Developing peptide inhibitors of Shp2 through a one-bead-one-compound approach

Kira Tomlinson Senior Honors Thesis Department of Chemistry Tufts University Spring 2017

Advisors: Professor Joshua Kritzer and Professor Krishna Kumar

Table of Contents

ABSTRACT ...... 3

CHAPTER 1: INTRODUCTION ...... 4 SHP2 IN HUMAN DISEASE ...... 4 Gain-of-function Shp2 mutations in ...... 4 Gain-of-function Shp2 mutations in myeloid malignancies ...... 5 Aberrant activation of Shp2 in cancer ...... 5 shRNA library validation of Shp2 target in RTK-driven cancers ...... 6 SHP2 IN CELL SIGNALING ...... , ...... 7 Activation of the Ras/ERK pathway ...... 8 SHP2 STRUCTURE AND REGULATION ...... 10 Catalytic domain ...... 10 SH2 domains and C-terminal tail ...... 11 Regulation through autoinhibition ...... 12 Gain-of-function mutations ...... 13 SHP2-INHIBITION STRATEGIES ...... 14 Direct Shp2 inhibition by targeting the PTP domain ...... 14 Allosteric Shp2 inhibition by stabilizing the autoinhibited conformation ...... 16 PEPTIDE INHIBITORS AS INVESTIGATIVE TOOLS ...... 17 Stapled peptides ...... 18 One-bead-one-compound libraries ...... 18 Peptidic phoshotyrosine mimicry ...... 19 PROJECT GOALS ...... 20

CHAPTER 2: EXPERIMENTAL DESIGN AND METHODS ...... 21 PEPTIDE DESIGN, SYNTHESIS, AND PURIFICATION ...... 21 SHP2-PTP EXPRESSION AND PURIFICATION ...... 23 ACTIVITY ASSAYS ...... 24 PNPP ...... 24 DiFMUP ...... 25 ONE-BEAD-ONE-COMPOUND LIBRARIES ...... 26

CHAPTER 3: RESULTS ...... 27 SHP2 CATALYTIC DOMAIN EXPRESSION AND PURIFICATION ...... 27 CONTROL INHIBITION ASSAYS ...... 32 PEPTIDE INHIBITION ASSAYS ...... 34

CHAPTER 4: DISCUSSION ...... 35

ACKNOWLEDGEMENTS ...... 39

REFERENCES ...... 40

APPENDIX ...... 42

2 Abstract

Shp2 is a protein tyrosine that is mutated in Noonan syndrome and myeloid neoplasms, and aberrantly activated in HER-positive breast cancers and H. pylori-induced gastric cancer. Shp2 has been found to act upstream of a variety of growth factor and cytokine signaling pathways, but its role within the cell has not been fully elucidated. Potent and specific inhibitors of Shp2 are needed to further investigate the role of Shp2 in these pathways and pathologies.

This project aims to develop peptide inhibitors of the Shp2 catalytic domain through rational design followed by one-bead-one-compound (OBOC) libraries. Novel peptides were derived from a loop in the auto-inhibited conformation of Shp2 that provided structural elements to circumvent the obstacles of specificity and phosphotyrosine mimicry, which are common in phosphatase inhibitor design. To confer a similar loop structure, the peptides were conformationally restrained through thiol-bisalkylation chemistry. When tested in a DiFMUP inhibition assay, the linear peptide, with an IC50 of 3.51 µM, was a better inhibitor of the Shp2

PTP domain than the bisalkylated peptides, with IC50s ~20 µM. Goals for the immediate future of this project include testing a serine-containing derivative of the linear D’-derived peptide, performing a mini-mutagenesis study of the DY motif, and subjecting the linear D’-derived peptide to a phosphotyrosine ELISA assay. Further iterations of the D’-derived peptide will be explored using OBOC libraries to improve activity and specificity.

3 Chapter 1: Introduction

Shp2 and human disease

Src-homology 2 (SH2) domain-containing phosphatase 2 (Shp2) is a non-receptor protein

tyrosine phosphatase encoded by PTPN11. PTPN11 was the first verified protein tyrosine

phosphatase . Gain-of-function mutations in PTPN11 have been found in cancers such

as myeloid malignancies and neuroblastoma, as well as in developmental diseases such as

Noonan syndrome (Figure 1a). Furthermore, aberrant activation of Shp2 is critical in the

pathology of H. pylori-induced gastric cancer and HER-positive (Figure 1b,c).2

a b c

Figure 1: Role of Shp2 in (a) Noonan syndrome, leukemia, (b) H. pylori-induced gastric cancer, and (c) HER-positive breast cancer. Adapted from Matozaki et al.1

Gain-of-function Shp2 mutations in Noonan Syndrome

Gain-of-function PTPN11 mutations occur in ~40% of patients with Noonan syndrome.2 Noonan

syndrome (NS) is an autosomal dominant developmental disorder that is characterized by

unusual facial features, short stature, heart defects, bleeding issues, skeletal malformations,

cognitive defects, and other symptoms with varying penetrance and expressivity.2 Noonan

syndrome is also associated with a 8-fold increased risk of developing leukemia.3 NS patients

who have wild-type PTPN11 instead have gain-of-function mutations in KRas, NRas, and other

4 members or regulators of Ras/ERK signaling, indicating that Noonan syndrome is caused by abnormal Ras/ERK pathway activation (Figure 1a).2

Gain-of-function Shp2 mutations in myeloid malignancies

Gain-of-function Shp2 mutants have also been found in a number of myeloid malignancies, which result from abnormal self-renewal, proliferation and differentiation of hematopoietic stem or progenitor cells.4 (AML) is a malignancy that results from clonal expansion of myeloid blasts in the peripheral blood and bone marrow, causing reduced production of normal red blood cells, platelets, and mature granulocytes.4 Other types of myeloid malignancies particularly relevant to Shp2 include myeloid neoplasms such as juvenile myelomonocytic leukemia (JMML), which is a pediatric myeloproliferative neoplasm characterized by persistent macrocytic anemia and hemaglobinemia.4 Gain-of-function PTPN11 mutations have been found in ~35% of sporadic JMML patients and with lower incidence in other myeloid neoplasms and AML.5

Although PTPN11 is rarely mutated in solid tumor cancers, gain-of-function PTPN11 is the third most common mutation in neuroblastoma, with an incidence of 3%.6

Aberrant activation of Shp2 in cancer

Wildtype Shp2 is aberrantly activated in the pathogenesis of H. pylori-induced gastric cancer and

HER-positive breast cancer. Gastric cancer manifests as ulcers, hyperplasia, and carcinoma in the stomach.7 The risk of non-cardia gastric cancer, which is located in all areas of the stomach except for in the vicinity of the esophagus, increases 6-fold after infection with H. pylori, a spiral-shaped bacterium that grows in the mucus layer of the stomach.7 CagA-positive H. pylori

5 injects CagA toxin into the cells of the stomach lining, altering cell morphology to produce a more mobile, “hummingbird” phenotype.8 In epithelial cells, CagA is phosphorylated by Src- family kinases, causing the recruitment and inappropriate activation of Shp2 (Figure 1b).

PTPN11 knockdown impairs the development of the “hummingbird” phenotype, affirming the critical role of Shp2 in CagA virulence.2 The CagA/Shp2 mechanism likely perturbs multiple pathways.2

HER-positive breast cancer is characterized by uncontrolled breast cell proliferation due to overexpression of human epidermal growth factor receptors (HERs). HER-positive breast cancer is more rapid, more likely to spread, and more likely to recur than HER-negative breast cancer.9 Gab2, a docking protein for signaling molecules, is overexpressed in 10-15% of breast tumors. Gab2 overexpression increases proliferation of the HER-positive cells by cooperating with HER overexpression in a Shp2-dependent manner (Figure 1c).2 The necessity of Shp2 in this pathology has been demonstrated by PTPN11 knockdown, which reduced the invasiveness of HER2/3-expressing cells.10

shRNA library validation of Shp2 target in RTK-driven cancers

Abnormal activation of Shp2 is critical in the pathology of the aforementioned diseases, indicating that inhibition of Shp2 could have therapeutic value. Novartis validated Shp2 as a therapeutic target for RTK-driven cancers with a deep-coverage short hairpin RNA (shRNA) library. Screening this shRNA library against 250 cancer cell lines, Chen et al.11 demonstrated that sensitivity to Shp2 depletion was correlated with RTK-dependent cell lines (Figure 2a).

Chen et al. further validated the Shp2-dependence of RTK-driven cancer cell lines in a complementation experiment, which first introduced doxycycline (dox)-inducible Shp2 shRNAs

6 into cancer cell lines and then provided shRNA-resistant alleles for wildtype Shp2 or catalytically inactive Shp2C459S. Shp2 depletion by the shRNAs caused a reduction in p-ERK levels and cell growth, which were restored upon expression of wildtype Shp2 but not catalytically inactive Shp2C459S (Figure 2b). These results indicate that Shp2 catalytic activity is necessary for the growth of RTK-dependent cancer cell lines.

a b

Figure 2: Genetic validation of Shp2 as a therapeutic target for RTK-driven cancers. (a) Waterfall plot showing correlation between shRNA-mediated RTK knockdown and shRNA-mediated Shp2 depletion. (b) Proliferation of GFP- expressing cells without an shRNA-resistant Shp2 allele (GFP), with an shRNA- resistant wildtype allele (SHP2), or with an shRNA-resistant, catalytically inactive allele (Shp2C459S) before and after dox-induced endogenous Shp2 depletion; data presented as mean ± s.d. (n = 3). Adapted from Chen et al.11

Shp2 in cell signaling

Though the Novartis genetic validation study places Shp2 downstream of RTKs and references to other common NS mutants place Shp2 more specifically in the Ras/ERK pathway, Shp2 plays a more versatile role in cell signaling. Upon growth factor or cytokine stimulation, Shp2 is recruited to receptor tyrosine kinases (RTKs), cytokine receptors, scaffolding adaptors, and/or immune inhibitory receptors. By binding and/or dephosphorylating these substrates, Shp2 regulates the Ras/ERK pathway, the Jak/STAT pathway, and the PI3K pathway, among others.2

7 This work will focus on the role of Shp2 in the Ras/ERK pathway, as it is the most studied and, based on current knowledge, most pertinent to the aforementioned pathologies. It is important to note that the role of Shp2 in the Ras/ERK pathway is multifaceted and has not been fully elucidated. Further investigation into the role of Shp2 in cellular signaling would benefit from potent and specific inhibitors that separately target the various functionalities of Shp2, including its catalytic activity and its phosphotyrosine-binding capabilities.

Activation of the Ras/ERK pathway

Shp2 is required for full activation of the Ras/ERK pathway, which propagates growth factor signals through a phosphorylation cascade. Upon growth factor receptor stimulation, the GTPase

Ras binds and activates the protein kinase Raf-1, leading to the sequential phosphorylation of two other protein kinases, MEK and ERK, the latter of which enters the nucleus and phosphorylates transcription factors.12 In some cell types, the absence of Shp2 results in no ERK activation, while in others, the absence of Shp2 results in ERK activation that is not sustained.13

Though there is general agreement that Shp2 acts upstream of Ras, the mechanism of

Shp2-mediated Ras activation is disputed. One general model suggests that RTK activation mediates the phosphorylation of tyrosine residues on the C-terminal tail of Shp2. This modification may serve a dual purpose: in addition to switching Shp2 from its basal, auto- inhibited conformation to a catalytically-active conformation, it could also provide phosphorylated residues as a docking site for Grb2. This recruitment of Grb2 would allow for

SOS-mediated Ras-guanine exchange, thus activating the Ras/ERK pathway.14

A second general model suggests that Shp2, after being switched out of its auto-inhibited conformation, dephosphorylates the Gap binding sites on RTKs such as the epithelial growth

8 factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2).15 By limiting the recruitment of Gap, which enhances the endogenous GTP-ase activity of Ras, Shp2 would prevent the inactivation of the Ras/ERK pathway (Figure 3a).

Additional models stem from other Shp2 substrates. Shp2 has been shown to interact with the binding proteins of Csk, a negative regulator of Src-family kinase (SFK) activity. These substrates suggest a model of Shp2-mediated Ras activation in which Shp2 dephosphorylates

Csk binding sites, limiting Csk recruitment and thus preventing the inhibition of SFK-mediated activation of the Ras/ERK pathway (Figure 3b).16 Another set of substrates are the Sprouty and

Spred proteins, inhibitors of the Ras/ERK pathway. Shp2 may mediate Ras activation by dephosphorylating Sprouty and Spred at tyrosine residues that are critical for Grb2 sequestration and, consequently, Ras/ERK inhibition (Figure 3c).17

a b c

Figure 3: Models of Shp2-mediated activation of the Ras/ERK pathway, which include (a) Shp2 prevention of GAP recruitment and subsequent Ras inactivation; (b) Shp2 prevention of Csk recruitment and subsequent inactivation of SFK, a Ras activator; and (c) Shp2 prevention of Sprouty sequestration of Grb2, a protein scaffold that recruits Sos, a Ras activator. Adapted from Matozaki et al.1

9 Shp2 structure and regulation

These models of Shp2-mediated Ras/Erk activation demonstrate that Shp2 can serve as both a phosphatase and a protein scaffold. This multifaceted role of Shp2 is mediated by its unique domain composition. Shp2 is a non-receptor protein tyrosine phosphatase with a classic PTP catalytic domain, two N-terminal SH2 domains, and a C-terminal tail (Figure 4). While the phosphotyrosine-binding capabilities of the SH2 domains are critical for Shp2 localization, they also allow Shp2 to serve as a protein scaffold. Furthermore, the SH2 domain at the N-terminus serves as a molecular switch between the basal auto-inhibited conformation and the open active conformation. Autoinhibition is the primary mechanism of Shp2 regulation and, unsurprisingly, many gain-of-function mutations destabilize the autoinhibited conformation.

Figure 4: Shp2 domains, labeled by border residues and phosphorylation sites on the C-terminal tail. Adapted from Matozaki et al.1

Catalytic domain

Shp2 has a classic PTP catalytic domain, which uses a highly conserved cysteine residue as a nucleophile to substitute the tyrosine hydroxyl group, forming a thiophosphoryl intermediate that is later resolved by activation of a water molecule for a second nucleophilic substitution. In addition to the catalytic cysteine (residue C459 in Shp2), the classic PTP domain has two other conserved structures that are critical for catalysis: a P-loop, which orients its backbone amide hydrogens inwards to create a partially positive microenvironment that stabilizes the phosphotyrosine substrate, and an acid-base loop, which has an aspartate residue

10 (residue D425 in Shp2) that abstracts a proton from the incoming water molecule to activate it as a nucleophile for resolution of the thiophosphoryl enzyme intermediate. Another important structural feature of the classic PTP domain is the conformational change that occurs upon ligand binding, which brings the catalytic acid residue near the phenolic oxygen of the phosphotyrosine substrate and sandwiches the phosphotyrosine ring between two aromatic amino acids.18

SH2 domains and C-terminal tail

Shp2 has two SH2 domains, which are N-terminal to the catalytic domain and named N-SH2 and

C-SH2 based on their location relative to each other. The SH2 domains are critical for Shp2 activity, as shown in an experiment where mutations that inactivated the N-SH2 and C-SH2 domains abolished myeloid cell transformation.19 Because of their ability to bind phosphorylated tyrosines on RTKs such as the PDGF receptor and scaffolding adapter proteins such as GAB2, the SH2 domains are thought to be recruitment domains. The SH2 domains are also involved in regulation of the PTP domain, however, as shown by an experiment where incubation of Shp2 with phosphopeptides derived from the SH2 domain on the PDGF receptor caused an increase in phosphatase activity.20 This autoinhibitory role of the SH2 domains has been further elucidated by x-ray-crystallography (discussed below).

The role of the C-terminal tail in Shp2 function is unclear, though it provides two tyrosine residues (Y542 and Y580), which can be phosphorylated by receptor and non-receptor tyrosine kinases.2 These phosphorylated tyrosine residues may serve as docking sites for scaffolding adapter proteins such as Grb2, thus mediating the role of Shp2 as a protein scaffold.

11 Regulation through autoinhibition

Shp2 is primarily regulated through an autoinhibitory mechanism that couples the recruitment of

Shp2 with activation of its catalytic domain. In this mechanism, the N-SH2 domain is lodged in the of the PTP domain with its own phosphotyrosine-binding site solvent-exposed; upon binding to phosphopeptides on RTKs, scaffold and adapter proteins, or other ligands, the

N-SH2 domain undergoes a conformational change and releases the catalytic domain.21

As mentioned above, this mechanism of autoinhibition was originally observed as an increase in Shp2 phosphatase activity after incubation with phosphopeptides derived from Shp2-

SH2 ligands.20 The roles of the SH2 domains in Shp2 auto-inhibition were further elucidated by x-ray crystallography. A structure published by Hof et al.21 shows Shp2 in a closed domain architecture, in which the N-SH2 domain forms an extensive polar interface with the PTP domain while its own phosphopeptide-binding site remains solvent-exposed (Figure 5a). While the phosphopeptide-binding site of the C-SH2 domain is also solvent exposed, there is little interface between the C-SH2 domain and the other two domains. The interaction between the N-

SH2 domain and PTP domain is dependent on the D’E loop (residues 58-63: NTGDYY), which extends deep into the active site but is bulky enough, due to the NTG residues, to keep the active site in the open, inactive conformation (Figure 5b). The D’E loop interacts with critical residues within the active site, such as the catalytic cysteine, through hydrogen bonding and water molecule coordination organized by N58, G60, and D61.21 Furthermore, residues D61 and Y62 mimic aspects of phosphotyrosine binding by respectively interacting with the catalytic cysteine and the hydrophobic residues critical for phosphotyrosine stabilization (Figure 5b).

It is important to note that Hof et al. also performed sequence alignments with other SH2 domains to show that the D’E loop (with the NxGDY/F motif) is specific to Shp2.21

12 a

Figure 5: Crystal structure of closed domain architecture of Shp2, in which (a) the N-SH2 domain makes extensive contacts with the PTP domain, particularly through the D’E loop, which (b) interacts with active site residues critical for catalysis and phosphotyrosine stabilization. Adapted from Hof et al.21

Gain-of-function mutations

Recalling the diseases discussed above, namely Noonan syndrome, JMML and AML, it is not surprising that the gain-of-function mutations at the root of these pathologies often disrupt the basal, autoinhibited conformation of Shp2. For example, an aspartate residue in the D’E loop

(D61) is critical in stabilizing the interface between the N-SH2 domain and the PTP active site because it interacts with the catalytic cysteine residue through coordination of a water molecule.

Mutation of this aspartate residue to a glycine (D61G) has been found in patients with Noonan syndrome while mutation of this aspartate to histidine, tyrosine, or valine (D61H, D61Y, D61V) has been found in patients with JMML and AML.19 It is important to note that the pathologies that result from substitution of this aspartate residue depend on the bulkiness and hydrophobicity of the mutated residue, indicating that severity of disease may be linked to degree of disruption of the autoinhibited conformation.19

13 Shp2-inhibition strategies

Two Shp2 inhibition strategies have been implemented to develop therapeutics for pathologies that result from abnormal activation of Shp2. One strategy is to inhibit the Shp2 PTP domain directly, quenching the phosphatase activity of Shp2 while allowing it to still serve as a protein scaffold. This direct inhibition strategy is complicated by phosphotyrosine mimicry and inhibitor specificity. The other Shp2-inhibition strategy is to allosterically stabilize the autoinhibited conformation. While this strategy circumvents the obstacles of phosphotyrosine mimicry and inhibitor specificity, it could disrupt the ability of Shp2 to perform its other, non-catalytic functions.

Direct Shp2 inhibition by targeting the PTP domain

Direct inhibition of the Shp2 PTP domain is impeded by phosphotyrosine mimicry and inhibitor specificity.21 Phosphotyrosine mimicry requires a careful balance of functional groups that will imitate the bulky, aromatic, and negatively charged phosphotyrosine without sacrificing pharmacokinetic properties like cell membrane permeability. This balance has not yet been achieved, as existing small molecule phosphotyrosine mimetics, such as sulfamic acid and phosphonodifluoromethyl phenylalanine, yield poor cellular efficacy.22

Attempts at inhibiting Shp2 are also hindered by the high degree of homology between phosphatase active sites. To find specificity, researchers have looked outside of the active site, designing bidentate inhibitors with a phosphotyrosine-mimetic and a second-site binding fragment.23 While this strategy does confer more specificity, it yields large molecules which can have impaired pharmacokinetic properties like bioavailability.

14 One example of a bidentate Shp2 inhibitor published by Grosskopf et al.23 uses benzene sulfonic acid as a phosphotyrosine mimetic and a 4-nitrophenyl group as a second binding fragment (Figure 6a,b). This inhibitor has an IC50 of 71 nM, with a 35-fold decrease in activity against Shp1, the phosphatase most homologous to Shp2, and a 45-fold decrease in activity against PTP1B, the quintessential protein tyrosine phosphatase. This inhibitor also blocked scattering of hepatocyte growth factor-induced pancreatic cancer cells and blocked growth of

Shp2-dependent human NSCL adenocarcinoma in a mouse xenograft model (Figure 6c,d).

a b

c d

Figure 6: (a) Compound 25, a bidentate inhibitor published by Grasskopf et al. which uses benzene sulfonic acid as a phosphotyrosine mimetic, as shown in (b) the model produced by a docking study with a crystal structure of the Shp2 catalytic domain (PDB ID:3ZM3). Compound 25 (c) reduced cell scattering (an indication of motility, invasiveness, and metastatic potential of cancer cells) in a dose-dependent manner and (d) counteracted tumor growth in a mouse xenograft model. Adapted from Grosskopf et al.2

15 Allosteric Shp2 inhibition by stabilizing the autoinhibited conformation

The obstacles of specificity and phosphotyrosine mimicry have been circumvented by designing allosteric inhibitors that stabilize the basal, autoinhibited conformation of Shp2. One example of an allosteric inhibitor discovered by Novartis and published by Chen et al.11 stabilizes the closed domain architecture of the autoinhibited conformation by forming hydrogen bonds and hydrophobic interactions at the interface of the PTP, N-SH2, and C-SH2 domains (Figure 7a,b).

Similar to the aforementioned bidentate inhibitor, this allosteric inhibitor has an IC50 of 71 nM but shows no activity against a panel of 66 kinases and 21 , including Shp1.

Administered at 100 mg/kg, this inhibitor slowed carcinoma growth and reduced leukemia burden in mice xenograft models (Figure 7c,d).

a b

c d

Figure 7: (a) SHP099, an allosteric inhibitor published by Chen et al., makes hydrogen bonds and hydrophobic interactions at the interface of the PTP, N-SH2, and C-SH2 domains as shown by (b) x-ray crystallography. SHP099 (c) slowed carcinoma growth at a rate comparable to the EGFR inhibitor Erlotinib and (d) reduced leukemia burden in mice xenograft models. Adapted from Chen et al.11

16

It is important to note that, while this allosteric inhibitor may be a successful therapeutic due to its inhibition of the multifaceted functions of Shp2, it is limited in its use as an investigative tool because it cannot distinguish between phosphatase and scaffold activity.

Peptide inhibitors as investigative tools

While the two inhibition strategies discussed above provide avenues for developing small molecule therapeutics, neither is ideal for developing investigative tools. The bivalent inhibitors allow for direct and selective inhibition of Shp2 catalytic activity but often have poor cell membrane permeability due to the phosphotyrosine mimic. The allosteric inhibitors allow for selective inhibition without sacrificing cell membrane permeability, but are not able to distinguish between the various functionalities of Shp2 in cell signaling.

A happy medium between these two strategies can be found by using constrained peptide inhibitors. Though linear peptide inhibitors are often less active, less cell-permeable, and easier to degrade than their small molecule counterparts,24 these weaknesses can be mitigated and even eradicated through the use of conformational constraints such as head-to-tail cyclization or side- chain stapling (discussed below). Constrained peptide inhibitors are advantageous because they form multiple contacts with their protein target, allowing for greater affinity and selectivity; they have little toxicity due to their easily degradable building blocks; and they are easily synthesized with automated technology.25 Furthermore, peptide inhibitors can be efficiently improved through the use of techniques such as one-bead-one-compound libraries (discussed below).

Another advantage that is particularly relevant to Shp2 is that peptides provide opportunities to mimic phosphotyrosine without sacrificing cell permeability (discussed below).

17 Stapled peptides

Peptides can be conformationally restrained by “molecular staples”, small molecules that form covalent bonds with the peptide backbone or side chains. Molecular stapling not only confers a more rigid structure that can improve activity, but also increases protease resistance and cellular uptake.26 One form of molecular stapling involves bisalkylation chemistry, which forms covalent bonds between cysteine side chains and the staple through nucleophilic thiol attack on bromylated sites on a hydrocarbon linker.27 Bisalkylation chemistry is an easy and efficient way to staple peptides, as the reaction is robust, fast, and clean and there is an abundance of commercially available, dibromo-aryl and dibromo-alkyl linkers.

One-bead-one-compound libraries

Peptide inhibitors can be efficiently optimized through derivative libraries, which simultaneously screen thousands to millions of library members that differ from a lead peptide by one or more residues.28 Though derivative libraries can be biological, using technology like phage display, or chemical, using combinatorial chemistry, most libraries follow three main steps: synthesis of the library, screening against the target, and decoding of the hits.29 One-bead-one-compound libraries, which are combinatorial libraries, offer advantages at each of these steps. Synthesis is performed through rapid and robust solid-phase peptide chemistry using a split-and-pool technique so that each bead will contain multiple copies of the same peptide sequence (Figure

8a).29 Screening can be done in solution or on bead: the latter option is more common and often involves associating the protein with a fluorophore or dye so that any beads carrying peptides that bind to the protein will light up with the visual signal (Figure 8b).29 Decoding can be done directly from the peptides or through encoded tags (Figure 8c).29

18 a b c

Figure 8: Schematic of one-bead-one-compound libraries, which entail (a) solid- phase split-and-pool synthesis, (b) screening against a target protein labeled with a visual indicator, and (c) cleavage and sequencing of peptides from the hit beads.

Peptidic phosphotyrosine mimicry

In designing phosphatase inhibitors, cell permeability is often sacrificed for phosphotyrosine mimicry. In peptides, however, phosphotyrosine can be emulated by natural amino acid side chains with minimal charge and bulkiness. For example, in a study of Grb2-binding peptides published by Quartararo et al.,30 bicyclization conferred a phosphotyrosine-mimicking epitope comprised of glutamate, leucine, and tyrosine. In this ELY motif, the leucine side chain provides steric hindrance to position the hydroxyphenyl and carboxylic acid groups of the tyrosine and glutamate side chains in the phosphotyrosine (pTyr) binding pocket (Figure 9).

Figure 9: Energy minimized model showing ELY motif in Grb2 pTyr-binding pocket. Adapted from Quartararo et al.30

19 Project Goals

This project aims to provide tools for further investigation of the roles of Shp2 in cell signaling by developing bisalkylated peptide inhibitors of the Shp2 catalytic domain through rational design followed by one-bead-one-compound libraries. Achieving this goal will require expression, purification, and activity-validation of the Shp2 PTP domain and establishment of an inhibition assay. After the rationally designed peptides are synthesized and purified, they will be tested in this inhibition assay. Successful inhibitors from the rationally designed series will be rapidly optimized through derivative one-bead-one-compound libraries, which will also be screened against the full Shp2 construct and Shp1 to improve inhibitor activity and selectivity.

20 Chapter 2: Experimental Design and Methods

Peptide design, synthesis, and purification

To rationally design peptide inhibitors of the Shp2 PTP domain, the obstacles of phosphotyrosine mimicry and specificity need to be considered. Interestingly, the crystal structure published by

Hof et al. suggests that the D’E loop of the N-terminal SH2 domain provides structural elements that may overcome these obstacles. In the D’E loop (residues 58-63: NTGDYY), residue D61 hydrogen bonds with the catalytic cysteine residue, while Y62 binds the hydrophobic residues in the catalytic pocket (Figure 10); this DY motif thus mimics some aspects of phosphotyrosine binding (Figure 10). Furthermore, residue N58, which is critical for establishing the hydrogen bonding network that interfaces the active site and the D’E loop, and for preventing closure of the water-activating WPD loop, is not found in any other SH2 domain; this lack of homology suggests that there may be inherent specificity built into the D’E loop.

Y279

C459 Y62 D61

G60

T59 N58

Figure 10: Crystal structure of the D’E loop in the PTP active site, in which the D61 and Y62 residues interact with the catalytic cysteine (C459) and residues responsible for stabilizing phosphotyrosine (Y279), and the N58, T59, and G60 residues keep the active site in the open conformation. Adapted from PDB: 2shp.

21

The D’E loop thus showed promise as a starting point for rational design. The NTGDYY sequence was flanked with cysteine residues to allow for bisalkylation with a variety of molecular staples in an attempt to confer a loop structure similar to that found in the autoinhibited conformation. It is important to note that in the crystal structure the residues at the ends of the loop are not only in close proximity to one another, but also have their side chains oriented in the same direction (Figure 10). This orientation indicates that molecular stapling of the cysteine residues flanking the NTGDYY sequence could be compatible with the loop conformation. It is difficult to predict from such as static structure, however, which molecular staples will promote the most active loop conformation, so the D’-derived peptide was stapled with o-, m-, and p-xylene to explore an incremental range of staple lengths.

These rationally designed peptides were synthesized through standard, solid-phase Fmoc chemistry. Briefly, Rink amide resin was swelled in DMF for 30 minutes. The resin was deprotected with 2 rounds of 10 minutes in 20% piperidine in DMF, then washed sequentially with DMF, DCM, and DMF. The first amino acid was coupled onto the resin for 30 minutes with

5 eq. of Fmoc-AA-OH, 5 eq. of PyBOP, and 5 eq. of HOBt in DMF, and 13 eq. of DIPEA. The resin was washed again with DMF, DCM, and DMF. Rounds of deprotection, washing, coupling, and washing were repeated until the last amino acid was coupled. The resin was then Fmoc- deprotected and acetyl-capped for 15 minutes in 10% acetic anhydride, 10% 2,6-lutidine in

DMF. The peptide was simultaneously cleaved from the resin and globally deprotected for 3 hours in a cocktail of 95% TFA, 2% water, 2% EDT, and 1% TiPS. After ether precipitation and lyophilization, a fraction of the linear peptide was redissolved in 50:50 acetonitrile:20 mM

NH4HCO3 buffer, pH 8.0 and bisalkylated for 1 hour with 1.5 eq. of linker dissolved in acetonitrile. The peptides were purified by HPLC (see appendix) and stored as DMSO stocks.

22 Shp2-PTP Expression And Purification

To ensure that the early discovery of catalytic domain inhibitors would not be complicated by competition with the N-SH2 domain or binding to the SH2 phosphopeptide-binding sites, a Shp2 construct of the Shp2 PTP domain was used to test the first series of rationally designed peptides.

Professor Dehua Pei’s lab at Ohio State University graciously provided a construct of

Shp2 PTP (residues 199-593) conjugated to a poly-His tag.31 As a preliminary step, this recombinant pET22(+) plasmid was amplified in E. coli NEB5 alpha cells and purified using the

Wizard Plus DNA Miniprep . The Shp2-PTP protein was then expressed using E. coli BL21

(DE3) cells, and purified using a batch Ni-NTA affinity column and an FPLC size-exclusion column. Briefly, 200 ng of plasmid was incubated with 50 µL of E. coli BL21(DE3) cells on ice for 30 min. The cells were heat-shocked for 30 seconds at 42°C and incubated on ice for 5 minutes. 500 µL of room-temperature SOC were added and the suspension was shaken for 60 minutes at 37°C. 50µL of the transformed suspension was plated on an ampicillin selection plate, which was incubated overnight at 37°C. Single colonies were selected for 37°C overnight cultures in 10 mL of LB supplemented with 100 µg/mL ampicillin. The overnight cultures were transferred to 1 L cultures, which were incubated at 37°C until the OD600 was between 0.6 and

0.8. Shp2 PTP expression was induced with IPTG and the induced cultures were shaken overnight at 37°C. The cells were the spun down at 5000 rpm for 10 minutes at 4° and resuspended in lysis buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 5 mM imidazole, 0.5 mM

TCEP, 1 unit of DNAase, a cOmpleteTM ULTRA protease inhibitor tablet, and 5% glycerol). The cells were lysed by sonication and the lysate was centrifuged at 10000 rpm for 1 hour at 4°C.

The supernatant was incubated with Ni-NTA affinity resin for 1 hour, then removed from the column. The batch affinity column was washed with 3 rounds of shaking for 10 minutes in wash

23 buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 30 mM imidazole, 0.5 mM TCEP). The polyHis tagged Shp2 PTP was eluted with 2 rounds of shaking for 15 minutes in elution buffer (50 mM

HEPES, pH 7.4, 500 mM NaCl, 250 mM imidazole, 0.5 mM TCEP). The elutions were pooled, loaded on a FPLC S200 column, and eluted in size-exclusion buffer (50 mM Tris, pH 7.4, 1 mM

MgCl2, 50 mM NaCl, 1 mM DTT). The FPLC fractions were analyzed by gel electrophoresis; the fractions containing Shp2-PTP were pooled and protein concentration was determined by a

BCA assay. The protein was stored at -80°C in 10% glycerol.

Activity Assays

To validate the activity of the Shp2 PTP construct and establish an inhibition assay to test the rationally designed peptides, two different phosphatase assays were used. Phosphatase assays monitor phosphatase activity by quantifying a dephosphorylated substrate with unique absorbance or fluorescence. For the activity validation assay, p-nitrophenyl phosphate (PNPP) was used as the substrate, while for the peptide inhibition assays, 6,8-difluoro-4- methylumbelliferyl phosphate (DiFMUP) was used as the substrate.

PNPP

A PNPP assay was used to validate the activity of the expressed and purified Shp2 PTP construct. Removal of the PNPP phosphate produces a water-soluble, yellow compound that can be quantified by absorbance at 405 nm (Figure 11). PNPP was used for initial activity validation of the Shp2 PTP construct because of availability of the substrate.

24

Figure 11: PNPP conversion by phosphatase; removal of the phosphate group and subsequent deprotonation of the remaining hydroxyl group creates resonance that causes the product to absorb light at 405 nm and appear yellow.

To determine the activity of the Shp2 PTP construct, a range of 12 protein concentrations was made by serially diluting the purified protein in size-exclusion buffer (50 mM Tris, pH 7.4,

1 mM MgCl2, 50 mM NaCl, 1 mM DTT). These dilutions were incubated in triplicate with

PNPP (0.25, 0.5, or 1 mM) for 45 minutes at 37°C. The absorbance was read at 405 nm using a

Tecan Infinite plate reader.

DiFMUP

To test the rationally designed peptides as inhibitors of the Shp2 PTP domain, a DiFMUP assay was used. Removal of the DiFMUP phosphate produces a compound that can be quantified by fluorescence at λexcitation=360 nm and λemission=450 nm (Figure 12). This compound has a high quantum yield (0.89), which makes the assay more sensitive, and is resistant to photobleaching.

DiFMUP was used for the peptide inhibition assays because of its sensitivity and reproducibility.

Figure 12: DiFMUP conversion by phosphatase; (a) removal of phosphate produces a compound with a λexcitation of 360 nm and a λemission of 450 nm.

25

To determine the activity of each peptide, a range of 8 concentrations was made by serially diluting a 400 µM stock solution (made from dilution of the DMSO stock) in size- exclusion buffer (50 mM Tris, pH 7.4, 1 mM MgCl2, 50 mM NaCl, 1 mM DTT). Each of these dilutions (ranging from 100 to 0.78 µM in the assay) was incubated in triplicate with Shp2 PTP

(0.08 to 0.8 µM in the assay, depending on the batch) and DiFMUP (75 µM in the assay) for 45 minutes at 37°C. The fluorescence was then monitored at an excitation wavelength of 360 nm and an emission wavelength of 465 nm (due to availability of filters) using a Tecan Infinite plate reader. A positive, vanadate control (with concentrations ranging from 5 µM to 0.039 µM in the assay) and a negative, buffer-only control were run in parallel with the peptides.

One-bead-one-compound libraries

To optimize the lead peptide inhibitor from the rational design series, a one-bead-one-compound

(OBOC) library will be used. The OBOC library will expedite this process by varying multiple positions within the lead peptide sequence with natural and unnatural amino acids to improve phosphotyrosine mimicry and inhibitor specificity. The library will be synthesized through split- and-pool solid phase synthesis, which will ensure that each microbead will carry multiple copies of just one peptide sequence. These beads will then be screened against biotinylated protein, which will be visualized with streptavidin-conjugated or quantum dots.

Successful binders will be selected, cleaved off of the microbeads, and identified. Unfortunately, current OBOC methods are not compatible with bisalkylation chemistry, as is difficult to decode the sequence of bisalkylated peptides through tandem mass spectrometry due to their quasi- cyclic nature. Non-degenerate libraries, however, can be designed so that each member has a unique mass and can thus be identified through mass spectrometry by its parent ion mass.

26 Chapter 3: Results

Shp2 catalytic domain expression and purification

To test the activity of the rationally designed peptide inhibitors, the Shp2 PTP domain first had to be expressed and purified. While the discussion below analyzes the purification of the most recent batch of Shp2 PTP, there were no obvious discrepancies between the purity of the 4 batches of Shp2 PTP that have been made thus far (see appendix, Figure A1).

Gel electrophoresis of samples taken post-induction and after each step of the affinity column indicated that Shp2 PTP was successfully expressed and purified, but not fully recovered. Though there was no pre-induction sample (because the purification was carried out from a frozen cell pellet), the post-induction sample showed an intense band just below 50 kDa, which likely represented the recombinant Shp2 PTP (46.3 kDa) (Figure 13). This band was seen in the flow-through that was collected after incubation of the cell-lysate on the affinity column, in each of the three column washes, and on the beads after the last elution, indicating that some of the expression product was lost at each of these steps (Figure 13). Though this expression product was also the most intense band in the elutions, there were 3 minor bands as well (Figure

13). To remove these impurities, the elutions were pooled and injected on an FPLC size- exclusion column. Gel electrophoresis of the FPLC fractions indicated that the lower molecular weight impurities were removed, but some of the ~70 kDa impurity remained (not shown).

After the Shp2 PTP-containing FPLC fractions were pooled, the protein concentration was determined by a BCA assay, and this protein concentration was used to calculate a yield of

4.40 mg/L culture. Interestingly, yield fluctuated from batch to batch, but was lowest when pLys cells were used for expression (see appendix, Table A1).

27

250 250

150 150

100 100 75 75

50 50 37 37

25 25 20 20

15 15 10 10

Figure 13: Electrophoretic analysis Shp2 PTP purification; there is no pre- induction sample because this batch was purified from a frozen cell pellet, but the post-induction sample shows an intense band just below the 50 kDa marker (Shp2 PTP is expected to be ~46.3 kDa), which is also present in the flowthrough, the washes, the elutions, and on the beads after the last elution, indicating that the recombinant Shp2 PTP was purified but not fully recovered.

28 After expression and purification, a PNPP assay was used to confirm that the purified protein was catalytically active Shp2 PTP. The first batch of purified protein yielded no activity after storage for a month at -80°C, so a second batch was expressed and purified. PNPP assays confirmed that the purified protein had phosphatase activity, indicating that it was Shp2 PTP. In an attempt to get a full activity curve within the linear range of the plate reader, three PNPP concentrations were used. While 0.25 mM PNPP did not yield a saturated curve, the seemingly saturated absorbances in the 0.5 mM and 1 mM PNPP curves were out of the linear range of the plate reader and were therefore not reliable (Figure 14). Because this PNPP assay could not provide a fully saturated activity curve, the DiFMUP assay was used to validate the activity of the second, third, and fourth batches of Shp2 PTP (see appendix, Figure A2).

0.25 mM 0.5 mM 1 mM

2

1.5

1 Absorbance(405 nm)

0.5

0 0.0001 0.001 0.01 0.1 1 10

log[protein(uM)]

Figure 14: Activity validation of the freshly purified Shp2 PTP; the activity curve appears to saturate at 0.5 mM PNPP and 1 mM PNPP, but these absorbances are not reliable because they are out of the linear range for the plate reader (0.1-1.0). Data from a single experiment, graphed as mean ± s.d. (n = 3).

29 The PNPP assay was used only once more to determine whether or not the second batch of Shp2 PTP would remain active after being stored at 4°C or at -80°C. Comparison of the 0.25 mM PNPP activity curves of the stored to the 0.25 mM PNPP activity curve of the freshly purified enzyme (Figure 14) showed that Shp2 PTP that was stored for a week at 4°C retained its activity, while Shp2 PTP that was stored for a week at -80°C lost ~80% of its activity

(Figure 15). Unfortunately, the Shp2 PTP that was stored at 4°C also lost its activity after a few weeks (not shown), requiring further investigation into how to store Shp2 PTP so that it would retain most of its activity over a long period of time.

4 degree -80 degree

1.4

1.2

1

0.8

0.6 Absorbance(405 nm)

0.4

0.2

0 0.0001 0.001 0.01 0.1 1 10

log[protein(uM)]

Figure 15: Loss in activity of the Shp2 PTP stored at -80°C; at 0.25 mM PNPP, the refrigerated Shp2 PTP seemed to retain its activity while the frozen Shp2 PTP lost ~80% of its activity when compared to the fresh Shp2 PTP. Data from a single experiment, graphed as mean ± s.d. (n = 3).

30 To determine if storage in glycerol would help the enzyme retain its activity, a third batch of Shp2 PTP was expressed and purified. As mentioned above, a DiFMUP activity assay was used to compare the activity of the fresh and stored enzyme because it provided a fully saturated, reproducible activity curve. Comparison of the DiFMUP activity curve of Shp2 PTP stored at

-80°C in 10% glycerol to the DiFMUP activity curve of the fresh Shp2 PTP showed that the addition of glycerol allowed the frozen enzyme to retain ~55% of its activity (Figure 16). It is important to note that this third batch of Shp2 PTP, which was the only batch expressed in pLys cells, had 5-10 fold lower activity than the second and fourth batches (see appendix, Figure A2).

a

4 104

3.5 104

3 104

2.5 104

2 104

4

Emission (465 Emission nm) 1.5 10

1 104

5000

0 0.001 0.01 0.1 1 10 log[protein(uM)]

b

5 104

4 104

3 104

2 104 Emission (465 Emission nm)

1 104

0 0.001 0.01 0.1 1 10 log[protein(uM)]

Figure 16: DiFMUP activity assay with 10% glycerol stock; 75 µM DiFMUP gave a fully saturated, reproducible curve, which showed that ~55% of the (a) fresh Shp2 PTP activity was retained by (b) Shp2 PTP stored in 10% glycerol at - 80°C. Data from a single experiment, graphed as mean ± s.d. (n = 3).

31 Control Inhibition Assays

To establish a Shp2 PTP inhibition assay, vanadate was used as a positive control and buffer was used as a negative control. At first, these controls gave unexpected results: the negative control gave a curve when it should have given a horizontal line representing maximum substrate turnover; the positive control gave a stunted curve with an upper end that was ceilinged by the negative control values in the same column on the assay plate (Figure 17). The dependence of the fluorescence measurement on location on the assay plate indicated that there was something wrong with the plate alignment within the reader. To find the correct plate alignment, bulk Shp2

PTP was incubated with DiFMUP for 45 minutes at 37°C, and then plated along the horizontal and vertical axes of a plate. The plate alignment that had been used gave increasingly reduced fluorescence measurements along the vertical and horizontal axes of the plate (not shown). It is important to note that, for the activity assays, an effective plate alignment had been used.

vanadate buffer

3.5 104

3 104

2.5 104

2 104

1.5 104 Emission (465 Emission nm)

1 104

5000

0 0.001 0.01 0.1 1 log[inhibitor(uM)]

Figure 17: Initial control Shp2 PTP inhibition assays; the negative, buffer-only control, gave a curve, while the positive, vanadate control gave a stunted curve. Data from a single experiment, graphed as mean ± s.d. (n = 3).

32 Using the plate alignment that gave the same fluorescence measurement for each well along the horizontal and vertical axes of the test plate, the buffer-only control gave the expected horizontal line representing maximum substrate turnover and the vanadate control gave a full inhibition curve indicating that vanadate inhibits Shp2 PTP with an IC50 of ~300 nm. This IC50 is higher than expected, considering that vanadate typically inhibits protein tyrosine phosphatases in the single digit to tens digit nanomolar range. It is important to note that, in these control inhibition assays, the third batch of Shp2 PTP was used. Because this batch was less active than the others, the final protein concentration in the assay was ~0.8 µM.

vanadate buffer

4 104

3.5 104

3 104

2.5 104

2 104 Emission (465 Emission nm)

1.5 104

1 104

5000 0.01 0.1 1 10

log[inhibitor(uM)]

Figure 18: Control inhibition assays with Shp2 PTP using the optimized plate alignment; the negative, buffer-only control and the positive, vanadate control gave the expected curves, though it is important to note that the vanadate curve may be shifted by two orders of magnitude because this control assay was run with a batch of Shp2 PTP that had lower activity than the other batches. Data from a single experiment, graphed as mean ± s.d. (n = 3).

33 Peptide Inhibition Assays

After the control inhibition assay reproduced the expected results, the rationally designed peptides were tested. In the D’-derived series, the linear peptide had an IC50 of 3.51 µM while the o-xylene-bisalkylated peptide had an IC50 of 19.1 µM and the m-xylene-bisalkylated peptide had an IC50 of 21.6 µM (Figure 19, Table 1). The p-xylene-bisalkylated peptide was not tested due to unavailability of the peptide. While the vanadate control did not give a full inhibition curve, the partial curve suggests that vanadate has an IC50 of 4.31 nM for the Shp2 PTP domain.

In this assay, the fourth batch of Shp2 PTP was used, at a final assay concentration ~0.4 µM.

linear o-xylene m-xylene vanadate

120

100

80

60

40 Normalized Emission (465 Emission nm) Normalized

20

0 0.001 0.01 0.1 1 10 100 log[inhibitor(uM)]

Figure 19: Inhibition assays with the D’-derived peptides; while the p-xylene variant was not tested, the other bisalkylated peptides were not as active as the linear peptide Data from a single, reproduced experiment, graphed as mean ± s.d. (n = 3); emission values were normalized based on a buffer-only control.

Table 1: IC50 values for the D’-derived peptides. Inhibitor IC50 (µM) * vanadate 4.31 E -3 linear 3.51 E 0 o-xylene 1.91 E 1 m-xylene 2.16 E 1 * IC50 values were calculated using KaleidaGraph (y=100/(1+(m0/m1)^m2))

34 Chapter 4: Discussion

In the preliminary stages of this project, the Shp2 catalytic domain has been expressed and purified, activity and inhibition assays have been developed using DiFMUP as a substrate, and a linear peptide inhibitor has been discovered that will serve as the starting point for derivative one-bead-one-compound libraries.

The Shp2 PTP domain has been expressed and purified four times. While the purification process seemed reproducible when analyzed by gel electrophoresis (see appendix, Figure A1), the yield and activity of the purified enzyme varied from batch to batch (see appendix, Table A1,

Figure A2). Some fluctuation in yield and activity was expected, but the third batch deviated notably from the other batches, with almost half the yield of the first and fourth batches and an order of magnitude less activity than the second batch. The third batch was expressed in pLysS cells, which contain a plasmid that carries the for T7 lysozyme to lower the background expression of the target gene. While the presence of the pLysS plasmid could contribute to the lower yield of the third batch, it would not account for the decrease in activity. This discrepancy was more likely a result of the expression and purification process itself, particularly because the activity of the Shp2 PTP domain is dependent on the catalytic cysteine, which can be easily oxidized. Though there were reducing agents present in the lysis, affinity column, and size- exclusion buffers, it is possible that the abundance of recombinant Shp2 PTP overwhelmed the reducing agents within the cell or that the enzyme was irreversibly oxidized. It is also possible that the low activity of the third batch is due to misfolding or unfolding of the enzyme, though the second batch of Shp2 PTP demonstrated that the purified enzyme maintained its structure for a few weeks when refrigerated.

35 Regardless of fluctuations in yield and activity, the second, third, and fourth batches of

Shp2 PTP were used to establish activity and inhibition assays. Though PNPP was used as a substrate to validate the activity of the first and second batches, it could not produce a full activity curve within the linear range of the instrument (Figure 14). To obtain a full, reproducible activity curve, DiFMUP was used instead. The DiFMUP activity assay was first implemented to find a storage solution that would allow Shp2 PTP to retain most of its activity over the longest period of time. After the PNPP assay demonstrated that freezing at -80°C caused the enzyme to lose ~80% of its activity and refrigeration at 4°C caused the enzyme to lose activity after just a few weeks (Figure 15), the DiFMUP assay demonstrated that storage in 10% glycerol allowed the enzyme to retain 55% of its activity after freezing at -80°C (Figure 16).

Once an activity assay was established to validate the activity of each batch of Shp2 PTP and to find an appropriate storage method, an inhibition assay was developed to test the activity of the rationally designed peptides. After finding the correct plate alignment, the buffer-only control gave the expected horizontal line that represented maximum substrate turnover and the vanadate control gave an inhibition curve with an IC50 of ~300 nM (Figure 18). This IC50 was higher than expected, as vanadate inhibits protein tyrosine phosphatases in the single digit to tens digit nanomolar range. Interestingly, when vanadate was run as a control in the peptide inhibition assay, it gave an inhibition curve with an IC50 of 4.31 nM (Figure 19, Table 1). The most notable difference between these two assays was that the third batch of Shp2 PTP was used in the control assay while the fourth batch of Shp2 PTP was used in the peptide assay. Though the assays were run at different protein concentrations—0.8 µM for the third batch and 0.4 µM for the fourth batch—this two-fold difference does not explain the two order of magnitude shift in IC50. This discrepancy is another indication of the strange activity of the third batch.

36 After the control inhibition assay reproduced the expected results, the D’-derived peptides were tested against the fourth batch of Shp2 PTP. Interestingly, the linear peptide was

~6-fold more active than the bisalkylated peptides, with an IC50 of 3.51 µM (Figure 19, Table 1).

The lower activity of the bisalkylated peptides indicates that the o-xylene and m-xylene linkers constrained the peptides in the wrong conformational space. It would be interesting to see the activity of the p-xylene peptide, to determine if the p-xylene linker would confer a more active conformation. It would also be interesting to test a wider range of conformations by using amino acids with sterically hindered alpha carbons, such as penicillamine, or by creating a tighter loop through head-to-tail cyclization. As mentioned in the introduction, cyclic and stapled peptide inhibitors are often more cell-penetrant and less degradable than their linear counterparts.

Considering the availability of one-bead-one-compound methods for linear peptides, however, it may be a better strategy to develop these properties through linear OBOC libraries.

The success of the novel D’-derived peptide revels numerous goals for the immediate future. First, a serine-containing variant of the D’-derived peptide should be tested, to ensure that the activity of the linear D’-derived peptide is not dependent on disulfide bond formation between the peptide and the catalytic cysteine. This serine series will also include a mini- mutagenesis study, which will investigate the importance of the DY motif by replacing the aspartate with an asparagine and the tyrosine with a phenylalanine. Because the D’-derived peptide has a similar sequence to that of the aforementioned ELY motif—an acidic residue, followed by a bulky residue, followed by a tyrosine—the second tyrosine in the D’-derived peptide will also be replaced with phenylalanine to investigate its role in inhibition. This series of peptides, along with the linear D’-derived peptide will also be tested in an ELISA assay to determine if the DY or DYY motif can be recognized by an anti-phosphotyrosine antibody.

37 These experiments pave the way to the long-term goals of this project. The most successful inhibitor from the D’-derived or serine series will be used as a lead sequence for derivative on-bead-one-compound libraries. Using natural and unnatural amino acids to vary side chain length and functionality, these libraries will expedite improvements in phosphotyrosine mimicry and overall inhibitor activity. Screening these libraries against the full construct of Shp2 and Shp1 will expedite the development of potent and specific peptide inhibitors of Shp2.

38 Acknowledgements

First and foremost, I would like to thank Professor Joshua Kritzer, who has always been willing to teach me new things and whose unwavering passion for science is infectious. I would also like to thank the Kritzer lab for their support, from double-checking my calculations to helping me finish purifications when I am running late for class. In particular, I would like to thank Leila

Peraro and Kaley Mientkiewicz; Leila has never stopped training me, from my very first day in lab, and Kaley has always been willing to collaborate and lend a hand. I thank Tufts Summer

Scholars for partially funding this project and Professor Dehua Pei’s lab for sharing their Shp2

PTP construct. Finally, I would like to thank my parents, who were my first research mentors, and who have instilled in me the awe for the unknown and the appetite for discovery that have kept me enamored with research.

39 References

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6. Martinelli S. et al. Activating PTPN11 mutations play a minor role in pediatric and adult solid tumors. Cancer Genet Cytogenet. 166(2), 124–9 (2006).

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8. “ and Cancer”. National Cancer Institute (2013).

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11. Chen, Y-N. P. et al. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases. Nature 535, 148–152 (2016).

12. Nelson, D.L. and Cox, M.M. Lehninger Principles of Biochemistry. 6th ed., New York, NY; W.H. Freeman and Co. (2013).

13. Shi, Z.Q. et al. The Shp-2 tyrosine phosphatase has opposite effects in mediating the activation of extracellular signal-regulated and c-Jun NH2-terminal mitogen-activated protein kinases. J Biol Chem. 273(9), 4904–8 (1998).

14. Li, W. et al. A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase. Mol Cell Biol. 14(1), 509–17 (1994).

15. Agazie, Y.M. et al. Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling. Mol Cell Biol. 23(21), 7875–86 (2003).

40 16. Zhang, S.Q. et al. Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment. Mol Cell. 13, 341–55 (2004).

17. Hanafusa, H. et al. Shp2, an SH2- containing protein-tyrosine phosphatase, positively regulates receptor tyrosine kinase signaling by dephosphorylating and inactivating the inhibitor Sprouty. J Biol Chem 279, 22992–5 (2004).

18. Zhang, J. et al. Functions of Shp2 in cancer. J. Cell. Mol. Med. 19, 2075–2083 (2015).

19. Ostman, A. et al. Protein-tyrosine phosphatases and cancer. Nat.Rev.Can. 6, 307–320 (2006).

20. Lechleider, R.J. et al. Activation of the SH2- containing phosphotyrosine phosphatase SH- PTP2 by its binding site, phosphotyrosine 1009, on the human platelet-derived growth factor receptor β. J Biol Chem. 268, 21478–81 (1993).

21. Hof, P. et al. Crystal structure of the tyrosine phosphatase SHP-2. Cell 92, 441–450 (1998).

22. He, R. et al. Protein tyrosine phosphatases as potential therapeutic targets. Acta Pharmacol. Sin. 35, 1227–46 (2014).

23. Grosskopf, S. et al. Selective inhibitors of the protein tyrosine phosphatase SHP2 block cellular motility and growth of cancer cells in vitro and in vivo. ChemMedChem 10, 815–826 (2015).

24. Adessi, C. et al. Converting a peptide into a drug: Strategies to improve stability and bioavailability. Curr. Med. Chem. 9, 963−978 (2002).

25. Zorzi, A. et al. Cyclic peptide therapeutics: past, present and future. Curr. Opin. Chem. Biol. 38, 24–29 (2017).

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27. Timmerman, P. et al. Rapid and quantitative cyclization of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces. ChemBioChem 6, 821-24 (2005).

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41 Appendix

Table A1: Concentration and yield of four batches of Shp2 PTP, as determined by a BCA assay. Batch Cell Type Concentration (mg/mL) Concentration (uM) Yield (mg/L culture) 1 BL21(DE3) 0.408 9.35 5.10 2 BL21(DE3) 0.104 2.38 3.12 3 pLys 0.216 4.96 2.87 4 BL21(DE3) 0.331 7.60 4.40

1 2

3 4

Figure A1: Analysis by gel electrophoresis of the affinity purification (for batches 1, 3, and 4) and size-exclusion purification (for batch 2) of the four different expressions of Shp2 PTP.

42 1 0.8 2 35000 30000 0.6 25000 20000 0.4 15000 0.2 10000 5000 0 0 Emission ( 465 nm) Absorbance (405 nm) 0.01 0.1 1 10 0.001 0.01 0.1 1 10 log[protein (uM)] log[protein (uM)]

3 50000 4 40000 40000 30000 30000 20000 20000

10000 10000 Emission (465 nm) Emission (465 nm) 0 0 0.001 0.01 0.1 1 10 0.001 0.01 0.1 1 10 log[protein (uM)] log[protein (uM)] Figure A2: Activity validation of the four different batches of Shp2 PTP; while batch 1 was only validated by a PNPP assay, batches 2, 3, and 4 were used to establish the control inhibition assays and thus were also validated by a DiFMUP assay. Data graphed as mean ± s.d. (n = 3).

Table A2: Masses of the purified linear and bisalkylated peptides determined by MALDI TOF, using α-cyano-4-hydroxycinnamic acid as a matrix and a linear mode for detection. Peptide Sequence Linker Expected Mass (Da) MALDI Mass (Da) Ac-CNTGDYYC none 979.04 976.73 Ac-CNTGDYYC o-xylene 1081.18 1081.07 Ac-CNTGDYYC m-xylene 1081.18 1080.83 Ac-CNTGDYYC p-xylene 1081.18 1080.79

43 a

b

c

d

Figure A3: HPLC traces of the (a) linear, (b) o-xylene bisalkylated, (c) m-xylene bisalkylated, and (d) p-xylene bisalkylated peptides in the D’-derived series when injected on a C8 analytical column and eluted with a gradient of 5-100% water in acetonitrile (with 0.1% TFA) over 30 minutes.

1

44 Table A3: Abbreviations

AML Acute Myeloid Leukemia BCA Bicinchoninic Acid BL21(DE3) A strain of Escherichia coli used for expression of recombinant proteins CagA Cytotoxin-associated gene A C-SH2 The carboxy-terminal Src-Homology 2 domain located within the protein: Src-homology 2 domain-containing phosphatase 2 Csk C-terminal Src kinase C-terminal/C-terminus The carboxy terminal region/end of a protein or peptide D' The structurally mapped D' strand within the amino proximal SH2 domain of SHP2 DCM Dichloromethane D'E loop The peptidic loop bridging the structurally mapped D' and E strands within the amino proximal SH2 domain of SHP2 DiFMUP 6,8-Difluoro-4-Methylumbelliferyl Phosphate DIPEA N-Ethyl-N-(propan-2-yl)propan-2-amine DMF N,N-Dimethylformamide DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid DNAase Dox Doxycycline DTT (2S,3S)-1,4-Bis(sulfanyl)butane-2,3-diol EDT Ethane-1,2-dithiol ELISA Enzyme-linked Immunosorbent Assay ERK Extracellular Signal-regulated Kinase Fmoc Fluorenylmethyloxycarbonyl FPLC Fast Protein Liquid Chromatography Gab2/GAB2 Gab2 (Growth factor receptor-bound protein 2) associated binding protein 2 GAP/Gap Guanasine triphophosphate phosphatase Activating Protein GFP Green Fluorescent Protein Grb2 Growth factor receptor-bound protein 2 GTPase Guanasine Triphosphate Phosphatase HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid HER Human Epidermal growth factor Receptor HOBt Benzotriazol-1-ol HPLC High-Performance Liquid Chromatography IC50 The concentration of an effector at which 50% of the enzyme activity is inhibited IPTG Isopropyl β-D-1-thiogalactopyranoside Jak Janus kinase JMML Juvenile Myelomonocytic Leukemia LB Lysogeny (Luria) Broth

45 MEK Mitogne-activated protein kinase kinase Ni-NTA Nickel-nitrilotriacetic Acid NS Noonan Syndrome NSCL Non-small Cell Lung N-SH2 The amino terminal Src-Homology 2 domain located within the protein: Src-homology 2 domain-containing phosphatase 2 N-terminal/N-terminus The amino terminal region/end of a protein or peptide OBOC One-Bead-One Compound PDB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) PDGF Platelet-derived Growth Factor PI3K Phosphatidylinositol-4,5-bisphosphate 3-kinase P-loop Phosphate-binding loop pLys A strain of Escherichia coli used for expression of recombinant proteins PNPP 4-Nitrophenylphosphate poly-His a polypeptide of Histidine residues PTP Protein-Tyrosine Phosphatase PTP1B Protein Tyrosine Phosphatase 1B PTPN11 Protein-tyrosine phosphatase, nonreceptor-type, 11; also, Src-homology 2 domain-containing phosphatase 2 pTyr phosophotyrosine PyBOP (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate Raf-1 Rapidly Accelerated Fibrosarcoma kinase 1 Ras small g-protein ras (name originating from similar gene product first discovered in Rat Sarcoma Virus [v-ras]) RTK Receptor Tyrosine Kinase s.d. standard deviation of the mean SH2 Src-Homology 2 Shp1 Src-homology 2 domain-containing phosphatase 1; also, Protein- tyrosine phosphatase, nonreceptor-type, 6 (PTPN6) Shp2 Src-homology 2 domain-containing phosphatase 2; also, Protein- tyrosine phosphatase, nonreceptor-type, 11 (PTPN11) Shp2 PTP A truncated version (amino acid residues 199-593) of the full length protein: Src-homology 2 domain-containing phosphatase 2; missing its Src-Homology 2 domains and C-terminal tail shRNA short hairpin Ribonucleic Acid SOC Super Optimal broth with Catabolite repression Sos STAT Signal Transducer and Activator of Transcription protein T7 T7 bacteriophage TCEP 3,3′,3′′-Phosphanetriyltripropanoic acid TFA Trifluoroacetic acid TiPS Triisopropylsilane Tris 2-Amino-2-(hydroxymethyl)propane-1,3-diol

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