylated pY peptide, biotin-A(β-

Ala)3IYpYANLI, was immobilized onto streptavidin-coated sensorchip SA as previously described [125]. GST-SH2(N) protein (10 µM) was preincubated with/without Cinn-GEE

(300 µM) for 10 min at room temperature and passed over the sensorchip for 3 min at a flow rate of 15 µL/min. A blank flow cell was used as a control to correct any signal generated due to the solvent bulk or non-specific interactions.

4.2.5 NMR Spectroscopy of [13C]Cinn-GEE and [13C]Cinn-GEE-PTP1B Complex.

All NMR experiments were performed for 6–12 h on a Bruker DMX-600 NMR spectrometer equipped with a triple-resonance and 3-axes gradient probe at 300 K. The

2D 1H-13C heteronuclear single-quantum coherence spectra (HSQC) were acquired using a constant-time sensitivity-enhanced method [83]. The N-SH2 domain used was a fusion protein with maltose-binding protein [MBP-SH2(N)], whereas the C-terminal SH2 domain contained a C-terminal histidine tag. All samples were dissolved in a buffer containing 5 mM Hepes (pH 7.4), 100 mM NaCl, 1 mM EDTA, and 1 mM β- mercaptoethanol (94:6 H2O:D2O). The samples were incubated at room temperature for

30 min prior to NMR experiments. The spectral widths were 9615 Hz in the 1H dimension and 19621 Hz in the 13C dimension, with carrier frequencies at 4.7 and 150

(for 13C at the aldehyde position), respectively. 68 4.2.6 Mass Spectrometry

MBP-SH2(N) (100 íM) was incubated with [13C]Cinn-GEE (140 μM) for 30 min in a buffer containing 10 mM Hepes (pH ) 7.4), 150 mM NaCl, 3 mM

EDTA, and 1 mM β-mercaptoethanol. After that, 1 μL of 200 mM sodium cyanoborohydride in water was added each hour for a total of 5 h. The resulting mixture was incubated for an additional 16 h. Prior to MS analysis, the mixture was dialyzed against a 30 mM ammonium acetate buffer for 12 h. Electrospray ionization (ESI) experiments were performed on a Micromass Q-Tof II (Micromass, Wythenshawe, U.K.) mass spectrometer equipped with an orthogonal electrospray source (Z-spray) operated in the positive ion mode. Sodium iodide was used for mass calibration for a calibration range of m/z 100-2500. Salt buffers from the protein samples were cleaned using manual syringe protein traps from Michrom BioResources (Auburn, CA). Proteins were prepared in a solution containing 50% acetonitrile/50% water and 0.1% formic acid at a concentration of 10 pmol/μL and infused into the electrospray source at a rate of 5-10

μL/min. Optimal ESI conditions were as follows: capillary voltage, 3000 V; source temperature, 110 °C; cone voltage, 60 V. The ESI gas was nitrogen. Q1 was set to optimally pass ions from m/z 500-1990, and all ions transmitted into the pusher region of the TOF analyzer were scanned over m/z 500-3000 with a 1 s integration time. Data were acquired in the continuum mode until acceptable averaged data were obtained (10-15 min). ESI data were deconvoluted using MaxEnt I provided by Micromass. A control experiment was carried out similarly except that no sodium cyanoborohydride was added.

69 4.2.7 Synthesis of Peptidyl Cinnamaldehyde Library and Screening

Peptide library was synthesized on TentaGel resins (130 μm) by using general

Fmoc based solid phase peptide synthesis as described in Figure 4.11. Activated cinnamaldehyde (0.1 equiv. compound 13) was attached at the N-terminal of the peptide library as described in chapter 2. To encode sequences of the peptides, only the peptides on the surface of the resins were modified with cinnamaldehyde by using a bead shaving methodology. This method also made it possible to decrease the peptide densities on the surface of the resins, which resulted in minimizing nonspecific binding of the protein.

Library screening against MBP-(N)SH2 domain of SHP-1 was performed as previously described. In a micro BioSpin column (0.8 mL, BioRad), 10 mg of the Cinn-

XXXX-NH2 library was swollen in dichloromethane, washed extensively with methanol,

H2O, and blocked for 1 h with 800 μL of the screening buffer containing 0.1% gelatin.

The resin was drained and resuspended in 800 μL of a biotinylated MBP-(N)SH2 domain of SHP-1 (0.1 μM − 2 μM final concentration) in the screening buffer plus 0.1% gelatin.

After 3−5h incubation at r.t. with gentle mixing, the resin was drained and re-suspended in 800 μL of the SA-AP buffer containing 1 μL of streptavidin-alkaline phosphatase (SA-

AP, ~1 mg/mL, Prozyme). After 15 min of gentle mixing at 4 ºC, the resin was rapidly drained and washed with the SA-AP buffer (800 μL × 6), and the SA-AP reaction buffer

(800 μL × 3). The resin was then transferred into a 35-mm Petri dish in 3 x 300 μL of the

SA-AP reaction buffer. Upon addition of 80 μL of 5 mg/mL BCIP in the SA-AP reaction buffer, blue color developed on positive beads in ~45 min, at which point the staining reaction was quenched by the addition of 3 mL of 8 M guanidine-HCl, pH 8.0. The resin was transferred back into the BioSpin column, extensively washed with water, and re- 70 plated in the Petri dish from which colored beads were picked manually using a pipette under a dissecting microscope.

4.3 Results

4.3.1 Stimulation of SHP-1 Activity by Peptidylaldehydes.

We have shown that peptidyl aldehydes act as slow-binding inhibitors of PTPs in

Chapter 2 and Chapter 3. They bind to the PTP active site as a pY mimetic to form an initial noncovalent complex, E•I, which is slowly converted into a covalent enamine adduct (E•I*) with an active-site arginine (Arg-221 in PTP1B). Arg-221 is a universally conserved residue among PTPs and is critical for pY substrate binding as well as transition-state stabilization by forming a pair of hydrogen bonds between the phosphate oxygen atoms and the guanidinium group [30, 82]. This finding led us to hypothesize that the aldehydes may also act as SH2 domain antagonists. Both PTPs and SH2 domains recognize pY peptides as their natural ligands and their active/binding sites have remarkable similarities. Like PTPs, SH2 domains utilize the guanidinium group of an absolutely conserved arginine (Arg βB5) to form a pair of hydrogen bonds with the phosphate group of pY [110]. A key difference is that the SH2 domains lack the catalytic cysteine in the PTP active site.

To test the above notion, we synthesized a peptidyl aldehyde, N-{2-[4-(3-oxo-1- propenyl)phenoxy]acetyl}-Ala-Arg-Leu (Cinn-ARL) (compound 21 in Figure 4.2 described in Figure 4.1). Our previous study showed that pY peptides of the consensus

LXpYARLI (X = any amino acid) bind to both N- and C-SH2 domains of SHP-1 with high affinity [124]. We employed Cinn-ARL to stimulate the phosphatase activity of

71 SHP-1 as a convenient test of its ability to bind the N-SH2 of SHP-1. Under physiological conditions, SHP-1 exists as an inactive form, in which the N-SH2 directly binds to and inactivates the PTP domain [125, 126]. Binding of a high-affinity ligand

(e.g., a pY peptide) to the N-SH2 disrupts the SH2/PTP interaction and increases its activity by up to 30-fold. As shown in Figure 2A, Cinn-ARL stimulated SHP-1 activity by up to 13-fold, with half-maximal activation (IC50) reached at ~500 µM. Assuming an equilibrium constant of ~30 between the open and closed states of SHP-1 [125, 126], we estimated a dissociation constant (KD) of ~17 µM for the interaction between Cinn-ARL and the isolated N-SH2 domain. Under the assay conditions, Cinn-ARL showed no significant inhibition toward the catalytic domain of SHP-1, SHP-1(∆SH2) [81], at up to

1 mM inhibitor concentration (data not shown). As a control, we also tested N-[4-(3-oxo-

1-propenyl)benzoyl]-Gly-Glu-Glu (Cinn-GEE) (compound 2 in Figure 4.2), which had previously been shown as a fairly potent slow-binding inhibitor of PTP1B and SHP-

1(∆SH2) (KI* = 5.4 and 11 µM, respectively). Surprisingly, Cinn-GEE is an even more potent activator of SHP-1 (with a maximum of ~30-fold stimulation) (Figure 4.3.A).

Activation of SHP-1 by Cinn-GEE is dose-dependent, with maximal activity reached at

150 µM and an IC50 of ~60 µM. Further increase in Cinn-GEE concentration resulted in decreased PTP activity, due to inhibition of the PTP active site. Neither the tripeptide

Gly-Glu-Glu nor cinnamaldehyde (compoubd 3 in Figure 4.2) alone showed significant stimulation of SHP-1 activity (<2-fold). The IC50 value suggests a KD of ~2 µM for the

Cinn-GEE/N-SH2 complex.

On the basis of the KI* value previously determined for SHP-1(ΔSH2) (11 µM), one would have expected that the PTP active site of SHP-1 be completely inhibited by

72 150 µM Cinn-GEE. A possible explanation for this apparent paradox is that the formation of the SH2/Cinn-GEE complex is much faster than that of the PTP/Cinn-GEE complex.

Consequently, there is no significant amount of E•I* complex (i.e., the enamine adduct) formed between the PTP active site and Cinn-GEE during the 30-min activation assay reaction. To test this notion, we added SHP-1 into an assay solution containing reaction buffer, pNPP (10 mM), and Cinn-GEE (45 µM) and monitored product formation at 405 nm as a function of time. The reaction progress curve has a sigmoidal shape, composed of an early delay region (<200 s), a linear region (200–700 s), followed by a tailing-off region (>700 s) (Figure 4.3.B). The delay in the early region indicates that activation of

SHP-1 (and thus binding of Cinn-GEE to the N-SH2 domain) is also time-dependent, as would be expected from a slow-binding mechanism similar to that of inhibition of PTPs.

However, the onset of the linear phase at ~200 s suggests that formation of the final

SH2–Cinn-GEE complex is complete within 3 min for the N-SH2 domain (at 45 µM

Cinn-GEE). Under similar conditions, formation of the E•I* complex between PTP and

Cinn-GEE typically takes >10 min (chapter 2). Therefore, the linear region represents a period during which SHP-1 activation is already complete whereas no significant amount of the covalent PTP–Cinn-GEE complex is yet formed. Finally, the rate of pNPP hydrolysis tails off as increasing amount of PTP is inactivated by Cinn-GEE. The control reaction in the absence of Cinn-GEE produced a straight line during the entire reaction time.

73 4.3.2 Direct Binding between Cinn-GEE and SH2 Domains.

Cinn-GEE was tested for binding to the isolated N- and C-SH2 domains of SHP-1 using the surface plasmon resonance technique (BIAcore). Immobilization of the aldehyde ligand onto streptavidin-coated sensorchips was effected by the addition of a biotin to the C-terminus of Cinn-GEE. To maximize the accessibility of the surface bound ligand to an incoming SH2 domain, a flexible linker of β-Ala-β-Ala-β-Ala-Lys

(BBBK) is added to the C-terminus of Cinn-GEE. The resulting ligand was synthesized by solid-phase chemistry, deprotected, and treated with N-hydroxysuccinimidobiotin to give compound 22 (Figure 4.2). For binding studies, we employed the N-SH2 domain as a fusion protein with glutathione-S-transferase [GST-SH2(N)], because the N-SH2 alone was not stable. The C-SH2 domain contained a C-terminal histidine tag. Figure 4.4.A shows the sensograms for the binding of GST-SH2(N) to the immobilized Cinn-GEE, after correction for nonspecific binding to the surface. The slow increase in response units over several minutes (after the initiation of sample injection) even at saturating concentrations of the SH2 protein confirms that the interaction is slow binding in nature.

In contrast, binding of GST-SH2(N) to immobilized pY peptides displays rapid kinetics

(ref. [124, 125] and Figure 4.5). Plot of equilibrium response units (RUeq = difference in response units prior to and at the end of sample injection) against SH2 concentration clearly showed saturation behavior and data fitting gave an equilibrium KD of 1.3 ± 0.2

µM (Figure 4.4.B). This value is similar to the KD value (~2 µM) estimated from the

SHP-1 activation profile. The C-SH2 domain also bound to the immobilized Cinn-GEE with an estimated KD of ~25 µM (the KD value could not be accurately determined due to significant nonspecific binding at high SH2 protein concentrations).

4.3.3 Cinn-GEE Is Active-Site Directed.

To determine whether Cinn-GEE binds to the canonical pY binding site of the

SH2 domains, competition experiments were performed with a pY peptide, IYpYANLI, which binds to the N-SH2 domain of SHP-1 with high affinity (KD = 0.6 µM) [124]. As shown in Figure 4.5, GST-SH2(N) protein alone (10 µM) bound very well to a sensorchip immobilized with the pY peptide. However, preincubation of the N-SH2 domain (10 µM) with excess Cinn-GEE (300 µM) completely abolished its binding to the sensorchip, under otherwise identical conditions. In the reverse competition experiment, the pY peptide also inhibited the binding of GST-SH2(N) to the immobilized Cinn-GEE

(data not shown). These results indicate that Cinn-GEE and IYpYANLI bind to the same site on the SH2 domain.

4.3.4 Cinn-GEE/SH2 Domain Interaction is Reversible

The fact that a pY peptide can displace GST-SH2(N) bound to the immobilized

Cinn-GEE indicates that the interaction between Cinn-GEE and the SH2 domain is reversible. To further demonstrate the reversibility of the interaction, SHP-1 was preincubated with excess Cinn-GEE to form the Cinn-GEE-SH2 domain complex, which was then rapidly diluted into an assay solution containing pNPP as substrate. The reaction progress as a function of time is shown in Figure 4.6. Initially, the rate of pNPP hydrolysis (as indicated by the slope at time 0) was relatively high; as time increased, the reaction rate decreased, eventually approaching the rate of pNPP hydrolysis by SHP-1 in the absence of Cinn-GEE. The simplest explanation is that, at time 0, the N-SH2 domain

75 was occupied by Cinn-GEE and SHP-1 was in the active form, whereas dilution caused time-dependent dissociation of the Cinn-GEE-SH2 domain complex and re-formation of the catalytically inactive SH2-PTP complex. Due to the complication of simultaneous inhibition of the PTP active site by Cinn-GEE, we have not been able to determine quantitatively the kinetics of Cinn-GEE-SH2 dissociation.

4.3.5 Mechanism of Cinn-GEE/SH2 Domain Interaction.

To determine the mechanism by which Cinn-GEE binds the SH2 domains, we synthesized Cinn-GEE labeled with 13C at the aldehyde carbonyl carbon and analyzed both the inhibitor (compound 14 in Figure 2.3 ) and the SH2 domain-inhibitor complexes by 1H–13C HSQC NMR spectroscopy. When dissolved in the assay buffer (pH 7.4),

[13C]Cinn-GEE alone showed a single cross-peak at δ 9.64 (1H) and δ 201 (13C) in the

NMR spectrum (Figure 4.7.A). This suggests that the free inhibitor existed predominantly in the aldehyde form in the aqueous solution. Addition of excess N-SH2 domain (with preincubation), resulted in the disappearance of the free inhibitor signal, with concomitant appearance of a single new cross-peak at δ 8.45/173 (Figure 4.7.B). No other signals were observed that could be attributed to either the free inhibitor or the enzyme-inhibitor complex. The HSQC spectrum for the C-SH2 domain/Cinn-GEE complex is somewhat different; in addition to a cross peak at δ 8.45/174, there is a second cross-peak of slightly lower intensity at δ 7.70/139 (Figure 4.7.C). The chemical shift values of the latter peak are very similar to those observed for the enamine adduct formed between Cinn-GEE and the active-site arginine of PTP1B (chapter 2). Our interpretation of these results is that Cinn-GEE binds to the N-SH2 domain by forming an imine adduct 76 with its active-site arginine (Arg βB5), whereas a mixture of imine and enamine adducts were formed when Cinn-GEE binds to the C-SH2 domain (see Discussion).

To gain further evidence for the proposed mechanism, we attempted to analyze the N-SH2 domain/Cinn-GEE complex by mass spectrometry. However, we have so far failed to observe the covalent adduct directly, probably due to the reversible nature of the adduct and the moderate affinity (KD = 1.3 μM). We next incubated the N-SH2 domain with Cinn-GEE under the same conditions but in the presence of a reducing agent, sodium cyanoborohydride. ESI-MS analysis of the reaction mixture revealed a new species with a molecular mass of 56102 ± 5 Da in the spectrum, in addition to the unmodified MBP-SH2(N) protein (molecular mass = 55626 ± 5 Da) (Figure 4.9). The difference in molecular mass between the peaks was ~ 476 Da, suggesting the addition of the single inhibitor molecule to the protein (491 - 18 + 2 = 475 Da). No peak corresponding to the addition of two or more inhibitors to the SH2 domain was observed.

These results are consistent with the formation of a reversible imine adduct between

Cinn-GEE and an amino group of the SH2 domain, which was reduced to a stable amine adduct by sodium cyanoborohydride. Unfortunately, all of our attempts to isolate and sequence a proteolytic fragment containing the covalently attached inhibitor have failed.

The role of Arg βB5 (Arg-30 in SHP-1) in inhibitor binding was assessed by constructing an R30K mutant of SHP-1 and examining its ability to be activated by Cinn-

GEE. Unlike the wild-type enzyme, whose activity is stimulated by up to 25-30-fold by

Cinn-GEE, the R30K enzyme is much less responsive to Cinn-GEE stimulation (<5-fold stimulation at maximum) (Figure 4.9). This shows that Arg-30 is intimately involved in inhibitor binding, most likely acting as the attacking nucleophile (see Discussion). A 77 possible explanation for the weaker binding of Cinn-GEE to the R30K mutant SH2 domain is that the lysine side chain may be too short to reach the inhibitor aldehyde in the initial noncovalent complex.

4.3.6. Screening of Cinn-XXXX-NH2 Library

For further insight of the binding preference of the SH2 domain toward peptidyl cinnamaldehydes, we generated Cinn-XXXX-NH2 library, and screened against the

(N)SH2 domain of SHP-1. Unfortunately, we could not observe any strong consensus sequences from the screening, thus far.

4.4 Discussion

We have shown that peptidyl aldehydes display slow-binding inhibition of SH2 domains derived from SHP-1. Although the SH2 domains have no catalytic activity, we will treat them as “enzyme entities (E)” in order to apply the kinetic models developed for enzymatic systems. Thus, the kinetics of SH2 inhibition can be described by the following equation,

KI k5 E + I E•I E•I* k6 as described in PTP inhibition kinetics (Chapter 2 and 3). The overall potency of the inhibitor is described by the overall equilibrium constant, KI* = KIk6/(k5 + k6). Due to technical difficulties, we have not yet been able to determine the KI, k5, and k6 values.

However, the KI* value, which equals to the apparent dissociation constant (KD) measured under equilibrium conditions, can be determined. Binding studies using surface

78 plasmon resonance gave KI* (or KD) values of 1.3 and ~25 µM for the binding of Cinn-

GEE to the N- and C-SH2 domains, respectively. The former value agrees quite well with the KD value (~ 2 µM) estimated from the SHP-1 activation profile. Therefore, the peptidyl aldehydes are fairly potent inhibitors of SH2 domains. We are confident that still more potent inhibitors can be developed by optimizing the peptide sequence or by attaching appropriate peptidomimetics to cinnamaldehyde.

Figure 4.10 shows our proposed mechanism for the observed slow-binding inhibition. The innamaldehyde moiety bindsto the pY-binding pocket to form the initial

EâI complex, in which the aldehyde carbonyl group is placed in close proximity to the guanidine group of Arg βB5. Nucleophilic addition on the carbonyl by one of the guanidino amine groups followed by dehydration generates a conjugated imine adduct.

The imine can be further converted into an enamine adduct (in the C-SH2 domain) through conjugate addition at the benzylic position by a yet unidentified nucleophile.

This mechanism is very similar to that proposed for the inhibition of PTP1B by Cinn-

GEE and is consistent with all of the experimental observations. The chemical shift values for the aldehyde-derived proton/carbon in the E·I* complex (δ 8.45/~ 174 and δ

7.70/139) indicate that the aldehyde carbonyl is no longer present but the carbon atom is still sp2 hybridized. This suggests that the aldehyde must have reacted with an active site residue to form an adduct involving a double bond at the carbonyl carbon. Further, the formation of this adduct must be reversible. Among the 20 proteinic amino acids, only lysine and arginine are capable of forming such a structure with an aldehyde (in the form of an imine or enamine). To determine which arginine or lysine residue is involved in the reaction, we modeled the structures of the N- and C-SH2 domains of SHP-1 based on the

79 X-ray crystal structure of the N-terminal SH2 domain of SHP-2, which has ~ 60% sequence identity to SHP-1 [111]. The results show that the pY-binding pocket of N-SH2 contains three arginine residues (at positions βB5, BC1, and βD6) and a lysine (at position BC2), whereas the C-SH2 domain contains only one arginine (at position βB5) and one lysine (at position βD6). Therefore, Arg βB5 is the only candidate that is common to both SH2 domains. Arg βB5 is similarly situated at the pY-binding pocket to

Arg-221 in the active site of PTP1B, which has been established as the residue modified by Cinn-GEE. Like Arg-221, Arg βB5 also forms a pair of hydrogen bonds with the phosphate group of the bound pY [110-113]. In contrast, the side chain of Lys βD6, the other candidate residue in the C-SH2 domain, is packed against the phenyl ring of bound pY and is more distant from the phosphate group [111]. Furthermore, the minor cross- peak (δ 7.70/139) in the HSQC spectrum of the C-SH2-Cinn-GEE complex has very similar chemical shift values to those of the enamine adducts formed between Arg- 221 and the inhibitor aldehyde in the PTP1B-Cinn-GEE complex (δ 7.6-7.8/130-137).

Finally, the reversible adduct can be reduced to a stable adduct by sodium eyword>

SH2 domains. Thus, we propose that the minor cross-peak at δ 7.70/139 (in the C-SH2 domain) represents the enamine adduct formed between the inhibitor and Arg βB5, whereas the major peak at δ 8.45/~ 174 (in both N- and C-SH2 domains) is the conjugatedThe imineproposed adduct mechanism (Figure 4.10). provides a sensible account for the activation of SHP-1 by peptidyl aldehydes. It is well established that pY peptides with high affinity to the N-

80 SH2 domain can disrupt the intramolecular complex between the N-SH2 domain and the

PTP active site [125, 126]. This relieves the autoinhibition by the N-SH2 domain and results in increased PTP activity. The peptidyl aldehydes bind to the same site as the pY ligands and are therefore expected to have the same stimulatory effect. The initial lag period (Figure 4.3) is also expected, since the formation of the SH2·I* complex is time- dependent and relatively slow. The lack of significant inhibition of the PTP active site during the activation assays is due to the still slower formation of the PTP·I* complex and substrate protection (10 mM pNPP). The peptidyl aldehydes presumably also bound to the C-SH2 domain during the activation assays. However, binding to the C-SH2 domain has no effect on the catalytic activity of SHP-1 [126].

An important issue in designing SH2 domain inhibitors is selectivity. Namely, will a peptidyl aldehyde designed for a given SH2 domain inhibit other SH2 domains or other enzymes such as PTPs? The preliminary data from this work suggest that it should be possible to develop selective SH2 inhibitors. First, high-affinity binding to an SH2 domain requires both the peptide and the cinnamaldehyde moieties, as neither component alone had significant effect on SHP-1 activity. Indeed, Cinn-ARL and Cinn-GEE have a

~ 10-fold difference in binding affinity to the N-SH2 domain. The reason for the unexpected high affinity of Cinn-GEE (relative to Cinn-ARL) is not yet clear. Given the specificity of the N-SH2 domain for pYAXL peptides, it is possible that the tripeptides

GEE and ARL bind to different sites near the pY-binding pocket. Second, Cinn-GEE showed a 20-fold difference in binding affinity to the N- vs C-SH2 domains of SHP-1, even though the two SH2 domains have similar specificity toward pY peptides. Finally, while Cinn-ARL showed respectable affinity to the N-SH2 domain, it had no detectable

81 inhibition of the PTP active site. In conclusion, we have demonstrated that peptidyl aldehydes function as covalent but reversible inhibitors of SH2 domains. The slow- binding kinetics is caused by the timedependent conversion of the initial noncovalent complex to a covalent imine/enamine adduct with the conserved arginine (βB5) in the pY-binding pocket. The peptidyl aldehydes represent a novel class of neutral SH2 domain inhibitors, which should overcome the permeability problems associated with the existing SH2 inhibitors.

Figure 4.1. Synthesis of N-{2-[4-(3-Oxo-1-propenyl)phenoxy]acetyl}-Ala-Arg-Leu-NH2

Figure 4.2. Structures of SH2 inhibitors.

A 35 GEE 30 Cinn Cinn-ARL 25 Cinn-GEE

10 Relative Activity Relative

0 1 10 100 1000 [I] (킡 ) B

Figure 4.3. Activation of SHP-1 by peptidyl aldehydes. (A) Plot of SHP-1 activity against SH2 domain inhibitor concentration. The data presented are phosphatase activities relative to that of SHP-1 in the absence of peptidyl aldehyde. (B) Time course of the activation of SHP-1 by Cinn-GEE. The reaction was initiated by the addition of SHP-1 (0.15 µM final) to a solution containing the reaction buffer (pH 7.4), 10 mM pNPP, and 0 or 45 µM Cinn-GEE, and monitored continuously at 405 nm on a UV-Vis spectrophotometer. The dashed straight line is drawn for comparison.

A 60 RUeq 50

30 RU 20

0 200 300 400 500 600 700 800 900 1000 Time (s) B

Figure 4.4. BIAcore analysis of the binding of GST-SH2(N) to immobilized Cinn-GEE (4). (A) Overlaid sensograms at increasing concentrations of GST-SH2(N) (0.23, 0.47, 0.94, 1.87, 3.75, 7.50, and 15 µM). (B) Plot of resonance signal under equilibrium binding conditions (signal difference between the points immediately before the initiation and the end of each injection) against SH2 concentration. Data were fitted to equation: RUeq = RUmax • [SH2]/(KD + [SH2]).

2500

2000

1500 ?Cinn-GEE RU 1000

500 + Cinn-GEE 0 150 200 250 300 350 400 450 500 550 Time (s)

Figure 4.5. Competition of Cinn-GEE and peptide IYpYANLI for binding to GST- SH2(N). The pY peptide was immobilized onto a streptavidin-coated BIAcore sensorchip as previously described (24). GST-SH2(N) (10 µM) was flowed over the surface in the absence or presence of Cinn-GEE (300 µM).

Figure 4.6. Time-dependent dissociation of the Cinn-GEE-SH2 domain complex. SHP-1 (1.5 µM) was preincubated with 0 or 600 µM Cinn-GEE for 3 min before being diluted 100-fold into an assay buffer containing 10 mM pNPP. The reaction progress curves as monitored at 405 nm are shown. Inset, the relative rate of pNPP hydrolysis (the ratio of reaction rate in the presence of Cinn-GEE over control) as a function of time.

Figure 4.7. HSQC spectra of 13C-labeled Cinn-GEE in the presence and absence of SH2 protein. (A) 200 µM Cinn-GEE only; (B) 200 µM Cinn-GEE and 400 µM N-SH2 domain; (C) 200 µM Cinn-GEE and 400 µM C-SH2 domain.

Figure 4.8. Deconvoluted spectra from LC-ESI MS analyses of the [13C]Cinn-GEE- MBP-SH2(N) complex before (A) and after treatment with sodium cyanoborohydride (B).

Figure 4.9. Comparison of the stimulatory effect of Cinn-GEE on wild-type and R30K SHP-1.

Figure 4.10. Proposed mechanism of inhibition of SH2 domains by peptidyl cinnamaldehyde derivatives. RXH, the yet unidentified nucleophile.

NH2 NHFmoc NHFmoc NH2 NH2 Met-Boc

H2N FmocHN FmocHN 0.6 eqv. FmocOSu in Boc-Met NH2 NH2 Met-Boc H2N DCM/Ether (55:45) H2N Boc-Met

NH2 NHFmoc NHFmoc

swell in H2O for 24 hrs

Ala-Ac Ala-Ac Met MRBBLXXXX-NH2 Ac-Ala General solid phase peptide synthesis by Ac-Ala using Fmoc-amino acids and HBTU/HOBt

Met MRBBLXXXXNH2

1. 20% piperidine in DMF Met H2NXXXXLBBRM 2. Ac-Ala/Boc-Met (99/1) 3. TFA

Met MRBBLXXXXNH2 1 % of the surface is availalbe for further synthesis.

O O

Ala-Ac MRBBLXXXX-NH2 Deprotection of amino acids O Ac-Ala side chains and cinnamaldehyde 10 % of available surface peptides will O O O by TFA be modified with cinnamaldehyde. MRBBLXXXXNH2 H2NXXXXLBBRM 90 % of inside peptides are available for 0.1 eqv. sequencing.

MRBBLXXXXNH - Cinn

Figure 4.11. Synthesis of peptidyl cinnamaldehyde library. TentaGel (130 μm) resins will be used as solid supports. Chemicals for library synthesis are commercially available from Advanced ChemTech and Aldrich. The activated cinnamaldehyde will be prepared as decscribed in chapter 2 (compound 13).

CHAPTER 5

TRANS-β-NITROSTYRENE DERIVATIVES AS SLOW-BINDING INHIBITORS

OF PROTEIN TYROSINE PHOSPAHTASES

5.1 Introduction

In the previous chapters, we reported that cinnamaldehyde is the neutral pY analog, and adding the tripeptide made it more potent inhibitor against PTPs, and DSPs.

Furthermore, we showed that cinnamaldehyde forms the imine/enamine adduct with

PTPs by interacting with the active-site arginine. In addition, it has been showed that cinnamicacid also inhibits PTP1B with moderate potency (IC50 ~ 20 μM, unpublished data). During these studies, we found that although the inhibition mechanisms of cinnamaldehyde and cinnammicacid are quite different from each other, the styrene structure of the both molecules is very important for the initial binding to the PTP active site, because without the stytrene structure (i.e. 3-phenylpropionic acid and 3- phenylpropionaldehyde), there is no inhibition observed against PTPs. Based on this information, we looked for another neutral pY analog that contains styrene moiety and tested against PTPs, and found that trans-β-nitrostyrene (TBNS) derivatives (Figure 5.1, compounds 27–32 and 34) act as slow-binding, reversible inhibitors of PTPs.

94 Mechanistic studies performed here suggest that TBNS directly interacts with the active site cysteine residue to form a covalent enzyme-TBNS adduct.

5.2 Experimental Procedures

5.2.1 Materials

All chemicals used for peptide synthesis were from Advanced ChemTech

(Louisville, KY). 1-Phenyl-2-nitroethane was purchased from APIN Chemical, whereas all nitrostyrene derivatives and other chemicals were obtained from Sigma-Aldrich.

Yersinia PTP Yop and alkaline phosphatase were purchased from New England Biolabs

(Beverly, MA). Protein concentrations were determined by Bradford assay using bovine serum albumin as standard.

5.2.2 Site Directed Mutagenesis of PTP1B

Plasmid pET-22(b)-PTP1B was generated by inserting a PCR fragment that codes for amino acids 1–321 of PTP1B into the NdeI and XhoI site of vector pET-22b

(Novagen, WI). Site-directed mutagenesis was carried out with this plasmid DNA as template using the QuickChange mutagenesis kit (Stratagene, CA). The DNA primers

(purchased from Integrated DNA Technologies, Inc., IW) used were as follows:

D181A, 5’-TATACCACATGGCCGGCCTTTGGAGT-3’;

C215A, 5’-GTTGTGGTGCACGCCAGTGCAGGCATC-3’;

C215S, 5’-CCCGTTGTGGTGCACAGCAGTGCAGC-3’;

C215D, 5’-CGTTGTGGTGCACGACAG TGCAGGCA-3’;

R225A, 5’-GCAGGCATCGGCGCGTCTGGAACCTTC-3’.

95 The identity of all DNA constructs was confirmed by DNA sequencing. Expression in

Escherichia coli and purification of recombinant PTP1B were performed as previously described (chapter 2). The expression and purification of recombinant SHP-1 has also previously been described [81].

5.2.3 Synthesis of trans-β-Nitrostyrene Derivatives

Synthesis of 4-Carboxy-trans-β-nitrostyrene (30) 4-Carboxy-TBNS was

NO2 prepared according to a modified literature method [127]. Nitromethane

(50.8 mg, 0.83 mmol) was added to 4-carboxy-benzaldehyde (100 mg,

0.67 mmol) in methanol (5 mL), and the resulting mixture was cooled to 4 CO2H oC. KOH (85 mg, 1.5 mmol) was dissolved in 1 mL of methanol, diluted into 5 mL of ice water, and slowly added to the above mixture through a syringe. After addition of the

KOH solution, the reaction was stirred for 15 min at 4 oC. The reaction mixture was diluted into 10 mL of ice water, and 100 mL of 5 M HCl was slowly added to the reaction mixture. The pale yellow precipitate formed was collected by filtration and recrystallized

1 from methanol (yield 21 mg, 16%). H NMR (250 MHz, DMSO-d6): δ 7.81-7.89 (m, 4H,

Ar), 8.04 (d, 1H, ArCH=CHNO2, J = 13.7 Hz), 8.16 (d, 1H, ArCH=CHNO2, J = 13.7

+ Hz), 13.18 (s, 1H, ArCO2H). HRESI-MS: Calcd for C9H7NO4 193.0370, found

4 -1 -1 193.0328. λmax and ε: 317 nm and 1.8 × 10 M cm .

Synthesis of N-{p-[(2-Nitroethenyl)benzoyl]}-L-Gly-L-Glu-L-Glu-NH2

(TBNS-GEE) (34) Tripeptide Gly-Glu-Glu-NH2 was synthesized on Rink resin by using standard Fmoc chemistry. Next, 4-carboxy-TBNS (30) was coupled to the resin bound 96 NO2 tripeptide by using O-benzotriazole-N,N,N’,N’-tetramethyluronium

hexafluorophosphate (HBTU) and N-hydroxybenzotriazole monohydrate

O N Gly-Glu-Glu H (HOBt) as coupling agents. The product (TBNS-GEE) was deprotected and released from the solid support by the treatment with 99% trifluoroacetic acid (TFA).

Excess TFA was removed under a gentle flow of nitrogen, and the residue was triturated with diethyl ether (5 x 3 mL). HPLC analysis of the crude product showed a purity of

+ + >90%. HRESI-MS: Calcd for C21H25N5O10Na ([M + Na] ) 530.1493, found 530.1474.

Synthesis of trans-[α-13C]-β-Nitrostyrene (35) This compound was prepared

13 NO2 from benzaldehyde-carbonyl- C by the same procedure as described for

* 1 3 4. H NMR (250 MHz, CDCl3): δ 8.01 (dd, 1H, ArCH=CHNO2, JH-H =

2 13.7 Hz, JC-H = 158 Hz), 7.62–7.42 (m, 6H). HRESI-MS: Calcd for

13 + + C7 CH7NO2Na ([M + Na] ) 173.0403, found 173.0405.

5.2.4 PTP assay

All assay reactions (1 mL) contained 50 mM HEPES (pH 7.4), 50 mM NaCl, 1 mM EDTA, 2% DMSO, varying concentrations of the PTP inhibitors, and 0.05–0.1 μM

PTP. The mixture was incubated for 2 h at room temperature, and the enzymatic reaction was then initiated by the addition of 10 μL of 100 mM pNPP and monitored continuously at 405 nm on a λ20 UV-Vis spectrophotometer. The IC50 values were determined by plotting the remaining activity as a function of inhibitor concentration, and the KI* values were obtained by fitting the data to the Michaelis-Menten equation. To obtain the KI value, the reaction was initiated by addition of enzyme (0.05 μM) as the last component 97 into the above reaction mixture, which also contained 1.0 mM pNPP. The reaction was monitored continuously on the UV-Vis spectrophotometer. The initial reaction rates were calculated from the early regions of the reaction progress curves (<30 s) and fitted to the

Michaelis-Menten equation to give the KI value. To determine the rate constant k6, the enzyme (0.1 μM) was incubated with 0–400 μM inhibitor in 100 μL of the above reaction buffer for 2 h at room temperature. After that, an aliquot of the mixture (25 μL) was withdrawn and rapidly diluted into 975 μL of the same buffer containing 1.0 mM pNPP

(without inhibitor). Reactivation of PTP activity was monitored at 405 nm, and the progress curves were fit to the equation:

-k t Abs405 = vs[t - (1 - e 6 )/k6]

where vs is the final steady-state velocity [87]. The rate constant k5 was calculated using the equation: KI* = KIk6/(k5 + k6).

5.2.5 Substrate Protection

PTP1B (0.05 μM) was added into the above assay buffer containing a fixed concentration of TBNS (20 μM) and varying concentrations of pNPP (0.5–20 mM). The reactions were continuously monitored at 405 nm and the pseudo-first-order inactivation rate constants (kobs) were obtained by fitting the individual reaction progress curves to the

-kobst equation: [P] = vst + [(vi – vs)/kobs](1 – e ) [87]. The obtained kobs were then plotted against [pNPP] according to the equation: kobs = kmax/(1 + [pNPP]/KM).

98 5.2.6 Competition between TBNS and Iodoacetic Acid

PTP1B (5.5 μM) was incubated for 1 h in the above assay buffer with or without the presence of 100 μM TBNS (no pNPP). Next, iodoacetic acid (2 mM final concentration) was added to the reaction and the mixture was incubated for an additional

20 min. An aliquot (10 μL) of the reaction mixture was withdrawn and diluted in 990 μL of the assay buffer containing 10 mM pNPP and 1 mM β-mercaptoethanol. The reaction was continuously monitored at 405 nm for ~15 min.

5.2.7 NMR Spectroscopy of [α-13C]TBNS and [α-13C]TBNS-PTP1B Complex

All NMR experiments were performed on a Bruker DMX-600 NMR spectrometer equipped with a triple-resonance and 3-axis gradient probe at 300 K. The 2D 1H-13C heteronuclear single-quantum coherence (HSQC) spectra were acquired using a standard

Bruker pulse sequence with sensitivity improvement [82, 128]. All samples were dissolved in a buffer containing 10 mM HEPES (pH 7.2), 150 mM NaCl, and 1 mM

1 EDTA (10:1 H2O/D2O). The spectral widths were 7,184 Hz in the H dimension and

21,129 Hz in the 13C dimension, with carrier frequencies at 4.7 ppm and 150 ppm.

5.3 Results

5.3.1 Inhibition of PTPs by TBNS and its Derivatives

A previous study showed that trans-cinnamic acid acts as a pY mimetic and, when attached to a tripeptide Gly-Glu-Glu, resulted in potent inhibition against PTP1B.

More recently, we showed that cinnamaldehyde also acts as a neutral pY mimetic, inhibiting PTPs, dual-specificity phosphatases, and Src homology 2 (SH2) domains. We 99 realized that TBNS (Figure 5.1, compound 1) is structurally similar to trans-cinnamic acid and cinnamaldehyde and may act as an inhibitor of PTPs. Since the nitro group is electronically neutral, TBNS should have the additional advantage of better membrane permeability over the negatively charged trans-cinnamic acid. We thus tested TBNS against cysteine-based classical PTPs, PTP1B, SHP-1, and Yersinia Yop for potential inhibition. Alkaline phosphatase, which is belong to LMWPs (refer to chapter 1), was also tested as a control. Since TBNS reacts readily with free thiols such as β- mercaptoethanol [129], we first tested TBNS against PTPs in the absence of any added free thiols. TBNS resulted in potent inhibition of PTP1B, with an IC50 of 2.5 μM (Table

5.1), which is 10-fold more potent than the negatively charged trans-cinnamic acid (IC50

≅ 20 μM, data not shown). It also inhibited Yop with similar potency (IC50 = 5 μM) but not alkaline phosphatase. To determine whether the observed inhibition is general for other TBNS derivatives, we tested several commercially available as well as synthetic

TBNS derivatives (Figure 5.1, compounds 2–6), which are modified with either electron- donating (2, 3, and 6) or electron-withdrawing groups (5) on the phenyl ring. All of the

TBNS derivatives inhibited PTP1B, with potencies comparable to that of the parent compound 1 (Table 5.1). In an attempt to improve the inhibitor potency, the tripeptide

Gly-Glu-Glu was attached to the para-position of TBNS to give TBNS-GEE (compound

8). TBNS-GEE has slightly improved potency (IC50 = 1.4 μM against PTP1B). This result suggests that TBNS binds PTPs in a manner different from cinnamic acid or cinnamaldehyde. However, it should be possible to convert TBNS into highly potent and selective PTP inhibitors by conjugating it to other peptides, peptidomimetics, or small molecules. 100 Assay against the catalytic domain of SHP-1, SHP-1(ΔSH2) [81], in the absence of free thiol was not possible, because this enzyme was rapidly inactivated under such conditions, presumably due to oxidation of its active-site cysteine. We therefore tested the TBNS derivatives against SHP-1(ΔSH2) in the presence of 1 mM β-mercaptoethanol.

All of the compounds showed inhibition, albeit with much lower potency (IC50 = 200–

450 μM) (Table 5.1). When PTP1B was tested in the presence of 1 mM free thiol, similar

IC50 values were obtained. This suggests that the poorer potency observed with SHP-

1(ΔSH2) is likely due to thiol inactivation of the TBNS derivatives. To test this notion, we examined the reaction of TBNS with β-mercaptoethanol by monitoring its absorption spectrum on a UV-Vis spectrophotometer. In the absence of free thiol, TBNS absorbed strongly at 320 nm and the absorption signal is quite stable (Figure 5.2.A). Upon the addition of ~1 equivalent of β-mercaptoethanol, the absorption maximum at 320 nm dramatically decreased (Figure 5.2.B). The absorption peak largely disappeared when 2 equivalents of thiols were added. However, further addition of excess HgCl2 restored the signal at 320 nm, whereas HgCl2 alone had no effect. These results are consistent with an earlier report that TBNS undergoes reversible conjugate addition with free thiols (Figure

5.2.C), which results in the loss of conjugation between the nitro group and the phenyl ring and therefore the absorption peak at 320 nm [129]. Moreover, these results suggest that the conjugate addition product does not inhibit PTPs or is a much poorer PTP inhibitor. Indeed, (2-nitroethyl)benzene (Figure 5.1, compound 7) showed no detectable inhibition of either PTP at 600 μM (Table 5.1).

101 5.3.2 Slow-Binding Inhibition of PTPs by TBNS

Compound 1 was selected for further kinetic characterization. It exhibited time- dependent inhibition toward PTP1B (Figure 5.3.A), which could be due to either slow- binding inhibition or time-dependent enzyme inactivation. However, since the observed inhibition is readily reversible (vide infra), it rules out the possibility of irreversible inactivation. Slow-binding inhibition generally results from the formation of an initial enzyme-inhibitor complex, E•I, which can slowly turn into a tighter enzyme-inhibitor complex, E•I* [87]. The inhibition kinetics can be described by the equation:

KI k5 E + I E•I E•I* k6

as described in the previous chapters. The equilibrium constant KI* represents the overall potency of the inhibitor, and its relationship with KI is described by the equation: KI* = KI

• k6/(k5 + k6). The KI and KI* values of compound 1 against PTP1B were determined to be

150 ± 16 μM and 1.3 ± 0.2 μM, respectively (see Experimental Procedures). To determine whether the inhibition is reversible as well as measuring the k6 value, PTP1B was preincubated with excess amount of TBNS to form the E•I* complex, which was then rapidly diluted into the reaction buffer containing pNPP as a substrate. The reaction progress curves as continuously monitored at 405 nm (for the release of p-nitrophenolate) showed time-dependent recovery of PTP1B activity (Figure 5.3.B). This result is consistent with the slow-binding mechanism. Curve fitting of this reactivation profiles

-1 produced the rate constant k6 (0.49 min ). The forward rate constant k5 was calculated

-1 from the KI, KI*, and k6 values (k5 = 56 min ). TBNS derivatives 2–6 also exhibited slow-binding behavior, although their KI, KI*, or k6 values were not determined. 102

5.3.3 TBNS Is Active-Site Directed

Two experiments were performed to determine whether TBNS is active site directed. First, substrate protection was tested by measuring kobs, the pseudo-first-order rate constant for onset of inhibition, at a fixed inhibitor concentration (20 μM) while varying the pNPP concentration (0.5–20 mM). There was clearly an inverse correlation between kobs and pNPP concentration (Figure 5.4.A). This result indicates that TBNS and pNPP compete for binding to the PTP1B active site. Second, TBNS was tested for its ability to protect PTP1B from covalent modification by iodoacetate (IDA). IDA had previously been shown to selectively alkylate the active-site cysteine of PTPs (Cys-215 in

PTP1B), causing their irreversible inactivation [37, 89]. PTP1B (5.5 μM) was preincubated with TBNS (100 μM) for 1 h prior to the addition of IDA (2 mM final concentration). After 20 min, an aliquot of the reaction mixture (10 μL) was diluted 100- fold into an assay buffer containing pNPP (1 mM) and β-mercaptoethanol (1 mM) and the reaction progress curve was monitored at 405 nm. Control reactions were carried out with either TBNS or IDA alone. The control reaction with IDA alone produced a linear progress line; its slope indicated a constant remaining PTP activity of 18 μmol/mg/min

(no PTP reactivation) (Figure 5.4.B). The TBNS only control exhibited slow enzyme reactivation, as observed previously (Figures 5.3.B), and a much higher final PTP activity

(93 μmol/mg/min at 800 s). The reaction with both TBNS and IDA also exhibited time- dependent reactivation, with a specific PTP activity approaching 75 μmol/mg/min at 800 s. The simplest explanation for these results is that TBNS bound to PTP1B active site,

103 preventing its covalent inactivation by IDA. Upon dilution, TBNS dissociated from the

PTP active site, restoring the enzymatic activity. Thus, the above data build a compelling case that TBNS inhibits PTP1B by binding to its active site.

5.3.4 Electronic Absorption Spectroscopy of E•I* Complex

The mechanism of PTP1B inhibition by TBNS was first examined by UV-Vis absorption spectroscopy. PTP1B (39 μM) and TBNS (50 μM) were rapidly mixed in a quartz cuvette and the absorption spectra were recorded every 2 min. At time 0 (or absence of PTP1B), TBNS produced a strong absorption peak at 320 nm (Figure 5.5).

The peak intensity decreased dramatically with time and reached minimum at 16 min, with a concomitant blue shift of the absorption maximum to ~300 nm. This spectral change occurred on a time scale similar to that of the formation of E•I* complex, suggesting that E•I* formation results in the loss of conjugation between the nitro group and the rest of the π system in TBNS. Note that reaction of TBNS with free thiols also causes the loss of the 320 nm signal (Figure 5.2). However, the reaction of TBNS with free thiols occurred at a much faster rate and did not result in any blue shift.

5.3.5 1H-13C HSQC NMR Spectroscopy of E•I* Complex

The nature of the E•I* complex was next examined by 1H-13C HSQC NMR spectroscopy. TBNS containing a specific 13C label at its benzylic position, [α-

13C]TBNS, was chemically synthesized. When dissolved in the PTP assay buffer (pH

7.4), [α-13C]TBNS alone showed three cross-peaks, which all have the same 13C chemical shift (δ 142.5) but different 1H chemical shift values (δ 8.17, 8.15, 7.89) (Figure 104 5.6.A). The doublet at δ 8.17–8.15 is due to signal splitting by the vinylic proton at the Cβ position (J = 13.8 Hz). The cross-peak at δ 7.89/142.5 is caused by long-range coupling

13 between the C at position Cα and the vinylic proton at Cβ position. These signals were confirmed by 2-D heteronuclear shift correlation experiments with 13C directing observation. One-dimensional 1H and 13C NMR (acquired in the same buffer), mass spectrometry, and thin-layer chromatography confirmed both the identity and purity of the compound, so the multiple peaks were not caused by the presence of impurities. Upon incubation with a molar excess of PTP1B, all of the free inhibitor signals were lost.

Instead, three new, broader cross-peaks were observed in the region of δ 7.6–7.8

(1H)/130–136 (13C) (Figure 5.6.B). As a control, the HSQC spectrum was recorded for the adduct of [α-13C]TBNS and β-mercaptoethanol under the same conditions. Two cross-peaks were observed at δ ~5.2/~46 and δ ~5.1/~46 but not in the region of δ

~7.7/~133, consistent with the conjugate addition of the thiol group to the Cα position and loss of the C=C bond (Figure 5.6.C). These results indicate that PTP1B interacts with

TBNS in a manner entirely different from β-mercaptoethanol. In fact, the chemical shift values of the Cα carbon (δ 130–136) suggest that, in the E•I* complex, the Cα carbon of

TBNS is still sp2 hybridization state and the C=C bond is retained. The presence of three cross-peaks for the E•I* complex was caused by the fact that the recombinant PTP1B was a mixture of multiple isoforms (see Discussion).

105 5.3.6 Effect of Active-Site Mutations on E•I* Complex

PTPs share a highly conserved active site, which contains the signature motif,

(H/V)C(X)5R(S/T). The cysteine residue (Cys-215 in PTP1B) is the catalytic nucleophile, whereas the arginine (R221 in PTP1B) is critical for substrate binding and transition state stabilization [30, 130]. In addition, a conserved aspartate (Asp-181 in PTP1B) on a mobile loop acts as a general acid/base during catalysis [43, 44]. To test the role of these residues in inhibitor binding, we performed the above HSQC experiments with D181A,

R221A, C215S, and C215A mutant forms of PTP1B. D181A and R221A mutants retained the ability to bind to TBNS and produced the same cross-peaks in their HSQC spectra as WT PTP1B (Figure 5.7.A and B). The weaker HSQC signals suggest that

TBNS binds less tightly to these mutants (as compared to WT PTP1B). In contrast, the

C215A and C215S mutants failed to show any of the three cross peaks (Figure 5.7.C).

Surprisingly, the peaks corresponding to the free inhibitor were also absent. These results indicate that Cys-215 is critical for TBNS binding; its mutation either abolished inhibitor binding or greatly reduced its binding affinity. The absence of free inhibitor signal is most likely due to conjugate addition of nucleophilic residues on PTP1B surface (e.g., cysteines, lysines) to the benzylic position of TBNS. Reaction with multiple residues would scatter the signals to levels beyond detection. To further evaluate the role of Cys-

215, we mutated it into an aspartate. Earlier study has shown that the C215D mutant of

PTP1B possesses weak but yet easily detectable catalytic activity toward pNPP (25). We repeated the HSQC experiment with this mutant and observed two weak peaks at δ

~7.6/~130 and δ ~7.8/~136, the same region where the three cross peaks were observed for the WT enzyme (Figure 7D). Thus, the C215D mutant also retains the ability to bind 106 TBNS. The same conclusions were also independently reached by electrosporay ionization mass spectrometric analysis of the mutants complexed with TBNS.

5.4 Discussion

In this work, we have demonstrated that TBNS and its derivatives act as neutral pY mimetics and slow-binding inhibitors of PTPs. In the absence of free thiols or other nucleophilic species that reacts with the Michael acceptor, these compounds exhibited respectable potency, with IC50 values in the low micromolar range. Considering their small sizes, the compounds probably only interact with the PTP active site. Thus, the observed potency is quite remarkable and may be further improved by tethering the pY mimetics to peptides, peptidomimetics, or small molecules that interact favorably with the surfaces near the PTP active site. Since the active site of PTPs is highly conserved and all of the compounds tested showed essentially equal potencies against PTP1B, SHP-

1, and Yop, we expect the TBNS derivatives to be effective against all PTPs. One drawback of the current TBNS derivatives is that they readily react with free thiols

(which are present in the cytoplasm of eukaryotic cells) and possibly other nucleophiles, resulting in dramatic reduction of their PTP inhibitory activity. However, the addition of thiols to TBNS is readily reversible. It should be possible to design TBNS analogues that are less prone to nucleophilic addition by thiols and therefore more available for PTP inhibition.

On the basis of experimental data describe above, we propose the following mechanism of inhibition by TBNS (Figure 5.8). TBNS first binds to the active site of

PTP and forms a noncovalent complex, E•I. During the conversion of the E•I complex to

107 the tighter E•I* complex, the active-site thiolate anion (Cys-215 in PTP1B) reacts with the TBNS nitro group to form a covalent adduct. Nucleophilic addition of sulfides to aliphatic and aromatic nitro compounds is well documented in the literature and is the first step in the Zinin reduction of nitro compounds into their corresponding amines

[131]. This mechanism is consistent with all of the experimental results. First, it provides a sensible explanation for the slow-binding behavior, as the chemical step is reversible and time-dependent. Second, mutation of Cys-215, which is the catalytic nucleophile for

PTP reaction and the proposed nucleophile attacking the nitro group, into either alanine or serine abolished both PTP activity and TBNS binding (Figure 5.7C). On the other hand, substitution of aspartate for Cys-215 retains partial catalytic activity [40] and the ability to bind TBNS (Figure 5.7D). This indicates that a powerful nucleophilic side chain at residue 215 is critical for inhibitor binding. In contrast, mutation of two other catalytic residues, Arg-221 and Asp-181, weakened but did not abolish the binding by

TBNS (Figure 5.7A and B). Third, HSQC spectra of the E•I complex revealed cross-

13 peaks at δ ~7.7/132. The C chemical shift values indicate that the Cα carbon is still in the sp2 hybridization state in the E•I* complex (the C=C bond is intact). Therefore, unlike its reaction with free thiols, binding of TBNS to the PTP1B active site involves no nucleophilic addition to the C=C bond. The upfield shift of the cross-peaks upon binding to PTP1B may be accounted for by the fact that addition of Cys-215 (or Asp-215) to the nitro group reduces the electron-withdrawing ability of the nitrogen and therefore the inductive effect on the Cα carbon and proton.

The above mechanism does not explain the presence of three cross-peaks in the

HSQC spectra. We noticed that these cross-peaks are remarkably similar to those 108 produced by the E•I* complex formed by PTP1B and peptidyl cinnamaldehyde inhibitors

(chapter 2). In the latter case, the cinnamaldehyde was labeled with 13C at the aldehyde carbonyl and the cinnamaldehyde forms an enamine adduct with the active-site arginine-

221 of PTP1B. It was proposed that the enamine could adopt different diastereomer forms, which gave different HSQC cross-peaks. However, the E•I* complex formed by

PTP1B and TBNS cannot assume these different diastereomeric forms. An alternative explanation is that the recombinant PTP1B protein may be a heterogeneous mixture of multiple species. Two-dimensional SDS-PAGE analysis of freshly prepared PTP1B samples revealed that it contained four different variants, all of which had molecular masses of ~37 kD but different pI values (5.98, 6.09, 6.24 and 6.39) (Figure 5.9). ESI-MS analysis of the free protein showed four peaks with molecular masses of 37,310, 37,335,

37,354, and 37,435 (data not shown here). Both SDS-PAGE and MS analyses gave a similar estimate for the relative intensities of the four species: MW 37,310 (100%), MW

37,335 (60%), MW 37,354 (25%), and MW 37,435 (15%). Analysis of multiple PTP1B samples purified at different times reproducibly gave the same pattern. Note that the intensity ratio is similar to that found for the three cross peaks in the HSQC spectra

(Figure 5.6). The peak at m/z 37,310 probably represents the unmodified PTP1B

(calculated molecular mass of PTP1B is 37,312), whereas the nature of modification in the other three species is yet unknown. MS analysis of the PTP1B-TBNS complexes showed that all four PTP1B variants were capable of binding TBNS.

In conclusion, we have discovered TBNS and its derivatives as a new class of reversible, covalent PTP inhibitors. Its electronically neutral nature may provide good membrane permeability for this class of inhibitors. Further studies to improve their

109 stability against thiols and their potency and selectivity are already underway in this laboratory.

110

IC50 (μM)

PTP1B SHP1(ΔSH2) Inhibitor No β- 1 mM β- 1 mM β- mercaptoethanol mercaptoethanol mercaptoethanol

27 2.5 ± 0.3 400 ± 133 423 ± 82

28 4.5 ± 0.5 270 ± 120 375 ± 75

29 3.0 ± 0.5 225 ± 25 ~340

30 2.7 ± 0.2 425 ± 75 ~450

31 23 ± 7 515 ± 65 225 ± 75

32 28 ± 8 390 ± 90 ~450

33 NA NA NA

34 1.4 ± 0.4 275 ± 25 ~350

Table 5.1. Inhibition constants of TBNS derivatives against PTP1B and SHP-1(ΔSH2)

111

NO2 NO2 NO2

CH3 OCH3

R OCH 31 3 R = 32 27 H 28 OCH3 29 CH3 NO2 30 CO2H NO2

O N Gly-Glu-Glu H 33 34

Figure 5.1. Structures of TBNS derivatized PTP inhibitors

112

A 0.7 0.6 0 min 0.5 30 min 0.4 0.3

Absorbance 0.2 0.1 0 280 320 360 400 440 Wavelength (nm) B 0.35 TBNS only 0.30 TBNS + RSH + HgCl 0.25 2 TBNS + HgCl 0.20 2 TBNS + 1 eq RSH 0.15 TBNS + 2 eq RSH Absorbance 0.10 0.05 0.00 280 320 360 400 440 Wavelength (nm)

NO2 NO2 RS RS

R' R'

Figure 5.2. Absorption spectra of TBNS in the absence (A) and presence of free thiols (B). UV/Vis absorption of 20 μM of TBNS was monitored in a range of 500 nm to 270 nm. All reactions are performed in the assay buffer in the presence of 0, 20, or 40 μM of β-mercaptoethanol. Excess amount of HgCl2 (200 μM) was added to quench β- mercaptoethanol. C. Reaction of thiols with TBNS.

113

0.7 [I], μM A 0 0.6 10 0.5 20 0.4 40 0.3 70 0.2 100 200

Absorbance (405 nm) Absorbance (405 0.1 400 0 0 100 200 300 400 500 600 700 Time (s) 0.30 B 0.25 a

0.20 b 0.15 c 0.10

0.05 Absorbance (405 nm) (405 Absorbance 0.00 0 200 400 600 800 Time (s)

Figure 5.3. Slow-binding inhibition of PTP1B by TBNS. (A) Reaction progress curves (monitored at 405 nm) for the hydrolysis of pNPP (1 mM) by PTP1B (0.05 μM) in the presence of indicated concentrations of TBNS (PTP1B was added as the last component). (B) Reaction progress curves for hydrolysis of pNPP by reactivated PTP1B. PTP1B (0.1 μM) was preincubated with varying concentrations of TBNS (0–400 μM) for 2 h before being diluted 40-fold into reaction buffer containing 1.0 mM pNPP. a, 20 μM TBNS. b, 40 μM TBNS; and c, 50 μM TBNS.

114

0.25 A 0.20 ) -1 0.15 (min 0.10 obs k 0.05

0.00 5101520 [pNPP] (mM)

0.25 B 0.20 TBNS only

0.15

0.10 TBNS + IDA 0.05 IDA only Absorbance (405 nm) Absorbance (405 0.00 0 100 200 300 400 500 600 700 800 Time (s)

Figure 5.4. Competition for PTP binding between TBNS and pNPP or IDA. (A) Effect of substrate concentration on the rate of onset of PTP inhibition by TBNS. The line was fitted to the data according to the equation: kobs = kmax/(1 + [pNPP]/KM). (B) Protection of PTP1B from IDA-mediated inactivation by TBNS. PTP1B (5.5 μM) was preincubated with TBNS (100 μM) for 1 h before IDA (2 mM final concentration) was added. After 20 min, the remaining PTP activity was assayed with pNPP (10 mM) as substrate.

115

0.5 0 min

0.4 2 min 4 min 0.3 6 min 16 min 0.2

Absorbance 30 min 0.1

0 280 320 360 400 440 Wavelength (nm)

Figure 5.5. Effect of PTP1B on the absorption spectra of TBNS. PTP1B (40 μM) and TBNS (50 μM) were rapidly mixed in a quartz cuvette and the absorption spectra of the mixture were recorded every 2 min.

116

Figure 5.6. HSQC spectra of [α-13C]TBNS (compound 35) in the absence and presence of PTP1B or free thiol. (A) 600 μM [α-13C]TBNS only; (B) 750 μM [α-13C]TBNS and 1.8 mM PTP1B; and (C) 600 μM [α-13C]TBNS and 2 mM β-mercaptoethanol.

117

Figure 5.7. HSQC spectra of [α-13C]TBNS (750 μM) in the presence of various PTP1B mutants (each at ~1.5 mM). (A) D181A; (B) R221A; (C) C215S; and (D) C215D.

118

S– O O S– N S O OH O O N N

KI k5 +

R R R

E I E•I E•I*

Figure 5.8. Proposed mechanism for the slow-binding inhibition of PTPs by TBNS derivatives.

119

pI = 5.98 6.09 6.24 6.39

37 KDa

Figure 5.9. 2-D SDS-PAGE analysis of freshly prepared PTP1B. The protein identities of the each spot were confirmed by in-gel trypsine digestion folled by MS analysis.

120

CHAPTER 6

DETERMINATION OF THE SUBSTRATE SPECIFICITY OF METHIONINE

AMINOPEPTIDASE BY COMBINATORIAL PEPTIDE LIBRARY SCREENING

6.1 Introduction

In living cells, protein synthesis is beginning with either methionine or N- formylmethionine [132]. In prokaryotes, after the removal of N-formyl moiety by peptide deformylase (PDF), the normal methionine is remained at the N-terminus of the proteins as in the eukaryotes [133, 134]. However, in both cases, only some of the proteins retain the methionine at their N-termini because the initial methionine is removed by enzymatic while the proteins are being synthesized [133, 134]. Methionine aminopeptidase (MetAP) is the enzyme that removes the N-terminal methionine by using a divalent metal ion as a cofactor [135-138]. Based on sequence alignment, two classes of MetAP exist (MetAP1 and MetAP2), and, so far, it is known that the substrate specificities of the both MetAPs are determined by the identity of the residue adjacent to the methionine [139-141]. In most cases, MetAP can completely hydrolyze the N-terminal methionine only when the adjacent residue bears a small side chain (e.g. Gly, Ala, Pro, Ser, Cyt, Thr, and Val)

[133].

121 In prokaryotes and yeast, complete deletion or inhibition of the all MetAP genes or MetAP genes products has been shown to be lethal [142, 143], while yeast cells deleted one of the MetAP genes can grow at a slower rate [141]. In human, both MetAP1 and MetAP2 exist [140], and recent findings suggested that the proliferation of the human endothelial cells can be blocked by the inhibition of the MetAP2 with the natural product, like fumagillin [144, 145]. Because proliferation of the endothelial cells plays a critical role in angiogenesis process, the MetAP2 provide a novel anticancer target [146].

As mentioned earlier, the specificity of the MetAP was relatively broad and it has been believed that two distinct classes of the MetAP share the similar specificities.

However, it is also true that no systematic researches have been performed to determine the MeaAP specificity in detail so far. In fact, the specificity of the enzyme can be used to not only find out its novel substrates in vivo, but also design its specific inhibitor; therefore, determination of the substrate specificity of the enzyme is one of the most important aspects in enzymology. Recently, our lab successfully determined the binding specificities of SH2 domains by screening the combinatorial pY peptide library [124,

147]. Here, we designed and synthesized new combinatorial peptide library by using 1-

(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) linker [148, 149] to try to determine the substrate specificity of the MetAP in detail.

6.2 Experimental Procedures

6.2.1 Materials

All peptide synthesis reagents, including Fmoc-protected amino acids, 2-(1H- benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-

122 hydroxybenzotriazole (HOBT), and resins were purchased from Advanced Chemtech

(Louisville, KY), Peptides International (Louisville, KY ), or Novabiochem (Darmstadt,

Germany). Streptavidin-Alkaline Phosphatase was obtained from PROzyme (San

Leandro, CA). All other chemicals were purchased from Aldrich (St. Louis, MO). E. coli

MetAP was kindly gifted by Dr. R. L. Holz (Utah State University).

6.2.2 Synthesis of Dde Linker on the TentaGel

Dde (1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl)-linker based TentaGel S

NH2 resin (90 µm) was prepared as described in Figure 6.1.

Coupling of Glutaric anhydride onto the TentaGel (36) The resins (1.5 g, 0.39 mmole) were swelled in 20 mL dichloromethane for 30 min at room temperature. Glutaric anhydride (134 mg, 1.17 mmole) was dissolved in 20 mL anhydrous dichloromethane

(DCM), and added into the swelled resin. Diisopropylethylamine (DIPEA, ~ 75 µL, 0.43 mmole) was added to the reaction mixture, and the reaction proceeded for 3 h, after which time the reaction was repeated to insure complete reaction. After the reaction the resins were washed with DCM (20 mL × 5 times).

Preparation of Dde linker (37) 5,5-Dimethyl-1,3-cyclohexanedione (328 mg, 2.34 mmole), 4-dimethylaminopyridine (DMAP, 143 mg, 1.17 mmole), and N-(3- dimethylaminopropyl)-N’-ethylcarboiimide hydrochloride (EDC, 224 mg, 1.17 mmole) were dissolved in the minimum amount of N,N-dimethylforamde (DMF), and added into the glutaric acid attached resins which were prepared in 20 mL anhydrous DCM. The

123 reaction proceeded for 36 h, followed by washing the resins with DCM, DMF, H2O,

DMF, and DCM (20 mL × 5 times each) again.

Coupling of 1,5-Diaminopentane onto the Dde linker (38) 1,5-Diaminopentane (~ 800 mg, 7.8 mmole) was dissolved in 20 mL anhydrous DCM, and added into the resins which contains Dde-linker. The reaction proceeded for 3h, and the resins were washed with DCM, DMF, and DCM (20 mL × 5 times each). After the reaction, Kaiser Test was performed by using a small amount of the dried beads to confirm the existing of the primary amine.

6.2.3 Synthesis of the Methionine (M) Library

The peptide library was prepared on the Dde-linker based TentaGel resins prepared above. Library synthesis was performed by using standard Fmoc chemistry employing HBTU/HOBt/DIPEA as the coupling reagents. The invariant positions

(LNββK+, and N-terminal M) were synthesized with 4 equiv. of the Fmoc amino acids and coupling reaction was terminated after Kaiser Test was negative. After the Fmoc was removed from the first lysine, 3-carboxy-propyltrimethyl ammonium chloride (4 equiv.) was added to the N-terminal of the lysine residue by using the HBTU/HOBt/DIPEA coupling, which produces the permanently charged lysine (K+). After then, the lysine side chain protecting group, Boc, was removed by treating 50 % TFA in DCM (20 mL) for 30 min, and the side chain amine was used for further synthesis of the library. The random positions were synthesized by using the split-synthesis method. The coupling reaction was performed with 5 equiv. of the Fmoc-amino acids and HBTU/HOBt/DIPEA for 1h,

124 and repeated one more time by using the same conditions. To make it easy to determine the sequences by mass spectrometry, 5 % Ac-Gly was added to the coupling reaction of

Leu, while 5 % Ac-Ala was added to the reaction of Nle [150, 151]. After removal of the terminal Fmoc group, the resins were washed with DCM, and the amino acids side chains were deprotected by treating the library with 20 mL of the Reagent K (1.5 g phenol, 1 mL

H2O, 1mL thioanisole, 0.2 mL anisole, 0.5 mL triisopropylsilane, and 18 mL TFA) for

+ 1h. The resultant NH2-MXXXXLNββK -resin library (X = 18 amino acids, excluded

Lys, Arg; Met and Cys replaced by Nle and Abu; X = 16 amino acids, exclude Ser and

Thr also; theoretically 93,312 peptides; called M library) was washed with DCM (20 mL

× 5times), and treated with 5 % DIPEA in DCM to neutralize the resins. After then, the resins were washed DCM, DMF, and DCM (20 mL × 5 times) before drying for storage at -20 ºC.

6.2.4. Library Screening

The M library was screened to determine the MetAP specificity by using the colorimetric method described previously with a few modifications [147]. The overall scheme of the screening procedures is described in Figure 6.2.

MetAP Reaction

In a micro BioSpin column (1.5 mL, BioRad), the M-library bound resins (10 mg, 2.6

µmole of the peptides) were prepared by washing with DCM, DMF, H2O and the MetAP reaction buffer (30 mM HEPES, pH 7.3, 150 mM NaCl, 0.1 mM CoCl2). MetAP was

+2 prepared by pre-charging with Co in the MetAP reaction buffer containing 1 mM CoCl2

125 for 20 min at 4 °C. The prepared enzyme (8−80 μg) was added to the resins in the MetAP reaction buffer (1.5 mL) and incubated for 20 min at r.t. (or 4 °C) . After the enzymatic reaction, the resins were washed with H2O (1.5 mL × 3 times), 300 mM EDTA (aq) (1.5 mL for 10 min), and H2O (1.5 mL × 3 times). For the control reaction, the resins were incubated in the MetAP reaction buffer without adding the enzyme.

Biotinylation of the Library

N-hydroxysuccinimido biotin (NHS-biotin, 0.4 μmole, 0.15 equiv.) was dissolved in

DMF (50 μL), and added into 50 mM sodium phosphate buffer (1.5 mL, pH 7.0). And then, the NHS-biotin containing solution was added into the MetAP treated library (10 mg, 2.6 µmole, 1 equiv.) to biotinylate onto the N-terminus of the peptides, and the reaction proceeded for 20 min. After 20 min, the reaction was quenched by the addition of 1.5 mL of Tris buffer (150 mM, pH 8.5), followed by washing with H2O (5 × 1.5 mL).

Cleavage of the Met

Before cleavage with CNBr, to reduce any oxidized Met, the biotinylated resins were treated with ~ 1 mL TFA containing ammonium iodide (5 mg) and methyl sulfide (20

μL) on ice for 20 min. After 20 min, the resins were washed with H2O (5 × 1.5 mL), and

70 % TFA (aq) (2 × 1.5 mL). 70 % TFA (1.5 mL) containing CNBr (600 mg) was added to the resins and the reaction proceeded for 24 h at room temperature in the dark. After

24h, the reaction was repeated one more time with the freshly prepared CNBr solution.

After CNBr cleavage, the resins were extensively washed with H2O (10 × 1.5 mL).

126 Colorimetric Screening

The resins prepared above were suspended in 1.5 mL of the SA-AP binding buffer

(30mM Tris, pH 7.6, 100 mM NaCl, 10 mM MgCl2, 0.07 mM ZnCl2, 20 mM potassium phosphate) containing 4 μL of streptavidin-alkaline phosphatase (SA-AP, ~ 1mg/mL,

Prozyme). After 15 min of gentle inverting at 4 °C, the solution was drained and the resins were washed with H2O (6 × 1.5 mL). After then, the resins were transferred into the 12 well cell culture cluster dish (Corning, NY) in 3 × 300 μL of the SA-AP reaction buffer (30 mM Tris, pH 8.5, 100 mM NaCl, 5 mM MgCl2, 0.02 mM ZnCl2), and 100 μL of 5 mg/mL 5-bromo-4-chloro-3-indolyl phosphate (BCIP) in the SA-AP reaction buffer was added to the resins. After adding BCIP, intense blue color was developed on the positive beads in ~ 40 min, at which the staining reaction was quenched by the addition of 3 mL of 8 M guanidine-HCl (aq). The resins were transferred into the Petri dish in 6 ×

1 mL of 8 M guanidine-HCl solution. Under a dissecting microscope, the colored beads were retransferred into the 35 mm Petri dish that contains 3 mL of H2O to wash away 8

M guanidine-HCl solution, and put into the 1.5 mL eppendorf tube with a minimum amount of H2O. The picked resins were stored at –20 °C in absolute methanol (~ 1 mL).

6.2.5. Partial Edman Degradation, Cleavage and Sequencing of Peptides

Partial Edman degradation of the peptides on the beads was performed as described in the previous report by using phenylisothiocyanate (PITC) and nicotinic acid

O-succimimide (Nic-OSu) [150, 151]. After finishing degradation, the beads were transferred into 35 mm Petri dish in methanol, and individually separated into the eppendorf tube. Remained methanol was completely dried by using a SpeedVac

127 Concentrator (Savant). To cleave the peptide from the resin, 10 μL of THF containing 3

% hydrazine and 6 % H2O was added into the resin and the reaction proceeded for 1h at room temperature [148]. After the reaction, the cleavage solution was completely evaporated by using a SpeedVac Concentrator for 3h. The cleaved peptide was dissolved in 5 μL of 0.1 % TFA (aq). One μL of the peptide solution was mixed with 2 μL of 0.1 %

TFA in 50 % acetonitrile saturated with 4-hydroxy-α-cyanocinnamic acid, and spotted onto a 96-well MALDI-TOF sample plate. MALDI-TOF spectrometry analysis was performed on a Bruker Reflex III instrument in an automated manner. Sequence determination by using a mass spectrum was performed manually.

6.3 Results and Discussion

6.3.1 M Library Design and Synthesis.

Previously, we determined the sequence specificities of the SH2 domains of

SHP1, SHP2 and SHIP by using combinatorial pY-peptide library [124, 147]. To extend this methodology, we applied it to determine the substrate specificity of the MetAP, and we synthesized the new library and design new screening procedures described in Figure

6.2. Compared to the previous library screening, there are two major differences are required for the MetAP specificity screening. First, the methionine should be positioned at the N-terminus of the library to be a substrate of the MetAP, while it was located at the

C-terminus in the previous pY library to cleave the peptide for MS analysis. Second, the substrate peptides rather than the MetAP should be biotinylated for colorimetric screening by SA-AP and BCIP because the enzyme is released from the substrate peptide

128 after the catalysis. For these requirements, we designed the new library and screening procedure.

First, because we could not put the methionine at the C-terminus of the library to cleave the peptide by CNBr, we needed to find out new way to release the peptides from the beads for MS analysis. For this purpose, we decide to put the linker between the peptide and the bead to release the peptides. The linker we want to use here should be stable under both strong acid (e.g. TFA) and base (e.g. piperidine and pyridine) conditions during the screening, and the cleavage of the peptide from the linker should be easy and efficient. We chose Dde linker [148], because it was known to stable under acid/base conditions, and the cleavage method is relatively easy and fast compared to the

HMB linker and the Photo-labile linker (Advanced ChemTech, KY) which are also known to be stable under acid/base conditions. Synthesis of the Dde linker was performed as described Figure 6.1. We performed synthesis of the linker on the solid beads directly, while in the previous reports [148], the linker was synthesized in solution phase and attached to the beads. The direct solid phase chemistry we use here is more convenient; in addition it is as efficient as the solution phase synthesis in term of the loading capacity. To determine the loading capacity, Fmoc was loaded onto the resins after the coupling of 1,5-diaminopentane, and deprotected by 2 mL (× 2) of 20 % piperidine in DMF, followed by measuring the absorbance of the released Fmoc group.

Based on the Abs301 of the cleavage solution, the loading capacity is ~ 0.23 mmole/g, which is ~ 87 % compared to the unmodified TentaGel S NH2 resins.

Second, because the MetAP is released from the peptide substrate after the enzymatic reaction, we could not use the labeled MetAP as we used labeled-SH2

129 domains in the previous studies. Thus we needed a new method to be able to distinguish the good substrate peptides from the bad substrate peptides. For this purpose, we decided to label the N-terminus of the peptide with biotin, after MetAP removes the N-terminal methionine. And then, we cleave the new N-terminal residues (X for the good substrate peptides, still M for the bad substrate peptides) by CNBr. If the peptides still contains the methionine after the enzymatic reaction (bad substrates), the N-terminal methionine will be biotinylated and cleaved by CNBr at the end. However, if the N-terminal methionine is removed by MetAP (good substrates), the new N-terminal residue, X, will be biotinylated and stable under the CNBr cleavage reaction. Therefore, at the end, only the peptides that are good substrates of MetAP contain biotin at their N-terminal, and they can be distinguished from others by using the previous colorimetric screening method.

One problem here is that the side chain of Lys and Arg could be biotinylated during the

N-terminal biotinylation, we needed to exclude them from the library. The other possible concern is whether we can completely cleave the methionine from the N-terminal of the peptides by using CNBr. In fact, it has been reported that CNBr cleavage efficiency is dependent on the adjacent C-terminal residue to the methionine. Especially, it was shown that if Ser or Thr is positioned at the C-terminal of the methionine, the cleavage yield was decreased to <50 % [152]. For this reason, we decided to temporarily excluded Ser and

Thr residue from the – 1 position, and tested the rest of the library members.

+ Based on these ideas, we synthesized the M library, NH2-MXXXXLNββK -resin

(X = 18 amino acids, excluded Lys, Arg; Met and Cys replaced by Nle and Abu; X = 16 amino acids, exclude Ser and Thr also; theoretically 93,312 peptides). We put a quaternary amine to enhance the ionization efficiency of the peptide for MS analysis, β-

130 Ala to increase the flexibility of the peptide chain, and Asn to increase the solubility of the peptide. For the random positions, we excluded Lys and Arg from the random positions, and we also excluded Ser and Thr at the adjacent position to the Met because of the reasons mentioned above. Therefore, the library is composed of the 93,312 different peptides, theoretically.

6.3.2 Cleavage and Sequencing of Peptides

As previously mentioned, Dde linker is superior to HMBA linker and Photo-libile linker because it is easy to be cleaved as well as stable in the presence of TFA. After completing the whole screening procedure and the Partial Edman Degradation, the peptides was released by treating the individual bead with 3 % of the hydrated hydrazine

(1:2 = hydrazine:H2O) solution in THF (10 μL) for 1 h. Compared to the CNBr cleavage method for the previous library, it is not only faster but also can provide a simpler spectrum. In the previous case [150, 151], during the Partial Edman Degradation of the peptide, Lys and Tyr side chains also capped with nicotinic acid by Nic-OSu, resulting in two sets of peptide ladder (M and M + 105) for Lys or Tyr containing peptides. However, because the side chain capping of Tyr can be hydrolyzed by hydrazine, only the single set of peptide ladder is detected in MS spectrum of the Tyr containing peptide. In fact, it could be applied to the previous library simply by treating 3 % of aqueous hydrazine solution right after the Partial Edman Degradation. A typical MALDI spectrum of the M library was shown in Figure 6.3. One interesting feature in this spectrum is that there are two sets of the peptide ladder observed (M and M – 51). Although the identity of the “M

– 51” peak is still unclear, thus far, we suspect that two sets of the peaks are caused by

131 the chemical modification of 3-carboxy-propyltrimethyl ammonium chloride, because it has been observed only in the libraries that also contain 3-carboxy-propyltrimethyl ammonium chloride.

6.3.3. Screening of E. coli. MetAP

Table 6.1 shows the peptide sequences we identified from E. coli. MetAP screening, and it is seemed that the enzyme prefer small side chain containg amino acids at the position next to the methionine, as we expected. Although, because of the lack of the data set, we cannot draw any further conclusion yet, we are still keep trying to optimize the screening condition, and getting more information from it. In addition, we are also trying different MetAPs, including human MetAP I and II, to compare their specificities.

132

O O O O O O OH NH2 N DIPEA H EDC, DMAP DCM O DCM/DMF 36

OH H2N NH2 N H DCM O O

O H N NH N 2 a.a/HBTU/HOBt H O O

Figure 6.1. Synthesis of Dde linker on the TentaGel S NH2 (90 μm)

133

XXXXM-NH2 XXXX-NH2

XXXXM-NH2 MetAP XXXXM-NH2 NHS-Biotin

XXXXM-NH2 XXXXM-NH2

XXXX-NH-biotin XXXX-NH-biotin

XXXXM-NH-biotin CNBr XXXX-NH2 in 70 % TFA (aq) XXXXM-NH-biotin XXXX-NH2

XXXXM-NH-biotin XXXX-NH2

XXXX-NH-biotin

SA-AP XXXX-NH2 BCIP XXXX-NH2

XXXX-NH2

: TentaGel S NH2 (90 um)

: Dde linker

X : 18 amino acids; excluded Arg, Lys; Met and Cys replaced by Nle and Abu X, X : 16 amino acids; excluded Arg, Lys, Ser, and Thr; Met and Cys replaced by Nle and Abu; X = good substarte, X = bad substarte

Figure 6.2. Scheme of the M library screening for MetAP

134

1000 * 989.6 831.5

* 1046.6 888.6 *

1145.7

G * TG V * 0 760 860 960 1060 1160

Figure 6.3. MALDI-TOF analysis of the Partial Edman Degradation Product for peptide sequencing. The peptide sequence is deduced to NH2-MVGTG-CO2H, where N-terminal methionine was cleaved by CNBr. G – glycine; T – threonine; V – valine; * the peaks correspond to M-51.

135

MYGQS MMQVT MAMPF MVIDP MGDCM MACSG MGAQD MAQAE MCGXT

Table 6.1. Peptides Selected from E. coli. MetAP Screening. M, norleucine; bold, small side chain containing amino acids.

136

APPENDIX

137

O O O H O H H O

N/A PTP1B; 15 % at 200 μM N/A PTP1B; 50 % at 1.1 mM SHP1; 50% at 1.5 mM

O O O

NH O O

PTP1B; 50 % at > 4 mM N/A N/A

O H O H

O NH2 O H

NO2

NO2 N

N/A PTP1B; 50 % at ~ 1mM PTP1B; 50 % at ~ 1mM PTP1B; 30 % at ~ 1mM

O O S O H O OH O

H OH

PTP1B; 50 % at ≥ 600 μM PTP1B; 30 % at ~ 960 μM N/A N/A

Figure A.1. Inhibition of the PTPs (PTP1B and SHP1) by small neutral molecules

138

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