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DEVELOPMENT OF PHOTOREVERSIBLE COVALENT INHIBITORS FOR PROTEIN TYROSINE PHOSPHATASES

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

The Degree Doctor of Philosophy in the

Graduate School of the Ohio State University

By

Gulnur Arabaci, M.S.

*****

The Ohio State University 2001

Dissertation Committee:

Professor Dehua Pei, Adviser Approved by

Professor Ross E. Dalbey

Professor Ming-Daw Tsai Adviser Department of Chemistry Professor Don W. Miller UMI Number: 3022438

UMI

UMI Microform 3022438 Copyright 2001 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.

Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT

Reversible protein tyrosine phosphorylation is a key determinant in eukaryotic signaling pathways. The level of phosphorylation is balanced by the opposing activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs).

Although a large number of PTPs have been identified, elucidating the in-vivo function of any one specific PTP is still a difficult task due to the lack of specific PTP agents.

This dissertation describes the development of photo-reversible and covalent inhibitors for specific PTPs. These inhibitors are a-haloacetophenone derivatives and inactive PTPs by alkylating the conserved active site catalytic cysteine residue. The inactivated PTPs can be conveniently reactivated by irradiation with UV light. Further, this class of PTP inhibitors can permeate human cells and affect tyrosine phosphorylation levels in certain proteins.

Initially, the a-haloacetophenone inhibitors tested possessed similar inhibition potency towards different PTPs. Previous results indicated that PTP IB inhibition by cinnamates could be improved by attaching peptide based specificity elements. First, a variety of a-haloacetophenones were tested for SHP-1(ASH2) and PTP IB inhibition.

u A combinatorial approach was then developed to screen for specificity elements that

improved PTP inhibition. This involved attaching a combinatorial peptide library to the inhibitor core and screening to identify elements that improved SHP-1(ASH2) and

PTP IB inhibition.

Ill Dedicated to my parents

IV ACKNOWLEDGMENTS

I would like to acknowledge my advisor. Dr. Dehua Pei for guidance and encouragement throughout my graduate study. Dr. Mark K. Coggeshall is thanked for carrying out the in-vivo PTP inhibition experiments. I would also like to thank the members of my dissertation committee, Dr. Ross E. Dalbey and Dr. Ming-Daw Tsai. I extend thanks to all members of the Pei group (past and present) for their help and also for providing a great work environment. Their support and assistance were crucial in my success. Special thanks are due to Dr. Kirk D. Beebe for providing enzymes, intellectual discussions about research and life, and in general, being a very good fiiend. I acknowledge the help of Kari Green-Church and Nanette Kleinholz with mass spectrometric experiments.

On a more personal note, I would like to thank my close friends, Latife Sahin,

Zebra Ayhan, Eser Tufekci, Elizabeth Guo and P.T. Ravi Rajagopalan. Their support and encouragement has always been there when I needed it the most. Finally, I thank my parents, my brother and his family for their love and encouragement of my quest for higher education. VITA

January 26, 1970 ...... Bom - Istanbul, Turkey.

June, 1991...... B.S. Chemistry, Istanbul Technical University, Istanbul, Turkey.

1992-1993...... Graduate student, Istanbul Technical University, Istanbul, Turkey.

1995-1997...... M.S. Chemistry, The Ohio State University.

1997-present ...... Graduate student. The Ohio State University.

PUBLICATIONS

1. Arabaci, G., Guo, X-C., Beebe, K. D., Coggeshall, K. M., and Pei, D. (1999) J. Am. Chem. Soc. 121, 5085-5086.

2. Beebe, K. D., Wang, P., Arabaci, G. and Pei, D. (2000) Biochemistry 39, 13251- 13260.

3. Wang, P., Arabaci, G. and Pei, D. (2001) J. Comb. Chem. in press.

FIELDS OF STUDY

Major Field: Chemistry

VI TABLE OF CONTENTS

Page

Abstract ...... ii

Dedication ...... iv

Acknowledgments ...... v

Vita ...... vi

List of Tables ...... ix

List of Figures ...... x

List of Abbreviations, Symbols and Nomenclature ...... xii

Chapters:

1. Introduction ...... 1 1.1 Phosphorylation in Signal Transduction ...... 1 1.2 Classification of Kinases and Phosphatases ...... 2 1.3 Structure and Mechanism of Protein Tyrosine Phosphatase ...... 5 1.4 Autoregulation and Biological Functions of SHP-1 and SHP-2 ...... 9 1.5 Inhibition of Protein Tyrosine Phosphatases ...... 12

2. Characterization of Photoreversible and Covalent Inhibition of Protein Tyrosine Phosphatases ...... 23 2.1 Introduction ...... 23 2.2 Experimental procedures ...... 25 2.2.1 Materials ...... 25 2.2.2 Purification of SHP-1 and SHP-1 (ASH2) ...... 25 2.2.3 Purification ofVHR phosphatase ...... 27 2.2.4 Activity assay for phosphatases ...... 28 2.2.5 Time dependent inhibition of phosphatases ...... 28 2.2.6 Determination of the reaction rate of Ic with free thiols ...... 31

vu 2.2.7 Mass spectrometric analysis of SHP-1 (ASH2) ...... 31 2.2.8 PTP reactivation ...... 33 2.2.9 Photolysis product determination ...... 34 2.2.10 Inhibition of B Cell PTPs ...... 35 2.3 Results ...... 36 2.3.1 Kinetics of time dependent inactivation of PTPs by a-haloaceto phenones ...... 36 2.3.2 Mode of PTP inhibition by a-bromoacetophenones ...... 38 2.3.3 Photo-reactivation of a-bromoacetophenone inactivated PTPs . 40 2.3.4 Analysis of photolytic reactivation ...... 41 2.3.5 Membrane permeability of a-bromoacetophenone ...... 43 2.4 Discussion ...... 44

3. Developing Specific PTP Inhibitors ...... 62 3.1 Introduction ...... 62 3.2 Experimental procedures ...... 63 3.2.1 Materials and general methods ...... 63 3.2.2 Biotinylation of proteins ...... 64 3.2.3 Library construction ...... 65 3.2.4 Library Screening and MALDI peptide Sequencing ...... 67 3.2.5 PTP inhibition assay ...... 69 3.3 Results ...... 69 3.3.1 Inhibition of SHP-1(ASH2) and PTPIB by a-haloketones ...... 69 3.3.2 Combinatorial approach to improve specificity of PTP inhibition 72 3.4 Discussion ...... 76

4 Summary and Conclusion ...... 96

Appendix ...... 98 A Substrate specificity of peptidedeformylases 98 B Substrate specificity of SHP-1 (ASH2) 109

List of References ...... 113

viu LIST OF TABLES

Table Page

2.1 Structures of a-haloacetophenone inhibitors ...... 49

2.2 Kinetic constants of PTP inhibition by la -d ...... 52

3.1 Kinetic constants of SHP-1(ASH2) by 1-14 81

3.2 Kinetic constants of PTP inhibition by 15-20 83

3.3 Kinetic constants of PTPs inhibition by aldehydes ...... 84

3.4 Sequences of peptides selected from Library 2 by SHP-1 (ASH2) .... 89

3.5 Sequences of peptides selected from Library 2 by PTPIB ...... 91

3.6 Sequences of peptides selected from Library 3 by SHP-1(ASH2) .... 93

3.7 Sequences of peptides selected from Library 3 by PTPIB ...... 95

A.l Sequences selected by P. yh/cr^arzM/K PDF ...... 103

A.2 Sequences selected by 5. PDF 105

A.3 Sequences selected by £■. co/z PDF ...... 107

IX LIST OF FIGURES

Figure Page

1.1 Sequence alignment of PTPs ...... 16

1.2 Structure of the PTP catalytic domain of SHP-1 ...... 17

1.3 Catalytic mechanism of PTPs ...... 18

1.4 Kinetic scheme for phosphate ester hydrolysis by PTPs ...... 19

1.5 Domain architecture of SHP-1 ...... 20

1.6 Autoregulation model of SHP-1 ...... 21

1.7 PTPs inhibitors ...... 22

2.1 Inhibition of PTPIB by la ...... 50

2.2 Inactivation of SP-1(ASH2) ...... 51

2.3 ESI/MS spectra of SHP-1(ASH2) ...... 53

2.4 Mechanism of PTP catalysis and inactivation ...... 54

2.5 Photolytic reactivation of SHP-1 ...... 55

2.6 HPLC analysis of photochemical products ...... 56

2.7 ESI/MS spectra for photoproduct ...... 57

2.8 MALDI-mass spectra for trypsin digested peptides ...... 58

2.9 Possible photoreversible products ...... 59

2.10 Possible mechanism of photochemical reactivation ...... 60

X 2.11 Westm blot analysis o f B cells phosphoproteins ...... 61

3.1 Compounds tested for PTP inhibition ...... 80

3.2 Inhibitors for PTPs ...... 82

3.3 Library constructions ...... 85

3.4 Model for library screening ...... 86

3.5 MALDI-mass spectrometry ...... 87

3.6 Specificiy of SHP-1 (ASH2) with library 2 88

3.7 Specificity of PTPIB with library 2 90

3.8 Specificiy of SHP-1 (ASH2) with hbrary 3 92

3.9 Specificity of PTP IB with library 3 94

A.l Specificity o f/*.yh/cipamm PDF ...... 104

A.2 Specificity of 5. PDF 106

A.3 Specificity of £■. co/z PDF ...... 108

XI LIST OF ABBREVIATIONS, SYMBOLS AND NOMENCLATURE

 Angstroms (10 '° meters) AMPSO 3-[(l, l-Dimethyl-2-hydroxyethyl)amino]-2-hyciroxypropanesulfonic acid BSA Bovine serum albumen B. subtilis Bacillus subtilis °C Degrees Celsius (temperature) PDF Peptide deformylase Da Daltons E. coli Escherichia coli EDTA Ethylenediaminetetraacetic acid EGFR Epidermal growth factor receptor ESI Electrospray ionization mass spectrometry f-MX N terminal formylated peptides FDH Formate dehydrogenase Fmoc 9-fluorenylmethyloxycarbonyl FPLC Fast protein liquid chromatography g Grams h Hours HBTU 2-( 1 H-benzotriazo 1-1 -y 1)-1,1,3,3 -tetramethyluronium hexafluoro phosphate HEPES N-[2-hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid] (His)ô Repeat of six histidine residues HOST 1 -hydroxybenzotriazole HPLC High performance liquid chromatography IC50 Inhibitor concentration required to reduce reaction rate by 50 % IPTG Isopropyl-P-D-thiogalactopyranoside Ki Equilibrium inhibition constant Kd Equilibrium dissociation constant k c a t Turnover number or the number of substrate molecules converted to product by an enzyme molecule per unit time (units = sec ') Km Michaelis constant, equal to the substrate concentration at half maximal rate (Vmax/2) (units = concentration units) k c a t / K m Specificity constant (units = M"'s ') L Liters EAR Leukocyte antigen related

XU LB Luria Bertani growth medium MALDI MS Matrix assisted laser desorption mass spectrometry MES 2-[N-MorphoIino]ethanesuIfonic acid min Minutes NAD^ Nicotinamide adenine dinucleotide (oxidized form) nm Nanometers NMR Nuclear magnetic resonance pH -Iog[Hl pKa -l0g[Ka] pY Phosphotyrosine PTP Proteintyrosinephosphatase PTK Proteintyrosinekinase [S] Substrate concentration SDS-PAGE Sodium dodecyl sulphate — polyacrylamide gel electrophoresis SH2 Src homology 2 TCEP T ris-(2-carboxyethyl)-phosphine TOP Time of flight Tris T ris-(hydroxymethy l)aminomethane UV-Vis Ultraviolet — Visible absorption spectroscopy V Specific activity (units = nmol/pg/min) Maximum rate when enzyme is saturated with substrate (dependent on enzyme concentration) VHR Vaccinia virus opened reading frame HI WT Wild type

xm CHAPTER 1

GENERAL INTRODUCTION

1.1 Phosphorylation in Signal Transduction

Enzymes are macromolecular proteins that contain amino acids joined by amide linkages. Each enzyme has a genetically mandated and unique primary structure, which results in a unique three-dimensional structure of precise orientation that possesses remarkable catalytic activity. Enzymes are the major agents that effect controlled chemical changes in biological systems. Organisms ranging from simple bacteria to mammals utilize enzymes in signal transduction processes to survive in changing environments (39). Signal transduction processes are involved with numerous mechanisms that provide reversible, continuous and dynamic regulation of intracellular pathways such as mitogenesis, motility, growth, metabolism, immune response and gene transcription (8, 9). A great majority of signal transduction pathways are mediated by covalent modification of various proteins by phosphorylation and dephosphorylation (10). These modifications are responsible for the activation, inactivation or localization of various proteins (12, 17, 18). Often these

1 changes in proteins cause the signal to be amplified, suppressed, interrupted or transferred to a specific region within the cell (20). Most importantly, understanding the mode of action o f enzymes involved in critical signaling pathways is often cmcial in studying abnormalities and diseases in living systems (39, 40). Typically, after isolating the proteins of interest important areas of enzyme characterization include delineation of substrate specificity, studying the kinetic mechanism and subsequently designing inhibitors (42). In eukaryotes, covalent modification of proteins by phosphorylation and dephosphorylation occur on specific amino acid residues namely serine, threonine, tyrosine and are mediated by protein kinases and phosphatases (9-

11). Protein kinases are enzymes that catalyze the transfer of the y-phosphoryl group of ATP to the specific amino acid residue concerned (30), whereas phosphatases reverse the effect of the kinases by removing the phosphate group firom phosphorylated cellular substrate proteins (9-10). The focus of this thesis is the development and refinement of specific photo reversible inliibitors for protein tyrosine phosphatases to assist in biological studies of these enzymes as well as evaluating their therapeutic potential.

1.2 Classification of Kinases and Phosphatases

Phosphate ester formation and hydrolysis are among the most crucial chemical reactions carried out by living organisms (18). Polymerization of our genetic material namely DNA involves these reactions as do the activation of substrates in many critical biosynthetic pathways (9). Phosphate ester chemistry is also central to the properties of biological membranes and often mediates key interactions between macromolecules (10-12). Phosphorylation and dephosphorylation regulate numerous biological processes and are catalyzed by protein kinases and phosphatases respectively (10-12, 33). In eukaryotic organisms, protein kinases and phosphatases are primarily classified based upon their substrate specificity (33). For protein kinases the classification system defines two groups: the serine/threonine kinases and the tyrosine kinases (33). In spite of differences in substrate specificity, the eukaryotic protein kinases share significant amino acid sequence homology as well as three- dimensional stmcture similarity (30, 33). In contrast, phosphatases present a diverse family that may be broadly classified into four groups: the non-specific phosphatases, the serine/threonine phosphatases, the protein tyrosine phosphatases (PTPs) and dual specificity phosphatases. These groups lack sequence or structure homology with each other (10-12, 30-33).

The non-specific phosphatases include enzymes such as the alkaline phosphatases and the acid phosphatases (10-12). These enzymes hydrolyze both proteinaceous and non-proteinaceous phosphate monoesters (33). The non-specific phosphatases typically function in degradative and catabolic pathways and are believed to serve as phosphate scavengers (10, 11, 33). From a mechanistic point of view, phosphate ester hydrolysis by alkaline phosphatases proceed through a phosphoserine intermediate while hydrolysis by some acid phosphatases proceed through a phosphohistidine intermediate (10). The serine and threonine phosphatases constitute a distinct class in which the

catalytic mechanism does not appear to involve a phosphoenzyme intermediate (37).

They require divalent metal ions for their function (33, 37). These enzymes have high

sequence homology in their catalytic domains but differ in the substrate specificity and

binding of their regulatory domains (37). They are often regulated by complex

mechanisms and have multiple biological functions. These include a wide range of

cellular processes, like cell cycle progression, protein synthesis, transcription regulation and neurotransmission (33, 39, 83, 84).

Protein tyrosine phosphatases or PTPs represent the third class of phosphatases

(8) and are further divided into two groups based on their cellular location: receptor like PTPs and intracellular PTPs (33, 39). In general, receptor like PTPs include an extracellular domain of variable length and composition, a single membrane-spanning region and one or two intracellular catalytic domains. The members of this group include CD45 and LAR (10-12, 33). The intracellular PTPs typically contain a single catalytic domain and various amino and carboxy terminal extensions. These extensions when present are believed to have targeting or regulatory functions.

Members of this group include PTPIB, TCPTP, PTPHl, SHP-1, SHP-2, PTP-PEP and

PTP-PEST (10-12).

The dual specificity phosphatases constitute a special class that share the greatest sequence similarity with the product of vaccinia virus opened reading frame

HI (64, 96). These enzymes are unique among PTPs in their ability to utilize phosphoserine and phosphothreonine in addition to phosphotyrosine as substrates (33, 45). They contain the same structural domains similar to all intracellular PTPs (64).

VHR and PAC-1 are the well-known members of this class (33). Without exception, all PTPs contain the active site signature motif (I/V)HCXAGXGR(S/T)G which contains the catalytic cysteine residue involved in the formation of the phosphoenzyme reaction intermediate (33, 45).

Functionally, these intracellular PTPs regulate cell growth and proliferation, cell cycle and cytoskeletal integrity in response to a variety of external stimuli (18, 40,

80). In-vivo, the relative level of tyrosine phosphorylation is balanced by the opposite actions of protein tyrosine kinases and PTPs (8). PTPs can function as tumor suppressors in some systems and have a demonstrated role in cell cycle regulation as well as T-cell activation (39). PTPs also play a role in bacterial pathogenesis (39). The

Yersinia genus, which is responsible for the bubonic plague or Black Death, encodes a

PTP that is essential for virulence and pathogenesis (40). These observations summarize the importance of the PTPs being examined in this dissertation. The thesis is focused mainly on two representative intracellular PTPs namely SHP-1 and PTPIB.

1.3 Structure and Mechanism of Protein Tyrosine Phosphatases

More than 100 PTPs had been identified to date (33) and genomic sequencing predicted that the human genome codes for 56 PTPs (104). Intracellular protein tyrosine phosphatases typically contain a PTP catalytic domain flanked by N and C terminal domains that have targeting and regulatory functions (11, 45). While the sequence homologies of the regulatory domains are low, significant homology exists among the catalytic domains of the PTPs (10-12). The sequence alignment of the

catalytic domains of SHP-1 and other intracellular PTPs are shown in figure 1.1 (72,

73). The catalytic domains are typically 250 amino acid residues long and contain the

signature motif (I/V)HCXAGXGR(S/T)G which contains the catalytic cysteine

residue (11, 27). Other areas of significant homology like the WPD loop are also displayed (73). Figure 1.2 shows the structure of the catalytic domain of SHP-1 in which the position of the catalytic cysteine, the WPD loop and the amino and carboxy termini are displayed (72, 73). The structure of the catalytic domain of SHP-1 can be divided into a core region and the extended N and C termini (72, 73). The structure contains 12 (3 strands and six a helices in which a highly twisted (3 sheet is formed by ten P strands spanning the entire core region. In addition four a helices form a four- helix bundle and are flanked over one side of the twisted sheet and the other two helices are located on the other side (72, 73).

Although the catalytic domain of SHP-1 has low sequence identity with the catalytic domain of PTPIB, its three-dimensional structure closely resembles that of

PTPIB (72, 73). In contrast to the PTPIB structure, the catalytic domain of SHP-1 has several insertions and deletions that occur mainly on surface loops (72). The binding site for phosphotyrosine is a deep pocket on the surface of the protein (72, 73). The active site formed by the PTP signature motif is located at the bottom of the phosphotyrosine binding pocket (72, 73). The structure also suggests that SHP-1 prefers substrates with acidic residues N terminal to the phosphotyrosine residue (73).

The position of the WPD loop is shown in figure 1.2 (73). The WPD loop was previously identified as part of the phosphotyrosine binding pocket (72). Comparison

of the catalytic domain stmcture of SHP-1 with other PTPs has identified a novel

confonnation change of the WPD loop after substrate binding relative to other PTPs

(72). The WPD loop is beheved to play a critical role in positioning the aspartate

residue, which acts as a general acid and general base during catalysis (7). The

movement of this WPD loop most probably depends on the identity of the amino acid

after the critical aspartate residue (72).

The structural details listed above have contributed to our understanding of the

catalytic mechanism of SHP-1 and PTPs in general. The catalytic mechanism

employed by SHP-1 and other PTPs is shown in figure 1.3 (24, 27). The kinetic

scheme for the reaction is shown in figure 1.4 (27, 33). Protein tyrosine phosphatases

employ nucleophihc catalysis to hydrolyze phosphotyrosyl peptides. The catalytic

nucleophile (cysteine-453 in SHP-1) is located at the bottom of a deep cleft in the binding pocket (72). This cysteine residue has been shown by x-ray crystallographic and chemical studies to be properly positioned for nucleophilic attack on the substrate phosphate (72).

Catalysis is initiated when the substrate binds to the enzyme and the binding is stabilized by the conformation change that brings a cmcial aspartate residue

(aspartate-419 in SHP-1) closer to the oxygen of the leaving group (22, 27). This aspartate residue has both general acid and general base roles in catalysis (27). The cysteine thiolate then attacks and cleaves the substrate, resulting in departure of the leaving group (dephosphorylated tyrosine) and transfer of the phosphate to the nucleophilic cysteine residue, thus forming a S-phosphocysteinyl covalent enzyme

intermediate (27). Next, the same aspartate residue that acted as a general acid in the

first step now acts as a general base by activating a water molecule for hydrolysis of the phosphoenzyme intermediate (27). The intermediate is thus hydrolyzed releasing

firee enzyme and inorganic phosphate. The conserved arginine residue in the PTP

signature motif plays a role both in substrate recognition and transition state stabilization in the first step of catalysis. The conserved serine and threonine residues in the PTP signature motif function in the breakdown of the phosphoenzyme intermediate (22, 27, 45).

From a kinetic point of view, the rate-limiting step of most PTPs is the breakdown of the phosphoenzyme intermediate (figure 1.4) (22). Often, the formation of the phosphoenzyme intermediate is much more rapid than its breakdown resulting in the observation o f a pre-steady state burst of product release in stopped-flow kinetic experiments (71). Rapid quench experiments with ^^P containing substrates have also demonstrated the existence of covalent phosphocysteine enzyme intermediates (22,

45).

The importance of aspartate-419 has been demonstrated in work presented towards my Master’s thesis. The kinetic properties o f selected SHP-1 mutant enzymes were determined with substituted phenyl phosphate substrates. The kcat values of the

H420Q and D419E mutant enzymes vary only 30 fold while the pKa values of the substrate leaving groups varied by almost three pH units. In contrast, the kcat values of the D419A mutant enzyme for the same substrates varied 900-fold suggesting that the

8 mutant enzyme had a different rate-limiting step. Namely, the rate-limiting step became the formation of the phosphoenzyme intermediate in contrast to the wild-type enzyme where the rate-limiting step is the breakdown of the phosphoenzyme intermediate. Other work done towards my Masters thesis include studying the inactivation of SHP-1 by iodoacetic acid derivatives. These compounds inactivate phosphatases by alkylating the essential cysteine residue. These studies laid the groundwork for the experiments presented in this dissertation, since the mode of inactivation of phosphatases by a-haloacetophenones are chemically and kinetically similar to that observed with iodoacetic acid derivatives.

1.4 Autoregulation and Biological Functions of SHP-1 and SHP-2

SHP-1 and SHP-2 are members o f a subfamily of cytosolic PTPs that contain two Src homology 2 (SH2) domains N-terminal to their catalytic domain (figure 1.5)

(13, 20). SH2 domains were originally identified among Src-like PTKs, but have now been found in only two of PTPs (13, 18, 41). These domains are approximately 100 amino acid residues long and were originally observed as conserved domains among cytoplasmic PTPs (20). SH2 domains were shown to bind phosphotyrosine residues important in protein-protein interactions in cell signaling events (21, 26). Both SHP-1 and SHP-2 are recruited to various cell surface receptors through specific interactions between their SH2 domains and phosphotyrosyl sites on the receptors (20, 21, 41, 45).

SHP-2 is expressed in most cell types and is involved in signaling stimulated by various ligands like growth factors, cytokines and hormones (57, 58). The C- terminally phosphorylated SHP-2 binds to Grb2 and is believed to play a role in

activation of the ras and mitogen activated protein kinase pathways (20, 57-58).

Therefore, SHP-2 acts as a positive regulator of cell proliferation (58, 92). Further, the

N-terminal SH2 domains of SHP-2 bind to phosphorylated growth factor receptors and

insulin receptor substrates and elevate the PTP activity of SHP-2 (56).

In contrast to SHP-2, SHP-1 is primarily expressed in hematopoietic and

epithelial cells (I). Like SHP-2, SHP-1 binds to growth factor receptors via its SH2

domains (2). In addition, SHP-1 has been reported to bind c-kit, the IL-3 receptor B-

chain, the erythropoeitin (EPO) receptor and the B-cell IgG Fc receptor (2, 5).

Physiologically, SHP-1 is implicated in various signaling pathways but is thought to be most important as a negative regulator of B-cell and T-cell signaling pathways (5,

39). In mutational experiments, the loss of PTP activity in SHP-1 results in sustained proliferation and a hyperactive response to antigens (40).

On a molecular level both SHP-1 and SHP-2 exhibit interesting biochemical properties. Initially SHP-1 was found to exhibit non-saturation kinetics towards both small molecule and phosphotyrosyl peptide substrates, displaying linear activity vs. substrate concentration plots (1). This behavior was later rationalized in a model, which explained the autoregulation of SHP-1 by its SH2 domains (figure 1.6) (2). The model proposed that the N-SH2 domain intramolecularly inhibits the catalytic domain activity in SHP-1 while the C-SH2 domain primarily serves to recruit the enzyme to an appropriate phosphotyrosyl peptide substrate (2, 5). The model was supported by the observation that deletion of the N-SH2 domain resulted in typical Michaehs-

10 Menten kinetic behavior in the truncated enzyme. Further, the inhibition of PTP

activity could be relieved by addition of a phosphotyrosyl peptide capable of binding

to the N-SH2 domain (2).

Structural data from x-ray crystallography also supported the model derived

from biochemical studies and yielded a specific explanation for the basis of the

auto inhibition (72). Specifically, a loop region in the N-SH2 domain of SHP-1 served

as a substrate mimic while simultaneously preventing the closure of a loop in the

catalytic domain that is critical for catalysis (5). Further, upon intramolecular binding

to the catalytic domain, the N-SH2 domain has a considerably lower affinity towards binding phosphotyrosyl peptides (1,2). The C-SH2 domain however is not involved in

autoinhibition and its phosphotyrosyl peptide binding site is entirely competent, thus

vaUdating biochemical data that proposes a role for the C-SH2 domain solely

involving recruitment of SHP-1 to an appropriate site of action (2, 5).

Dual phosphorylated peptides separated by approximately 40 A° (105) or 23-

25 amino acids most effectively bind SHP-1 (106). A scenario may be envisioned in which the C-SH2 domain recruits the enzyme to an appropriate dual phosphorylated receptor and thus cooperatively enhancing the binding of phosphotyrosyl peptides by the N-SH2 domain, which has a weakened affinity for phosphotyrosyl peptides in the

PTP bound state (5). Once the phosphotyrosyl binding site in the N-SH2 domain is occupied, inhibition of the PTP active site is relieved and catalysis significantly enhanced (5).

11 1.5 Inhibition of Protein Tyrosine Phosphatases

Protein tyrosine kinases and protein tyrosine phosphatases play important roles

in eukaryotic signal transduction as discussed in earlier sections. Comprehension of

mechanisms behind the reversible phospho-tyrosine dependent modulation of protein

function and cell physiology must thereby involve characterization of PTPs and PTKs

(8?K

In spite of the large number o f PTPs identified to date and the emerging role

played by PTPs in disease, a detailed understanding of the role played by PTPs in

signaling pathways has been hampered by the absence of PTP specific agents. PTP

specific agents could serve useful roles in determining the physiological significance

of protein tyrosine phosphorylation in complex signaling pathways and can also

provide valuable therapeutic lead compounds in the treatment of several human

diseases (89). Recently, a chemical genetic strategy that allows for the rapid functional

characterization of kinases was described (100). The strategy involved sensitizing the

protein kinase of interest to cell permeable molecules that do not inhibit wild type kinases. While this strategy allows in theory thein-vivo characterization of any kinase

involved in signaling, it requires considerable effort and investment of resources

(100). Like in the case of kinases, there is a paucity of both potent and specific phosphatase inhibitors.

The major goal of the work presented in this thesis is the development of photo-reversible and specific PTP inhibitors that could be employed in biological

12 experiments to study the role of specific PTPs in signal transduction as well as

providing possible lead compounds for therapeutic purposes. In this section, a general

summary of the literature regarding the inhibition of PTPs is presented.

Inhibitors of PTPs discovered so far can be divided into different classes namely non-specific inhibitors, inhibitors obtained by screening methods and inhibitors developed by rational structure based design. A large number of non­ specific small molecule and metal containing inhibitors of PTPs and phosphatases in general are available. The most widely used non-specific but effective PTP inhibitor is vanadate because of its high similarity to phosphate. Vanadate species are widely used pharmacologically for the non-specific inhibition of PTPs (89). It should be noted however that while being effective, vanadate inhibition is not specific to phosphatases and is also observed in a variety of enzymes such as ATPases and nucleases (101).

Other non-specific PTP inhibitors are phenylarsine oxide and gallium nitrate. The nitroso containing natural product dephostatin (figure 1.7 (89)) was shown to exhibit non-specific inhibition and subsequent studies indicated the importance of the nitroso functionality for the potency of PTP inhibition. Nitric oxide itself is known to inactivate PTPs by oxidizing the critical cysteine residue and inactivation of PTPs has been suggested as one possible mechanism for action of nitric oxide in the regulation of cellular processes (89).

In addition to dephostatin, a diverse range of PTP inhibitors have resulted firom natural product screens. In most cases, the maimer in which these compounds interact with the target PTPs are unclear, rendering structure based design of new analogues

13 difficult. These natural product inhibitors typified by nomuciferine (figure 1.7 (89))

exhibit potent inhibition of various phosphatases as demonstrated by their low IC 50

values. While these compounds display potent inhibition and moderate selectivity, a

majority of them are of limited used due to low cell membrane permeability.

Synthetic-library derived compounds have also been screened for phosphatase

inhibition (86). Combinatorial solid-phase library synthesis finds wide applicability in

the pharmaceutical industry and has been begun to yield results in the area of PTP

inhibition. Using a radio frequency tag encoded library of ciimamoyl tripeptides,

potent inhibitors of PTP IB (K,- values o f0.079 pM) have been discovered though their

specificity has not been evaluated (99).

Mechanism based irreversible inhibitors represent yet another class of PTP

inhibitors (89). Attempts have been made to design irreversible PTP inhibitors utilizing the architecture of the PTP catalytic site and the nature of the thiol mediated phosphate hydrolysis. Representative compounds such as 4-fluoromethylphenyl phosphate (figure 1.7 (89)) are envisioned to form reactive quinone methides following enzyme mediated phosphate ester cleavage (89). Subsequent attack by the enzyme nucleophile results in covalent attachment. While offering conceptually interesting approaches towards enzyme inhibition, these mechanism based irreversible inhibitors suffer from low potency and possible degradation or non-specific interaction with other cellular enzymes (89).

The final classes of phosphatase inhibitors are designed based on the substrate structure and enzyme-substrate interactions (89). Typically they are reversible and

14 competitive inhibitors of PTPs and are either peptide or non-peptide based. Peptide

based inhibitors have been designed which mimic the natural phosphotyrosyl peptide

substrate and replace the tyrosine phosphate group with non-hydrolysable or poorly

hydrolysable substituents such as thiophosphate and phosphonate groups (figure 1.7

(89)) (90-91). While many of these inhibitors have remarkable potency their use is

limited due to proteolytic susceptibility and weak membrane permeability. The design

of nonpeptide analogue inhibitors of PTP has also been attempted. While these

compounds are stable to proteolysis, they are of limited use due to poor membrane

permeability (90).

This thesis presents the development and characterization of specific and

covalent PTP inhibitors that are both membrane permeable and photo-reversible.

These inhibitors are a-bromo and a-chloroacetophenones which bind to the PTP

active site and inactivate it by forming a covalent and irreversible adduct with the

essential cysteine residue. The inactivated phosphatases can then be photo-reactivated by irradiation with 350 nm UV light. These compounds in theory could be used to provide a novel and useful class of on-off switch molecules to study signal transduction events in-vivo.

15 al

SHP-I 245 -GFVOa S ESLQXQEVlXILHQRLEGQRPKIIKOKli B i B /X lfQ G R SHP-2 246 E BTZ.QQQECXLLYSXXKGQRQSIXN10 BIPKVLHDG YPTP 164 ------REKPKTSGBHQAGKARATAPSTVSl Y SPEARAXLSSRLTTLRMTLAPAIMDf LQACGCEpŒJ G PHD:I Q C PTP la 001 ------MDfEKEFBQIDK SG-SMAX: YPDZRHBASSPPCRVAIC—LPKNKUM H2 G CKLHI[QK FTP-AX.PHA 202 SPSTMKKYPPLPVDXLEEBZNRKIIXD]lOHKLPlUn E aX LP ACPZOATCKAAS—KHPfKXJGi B£ G mziiT P V PTP-MU 079 ------AZPVADLLQBZTQNKCASGYIGFKEI Y ES:FFXC-QSAPWOSAX—RDHNMOER H^fRLQTZ

SHP - 1 2 9 5 D S N Z P G S D i d ILLGPDOI A m SCLKATVMDm * UffQ] ; Q — R M X C V I t « P C SHP - 2 2 9 4 DPMXPVSDY CFCTKCHNSKPiaCSI 3CLQMTVHDPW] y PFQ] :Q — K S X C V I ï « P D YPTP 2 3 5 R R Q T A V R A d —— — — — —R " rPLQSQLXSHPi y u h a a t |E|ZAHQRPGM PI Y fRQ PTPIB 0 6 3 DH——— 0 » —QR————SI 3PLPMTCGHPW1 G W EQK ;G— SLKCM ï «PQ PTP-ALPHA 2 7 9 CG-VPDSDY ■— K M — — K l spKXKTVMDnn y c w r q » — C C K C A ( t « P O PTP-MU 9 4 6 b g - d t n s d y | —Ptl——H1 SPMQXTXYDPWI^mHB^ G—RVKCC%yPD pio [ill wPD loop o3

SHP-1 370 V 6 MQRAYGPYSVTMCGRHDTTCYlCkTL< V SPLDNGDLZR ------8 A«HTÉ3 TLÿfP&GVPSCPG— GVLSfbQIM QR SHP-2 374 S Y A L X E Y 6 VMRVRMVXSSAAKDY 1 L R X L l C SKVOQGNTCR T . WQl l PR* WPD IGVPSOPG— GVLOl [ E C V H H K YPTP 305 SG-TYGSZTVRSXMTQQVCL 6 D 6 ZNA 1 [• Y T L I ] RXAGQKTZS ------, ? Y M v a MPI ^TAVSSCVTKALASl t D Q T A C T PTPIB 128 KKRXXKZFKDTM LKLTLZSXDZXSYYl V RQL 1 I DILTTQBTR 1 lléHMltV. MPC GVPCSPA—SPLNI £ P K V R S S PTP-ALPHA 348 QG ------CWTYOKVRVSVIDVTVLVDYI RKP( J QQVGOVTMRXPQRZ ! PQ] £ PTl WPC PGVPPTPZ— GMLKI £ R3CVXAC PTP-MU lois D ------TSZYRDZ]CVTLZXTBLLAKy\|ÿTPJVKRGVBXZR ------ffrT^WPEhCVPYHAT—QLLGl|^QV1CSK a4 SHP-1 QCSL-PHI ?qOTZZVZDKLMBNZSTKQLDClI c 1 am 01 vp kl Ç n u Ç Y-K SHP-2 ÛCSZ-HD ITPZVZDZLZDZZRCKGVDa i[\pKTI a I VE 5 c r - R YPTP 2RZ^QLIGAWCIOfDSRMS -< mm • soHFfr PTPIB G S L S -P C a ITPCLADTCLLLMDXRXDPSi PTP-ALPHA 422 MP QY ITrWZDAMLDMIBS 1 PTP-MU 1084 SP P ^SCPrVTDIMLDKABR CG%

a6

SHP-1 509 PZY% a CAQPZETTKKKLCVLQSQKGQBSCYGMITY------SHP-2 513 P IY Ï a /QHYZBTLQRRZZKBQKSICRKGHCyTMXKY------YPTP 454 LZKI a BGOGRPLLNS------PTPIB 269 PSYI a /ZSGAKFINGDSSVQDQWXELSHEDLKPPPCBZPPPPRPPXRZLEPBIf PTP-ALPHA 484 PLYC a v T f ————------——------————— ——— PTP-MU 1146

Figure 1.1. Sequence alignment of protein tyrosine phosphatases. Boxed regions indicate areas of high sequence conservation (72).

16 WPD loop Cy»453

C4eemmns

L K-tammiis

Figure 1.2. Structure of the PTP catalytic domain of SHP-1 (73).

17 " 3 Cys t) r 0 ^ 0

H o —A -P-— S~^ys

r H- ^ r O .P - S - ^ y . H _o o- AH i ? ^ Q-— S—Cys h o / " A -oV

|Sp ~S Cys AoH

Figure 1.3. Catalytic mechanism of protein tyrosine phosphatases (22).

18 k2 k3 E-SH + R0P03^-i=^ E-SHJÎ.OPO32- E-S-POg^-—p*-E-SH + Pi ROE H,07

Figure 1.4. Kinetic scheme for phosphate ester hydrolysis catalyzed by PTPs (22).

19 4 1Q2 110 210 270 512 S95 N — I N-SH2I— I C-SH2I— r FT? I C-4all |— C

Figure 1.5. Domain architecture of SHP-1. N-SH2 and C-SH2 domains are the Src homology 2 domains, PTP represents the catalytic domain and C-tail the C-terminal domain (13).

20 SM2 PTP PTP ;h 2 [smÆ /-N PTP N OH-

-C

a c t i v e pH 7.4 p H < S .S in a c tiv e a c tiv e

Figure 1.6. Autoregulation model in SHP-1 (2). At neutral pH, SHP-1 is negatively autoregulated by interaction of the N-SH2 domain with the catalytic PTP domain (in the absence of a specific phosphotyrosyl ligand). The inhibition is relieved at low pH or by engaging the N-SH2 domain with a specific phosphotyrosyl ligand.

21 OH Me MeO NH MeO

OH

dephostatin nomuciferine

. /O H :p — OH

4-fluoromethylphenyl 2-difluoromethylnaphthyl phosphate phosphonate

Figure 1.7. Protein phosphatase inhibitors (89).

22 CHAPTER 2

CHARACTERIZATION OF PHOTOREVERSIBLE AND COVALENT

INHIBITION OF PROTEIN TYROSINE PHOSPHATASES

2.1 Introduction

Signaling events regulate critical cell functions like growth, mitogenesis, cell­ cell interactions, gene transcription and the immune response (83, 85). One of the most important post-translational modification events in eukaryotic cells is the reversible phosphorylation of proteins on tyrosine residues (83). Two different enzyme families, protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) balance the level of reversible tyrosine phosphorylation in vivo (8). The defective or inappropriate operation of these enzymes is implicated in diseases such as diabetes, cancer and immune dysfunctions (45, 50). About 100 PTPs have already been identified in various organism (33) and around 56 PTPs are estimated to be coded in the human genome (104). In spite of the large number of PTPs identified to date, the role played by PTPs in signaling pathways and disease are still largely unknown due to the absence of PTP specific agents. Consequently, characterization of individual

23 PTPs and development of specific PTP inhibitors could prove useful in determining

physiological function of the specific PTP of interest in a complex cellular signal

transduction pathway as well as leading to the development of valuable therapeutic

agents for the treatment of several human diseases (89).

Efforts to develop specific PTP inhibitors have intensified recently but have

met with limited success. The following is a brief summary o f the various strategies

available for the inhibition of PTPs. Non-specific PTP inhibitors like vanadates,

phenylarsine oxide, and gallium nitrate have been known for a longtime and are still

used in some biological studies (89). These agents are known to inhibit other targets

like ATPases and nucleases (89). Mechanism based irreversible inhibitors such as 4-

fluoro-methylphenylphosphates and a-bromophosphonate analogs while offering

conceptually interesting approaches towards PTP inhibition, suffer fi’om low potency

and possible degradation or non-specific interaction with other cellular nucleophiles

(89). Phosphonate inhibitors while binding to PTPs with high affinity are limited by

poor cell membrane permeability. Natural product screen derived PTP inhibitors like

dephostatin and nomuciferine have been discovered but are difficult to develop in a

rational stmcture based manner because the manner in which they interact with the

target PTPs are unclear (89).

This chapter describes the development and characterization of specific,

covalent, photo-reversible and cell membrane permeable a-haloacetophenone

derivative inhibitors of PTPs. The time dependent inhibition kinetics of six different phosphatases (acid phosphatase, alkaline phosphatase, dual specificity phosphatase

24 VHR, prototypical phosphatase PTPIB, Src homology 2 (SH2) domain containing

phosphatase (SHP-1), and the catalytic PTPase domain of SHP-1) by several a-

haloacetophenone derivatives was studied. The mode of inhibition and photochemical

reactivation was also assessed by chromatography and mass spectrometry. Further, membrane permeability and the potential for use of these a-haloacetophenone derivative as tools in cellular studies was examined.

2.2 Experimental Procedures

2.2.1 Materials

jp-hydroxyacetophenone, p-hydroxyphenylaceticacid and inhibitors lb, bromo aceticacid were purchased from Sigma-Aldrich Chemical Company. Inhibitors la, Ic,

Id were synthesized by Dr Guo in this lab as described previously (Arabaci, G. et al.,

1999). Trypsin protease. Acid and alkaline phosphatases and the sodium salt of p- nitrophenylphosphate (p-NPP) were purchased from Sigma Chemical Company.

2.2.2 Purification of SHP-1 and SHP-1(ASH2)

Both wild type SHP-1 and SHP-1 (ASH2) were expressed and purified from E. coli using pET-lla vector. BL21(DE3) cells carrying the appropriate plasmids were grown in 3 liters of LB media containing ampicillin (75 mg/L) at 37°C until the absorbance at 600 nm of the media reached a value of about 0.5 to 0.6. The cells were

25 then induced with 0.1 mM isopropyl-p-D-thiogalactopyranoside for 5 hours at 30°C.

The cells were then harvested by centrifugation and resuspended in 60 ml of 20 mM

Tris, pH 8.0 buffer containing 10 mM EDTA, 10 mM p~mercaptoethanol, 50 mM

NaCl, 1% TritonX-100, 0.5 mM o-phenanthroline, 0.64 mM benzamidine, 0.29 mM

phenylmethylsulfonyl fluoride, 20 pg/ml each of trypsin inhibitor, leupeptin and

pepstatin-A. The cells were lysed at 4°C by the addition of 6 mg of chicken egg white

lysozyme and occasional stirring over 30 min.

Sonication was then done and the lysate centrifuged at 15000 rpm for 15 min

in a Sorvall SS-34 rotor. The supernatant was diluted 2 fold in 20 mM HEPES, pH 7.2

(buffer B) containing 1 mM EDTA, 10 mM P-mercaptoethanol, 10 mM NaCl and loaded onto a pre-equilibrated SP-Sepharose (Pharmacia) column (8x2.5cm). The column was washed with 50 ml of the same buffer B and the bound protein eluted with 200 ml of buffer B and a gradient of 10 mM to 2 M NaCl at 1 ml/min. The eluted fractions were analyzed by activity assays and SDS-PAGE. The selected fractions were combined and concentrated in a Centriprep-30 concentrator (Amicon) to a volume of 2 ml. This was then diluted to 70 ml with 20 mM Tris pH 8.0 (buffer C) containing 1 mM EDTA, 10 mM P-mercaptoethanol, 10 mM NaCl and loaded on to a pre-equilibrated Q-Sepharose (Pharmacia) column. Bound protein was eluted in 200 ml buffer C and a gradient of 10 mM to 1 M NaCl at 2 ml/min.

26 The fractions containing purified protein were combined and concentrated to

about 1 ml. Glycerol was then added to a final concentration of 33% (vol/vol)

followed by quick-freezing in liquid nitrogen and storage at -80 °C.

2.2.3 Purification of VHR Phosphatase

VHR phosphatase expressing plasmid (pET-llb based) obtained from Dehua

Pei was transformed into E. coli BL21(DE3) cells. These cells were grown in LB

media supplemented with 75 mg/L ampicillin at 37°C till the optical density at 600 nm

reached a value of 0.6. The cells were induced by the addition of 0.1 mM isopropyl-p-

D-thiogalactopyranoside for 5 hours at 30°C and harvested by centrifugation. The cell

pellet was resuspended with 20 mM HEPES buffer, pH 7.0 containing a mixture of 1

mM EDTA, 1 mM P-mercaptoethanol, 1% Triton X-100, 0.29 mM phenylmethylsulfonyl fluoride, 20 pg/ml each of trypsin inhibitor, leupeptin and pepstatin-A. The cells were lysed at 4°C by the addition of 6 mg of chicken egg white lysozyme with occasional stirring over 30 min.

Sonication was then done and the lysate centrifuged at 15000 rpm for 15 min in a Sorvall SS-34 rotor. The supernatant was diluted 2 fold in 20 mM MES, pH 6.0 buffer containing 10 m NaCl, ImM EDTA, 1 mM p-mercaptoethanol and loaded onto a pre-equilibrated SP-Sepharose (Pharmacia) column (8x2.5cm). The column was washed with 50 ml of the same MES buffer and the bound protein eluted with 200 ml of MES buffer with a gradient of 10 mM to 1 M NaCl at 1 ml/min. The eluted

27 fractions were analyzed by activity assays and SDS-PAGE. The fractions containing

purified protein were combined and concentrated to about 1 ml. Glycerol was then

added to a final concentration of 33% (vol/vol) followed by quick-freezing in liquid

nitrogen and storage at -80 °C.

The other phosphatases used were PTP-IB, acid and alkaline phosphatases.

PTP-IB was provided by Dr. Z.-Y. Zhang.

2.2.4 Activity Assay for Phosphatases

Activity assays with pNPP as a substrate were performed at room temperature in 50 pi reaction volume. The solution typically contained 0-50 mM pNPP, 100 mM

HEPES at pH 7.4, 1 mM P-mercaptoethanol, 1 mM EDTA, 150 mM NaCl, and phosphatases in desired amounts (l-5pM). Enzyme dilutions were made in a buffer of

20 mM HEPES pH 7.4, 10 mM NaCl and 100 pg/ml BSA. The reaction time was typically 10 min after which the reaction was quenched by the addition of 950 pi of 1

M NaOH. The absorbance of the released pNP at 405 nm was then measured and nanomoles of product (pNP) released calculated by comparison with a standard curve.

The specific phosphatase activity was then calculated.

2.2.5 Time Dependent Inhibition of Phosphatases

Several a-haloacetophenone inhibitors were tested for time dependent inhibition of phosphatases either by an end point assay or by a continuos assay (31).

28 (i) End Point Inhibition Assay. This method was used to study the inhibition of phosphatases by la and lb only. Enzyme stock solutions were freshly prepared in each case by diluting a frozen stock in buffer A (20mM HEPES, pH 7.4, 10 mM NaCl and 100 pg/ml bovine serum albumin) to a final concentration of 1-2 mg/ml. Inhibitor stock solutions were prepared by weighing exact amounts of inhibitors into accurate volumes of N,N-dimethylformamide and deionized water (typical stock concentrations o f 200-500 mM).

A 90 p.1 volume of buffer (50 mM HEPES, pH 7.4) containing the required concentration of inhibitors la and lb (0-10 mM) was prepared and 10 pi of phosphatase (10-20 pg) was added at time zero. At various incubation times 10 pi aliquots were withdrawn from this reaction and added to a 40 pi solution containing

10 mM /?NPP, 1 M p-mercaptoethanol and either buffer B (100 mM Bis-Tris, pH 5.5,

150 mM NaCl and 1 mM EDTA for SHP-1) or buffer C (100 mM HEPES, pH 7.4,

150 mM NaCl, 1 mM EDTA for all other enzymes). The excess P-mercaptoethanol ensures the quenching of excess la and lb . This reaction was incubated at room temperature for 10-60 min aind quenched with 950 pi of 1 N NaOH. Absorbance measurements were done at 405 nm from which the phosphatase activity for each time point was determined. The substrate to product conversion was kept less than 10 %. A plot of log(A/Ao) against time gave a straight line, where Ao is the absorbance produced by the enzyme at time zero (no inactivation) and A is the absorbance from

29 the enzyme aliquot withdrawn at time t. The apparent inactivation rate Aobs is calculated from the slope of the line. Finally, a secondary plot o f against inhibitor concentration [I], and data fitting against equation (61)

Arobs = A'inact X [I]/(^I + [I]) gave the equilibrium inhibition constant K\, and the first-order rate constant ^inact-

Figure 2.1 shows a typical data set (determined with la and PTPIB).

(ii) Continuos Inhibition Assay. This method was used for inhibitors lc,d and bromoacetic acid . Because these inhibitors are less reactive towards free thiols (e.g.

Id) and/or have relatively high Kj values, very high inhibitor concentrations were used in the inactivation assays. Quenching with IM P-mercaptoethanol was found not to be instantaneous, complicating the kinetic determination. In this method, enzyme from the frozen stock was diluted to a final concentration of 1 mg/ml in buffer A plus 1.5 mM tris(carboxyethyl)phosphine (TCEP) and incubated in ice for 10 min.

At time zero, an aliquot of the treated enzyme (2-3 pi) was added into a cuvette containing 1 ml of 50 mM HEPES, pH 7.4, 10 mM pNPP, and 0-100 mM inhibitor.

The solution was rapidly mixed and the reaction progress was monitored on a UV-vis spectrophotometer at 405 in a continuous fashion (figure 2.2). The absorbance (A) versus time (t) data were fitted against equation (61)

A = Vo(l-e-yk where Vg is the initial reaction rate and k is the observed inactivation constant (figure

2.2, A )(61). A secondary plot of the obtained k values against each different inhibitor

30 concentration [I] in the double reciprocal fashion produced a linear line and the data fitting against equation (figure 2.2, B) (61)

\ ! k = 1/kinact + (1+ [Sj/ATw) X Ki/(Â:,„a«x [I]) gave the equilibrium constant AT/ and the first-order inactivation constant kinaa-

2.2.6 Determination of The Reaction Rate of Ic with Free Thiols

At time zero, Ic (8 mM) and P-mercaptoethanol (16 mM) were dissolved in 20 mM sodium phosphate, pH 7.4, 20 mM NaCl 50% (v/v) DiO/deureated acetone. The

*H-NMR spectra of the mixture were recorded at appropriate time intervals (0-10 hours). Plot of remaining Ic concentration against time produced a reaction progress curve, and the initial rate was calculated fi'om the early part of the curve. The second order rate constant, kz, was then calculated using the equation: Vo= kz x [lc]o x

[RSH]o. A control experiment was also performed in the absence of any free thiol, p- mercaptoethanol and the pseudo first-order rate constant for hydrolysis of Ic at pH 7.4 was determined as 2.3x10"^ m in'\ corresponding to a half life (ti/ 2 ) o f 51 hours for Ic under those conditions.

2.2.7 Mass Spectrometric Analysis of SHP-1(ASH2)

(i) Stoichiometry of inactivation. 57pM SHP-1 (ASH2) was added to 50 mM

HEPES buffer (pH 7.4) containing 200 pM la, and the resulting mixture (90 pi) was incubated for 7-8 min at room temperature. The reaction was then quenched with P-

31 mercaptoethanol to a final concentration of 100 mM. A small aliquot was checked for remaining activity against pNPP and was found to be essentially inactive. For mass analysis, a few pi of the treated enzyme (usually 10-50 pg) was dissolved in 0.1% trifluoroacetic acid and passed through a C-8 reversed-phase HPLC column. The sample was eluted with 0.1% trifluoroacetic acid in acetonitrile just before injecting into a Perkin-Ehner Model API-300 mass spectrometer (electrospray). As a control experiment, C453S mutant o f SHP-1 (ASH2) and the mutant-inhibitor complex were prepared and analyzed by ESI MS in the same fashion above.

(ii) Location of the modified site. To locate the site of modification, the inactivated SHP-1 (ASH2) (100 pg) was digested with trypsin (1.5 pg) in 50 mM

HEPES, pH 7.4 buffer for 12 hours at room temperature. The resulting peptide mixture was purified on a Sep-Pak, column (C-18) to remove the small molecule contaminants. The bound peptides were eluted with 500 pi o f 50% methanol and 500 pi of 95% methanol. After removal of the solvent in vacuo, the peptides were dissolved in —10 pL of 0.1% trifluoroacetic acid (TEA). A 1 pi aliquot of this mixture was mixed with 2 pL of a saturated solution of a-cyano4-hydroxycinnamic acid, and 1 pi of the mixture was applied to the MALDI spectrometer plate. MALDI mass analysis was performed on a Kratos Kompact MALDI HI mass spectrometer in the positive ion mode.

(Hi) Differential reactivation o f SHP-1 v^. SHP-1(ASH2). To investigate why

SHP-1 (ASH2) has only 30% photo-reactivation although SHP-1 has 80% under the

32 same conditions, 100 jxg of enzyme was incubated with 300 fxM of the inhibitor la in the presence of 50 mM HEPES, pH 7.4 buffer for 10 min in the dark at room temperature. The excess inhibitor and P-mercaptoethanol were removed by loading the inactivated enzyme onto an SP-Sepharose column equilibrated with 20 mM

HEPES, pH 7.0, (buffer A) containing 10 mM NaCl and ImM p—mercaptoethanol.

The column was washed with buffer A and eluted with 20 mM HEPES, pH 7.0, buffer and 400 mM NaCl. The eluted sample was transferred into a cold room (4°C) and incubated in a photochemical reactor equipped with 4x350 nm lamps (Southern New

England Ultraviolet Co.) for 45 min. Aliquots (10 pi) were removed after 30 min and the amount of recovered PTP activity was measured with /?NPP as substrate as described earlier. The reactivated SHP-1 (ASH2) was digested with trypsin (1.5 pg) in

50 mM HEPES, pH 7.4 buffer for 12 hours at room temperature and prepared for

MALDI mass analysis.

2.2.8 PTP Reactivation

Freshly thawed SHP-1 (final concentration 4.7 pM) was added to 50 mM

HEPES buffer (pH 7.4) containing 100 pM inhibitor (e.g. la), and the resulting mixture was incubated in the dark for 8 min at room temperature. After quenching any excess inhibitor with P-mercaptoethanol (final 100 mM), the sample was transferred into a cold room (4°C) and incubated in a photochemical reactor equipped with 4x350 nm lamps (Southern New England Ultraviolet Co.). Aliquots (10 pi) were removed

33 every 2 to 5 min and the amount of recovered PTP activity was measured with /?NPP

as substrate as described above. Reactivation of SHP-1 (ASH2) and PTPIB that had been inactivated by la-d was carried out in a similar fashion except for the differences

in PTP and inhibitor concentrations.

2.2.9 Photolysis product determination

SHP-1 (10 pM) was added to 50 mM HEPES pH 7.4 containing 100 pM inhibitor la, and the resulting mixture was incubated in the dark for 8 min at room temperature. After any excess inhibitor was quenched with P-mercaptoethanol (final concentration lOOmM), a 10 pi ahquot was removed from this mixture and the amount of remaining PTP activity was measured with 10 mM pNPP as substrate as described earlier (Section 2.2.3). The rest of the mixture was applied onto a pre-equilibrated SP-

Sepharose column and washed with 1 ml buffer A (containing 20 mM HEPES, pH

7.2, 10 mM NaCl, and I mM P-mercaptoethanol) to remove any excess inhibitor and

P-mercaptoethanol at 4°C. The SHP-1-inhibitor complex was eluted with 500 pL, 400 mM NaCl, 20 mM HEPES, pH 7.4, and 1 mM P-mercaptoethanol and incubated in a photochemical reactor equipped with 4x350 nm lamps (Southern New England

Ultraviolet Co.) for 45 min. Aliquot (10 pi) were removed at the end of 45 min and the amount of recovered SHP-1 activity was measured with pNPP as substrate as described in section 2.2.3. As soon as the irradiation of SHP-1 was completed, the reactivated SHP-1 mixture was applied to a centricon apparatus (Amicon) to separate

34 the photolysis products from SHP-1 and the filtered solution collected. All the above purification and irradiation experiments were performed in the dark and at 4°C. The collected sample was applied to an analytical reverse phase HPLC column for detection of the photolysis products. Vydac C-18 protein/peptide column using a gradient o f 5-65% acetonitrile over 30 min (with 0.05% TFA) at a flow rate of 1 ml/min was used in the HPLC system. For comparison authentic standard samples were also analyzed using the same conditions. Further analysis of the photoysis products were done by collecting product peaks from the HPLC and submitting for

ESI-MS analysis.

2.2.10 Inhibition ofB Cell FTPS

5x10® Ramos human B cells were incubated at 37°C for 3 min with nothing,

0.1% DMSO, or with various concentrations (30-1000 pM) of inhibitor la in 0.1%

DMSO. A 3 min incubation of Ramos B cells with 100 pM phenylarsine oxide was used as a positive control. The resulting Ramos cell lysates were separated by SDS-

PAGE, transferred to nitrocellulose filters, and probed with anti-phosphotyrosine antibody as described earlier (31). These experiments were done by Dr. Coggeshall.

35 2.3 Results

2.3.1 Kinetics of time dependent inactivation of PTPs by a-haloacetophenones.

PTPs dephosphorylate phospho-tyrosine residues on proteins involved in a variety of signaling pathways (83). Our broad goal was to obtain specific PTP inhibitors to assist as biological tools and for therapeutic intervention. The potential of a-haloacetophenone compounds as specific covalent inhibitors for PTPs were assessed, a-chloroacetophenone, a-bromoacetophenone and bromoacetic acid were assayed for inhibition o f the catalytic domain o f SHP-1, SHP-1 (ASH2). Further, a-bromoacetophenone was assayed for inhibition of different phosphatases. The phosphatases tested were SHP-1 (ASH2), SHP-1, PTPIB, dual specificity phosphatase

VHR, acid phosphatase and alkaline phosphatase.

All compounds displayed time dependent inhibition of phosphatases. Their inhibition kinetics follow a two-step mechanism where the first step is a rapid reversible non-covalent binding of the inhibitor to the active site of the enzyme and the second an Sn 2 reaction between the inhibitor and the cysteine thiol (figure 2.4).

This results in an irreversibly and covalently modified inactive enzyme inhibitor complex (E.I*, see below) (61).

E + I ► EJ ► EJ*

36 Kl is the dissociation constant of the non-covalent complex E.I, and Ar,nact the first order rate constant for conversion of the E.I complex into the covalent complex, E.I*.

Two different inhibition assay methods were employed (section 2.2.3, Figures

2.1 and 2.2). For inhibitors Ic, Id and bromoacetic acid, a continuous assay was necessary. These inhibitors were less reactive towards thiols and had relatively high K\ values. The higli K\ values required the use of very high inhibitor concentrations in the assay and quenching with IM 2-mercaptoethanol was not immediate. To avoid complications, the continuous assay method was used for these inhibitors.

Table 2.2 lists the inhibition constants determined for the inhibition of various phosphatases by compounds listed in Table 2.1. The important trends noted are discussed as follows. Inhibitor la binds to SHP-1(ASH2) and PTPIB with the highest affinity, as judged by similar Ki and Arinact values. The inhibition of full length SHP-1 by la was found to have a lower Ki relative to inhibition of its catalytic domain. This may be due to the SH2 domains present in SHP-1 blocking access of the inhibitor to the PTPase active site under physiological conditions. Compounds la, lb and Ic have different substituents at the para position of the phenyl ring in the inhibitor. Relative to inhibitor la, compounds lb and Ic bind to SHP-1 (ASH2) with 3 and 5 fold lower affinity, having K\ values of 128 pM and 193 pM respectively. They have 4 and 6 fold higher kinact values than inhibitor la, thereby retaining similar overall potency as la.

Inhibitor Id (a-chloro derivative) binds to SHP-1 (ASH2) with 13 fold lower affinity

(higher Ki value) than its counterpart Ic (a-bromo derivative).

37 The specificity of inhibition among various phosphatases was examined by

studying the inhibition of acid phosphatase, alkaline phosphatase, dual-specificity

phosphatase VHR, PTPIB, SHP-1 and SHP-1 (ASH2) by compound la. No significant

inhibition of acid and alkaline phosphatases was detected up to 10 mM. However la

inhibited VHR phosphatase weakly due to a very low binding affinity (^i=8.9 mM).

This affinity is 200 fold lower than that displayed with SHP-1 (ASH2) and PTPIB. As another control, bromoacetic acid was assayed against SHP-1 (ASH2), and the inhibition was found to have a K\ of 77 mM. This means a 200 to 2000 fold lower binding affinity compared to all a-bromoacetophenone derivatives. In another control experiment, the reaction rate of Ic was determined with 2-mercaptoethanol at pH 7.4.

The reaction rate was 0.21 M'^min'* which is 45000 fold slower than that with SHP-

1(ASH2) (Â:inact/^i=9300 M'^min'*). These kinetic results suggest that a-bromo and chloroacetophenone derivatives (la-d) serve as new PTP inhibitors.

2.3.2 Mode of PTP inhibition by a-bromoacetophenones.

The mode of PTP inhibition by la was investigated by mass spectrometry.

First, the molecular weight of the catalytic domain of SHP-1, SHP-1 (ASH2) was determined by electro-spray ionization mass spectrometry (ESI-MS). This gave a molecular mass of 45757 ± 5 Da which is close to the sequence predicted mass of

45744 Da (figure 2.3). SHP-1 (ASH2) was then inactivated by excess la for 7 minutes at room temperature. ESI-MS analysis of the inactivated SHP-1 (ASH2) gave a major

38 peak at 45891 + 5 Da (figure 2.3). The increase in molecular mass relative to uninhibited SHP-1(ASH2) 133.9 + 0.5 Da, suggests that one /7-hydroxyphenacyl (the calculated mass is 135) group is attached to the protein with removal of one hydrogen most hkely firom the active site Cys-453 residue (figure 2.4). The absence of M+268 or M+402 peaks in the inactivated protein suggests that only one p-hydroxyphenacyl molecule was attached to the protein. The stoichiometry between the inhibitor and the protein is thus 1:1, although there are 4 cysteine residues in the protein and an excess of inhibitor la was used. As a control, the mutant SHP-1 (ASH2), C453S was analyzed similarly in the presence and absence of la. No mass difference was found before and after treatment with la.

Further confirmation was provided by matrix assisted laser desorption ionization mass spectroscopy (MALDI) analysis of the trypsin digested SHP-1 (ASH2) before and after inactivation with la. In the absence of la treatment, a mass of 2044 was obtained for a tryptic digest fragment corresponding to the active site region

Q44oESLPHAGPnVHCSAGIGR 459 (A, figure 2.8). The inactivated protein showed a disappearance of the peak at m/z — 2044 and appearance of a new peak at m/z = 2178

(B, figure 2.8). This result confirms that only one inhibitor molecule is attached to each molecule of SHP-1 (ASH2) and the site of attachment is the active site Cys-453 residue (figure 2.4).

39 2.3.3 Photo-reactivation of a-bromoacetophenone inactivated PTPs

Earlier work by Givens et al showed that /7-hydroxyphenacyI and p-

methoxyphenacyl groups could be used as photo-labile protecting groups to generate

caged molecules useful in biochemistry, physiology and synthesis (62, 63). The p-

hydroxy and p-methoxy derivative compounds were attached to the C-termini of

amino acids and peptides and irradiated with 350 nm UV light (62). The irradiation resulted in release of the amino acids and p-hydroxyphenylacetic acid or p- methoxyphenylacetone (2a-d, 3a, b, d, figure 2.9 (62-63)). This suggested that inactivation by a-haloacetophenones observed in the previous section could be reversed by irradiation with UV light.

To demonstrate the reversibility SHP-1 (47pM) was inactivated with 100 pM of inhibitor la for 7 minutes at room temperature in the dark. The inactivated sample was directly irradiated with 350 nm light at 4 °C for up to 1 hour. The irradiation resulted in 80% recovery o f original enzymatic activity of SHP-1 after 15 minutes.

The irradiation when applied to inactivated SHP-1 (ASH2) and PTPIB under the same conditions resulted in recovery of only 30% of original activity even after an hour of irradiation (figure 2.5). In a control experiment, the irradiation had almost no effect on the native enzyme activity. Further experimental details are provided in section 2.2.7.

These results demonstrate that inactivation of PTPs by a-haloacetophenones can be conveniently photo-reversed.

40 2.3.4 Analysis of photolytic reactivation.

Earlier work by Givens et al identified the major products of photo-activation

of p-hydroxyphenacyl caged peptides (62, 63). The major products identified were p-

hydroxyphenylacetic acid and p-hydroxyacetophenone (2a-d, 3a, b, d, (62)).

Mechanistic explanations were suggested (figure 2.10 (62, 63)). For characterization

purposes, products of the photo-reactivation of inactivated PTPs were determined. The

results are presented in this section.

SHP-1 was chosen for these experiments due to maximal recovery of activity

upon irradiation, thereby likely producing largest amounts of the reactivation products

to be identified. Inhibitor la was used to inactivate SHP-1 and this mixture was

purified through an SP-Sepharose column to remove excess la. The purified and

inactivated SHP-1 was irradiated for 45 minutes at 350 nm and 4°C. The irradiated

mixture was applied to a Centricon apparatus, thus separating the photolysis products

from reactivated SHP-1. The filtered solution was collected and analyzed by C-18

reverse phase HPLC. The observation of only one major peak (retention time of 14

minutes) suggested only one major product was being released. To identify the product, authentic samples ofp-hydroxyphenylacetic acid and /?-hydroxyacetophenone

(expected major products) were also analyzed. Their retention times under identical conditions were 11 and 14 minutes respectively (figure 2.6). This suggested that the photolysis product was most probably p-hydroxyacetophenone. For further analysis, this peak was collected and submitted for mass spectrometric (ESI-MS) analysis. A single peak at m/z 137 was noted, confirming the product to be p-

41 hydroxyacetophenone (figure 2.7). Possible reasons for observing only the ketone

product in contrast to the results o f Givens et al are discussed in Section 2.4.

In order to understand the limited recovery of activity upon irradiation of the

inactivated SHP-1(ASH2) and PTPIB, the irradiated en 2:yme-inhibitor complexes

were digested with trypsin and analyzed by MALDI-MS. Similar control experiments

were done with the active enzymes and inactivated enzyme-inhibitor complexes that

were not irradiated. The trypsin digest of active SHP-1 (ASH2) (not treated with la)

showed a peak of mass 2044 Da which corresponds to the calculated mass of the

active site firagment Q 44oESLPHAGPnVHCSAGIGR 459 (A, figure 2.8). Similar

experiments with the irradiated SHP-l(ASH2)-la complex showed new peaks with

masses of 2178 Da and 2121 Da along with a small amount o f unreacted original

active side firagment peak (C, figure 2.8). The peak at 2178 corresponds to the

covalent attachment of the thiol o f Cys-453 and the a-acetophenone molecule

(m/z=134). The mass peak at 2121 Da most probably corresponds to the formation of a disulfide bond between Cys-453 and 2-mercaptoethanol (m/z=77). These results suggest that the inhibitor is still attached to the Cys-453 containing active site peptide and prevents higher recovery of activity upon irradiation. Possible reasons are discussed further in Section 2.4.

42 2.3.5 Membrane permeability of a-bromoacetophenone.

Non-hydrolysable pY mimetic inhibitors such as phosphonates, malonates and cinnamates have high binding affinities to PTPs (89). They are however significantly limited for pharmacological or therapeutic use because of their poor cellular penetration (89). a-haloacetophenones (la-d) are not charged and are pY mimics. The potential of these inhibitors in cellular studies was tested with human B cells as follows. The experiments described in this subsection were done by Dr. Coggeshall.

Human B cells were incubated with varying concentrations of hihibitor la for 3 minutes. Cells were then lysed and cellular proteins separated by SDS-PAGE followed by western blot analysis using an anti-pY antibody (figure 2.8). Treatment of cells with around 300 pM of la showed hyperphosphorylation o f a protein at 110 kDa. This result was consistent with the inactivation of a PTP that would have normally dephosphorylated this protein. Another interesting result was that in the presence of la, the phosphorylation level of a 50 kDa protein was decreased. This may be explained by that inactivation of a specific PTP by la caused the inactivation of a downstream PTK that in turn caused the decreased phosphorylation of the 50 kDa protein. It is known that the activation of some PTKs require dephosphorylation of pY residues by a PTP. These results suggest that the inhibitor la can penetrate into cells and inhibit PTPs thus demonstrating the potential of a-haloacetophenones in biological studies.

43 2.4 Discussion

Protein tyrosine phosphatases dephosphorylate phosphotyrosine residues on proteins involved in a variety of signaling pathways (83). The broad goal in this chapter was to obtain specific PTP inhibitors to assist in biological experiments and for therapeutic intervention. Specifically, the potential of a-haloacetophenone compounds (Table 2.1) as specific covalent inhibitors of PTPs were assessed. We envisioned that a-haloacetophenones could bind to the active site of protein tyrosine phosphatases as a phosphotyrosine mimic. The phenyl ring could engage in hydrophobic interactions with the PTP active site as the phenyl ring in the substrate does, and the electronic rich halogen atom could mimic the negatively charged phosphate oxyanions. The binding of the inhibitor to the active site in such a manner would place the a carbon, which is highly susceptible to nucleophilic attack next to the essential catalytic cysteine thiolate. An Sn 2 type reaction between the inhibitor and the thiolate results in the formation of a covalent enzyme-inhibitor adduct through a stable thioether linkage and loss of phosphatase activity (figure 2.4).

All compounds tested displayed time dependent inhibition of phosphatases.

Their inactivation kinetics were well described by a two step mechanism where the first step is the reversible and rapid non-covalent binding of the inhibitor to the active site of the enzyme and the second and a Sn 2 reaction between the inhibitor and the active site cysteine thiol (figure 2.1 and 2.2). In every case an equilibrium inhibition

44 constant and a first order inactivation rate constant was determined (Table 2.2). Mass spectral analysis o f the active and inactivated protein aided by tryptic digests confirms stoichiometric modification of the enzyme (1:1) at the essential cysteine (figure 2.3 and 2.4). While the mode of inhibition is the same for all inhibitors studied in a quahtative sense, the potency of each compound and its selectivity vary. The following is a brief discussion of the results obtained with regard to the potency of each compound and the selectivity of inhibition among different phosphatases.

The effect of different substituents at the para position was evaluated by studying inhibitors la, lb and Ic (Table 2.2). The substituent changes cause a marginal effect of increasing the equihbrium inhibition constant (Ki) 4-fold. This however is opposed by a counteracting change in the first order inactivation rate constant (kinact) so that the overall potency of compounds la, lb and Ic are similar to each other. The effect of replacing bromine with chlorine at the reaction center was also evaluated by comparing the inhibition constants of compounds Ic and Id.The chloro compound has a 13-fold higher inhibition constant and an identical kinact value relative the bromo analog. This maybe due to the smaller size of the a-chloroacetyl group which is a less effective mimic of the phosphate moiety than the corresponding bromo group. The above trends were determined using the catalytic domain of SHP-1

(SHP-1 (ASH2)).

The selectivity of inhibition is of critical importance in determining the usefulness of the compounds tested for biological experiments. To address selectivity

45 issues, inhibitor la was assayed against six different phosphatases (SHP-1 (ASH2),

SHP-1, PTPIB, VHR phosphatase, acid phosphatase and alkaline phosphatase). Acid

and alkaline phosphatases employ a completely different mechanism for phosphate

ester hydrolysis (37) and no significant inhibition of either was detected up to a concentration o f 10 mM la. Inhibition of PTPIB was essentially the same as inhibition of SHP-1 (ASH2) in potency.

Inhibition of VHR phosphatase has a 200-fold lower equilibrium inhibition constant compared to that of SHP-1 (ASH2) and PTPIB. VHR phosphatase is able to dephosphorylate phosphoserine and phosphothreonine groups in addition to phosphotyrosine groups (96). While employing the same catalytic mechanism, VHR phosphatase has limited sequence and structure similarity relative to PTPs (64, 96).

The weaker inhibition of VHR phosphatase maybe due to a much more shallow active

(64) site relative to that in PTPs and a conresponding weaker hydrophobic interaction with the phenyl ring. The phenyl ring is crucial in providing binding affinity as demonstrated by the 2000 fold lower affinity for bromoacetic acid relative to compound la. These results suggest that a-haloacetophenones are potent and selective inhibitors for protein tyrosine phosphatases. The next chapter presents and discusses methods to improve binding affinity and specificity by attaching specific ligands to the para position of the inhibitors.

Mass spectral results presented in section 2.3 and discussed earlier in this section conclusively point to the stoichiometric labeling of the enzyme at the catalytic

46 cysteine. Photo-reactivation of the inactivated enzyme was then achieved by

irradiation under 350 nm UV light. In earlier work, p-hydroxyphenacyl and p-

methoxyphenacyl groups were used as photolabile protecting groups in generating

caged amino acids (62, 63). When irradiated, they released the free amino acids and

two other major products namely, p-hydroxyacetophenone (2a-d, figure 2.9) and p-

hydroxyphenylacetic acid (3a, b, d, figure 2.9) (62, 63). Possible mechanisms have

been proposed to account for their formation (figure 2.10) (62, 63). The products of

photoreactivation of inactivated SHP-1 were examined by HPLC and mass spectral

analysis (figure 2.6). Only the ketone product (2b, figure 2.9) was detected. Notable

differences from previous studies are as follows. The experiments here involved photoreactivation within an active site of a protein unlike previous studies, which were

carried out in solution. Further, the reactivation in this study involved a CHz-S bond unlike the CH2-O bond in previous work. These differences may account for the observation of the keto product only.

Maximal extent of photoreactivation was observed with the full length SHP-1

(~ 80%) whereas only 30% reactivation was observed with the catalytic domain (SHP-

1(ASH2) and PTPIB (figure 2.5). The reason for the differing extent of reactivation is not readily apparent. MALDI mass analysis after irradiation of the inactivated SHP-

l(ASH2)-la adduct shows the inhibitor group still attached to the active site peptide

(C figure 2.8). This maybe due to the inhibitor derived reactive species attaching covalently to the active site resulting in permanent loss of activity.

47 Finally, in order to address cell permeability, bioavailability and in-vivo specificity issues, human B-cells were treated with inhibitor la. Appropriate control experiments were carried out also. Western blot analysis of cellular proteins using an anti-phosphotyrosine antibody showed dephosphorylation and hyperphosphorylation of specific proteins suggesting specific inhibition of PTPs that control the phosphorylation state of the observed proteins.

48 OR

l a X = Br R = H 1b Br CH3 Ic Br CH2 CO2 H Id Cl CH2 CO2 H

Table 2.1: Structures of a-haloacetophenone inhibitors.

49 - 0. 2 -

20

? -0.6 H 320 (iM 240 ■0-8 - 80 uM laOuM

Time (min)

B 0.5-

0.4-

0.3-

0. 2-

0 . 1-

0 50 100 150 200 250 300 350

Figure 2.1: Inhibition of PTPIB by la. (A) Plot of log(A/Ao) against time. The linearity indicates first-order inactivation kinetics. (B) Secondary plot of slopes from A (kobs) vs inhibitor concentration [I].

50 IOO mM laouM ISOuM 200 uM 3S0uM 350 uM

1 ' r 100 200 300 400 600 Time (s)

B 200

1 5 0 -

^ 100-

5 0 -

0 0.002 0.004 0.006 0.008 0.01 0.012 1/IIJ (nM-’)

Figure 2.2: Inactivation of SHP-I(ASH2) by Ic. (A) Reaction progress curves for SHP-1 (ASH2) catalyzed hydrolysis of pNPP in the presence of varying concentrations of inhibitor Ic. (B) Secondary plot of observed inactivation rate constants (1/k) vs inhibitor concentration (1/[I]).

51 Enzyme Inhibitor Ki (pM) kinact (min ')

SHP-1(ASH2) la 43± 10 0.40 ±0.10 lb 128 ± 10 2-4 ± 0.2 Ic 193 ±38 1.8 ±0.3 Id 2540 ± 610 1.8 ± 0.4 BrCHzCOzH 77000 ± 14000 1.4 ±0.2 SHP-1 la 530± 120 2.6 ± 0.2 PTPIB la 42 ± 5 0.57 ± 0.05 VHR la 8900 ± 4500 3.4 ± 1.6

Table 2.2: Kinetic constants of PTP inhibition by la-d. Data reported are the mean ± SD from three or more independent experiments carried out at pH 7.4 and at room temperature.

52 a 145757

JK. 45891

45757 45971 I r A -J 45000 45500 46000 46500 47000 Molecular Mass (Da)

Figure 2.3: Reconstructed ESI/MS spectra for (a) native SHP-1(ASH2); and (b) la inactivated SHP-1(ASH2).

53 Tyr Pi -Z.

OR

Figure 2.4: Mechanism of PTP catalysis and inactivation by a-haloacetophenones (31).

54 100 SHP-1 + hv

80-

SHP-1 + la + hv 60-

40- SHP-1(ASH2) + la + hv

2 0 - SHP-1 + l a (no light)

0 10 20 30 40 50 Time(min)

Figure 2.5: Photolytic reactivation of la inactivated SHP-1 and SHP-1(ASH2). All activities are relative to those of the untreated enzymes.

55 8 12 16 20 24 Retention Time (min)

Figure 2.6: HPLC analysis of the products of photochemical reactivation, a) photo­ reactivation reaction; b) photo-reactivation reaction mixed with 2b (figure 2.9); c) mixture of 2b and 3b (figure 2.9).

56 120

137.0 100

158.9 40 121.1

750.0 851.0

60 80 100 120 140 160 180 200 m/z

Figure 2.7. Reconstructed ESI/MS spectra for photoproduct of irradiated-inactivated SHP-1(ASH2).

57 c 2 1 7 8

19 50 2000 2050 2 100 2 150 2200 2 2 50

M o !e c u la r M a ss (Oa)

Figure 2.8. MALDI-TOF spectra for trypsin digested (A) native SHP-1 (ASH2); (B) la inactivated SHP-1(ASH2), and (C) UV light treated la inactivated SHP-I(ASH2).

58 hv K-OH H.CH).or(Cfl3hC

UY«oa^;X“ a 4Y-OCH,.X«=QiP(CRh 2 ii-d 3«.b.d hY=c%x-q K.Y=Ot X=» c.Y-C)CH,,X-=OiCR. tY -C % X-CH (eq 1)

Figure 2.9. Possible products of photochemical reactivation by Givens et al. (62).

59 ,OCOR h v ^ ST DufEif* 2TC HO' 4a, R = CHzCHiCHzNH,^ h,R-CH2CH2CH(NH3*)COi* C, R = CH(CH3)NHCOCH(NH3 XZH]

O f^pCOR s r e u p SET

-RCO2*

GABA L-glutamate. or ' j o r r . ala-ala

Figure 2.10. Possible mechanisms for photochemical reactivation by Givens et al. (62, 63).

60 kOa 3 4 6 7 208- - * X r f 127-

... 0 85- a « #8% 4.#W % # ?3' :# 0 g 1Æ.V*' ^ , 45- ^ 33-

Figure 2.11: Western blot analysis of B cell phosphoproteins. 5 x 10*^ Ramos human B cells were incubated at 37 °C for 3 min with nothing (lane 1), 0.1% DMSO (lane 2), or with 30 pM (lane 3), lOOp M (lane 4), 300 pM (lane 5), or 1000 pM (lane 6) la in 0.1% DMSO. Lane 7 is a positive control with 100 pM phenylarsine oxide. The resulting cell lysates were separated by SDS-PAGE, transferred to nitrocellulose filters, and probed with an anti-pY antibody (31).

61 CHAPTERS

DEVELOPING SPECIFIC PTP INHIBITORS

3.1 Introduction

Tyrosine phosphorylation regulated by the action of PTKs and FTPs are crucial in eukaryotic signal transduction mechanisms (83). As a result of genome sequencing efforts several PTP genes have been identified (83). To study the role of a specific

PTP enzyme in signaling easily, it is necessary to have an inhibitor that is specific to that PTP (31, 89). These isoform specific inhibitors in addition to aiding biological experiments, may provide therapeutic lead compounds.

The previous chapter described the discovery of a-haloacetophenones as photoreversible inactivators of PTPs. The compounds covalently attached to the catalytic cysteine and thereby removed enzyme activity. The activity could be recovered by irradiating with UV light at 350 run. Further, the inhibitors were membrane permeable and demonstrated their effect on in-vivo tyrosine phosphorylation levels. However, the inhibitors tested showed almost equal inhibition of SHP-1 (ASH2) and PTP 1B .

62 In order to develop an inhibitor for a specific isoform PTP, the following

experiments were proposed. First, a more comprehensive study of inhibition potency

as it relates to a-haloketone structure would be undertaken. After the best inhibitor

was identified, the derivatization of the inhibitor was planned to improve binding

towards a specific PTP. Earlier work showed that the inhibition of PTP IB by

cirmamates could be improved greatly by adding tripeptides to the para position (99).

We therefore proposed to similarly add specificity elements to a-haloketones to

improve SHP-1(ASH2) and PTP IB inhibition. The identification of these specificity

elements was to be done by a combinatorial approach.

3.2 Experimental Procedures

3.2.1 Materials and General Methods

Inhibitors (1-4, 6, 10-14) and aldehyde derivative inhibitors were purchased firom Aldrich-Sigma Chemical Company. The rest of the inhibitors were synthesized in this laboratory by Dr. Y. Tian and Dr. Fu unpublished data. The kinetic assay and analysis of the inhibitors (1 through 20) against SHP-I(ASH2) and PTP IB were performed as described in chapter 2, section 2.2.5 as a continuos fashion. Inhibitors (7-

14) were tested by Marry Porter who was an undergraduate student.

63 Bovine serum albumin (BSA), 5-bromo-4-chloro-indolyl-phosphate (BCEP),

biotin-hydroxysuccinamide, and streptavidin-alkaline pbospbatase conjugate were

purchased firom Sigma Chemical Company. All peptide synthesis regents and resins

were purchased firom Advance Chem Tech (Louisville, KY). Soluble peptides with

inhibitors were synthesized on a Wang resin using standard Fmoc chemistry on 0.2

mmole scale by Kirk D. Beebe (as described in 46). SHP-1, SHP-1(ASH2), VHR were

purified as described previous Chapter (Section 2.2.2-2.2.3). PTPIB were purified and

supplied by Kirk D. Beebe.

3.2.2 Biotinylation of Proteins

SHP-1(SH2) (444 fiM) and PTPIB (513 pM) were treated with 1.5-2.0 molar equivalents of N-hydroxysuccinimidobiotin in the buffer A containing 20 mM

HEPES, pH 7.4, 150 mM NaCl, 10 mM P-mercaptoethanol for 30 min at room temperature. Dry N-hydroxysuccinimidobiotin was dissolved first in DMF and immediately before addition to the protein was diluted into buffer A. The excess of biotin was quenched with 200 mM Tris-HCL, pH 8.0 buffer and the samples passed through s Pharmacia G-25 Fat Desalting column in 20 mM Tris-HCL, pH 7.5, 150 mM NaCl, 10 mM P-mercaptoethanol, and 1 mM EDTA to remove excess biotin. The protein was collected, concentrated in a Centriprep-10 concentrator, and quickly firozen in the presence of 33% glycerol. To a quantitative check whether the protein was biotinylated, the sample was loaded onto a streptavidin-agarose column (from

64 Sigma). As a control experiment, an imbiotinylated protein sample was also loaded

onto a streptavidin-agarose column. Both of the samples were washed with the buffer

A and the immobilized protein was detected with Bradford reagent with a strong

increase in a blue color compared to the control. In addition, the activity of

biotinylated protein was detected with a pNPP assay as a substrate as described in

chapter 2 (section 2.2.4).

3.2.3 L ibrary Construction

The inhibitor library was constructed as an N-terminal a-bromoacetophenone derivative inhibitor peptide consisting of four or three random positions and a standard

BBRM sequence, Inh-XXXXBBRM and Inh-XXXBBRM (figure 3.3, library 2, 3). 17 different amino acids were used in the random portion of the library. Lysine, arginine and cysteine residues were excluded and norleucine (Nle) was used as a replacement for methionine. TentaGel-S NH 2 resin (100 pm, 0.26 mmol/g, 2.86x10^ beads/g) was used as the solid support for the peptide libraries. Synthesis was performed on a 1.0 g scale using standard Fmoc/HBTU/HOBt chemistry (74-76). For the peptide libraries, a common four amino acid linker BBRM (B=|3-alanine residue) was first synthesized on the resin. The coupling efficiency was monitored with ninhydrin test (74-76).

Four and three randomized positions were generated by split-pool synthesis method to construct the one-bead one-sequence peptide libraries (74, 77). Fmoc groups of the standard residues were deblocked with 20% (v/v) piperidine in DMF

65 (twice, 5 and 15 min) and washed 5 times with 5 ml DMF to remove the excess of piperidine. The resin was then evenly divided into 17 aliquots and placed into the 17 separate vessels. A different amino acid (5 fold excess relative to the resin) was coupled to the resin in each of the reaction vessels for 3-4 hours. The same coupling reaction was repeated one more time to complete coupling. To produce a small amount of chain termination for later sequencing analysis, 10% (mol/mol) acetylglycine was added to the amino acid solutions used for coupling at the randomized positions with the exception of norleucine and isoleucine residues (48).

10% AT-acety-D,L-alanine (mol/mol) was used for norleucine (Nle) and a mixture of

5% A-acetyl-D, L-alanine (mol/mol) and 5% A^acetylglycine (mol/mol) was used for isoleucine (He) instead of the acetylglycine capping reagent. After the coupling is completed, the resin from the 17 different vessels were combined together and washed five times with 5 ml of DMF and then, deblocking of the resin-bound peptides was performed twice with 20% piperidine in DMF (48, 76). After deprotection, the resin was redistributed to 17 portions, coupled with 17 different amino acids in the presence of 10% capping reagents and this procedure was repeated until tetrapeptide libraries formed. After coupling at all four randomized positions and deblocking with piperidine. The side-chain deprotection of the resin-bound peptides was performed with a typical cleavage cocktail containing 4.75 ml of trifluoroaceticacid, 0.1 ml of anisole, 0.1 ml of ethanedithiol, and 0.25 ml of thioanisole for 1 hour at room temperature (76). The resin was washed 5 times with 5 ml CH2CI2, 5 ml DMF and

66 ether to remove any excess thiol compounds, which may cause problems by reacting

the inhibitor. Then, the resin was dried under the vacuum and stored at -20°C.

3.2.4 Library Screening and MALDI Peptide Sequencing

The library screening was performed in a I ml plastic chromatography column at room temperature in the dark. Twenty five mg of resin was washed 5 times with 1 ml ether, 1 ml DMF and 0.5 molar equivalent of the N-hydroxylsuccinimido-ester of the inhibitor 17 (synthesized by Dr. Fu unpublished data) was added to the N-terminus of the peptide library in 1 ml DMF solution for 45 min in the dark (figure 3.3, library

2, 3). After, the reaction the library beads were washed 5 times with DMF and 10 times with deionized water. The beads were then incubated with 1 ml TBS buffer (25 mM Tris-HCL, pH 8.0, 150 mM NaCl, and 0.1 % Tween 20) containing 2 mg bovine serum albumin for 1 hour at room temperature to block any nonspecific protein binding sites. The beads were next suspended in fresh TBS buffer containing 4-5 pg biotinylated SHP-1(ASH2) or PTPIB in the presence of 1 mM P-mercaptoethanol at room temperature for 20-30 min. Then, the solution was drained with vacuum suction, and the resin was washed once with 1 ml of TBS buffer. The beads were incubated with 1 ml TBS buffer containing 1 pg straptavidine alkaline phosphatase conjugate for

10 min and the solution was removed from the beads and washed once with PBST (40 mM sodium phosphate, pH 7.4, 280 mM NaCl and 0.2% Tween 20), twice with low salt buffer (20 mM Tris-HCl, pH 8.5 and 10 mM NaCl). The beads were suspended in

67 1 ml low salt buffer with 0.5 mg of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) in a

petri-dish while gently shaking at room temperature for 2-4 hours. The placement of

the beads in a petri dish allows for convenient periodic monitoring of staining reaction

under a microscope. The staining procedure was quenched by incubation in 6 M

guanidine hydrochloride for 20 min and the beads were washed exhaustively (20-30

times 1 ml) with deionized water. Positive beads were identified by their intense

turquoise color (figure 3.4) and were manually removed fi-om the library with a

micropipette under a low power microscope. A control screening reaction was

performed with the inactive biotinylated C453S mutant of SHP-1(ASH2) under the

same conditions as the positive reaction. Another control reaction for PTPIB was also

carried out with the biotinylated-inactived PTPIB (by inhibitor la) at the same

conditions as positive reaction. Both control screenings resulted in no colored beads.

Individually selected beads were dried and incubated with 25 pi of 30 mg/ml

cyanogen bromide (CNBr) in 70% formic acid for 20-24 hours in the dark. The

peptides cleaved firom beads were dried under vacuum to remove the excess CNBr and

the dried peptides were dissolved in 3.5 pi of 0.1% TFA in deionized water. For

sequence analysis, 1 pi of dissolved peptide was mixed with 2 pi of a saturated

solution of 2,5-dihydroxybenzoicacid (DHB in 0.1% TFA), mixed quickly followed by spotting 1 pi from the mixture on the MALDI sample plate. The analysis of the

sample was performed on a Kratos Kompact MALDI-UI mass spectrometer in the positive ion mode as described previously (48).

68 3.2.5 PTP inhibition assay

Continuous (for SHP-1(ASH2) and PTPIB) and end-point (for VHR) inhibition assays described in section 2.2.5 were performed for phenylacetaldehyde and benzaldehyde compounds. Different inhibitor concentrations (10-1000 pM) were used to determine the inhibition rates with j?-NPP as a substrate in 50 mM HEPES buffer, pH 7.4. The reactions were initiated by the addition o f 0.1-0.5 pg of phosphatase and monitored at 405 run on a UV-Vis spectrometer. The initial rates were obtained from the first 100 sec of the reaction curves. The same reaction was also performed in the absence of inhibitors. These rates were converted to percentages of the maximal rate (in the absence of inhibitor). The IC 50 values were determined by plotting these percentages as a function of inhibitor concentrations (Table 3.3)

3.3 Results

3.3.1 Inhibition of SHP-1(ASH2) and PTPIB by a-haloketones

The previous chapter described the inactivation of PTPs by a- haloacetophenones. Here the study was extended to several commercially available and some synthesized compounds in order to better characterize the relationship between structure and potency of inhibition. These compounds were tested for inhibition of SHP-1 (ASH2). Structures of the compounds tested are listed in figures

69 3.1 & 3.2. Four of the compounds tested (figure 3.1 & 3.2; 5, 7-9, 17, 18) were synthesized in the Pei laboratory by Drs. Yi and Fu. Kinetic experiments and analyses were carried out as described in section 2.2. Table 3.1 lists the equilibrium inhibition constants (K,-) and first order inactivation rate constants (kmact) for the inhibition of

SHP-1(ASH2) by the compounds tested.

In chapter 2, a-bromo-parahydroxyacetophenone (figure 2.1, la) was identified as a potent inhibitor of PTPIB and SHP-1(ASH2). The inhibition constant measured was 43 pM. First, compounds 1-6 (figure 3.1) were tested in order to determine the effect of the para group substituent on inhibition potency. The lowest inhibition constant was observed for compounds 5 and 6 . The weakest binding was observed for compounds 2 and 4 containing nitro and bromo groups at their para positions. However compounds 2 and 4 possessed highest values for the first order inactivation rate constant suggesting possible electronic effects.

Comparing the data for compounds 7, 8 and 9, no significant variation in inhibition potency was observed. When chloride replaces bromide as the leaving group at the a position (figure 3.1; 10-14), at least a 10-fold reduction in binding affinity is observed. This trend was previously noted in chapter 2. Compounds 13 and

14 displayed considerably weakened binding and no appreciable increase in the first order inactivation rate constant.

Since para substituted a-bromoacetophenones inhibit SHP-1(ASH2) and

PTPIB with similar potency (table 2.2), it was suggested that specificity could be

70 induced by attaching specificity elements to the para position of the inhibitor nucleus.

Earlier work on the inhibition of PTPIB by cinnamic acid (99) derivatives showed that the inhibition could be greatly improved by attaching specificity elements namely the tripeptide GEE to the para position (99). We rationalized that a similar strategy to attach specificity elements to the para position of a-bromoacetophenones might confer the ability to bind a particular PTP with greater potency. Table 3.2 summarizes the effect of adding potential specificity elements to improve the inhibition of SHP-

1(ASH2) and PTPIB. For SHP-1 (ASH2), the addition of GEE and EE displayed a slight increase in binding affinity relative to compound Ic (tables 2.2 & 3.2). The addition of GEE and EE caused the potency of PTPIB inhibition to improve 10-50 fold, similar to the effect caused in cinnamic acid derivatives (99). Compounds 17-20 were tested to explore if restricted rotation could improve the potency of inhibition.

Such conformationally restricted linkers can improve inhibition by significantly reducing the unfavorable entropy loss on binding. However, this strategy provided no increase in binding affinity.

Inhibition by Aldehydes. Recently it has been shown that calpeptin (a peptide aldehyde inhibitor of the cysteine protease calpain) acts as a reversible inhibitor for

PTPs (78, 79). Thus, we decided to test some aldehyde containing compounds for inhibition of PTPs. Benzaldehyde and phenylacetaldehyde were tested for inhibition of

SHP-1(ASH2), PTPIB and dual specificity phosphatase VHR (table 3.3). The aldehyde functionality in these compounds could potentially trap the catalytic cysteine

71 and provide a basis for inactivation and covalent attachment. However, benzaldehyde provided no inhibition of all three phosphatases up to 500 |oM. Phenylacetaldehyde inhibited PTPIB and SHP-1(ASH2) with IC 50 values of 150 pM and 500 pM respectively. A more detailed kinetic study was carried out for the inhibition of

PTPIB. The type of inhibition (usually classified as competitive, uncompetitive or noncompetitive) was reversible and mixed type.

3.3.2 Combinatorial Approach to Improve Specificity of PTP Inhibition

We proposed to combinatorially identify ligands that would confer specificity and potency of inhibition to a-bromoacetophenones. This subsection describes the approach used and the results obtained for the screening o f different peptide libraries

(containing a-bromoacetophenones) against SHP-1(ASH2) and PTPIB.

Library Synthesis. Combinatorial peptide libraries (figure 3.3; library 1-3) with four randomized positions were synthesized on TentaGel S resin using the split-pool synthesis method (section 3.2). This synthesis provided approximately 100 pmol of a unique peptide sequence on each bead. This method gives equal representation of all amino acids used at each of the randomized positions. In this work, each randomized position included 17 amino acids. Arginine, lysine and cysteine were excluded and methionine replaced by its isosteric analog norleucine. The side chains of arginine and lysine posed the risk of possible side reactions with the activated inhibitor-NHS ester during library synthesis. Possible non-specific biotinylation was also another reason

72 for excluding arginine and lysine. The theoretical diversity of the libraries 2 and 3 is

\1^ or 83521 and 17^ or 4913 unique sequences. The peptide linker BBRM, was added

to the C-terminus of the random region for the following reasons. The C-terminal

methionine allows for the bound peptides to be cleaved from the resin by cyanogen bromide treatment prior to sequencing. Omission of methionine from the randomized positions does not allow internal cleavage. Arginine improves the solubility and ensures greater efficiency of ionization, improving the sensitivity for MS analysis. The

P-alanine (B in BBRM) residues add flexibility to the peptides making them more accessible to the enzyme.

Sequence Analysis. For sequencing purposes we used a technique, which enables rapid determination by MALDI mass spectrometry (48, 76). This method encodes the peptide sequence on each bead by generating a set of chain termination products as the peptide is being synthesized (figure 3.5 (48)). This was achieved in our case by the addition of a small amount of a capping agent (10% A-acetyl-D,L-alanine

(for norleucine); 5% each of A-acetyl-D,L-alanine and A'-acetylglycine (for iso leucine); 10% AT-acetylglycine (for all other amino acids) to each reaction vessel along with the individual amino acid (90%) during the synthesis of the randomized region. For the libraries tested, each bead contained a full-length peptide and four or three (in library 2 or 3) chain-termination products. The sequence of the full-length peptide was determined from the mass differences between these termination products.

73 Bead Selection. Typically, 25 mg of resin was used in each screening which was sufficient to statistically represent each sequence once. Screening was based on the phosphatase binding followed by inactivation and covalent attachment to the bead selected (figure 3.4). Previously, the PTP being screened was biotinylated at surface lysine residues by treatment with NHS-biotin. This allows a streptavidin-alkaline phosphatase conjugate to be recruited to the bead surface. Addition of 5-bromo-4- chloro-3-indolyl phosphate (BCIP), which is a substrate for alkaline phosphatase results in hydrolysis on the surface of selected beads. BCIP hydrolysis gives indole, which dimerizes in air to indigo dye ultimately resulting in a turquoise precipitate being deposited on the bead surface (figure 3.4). These colored positive beads carrying sequences selected by the PTP used, were individually removed firom the library, washed with water, and treated with cyanogen bromide. This cleaved the peptide off the solid support at the C-terminal methionine residue, generating a homoserine lactone. MALDI analysis of the cleavage mixture generated a peptide ladder in the mass spectrum firom which the sequence was deduced (figure 3.5).

Specificity of SHP-I(ASH2) and PTPIB Inhibition. The combinatorial approach described above was proposed to identify specific amino acid residues which when attached to inhibitor Ic (figiure 2 .1 ) would improve significantly the specificity and potency of inliibition of a particular PTP. Towards this end. Dr. Kirk Beebe

(graduate student in the Pei laboratory) screened library 1 (figure 3.3) against SHP-

1(ASH2). However, no selectivity was observed at any of the four positions and it was

74 suggested that a conformationally restricted inhibitor nucleus might provide tighter

binding. Thus, libraries 2 and 3 were prepared and screened against SHP-1(ASH2) and

PTPIB. The results obtained by screening library 2 with SHP-1(ASH2) and PTPIB

are presented here. Figure 3.6 shows the positional selectivity displayed by SHP-

1(ASH2). Surprisingly, no significant selectivity is displayed at position Pi as evidenced by the selection of acidic, hydrophobic and aromatic residues at that position. Positions and P 3 display moderate selectivity for glutamate and tyrosine respectively. Pro line is selected for at position P 4 but not significantly. Table 3.4 lists the sequences identified by SHP-1(ASH2). They have been arbitrarily classified into three parts based on identity at the Pi position. Similarly, figure 3.5 and table 3.5 summarizes the results obtained by screening library 2 with PTPIB. Again, little selectivity is displayed at all four positions. Possible reasons for this apparent lack of specificity in SHP-1(ASH2) and PTPIB are discussed in section 3.4.

The attachment of the randomized position was changed in library 3 (figure

3.8), to examine if the selectivity was influenced. This library had a reduced size (17^

= 4913 unique sequences). The sequences selected by PTPIB and SHP-1(ASH2) are listed in Tables 3.6 and 3.7 respectively. Analysis for PTPIB showed selectivity for leucine at the P 3 position with no significant selectivity at Pi and P] The selectivity

for SHP-1(ASH2) was again most noticeable at P 3 with selection of leucine, isoleucine, phenylalanine and tyrosine.

75 3.4 Discussion

The broad goal of the experiments outlined in this chapter were to derivatize the para position of the a-haloketone inhibitor nucleus with various specificity elements so that by interacting with residues outside the active site the derivatized inhibitor would exhibit selectivity towards a specific PTP. Initial efforts were focused on testing various a-haloketones to build a structure vs. activity relationship. Later, a combinatorial approach was employed to screen for peptide based specificity elements that would increase the specificity and potency of inhibition towards a particular PTP.

The catalytic domain of SHP-1 (SHP-1(ASH2)) and PTPIB were used in the study.

The results obtained in chapter 2 indicated that a-bromoacetophenones acted as potent, time-dependent inactivators of PTPs. In this section, the data set was expanded to 20 compounds (figures 3.1 & 3.2 and tables 3.1 & 3.2). The kinetics of

PTP inactivation for these compounds was examined using the methods described in chapter 2. The reaction between the catalytic cysteine nucleophile in both PTPs (SHP-

1(ASH2) and PTPIB) and the electrophilic a-carbon center is most likely a Sn2 type reaction. This is supported by a lack of significant variation in the first order inactivation rate constant (kmact) in the presence of significant variations in electronic effects. However, these variations in electronic effects cause significant changes in the equilibrium binding constant measured (K,). Specifically, compounds 2 and 7 contain significantly deactivated aromatic rings and maybe poorer mimics of the physiological tyrosine ring. Variation due to different types of aromatic rings (compounds 6 , 8 and

76 9) produce little change in inhibition potency. Comparing compounds 1 with 10 and 3 with 1 1 shows a weaker binding for a-chloroketones relative to the bromo analogs.

This may be because the bromo compounds are better mimics of the phosphate reaction center than the corresponding chloro compounds. In summary, apart from the generally higher potency of bromides relative to chlorides, no significant enhancement in potency of inhibition was observed that correlated with any obvious molecular or structural properties. Briefly, aldehyde containing compounds were tested for PTP inhibition. Phenylacetaldehyde provided moderate inhibition of PTPIB and SHP-

1(AHS2) that in the future may be studied in greater detail.

Previous results indicated that the potency of PTPIB inhibition by cirmamates could be improved significantly by the attachment of the tripeptide GEE to the para position (99). Thus, we envisioned a similar extension to the a-bromoacetophenones.

Addition of these potential specificity elements could mimic amino acid residues flanking the pY site of physiological PTP substrates. The specificity elements were identified by a combinatorial approach. Initially, the library was synthesized by attaching the peptide library part with three or four randomized positions to the inhibitor nucleus. Synthesis of the library using the split-mix strategy (48) provides a unique sequence on each bead. The amount of beads used in a screening experiment ensured that each unique sequence was represented at least once (76). After derivatization with the a-haloketone inhibitor and selection with a PTP, sequences of the positive beads could be determined rapidly by MALDI mass spectrometry. The

77 major advantage of this method is that individual sequences can be obtained, allowing for the identification of any specific inhibitors regardless of their abundance in the library. In control experiments, specificity elements GEE, EE etc were added to a- bromoketones and the kinetics of SHP-1(ASH2) and PTPIB inactivation recorded.

The addition served to improve the potency of PTPIB inhibition in line with the results obtained fi-om cirmamates (99). However, no enhancement of SHP-1(ASH2) inactivation was observed.

Initially, library 1 was screened against SHP-1(ASH2). No selectivity was observed at any of the four randomized positions. It is possible that excessive flexibility at the linkage between the inhibitor ring and peptide portion served to reduce the potency of inhibition due to unfavorable entropy loss upon inhibitor binding. Therefore, libraries 2 and 3 were constructed in which a conformationally restricted biphenyl group was used. Other methods of conformation restriction could use double bonds or substituted amide linkages to serve the same purpose. Results obtained by screening library 2 with SHP-I(ASH2) and PTPIB are presented in this chapter.

With the exception of the P3 position in library 3, significant selectivity at any randomized position (for both SHP-l(ASH2) and PTPIB) was not displayed. Further, no discernable trend like selection of acidic, hydrophobic or aromatic residues at any of the positions was noted. There could be different reasons for this apparent lack of selectivity. The lack of selectivity may originate from technical and experimental

78 factors. Control experiments that maybe performed could include the use of other resins in library synthesis. Others have noted that various resins have different properties affecting enzyme accessibility and other factors (60).

One obvious reason could be the general lack of specificity in both enzymes.

Particularly, in SHP-1 the two SH2 domains may play a crucial role in determining specificity. Their absence in SHP-1 (ASH2) may have caused a lack o f observed selectivity. It must however be pointed out that screening with SHP-1 would be complicated by the loss in potency of inhibition. Another reason for the lack of selectivity could be that the randomized peptide library represents only the C-terminal region of a pY peptide substrate. Structural studies with SHP-1 and PTPIB have indicated a preference for acidic residues N-terminal to the pY position in a substrate

(72). Therefore, a more physiologically relevant library to screen would contain the inhibitor nucleus and flanking peptide sequences on both sides. However, the synthesis of such a library at this time poses significant technical difficulties. Careful and detailed analysis of a crystal structure of the PTP with a substrate analog bound in its active site would aid tremendously. Identification of critical contacts and other structural details followed by molecular modeling are needed.

79 Br Br 'B r

(3) 2 <1 ) NO (2)

Br Br

(5) Br (4) CF,

Br Br Br

(7) (8) (9)

Cl

(10) (11) (12)

O. Cl F

OH F OH

(13) (14)

Figure 3.1. Compounds tested for PTP inhibition.

80 Inhibitors (fig 3.1) Ki(pM) kinact (min")

1 81+2 0.58+0.02

2 134±35 2.133±1.29

3 56.3±0.34 0.51±0.03

4 100±8.2 1.85+0.7

5 14.2±0.76 0.37±0.01

6 14.36±7.5 0.59±0.16

7 173+19.2 1.3+0.20

8 91.9+4.1 0.86±0.03

9 24.6+8.7 0.48+0.04

1 0 4 8 0 + 2 5 0.21+0.04

1 1 380±48 0.22±0.07

1 2 N D ND

1 3 17491±2300 2.1+0.01

1 4 1394±990 0.59±0.24

ND = not determined

Table 3.1. Kinetic constants of SHP-1(ASH2) by 1-14. Data reported are the mean ± SD from three or more independent experiments carried out at pH 7.4 and at room temperature.

81 HO HO

OH NHj

HO HO

OH

18 o

HO OH

OH NH.

HO HO

Figure 3.2. Compounds tested for the inhibition of SHP-1(ASH2 ) and PTPIB.

82 En 2ymes Inhibitors K i(p M ) kinact (min ')

SHP-I(ASH2) 15 29.4±4 6.6±0.4 16 22 6.0 17 241±14 1.3+0.2 18 152±12 3.0±0.3 19 1587±137 2.5± 0.1 20 >200 >0.13

PTPIB 15 2.8+0.5 1.2+0.1 16 12 3.9 17 3.5±0.2 0.4+0.1 18 I.0±0.02 0.5±0.1 19 0.8± 0.02 0.4+0.1 20 2.0+0.3 0.5±0.1

Table 3.2. Kinetic constants of PTP inhibition by 15-20 (Inhibitor 15-16 were from Kirk D. Beebe’s unpublished results). Data reported are the mean ± SD from three or more independent experiments carried out pH 7.4 and at room temperature.

83 Enzymes Inhibitor IC50 (l-iM)

PTPIB CfiHsCHzCHO 150 SHP-UASH2) 500 VHRNI PTPIB CfiHsCHO NI SHP-1(ASH2) NI VHR NI

NI= No Inhibition up to 1 mM of inhibitors

Table 3.3. Kinetic constants of PTPs inhibition by aldehydes. CgHgCHzCHO is pheylacetaldehyde. CgHgCHO is benzaldehyde.

84 Library 1

Br

X,X,X,X^BBRM

Library 2

Library 3

Figure 3.3. Library 1 Inhibitor Ic attached to N-terminal peptide library (unpublished data by Krik D. Beebe). Library 2 Inhibitor 17 attached to N terminal peptide library. Library 3 Inhibitor 18 attached to N terminal peptide library.

85 FTP (3"MRBBXXXInh (^hMRBBXXXInh

PTP

1) SA-AP c o n ju g a te 2) BCIP

Biotin

InhXXXBBRM

[O]

Br

Br Blue color

Figure 3.4. A model for library screening of PTPs. (SA= streptavidin, AP= alkaline phosphatase).

86 MRBBLFE-Inh 2000 MRBBcap MRBBXjCap MRBBXgXgCap 1500 - MRBBX3X2X, cap

1000 -

500 -

J _”1“ l £ 500 600 700 800 m/z

Figure 3.5. MALDI-mass spectrometry of typical peptide mixture derived from selected TentaGel S NH 2 bead carrying library 3 (figure 3.3) (B=P-Alanine).

87 P 1

ADEÉL lFGH 1L LMNPQSTWYV . Jl

"z 1 J ■ u(I) iLiADE FGH 1L MNPQST WYV C m mCD Q.a (C V-o o Z ■ ADE■1 FGHMHwdhjLILMNPQSrWY

ADE FGH IL MNPQSTWYV

Figure 3.6.Specificity, as described by the screening of Library 2 (figure 3.3) of SHP- 1(ASH2). Displayed are the amino acids identified each position from Pi which is next to the Inhibitor 17 to P 4 which is far to the inhibitor 17. Abundance on y axis represents the number of selected sequences that contain a particular amino acid at a certain position.

88 peptide peptide sequence sequence

Class I Inh-YNIL Inh-EAYP Inh-YEPA Inh-SIYQ Inh-YEMP Inh-YGN Class III Inh-YGNV Inh-XDQV Inh-FTQF Inh-XDSH Inh-FPSW Inh-XILN Inh-WEGP Inh-XAFQ Inh-VEVS Class II Inh-VEPP Inh-DEDI Inh-VIAS Inh-DIWP Inh-AEYN Inh-DPYT Inh-APQH Inh-DAYA Inh-AFHL

X could be iso leucine, leucine or norleucine, M is norleucine

Table 3.4.Sequences of peptides selected from Library 2 (figure 3.3) by SHP- 1(ASH2).

89 à lLADEFGH IL lMNPQSTWYV

c 2 ADEFGH IL MNPQSTWYV O)c5 a . CL CO 'o o l u . k j I ADEFGH IL MNPQSTWYV

ADEFGH IL MNPQSTWYV

Figure 3.7. Specificity, as described by the screening of Library 2 (figure 3.3) of PTPIB. Displayed are the amino acids identified each position fi-om Pi which is next to the inhibitor 17 to P 4 which is far to the inhibitor 17. Abundance on y axis represents the number of selected sequences that contain a particular amino acid at a certain position.

90 peptide peptide sequence sequence

Class I Inh-EPHY Inh-GLAT frih-EPLD Inh-GLAY Inh-EEPQ Inh-GWHY Inh-EDDQ Inh-APQN Inh-EVFA Inh-AVDV Inh-ENAQ Inh-ADLM Inh-DMPA Inh-XLWL Inh-XHTN Class n Inh-XHDP Inh-YQYM Inh-XYGN Inh-YNFA Inh-XPQA Inh-FSIW Inh-VDVL Inh-FEHH Inh-VFFA Inh-FYVP Inh-VMVT Inh-WYDM Inh-VLIL Inh-VLEF Class in Inh-PAAW Inh-GGHY Inh-NSNH

X could be isoleucine, leucine or norleucine, M is norleucine

Table 3.5.Sequences of peptides selected from Library 2 (figure 3.3) by PTPIB.

91 A D E F G H N PQ STWYV

12 10 0) 0 8 c CD2 6 (U Q. 4 Q. «3 2 "o 0 0 z ADEFGH ILMN PQ STWYV

ADEFGH ILMN PQ STWYV

Figure 3.8. Specificity, as described by the screening of Library 3 (figure 3.3) of SHP- 1(ASH2). Displayed are the amino acids identified each position fi-om Pi which is next to the Inhibitor 18 to P 3 which is far to the inhibitor 18. Abundance on y axis represents the number of selected sequences that contain a particular amino acid at a certain position. (M is for Nleu, X colud be I, L or M).

92 Peptide Peptide sequence Sequence Class I Inh-GFL Inh-YMY Inh-GHL Inh-YVH Inh-G II Inh-YIQ Inh-GVW Inh-WYL Inh-XDF Inh-XTF Class n Inh-XDI Inh-DYV Inh-XMF Inh-DLY Inh-XLF Inh-DGF Inh-XQY Inh-EFL Inh-XIY Inh-HDP Inh-XEF Inh-HDL Inh-VLL Inh-HGL Inh-VEL Inh-HNI Inh-AFL Inh-HHL Inh-AFI Inh-SHH Inh-AQY Inh-TEI Inh-NQN Inh-NVE Class III Inh-NVF Inh-GLN Inh-QGL Inh-GLF Inh-QFI Inh-GPS Inh-VPF X could be isoleucine, leucine or norleucine, M is norleucine

Table 3.6 . Sequences of peptides selected from Library 3 (figure 3.3) by SHP- 1(ASH2).

93 DEFGH X N PQ STWYV

12 10 (U Ü 8 «Cc 6 I 4 2 'oI o 0 ADEFGH I L MNPQSTWYV

DEFGH I L MNPQSTWYV

Figure 3.9. Specificity, as described by the screening of Library 3 (figure 3.3) of PTPIB. Displayed are the amino acids identified each position from P, which is next to the Inhibitor 18 to P 3 which is far to the inhibitor 18. Abundance on y axis represents the number of selected sequences that contain a particular amino acid at a certain position. (M is for Nleu, X colud be I, L or M)

94 Peptide Peptide sequence Sequence Class I Inh-AML Inh-DAL Inh-XLV Inh-DPS Inh-XLL Inh-DHV Inh-XVF Inh-DHV Inh-XIF Inh-DWL Inh-VAL Inh-ELP Inh-VWV Inh-VQQ Class II Inh-VLQ Inh-YQL Inh-VLL Inh-YTF Inh-VDL Inh-YHL Inh-VDG Inh-VDN Class III Inh-WVS Inh-GQD Inh-HHY Inh-GPS Inh-QFL Inh-PYI Inh-TQG Inh-AQE Inh-TFL X could be isoleucine, leucine or norleucine, M is norleucine

Table 3.7. Sequences of peptides selected from Library 3 (figure 3.3) by PTPIB.

95 CHAPTER 4

SUMMARY AND CONCLUSION

Signal transduction processes are important biochemical mechanisms that regulate mitogenesis, motility, growth, metabolism, immune response and gene transcription (8, 9). Most eukaryotic signal transduction pathways are mediated by kinase dependent phosphorylation and phosphatase dependent dephosphorylation of target proteins (10), causing activation, inactivation or localization of various proteins

(12, 17, 18). The biochemical signals can also be amplified, suppressed, interrupted or communicated to specific cellular regions (20). Importantly, enzymes involved in key signaling pathways are often implicated in studies of human diseases and abnormalities (39, 40). Detailed biochemical studies of these signaling pathways are hampered by the lack of specific inhibitors for the signaling enzymes. Such specific inhibitors in addition to providing important biological tools help develop possible therapeutic compounds.

This dissertation focuses on the development of specific inhibitors for an important subclass of signaling phosphatases called protein tyrosine phosphatases

(PTPs). Chapter 2 describes the discovery and characterization of a-

96 haloacetophenones as novel covalent and photo-reversible PTP inhibitors. The kinetic

constants for the time-dependent nature of PTP inhibition were measured. Mass

spectrometric experiments confirmed that PTP inhibition occurred by the covalent

attachment of the inhibitor to the active site cysteine. The inactivated PTP could be

reactivated by irradiation with UV light, thereby providing a means to turn off and on

the PTP activity. The in-vivo effect on tyrosine phosphorylation levels was also

demonstrated.

The a-bromoacetophenones studied thus far provided moderate PTP inhibition potency and selectivity. An improvement in these parameters was needed before the ultimate goal of developing specific inhibitors for a given PTP could be realized.

Previous experiments showed that the potency of PTPIB inhibition by cinnamates could be improved considerably by the attachment of a peptide based specificity element. Therefore, a combinatorial strategy was developed and employed to identify similar peptide specificity elements to improve PTPIB and SHP-1(AHS2) inhibition.

The inhibitor library consisted of the selected a-bromoketone compound attached to a library of peptide sequences. After screening with the PTPs and sequence analysis, peptide elements were identified that moderately improved PTPIB and SHP-1(ASH2) inhibition. Further improvements may be achieved by detailed structure analysis and by including both N and C terminal sequences that flank the inhibitor core.

97 APPENDIX A

SUBSTRATE SPECIFICITY OF PEPTIDE DEFORMYLASES

Introduction

Peptide deformylase (PDF) is an essential Fe^^ metalloenzyme that catalyzes

the removal of the N-terminal formyl group from nascent ribosomally synthesized

polypeptides in eubacteria (47, 52). Its important role in post-translational

modification and protein maturation has made it an attractive target for antibiotic

design (52). Nearly 100 PDF sequences have been identified from bacterial genomes to date. PDFs exist generally as monomers of around 200 amino acids and PDF from

E. coli has been characterized structurally and mechanistically (53). Fe^^ has been suggested as the metal ion used in-vivo and explanations provided for conflicting reports in the literature (54). Stable Ni^% Co^^ and Zn^"^ substituted PDFs have also been prepared and used (53).

Substrate specificity studies of E. coli PDF began by synthesizing and testing individual N-formylmethionyl peptides (48). Individual kcat/Km values were measured and conclusions drawn. Later, the substrate specificity o f E. coli PDF was examined elegantly and in greater detail by a combinatorial approach (48). A N-formyl peptide library with four randomized positions was synthesized by the split-pool method and

98 screened against the Fe^^-PDF (48). The consensus sequence derived was f-

MX(F/Y)Y where X could represent any amino acid except aspartate and glutamate.

The specificity for methionine at the first position is not surprising given that the physiological substrates are N-formylraethionyl peptides. Little selectivity was found at the second position. The third position showed a preference for phenylalanine and tyrosine and the fourth for tyrosine (48).

The discovery of a sequence in the eukaryotic organism Plasmodium falciparum (P. falciparum) with 33% homology to the E. coli PDF prompted its investigation (103). Initial biochemical characterization of this P. falciparum protein revealed a few fold lower PDF activity relative to E. coli PDF. We proposed to examine the substrate specificity ofP. falciparum PDF and also B. subtilis PDF by a similar combinatorial approach.

Strategy and Methods

Method A. In this method the library synthesis was carried out as described in chapter 3 except the N-terminal position was formylated using excess formic acid and diraethylamino pyridine (DMAP). The library was screened with 1-2 pg B. substilis

Co^^-PDF or P. falciparum Ni^^-PDF enzymes in 1 ml buffer (50 mM sodium phosphate, pH 7.4 and 150 mM NaCl). Details have been published elsewhere (48).

After removing the enzymes any fi-ee N-termini were biotinylated using N- hydroxysuccinimidobiotin/DMF for 30 minutes and followed by incubation with streptavidin-alkaline phosphatase conjugate for 10 minutes. After staining with BCIP

99 and bead selection, MALDI based sequence analysis was performed as described in chapter 3.

Method B. This method utilizes a novel means of sequence determination by partial Edman degradation (13). The library was synthesized in a similar manner to the previous method except that capping was done only for norleucine and isoleucine in order to distinguish between amino acids of the same molecular weight (norleucine, leucine and isoleucine).

PDF Activity Assay. Formate release from N-formylated peptides catalyzed by

PDF was monitored by a formate dehydrogenase assay (52). A standard line was generated using the oxidation of 20-200 pM sodium formate by 0.25 units of

Pseudomonas oxalaticas formate dehydrogenase (Sigma) in the presence of 1 mM

NAD’*’ and 50 mM sodium phosphate (pH 7.0) in a total volume of 1 ml. The reactions were incubated at 0"^C for 25 minutes and the absorbance at 365 nm measured and plotted against concentration of sodium formate. PDF reactions in a volume of 0.5 ml with 0-2 mM substrate were allowed to react till ~ 10% product conversion was reached. The reaction was quenched by heating and 10 pi of 100 mM NAD’*' and 0.25 units of formate dehydrogenase were added. The total volume was then adjusted to 1 ml and the reaction incubated at 0°C for 20-25 minutes. Absorbance at 365 nm was measured and the formate released calculated.

100 Results and Discussion

Amino acids lysine and arginine were omitted from the library as their side chains would be biotinylated and lead to false positives. Cysteine and methionine were excluded from the randomized positions due to possible oxidation and internal cleavage, though norleucine was used to substitute for methionine. The theoretical diversity of the library used was \1^ or 83521 different sequences. The peptide linker

BBRM was added to the C-terminus to make the peptides more flexible and allow more access to the screening enzyme. Arginine was incorporated to increase the detection sensitivity for mass analysis because of its high ionization ability.

Methionine was used to allow cleavage from the bead using cyanogen bromide.

Typically, 80-150 blue beads were selected and sequence analysis done. Generally,

65% of positive beads could be sequenced completely, while 35% had one or two peaks missing. The partial Edman degradation sequencing method allowed successful sequencing of 80% of positives. A detailed discussion of the advantages and problems of this method can be found elsewhere (13).

The sequences selected by each enzyme were grouped into different classes based on the identity of the N-terminal residue. The sequences selected are listed in tables Al, A2 and A3. Figures Al, A2 and A3 summarize the selectivity at each position graphically. The P. falciparum and B. subtilis PDFs displayed marginal selectivity for methionine at the first position (Pi) and Uttle selectivity at P 2 , P 3 and P 4 positions. As a control, similar selection was performed with E. coli PDF and strong selection for norleucine was noted at Pi This is consistent with earlier results (48).

101 Some of the sequences selected by P. falciparum PDF were synthesized and their

kcat/Km values determined. The kinetic properties were significantly different firom E.

coli PDF, but a more detailed analysis is required before any definitive conclusions

can be drawn. Summarizing, the substrate specificity for P. falciparum and B. subtilis

PDFs are most likely different from the E. coli PDF though more experiments have to be done for confirmation.

102 Peptide sequence Peptide sequence Peptide sequence Class I f-FHLI Class V f-AFQW f-FYGL f-SAVA f-AFNP f-FFHP f-SAGW f-AFHY f-FSQY f-SVQW f-AYQI f-FSEA f-SVML f-ADVW f-FEFS f-SNWW f-ADVF f-SPWH f-ADYW Class m f-SFLF f-ADYI f-MMYS f-SEME f-AEHG f-MMHY f-HNHF f-AHPF f-MLAI f-HDHF f-AHPS f-MIMH f-HSTY f-AAHA f-MVTN f-HVHH f-AAYT f-MVGQ f-HYHP f-APGF f-MWLA f-WDQI f-ANIP f-MWFF f-WHMI f-ASAW f-MTSY f-WNPH f-ASHL f-MSHQ f-PEVS f-AWFY f-MEYL f-PHMA f-MFGS f-PYMI Class n f-MFHF f-GFHH f-YAVP f-MYSQ f-GTWH f-YAAV f-MYTY f-GPLQ f-Y W I f-MHAYF f-LHFL f-YNWG f-MHLL f-LHWH f-YLGW f-LFGW f-YQYQ Class IV f-NFFF f-YYQQ f-DYGL f-NYQH f-YYMN f-DFFF f-NDIG f-YFYH f-DAPI f-NEWL f-YMWA f-DPMD f-NHVA f-YMFY f-DWMH f-NQMT f-YENG f-ETMV f-QAMF f-YHTH f-ETGI f-QGIY f-FHHH f-EPYF f-QHVF

Table A l Sequences selected by P.falciparium PDF. (M = Norleucine).

103 ANDQEGHI LMFPSTWYV

20

15

10 1

ANDQEGHI LMFPSTWYV

20

15

10

ijiiijyANDQEGHI LMFPSTWYV

ANDQEGHI LMFPSTWYV Figure A.I. Positional selectivity of P. falciparum PDF. (M = Norleucine).

104 Peptide sequence Peptide sequence Peptide sequence

Class I Class III Class IV f-MHSY f-HQYL f-YLNH f-MFFY f-HEWH f-YHHY f-MFWG f-HGYF f-YHYP f-MDWY f-HYSH f-YYYA f-MMML f-HYAN f-YALY f-MWIF f-HLHI f-YHDY f-MHSN f-HPAP f-YVFW f-MYGN f-YTVF f-MQHL Class IV f-YYPF f-MHSH f-FHMF f-YNFL f-MHSP f-FSHF f-YHQF f-MDHG f-FGNL f-YQTN f-MIHM f-FQPH f-YFLL f-MPMY f-FQGN f-YQYF f-MNHH f-FVNP f-YYLW f-MLML f-F\TWT f-MHYY f-FYFW Class V f-MPNA f-NAVQ f-MPFY Class V f-NQHT f-MTMI f-QSWH f-NSPQ f-MLHG f-QEYY f-NFHF f-MWFI f-QQHF f-NYPW f-MSQP f-QHHW f-NHHM f-MHVF f-QSPQ f-NQAL f-QQYE f-SIMI Class n f-QHMV f-SFDH f-ANm f-QHYN f-SAYF f-AEIA f-PLYA f-SFHI f-ANPH f-PFHW f-SFMH f-AHFY f-PWYH f-GFNF f-APHW f-PFYY f-GLEL f-AQFH f-PFMH f-GMYH f-AHML f-LHVY f-GADT f-AHMY f-LAIP f-WAMF f-AGLY f-LNDG f-WINH f-ALLQ f-LHNL f-DLAL f-ASIP f-LLYH f-ESNS

Table A.2. Sequences selected by B. subtilis PDF. (M = Norleucine).

105 DEFGHILMNPQSTWYV

Figure A.2. Positional selectivity of B. subtilis PDF. (M = Norleucine).

106 Peptide sequence Peptide sequence Peptide sequence Class I f-MLVP f-MPAE f-WIVH f-MLGH f-MPDP f-WHNT f-MLTY f-MPLI f-MLAS f-MPSF Class m f-MLLP f-MPME f-LHSV f-MLTY f-MPTG f-LNLG f-MYPG f-MMEL f-LTDP f-MYYD f-MMFH f-LGHT f-MYEQ f-MMMP f-IHFF f-MYVP f-MSFP f-IVPY f-MYLT f-MSYL f-IESF f-MYGL f-MAYV f-ATLA f-MYTI f-MGLF f-AAEY f-MYVP f-GWSA f-MFWE Class n f-VALG f-MFTH f-YHGP f-NEQA f-MFWE f-YHAS f-SYGH f-MVYP f-YAHY f-HGHS f-MHMP f-FLHG f-HNTL

Table A 3 Sequences selected by E. coli PDF. (M = Norleucine).

107 40

30

20

10

0

40

30

20

10

0 AOEFGHILMNPQSTVWY

40

30

20 -

10

0 ADEFGHILMNPQSTVWY

40

30

20

10

0 ADEFGHILMNPQSTVWY

Figure A 3 Positional selectivity oïE . coli PDF. (M = Norleucine).

108 APPENDIX B

SUBSTRATE SPECIFICITY OF SHP-1(ASH2)

Introduction

PTP substrate specificity can originate firom to two factors. First, the PTP catalytic site could demonstrate specificity by binding and dephosphorylating certain pY peptides better than others (44-46). Also, the targeting and regulatory domains of

PTPs could localize it to act on a particular target protein thereby giving rise to selectivity (45, 95). As a first step in determining the relative contribution of these two factors to SHP-1 specificity, the experiments in this section were performed. The goal was to evaluate specificity of the PTP catalytic site in SHP-1 by a combinatorial approach.

In previous work, ligands for SH2 domains were identified by sequencing a pool of peptides enriched by binding to SH2 domain proteins (46). The methodology has also been applied to other domains like the PDZ domain (49). The substrate specificity of LAR PTPs and PTPIB have also been evaluated by combinatorial approaches using on-bead screening strategies (60). In many cases, the compound on the bead contained a non-hydrolyzable pY analog attached to randomized positions

(88). However, the libraries used contained low sequence diversity and did not yield

109 information regarding the importance of residues C-terminal to the pY position (88).

Here, we attempted to identify the substrate specificity of the catalytic domain of

SHP-1 (SHP-1(ASH2)) using a 2.5x10^ sequence containing peptide library. The

library was screened for binding of the inactive D419A mutant of SHP-1 (ASH2) to

specific sequences.

Strategy and Methods

Library (Ac-DEXXpYXXXEBBRM) synthesis and sequencing methods were

performed as described in chapter 3 with the addition of lysine, arginine and

phosphotyrosine (pY). The library was synthesized by Peng Wang (graduate student in

the Pei laboratory). Protein purification was performed as described in chapter 2.

Bio tiny lation of the protein was carried out by addition of 1-2 equivalents of N-

hydroxysuccinimidobiotin as detailed in chapter 3. The pentapeptide library

(theoretical diversity is 19^ = 2.5x10^) was screened against the D419A mutant of

SHP-1 (ASH2). The library beads (100 mg) were exhaustively washed with CH2CI2,

DMF, ether, water and TBS buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.1%

Tween-20) containing 0.1% (w/v) gelatin. The beads were incubated for 1 hour in

TBS buffer to block any nonspecific protein binding. The beads were then incubated with firesh TBS buffer containing 1-5 pM biotinylated D419A-SHP-1(ASH2) for 6-12 hours at room temperature with constant mixing. After the incubation was completed,

10-15 nM of streptavidine-alkaline phosphatase was added to form a complex with the

110 bound biotinylated protein. After incubation for 10-15 minutes the solution was

removed from the beads by vacuum and the beads washed three times with 3 ml of

PB ST buffer (20 mM sodium phosphate, pH 7.4, 150 mM NaCl and 0.1% Tween).

This was followed by washing three times with 3 ml of PBS buffer and three times

with 3 ml of TBS buffer. The beads were then resuspended in 12 ml of TBS buffer, pH

8.5, and incubated with 1 mg of BCfP while mixing gently. The selection of blue

beads and sequence analysis were carried out as described in chapter 3.

Results and Discussion

This library with five randomized positions and a theoretical diversity of

2.5x10^ was screened against D419A-SHP-1(ASH2). Aspartate-419 is conserved among PTPs and acts a general acid and base during catalysis (19, 82). The D419A mutant was chosen because earlier work showed that it had very low activity and high binding affinity for substrates. The activity of this D419A mutant was 2000 times lower than the catalytic domain of wild type SHP-1. The wild type derived SHP-

1(ASH2) was not used as it would have dephosphorylated the pY group and dissociated from the bead. The C453S mutant o f SHP-1 (ASH2) was not used due to the possibility of structural changes in the active site causing a change in specificity.

The screening showed little selectivity at all five randomized positions. There could be various reasons for this result. One reason might be that the SH2 domains contribute to PTP selectivity and their absence in D419A-SHP-1 (ASH2) results in no

111 selectivity being observed. Previous work indicated that PTP IB showed selectivity N- tenninal to the pY position (32, 60). It is surprising that SHP-1 (ASH2) shows no selectivity given the sequence similarity to PTP IB. However, a possible reason might be the difference in properties of the WPD loop, which plays an important role in substrate binding (72). Yet another explanation for the lack of selectivity is that biotinylation of the surface lysines may have caused structural changes and vitiated the experiment. The use of fusion proteins with biotinylation on the non-PTP portion would prevent such a problem.

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