Discovery, Development and Cellular Delivery of Potent and Selective

Article Cite This: J. Med. Chem. 2017, 60, 9349-9359

pubs.acs.org/jmc

Discovery, Development, and Cellular Delivery of Potent and Selective Bicyclic Peptide Inhibitors of Grb7 Cancer Target Gabrielle M. Watson,† Ketav Kulkarni,† Jianrong Sang,†,‡ Xiuquan Ma,† Menachem J. Gunzburg,† Patrick Perlmutter,§ Matthew C.J. Wilce,† and Jacqueline A. Wilce*,† †

Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, VIC 3800, Australia ‡ Department of Physiology, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China § School of Chemistry, Monash University, Wellington Road, Clayton, VIC 3800, Australia S Supporting Information *

ABSTRACT: Grb7 is a signaling protein with critical roles in tumor cell proliferation and migration and an established cancer therapeutic target. Here we explore chemical space to develop a new bicyclic peptide inhibitor, incorporating thioether and lactam linkers that binds with affinity (KD = 1.1 μM) and specificity to the Grb7−SH2 domain. Structural analysis of the Grb7−SH2/peptide complex revealed an unexpected binding orientation underlying the binding selectivity by this new scaffold. We further incorporated carboxymethylphenylalanine and carboxyphenylalanine phosphotyrosine mimetics and arrived at an optimized inhibitor that potently binds Grb7−SH2 (KD = 0.13 μM) under physiological conditions. X-ray crystal structures of these Grb7−SH2/peptide complexes reveal the structural basis for the most potent and specific inhibitors of Grb7 developed to date. Finally, we demonstrate that cell permeable versions of these peptides successfully block Grb7 mediated interactions in a breast cancer cell line, establishing the potential of these peptides in the development of novel therapeutics targeted to Grb7.

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INTRODUCTION Targeting protein−protein interactions (PPIs) in signaling pathways is an attractive strategy in the development of anticancer therapeutics.1 Signaling pathways regulating fundamental cellular processes (such as proliferation, migration, and survival) utilize PPIs to rapidly and specifically transmit external signals to the cell, and it is often the deregulation of these pathways that leads to the development of tumors and their advancement into a metastatic state.2,3 However, targeting PPIs requires the development of inhibitors able to form interactions with large and relatively featureless protein surfaces. Here, the use of peptides can overcome this challenge by spatially and functionally mimicking the natural binding partner, leading to potent and selective inhibitors.4 Growth factor receptor bound protein 7 (Grb7) is an intracellular signaling protein with critical roles in cancer cell proliferation and migration that has been found to be overexpressed in cancers of the breast, pancreas, ovaries, and esophagus.5−8 Grb7 propagates proliferation and survival signals from © 2017 American Chemical Society

phosphorylated tyrosine kinases including HER2/3, the well-known predictor of poor prognosis in breast cancer patients.9,10 Grb7 also interacts with focal adhesion kinase (FAK) promoting cell migration and thus, when overexpressed, contributes to the metastatic potential of the cell.11 Overexpression of Grb7 has now been identified as a more significant predictor than HER2 overexpression for reduced cancerfree periods and a worse prognosis for breast cancer patients.12 Consistent with its effects when overexpressed, Grb7 knockdown results in inhibited proliferation and growth in HER2+ breast cancer cell lines,10,13 as well as inhibited migration, invasion, and colony growth in triple negative breast cancer cell lines.14,15 Grb7 is thus identified as a therapeutic target for the treatment of a number of cancers, including HER2+ breast cancer and triple negative breast cancer. Received: September 7, 2017 Published: October 30, 2017 9349

DOI: 10.1021/acs.jmedchem.7b01320 J. Med. Chem. 2017, 60, 9349−9359

Journal of Medicinal Chemistry

Article

Figure 1. Redesigning Grb7 targeted bicyclics. (A) X-ray crystal structure of peptide 2 (blue sticks) bound to the Grb7−SH2 (gray cartoon and surface representation) identifying a surface exposed aspartic acid (orange sticks) in the Grb7−SH2 EF loop (PDB ID 5EEL), (B) Schematic and chemical structure of peptides 1−5. For the schematics, the amino acid code is displayed in color and the remainder of the schematic conveys the chemical structure including the amidated C-terminus.

peptide interaction surface.18 This unexpected orientation exemplifies one of the challenges of rational inhibitor design. Building on the success of this bicyclic peptide strategy, we then embarked on further exploration of chemical space to discover even more potent Grb7−SH2 domain bicyclic inhibitors. In this report, we describe the synthesis and analysis of triazole- (peptide 3) and lactam-linked (peptides 4 and 5) bicyclic Grb7−SH2 inhibitors that are specific for Grb7 and achieve binding affinities up to KD = 270 nM. The X-ray crystal structure of the highest affinity peptide 5/Grb7−SH2 complex revealed, yet again, a rearranged mode of peptide binding and identified the importance of engaging the pY binding pocket of the Grb7−SH2 domain. This led to the further modification of the bicyclic inhibitors to incorporate carboxymethylphenylalanine (cmF) or carboxyphenylalanine (cF) pY mimetics (peptides 6 and 7). These inhibitors bound Grb7−SH2 with affinities up to KD = 130 nM, and the X-ray crystal structures provided insight into the molecular basis for the enhanced affinity of the slightly shorter cF pY mimetic. Finally, cell-permeable versions of the optimized peptide inhibitors were shown to block Grb7 interactions with HER2, SHC, and FAK upstream binding partners in a HER2+ breast cancer cell line. Together this work demonstrates the rational structure-based progression toward the most potent Grb7−SH2 domain peptides developed to date. These peptides represent invaluable tools for understanding Grb7 function and are a significant advancement in the development of Grb7targeted anticancer agents.

Grb7 is a 532 amino acid modular protein consisting of an N-terminal proline-rich region, a central GM (for Grb and Mig) domain, and a C-terminal SH2 domain. The central GM region encompasses the Ras-associating (RA) domain, pleckstrin homology (PH) domain, and the between PH and SH2 (BPS) domain. It is the C-terminal SH2 domain that interacts with the HER2/3 receptors and the focal adhesion kinase (FAK) through binding phosphorylated tyrosines (pY), and it is therefore this domain that is currently being targeted for therapeutic development.16−18 A nonphosphorylated thioether-linked cyclic peptide 1, named G7-18NATE (cyclo-(CH 2CO-WFEGYDNTFPC)amide), was identified via phage display to inhibit interactions between Grb7 and HER3 in breast cancer cell extracts.16 When peptide 1 was attached to the penetratin cell permeability sequence (1P), it was shown to inhibit migration and proliferation in breast and pancreatic cancer cell lines, as well as reduce tumor metastasis in a pancreatic mouse model derived from Grb7 overexpressing cells.7,10,14 Furthermore, peptide 1P (as well as other cell permeable forms of peptide 1) was identified to have synergistic effects with the currently available therapeutics doxorubicin and trastuzumab in inhibiting proliferation and migration in breast cancer cell lines.10,19 Subsequent biophysical analyses of the Grb7−SH2/peptide 1 interaction identified, however, that the interaction was only of modest affinity with a KD of 4.1 μM in a buffer of high phosphate concentration (50 mM) and 18.1 μM in buffer containing a physiological concentration of phosphate (1 mM).20 To improve on this, the peptide backbone was further constrained through the introduction of a second linker (via incorporation of an O-allylserine-based olefin linkage) and removal of two amino acids residues to arrive at peptide 2 (cyclo-(CH2CO-XFEGYDNXC)-amide where X= O-allylserine) with a KD of 0.83 μM in high phosphate concentration buffer.18 Interestingly, the X-ray crystal structure of the Grb7−SH2/peptide 2 complex revealed that the cyclic inhibitor bound with a rearrangement of the

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RESULTS

Redesign of Bicyclic Grb7 Targeting Inhibitors. The X-ray crystal structure of the Grb7−SH2/peptide 2 complex was recently solved to 2.47 Å resolution and revealed the structural basis for the KD = 0.83 μM binding affinity observed between the bicyclic peptide and the Grb7−SH2 domain (PDB ID: 5EEL).18 Note that the nomenclature used to describe SH2 domain secondary structure is as derived by Eck et al. (Figure 1A).21 For clarity SH2 domain amino acids will be 9350



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Figure 2. Binding studies of bicyclic inhibitors for Grb7−SH2. (A) Equilibrium binding curves for peptides 3−5 binding to Grb7−SH2 in high phosphate buffer (50 mM, +PO43−), (B−D) corresponding SPR sensorgrams, (E) equilibrium binding curves for peptides 3−5 binding to the Grb7−SH2 in low phosphate buffer (1 mM; −PO43−), (F−H) Representative SPR sensorgrams. The peptide concentrations utilized are listed on the left of the sensorgrams, with lower concentrations omitted for clarity.

denoted by the three letter code, and peptide amino acids by the single letter code. Close inspection of this X-ray structure revealed that Asp497 in the Grb7−SH2 EF loop is surface exposed and 4-fold when the phosphate concentration was decreased to 1 mM (a concentration representative of physiological levels).20 As peptides 3−5 were not designed to engage the pY binding pocket, this reliance on high concentrations of phosphate was also anticipated for these peptides. To determine this, the SPR experiments were repeated in a buffer containing 1 mM phosphate. As expected, binding of all three inhibitors to the Grb7−SH2 decreased >2.5-fold as evidenced by a shift to the right in the binding curve (Figure 2E; Table 2). While the sensorgrams did not markedly differ for the peptide 3 and 4 interactions with the Grb7−SH2, the Grb7−SH2/peptide 5 interaction no longer displayed the slower off-rate (Figure 2F−H). This suggested that the engagement of the pY binding pocket via a phosphate was required for formation of the more stable complex. The Crystal Structure of the Grb7−SH2/Peptide 5 Complex Revealed an Unexpected Binding Orientation of the Lactam. To understand the structural basis for the 9351



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in the EF loop (Figure 3B). This binding mode is reminiscent of peptide 1 binding to the Grb7−SH2; however, the removal of F9 and P10 from peptide 1 enables 5 to have improved shape complementarity along the groove formed by the EF loop (Figure S1). It was also noted that the BC loop of the Grb7−SH2 adopted two conformations in the asymmetric unit of the peptide 5 bound crystal structure. In two of the four chains, a chloride and water molecule were positioned in the pY-binding pocket, whereas the pY-binding pocket of the other two chains were “empty”. The chloride forms ionic bonds with the guanidino side chains of two essential SH2 domain pY-binding pocket residues, Arg438 and Arg458 (Figure 3C). The adjacent water molecule in the binding pocket also coordinates the chloride, with the water forming hydrogen bonds with the Ser460 side chain hydroxyl and Gln461 backbone amine (from the Grb7−SH2) as well as the Y5 hydroxyl from the peptide 5. In the remaining two chains of the Grb7−SH2 pY-binding pocket the coordinating chloride is not present and the BC loop exists in a more “open state” and is less engaged with the peptide (Figure 3D). Thus, the Grb7−SH2/peptide 5 crystal structure provides insight into two possible conformational states of the Grb7−SH2 domain pY binding pocket, one in which electronegative ions act to coordinate additional interactions between the protein and ligand forming a “closed”

Table 2. Binding Parameters of Peptides 3−5 for the Grb7−SH2 Domain in a Buffer Reflecting Physiological Phosphate Conditions (1 mM) peptide

KDa (μM)

Bmax/theoretical Bmaxb (%)

3 4 5

7.81 ± 0.07 15.7 ± 0.06 1.10 ± 0.01

55 37 29−39

Equilibrium dissociation constants calculated from fits by a single-site binding model. The errors shown are the standard error arising from the fits. bThe range of Bmax values that were derived from fits by a single-site binding model are displayed as a % of the calculated theoretical Bmax. a

improved affinity of the Grb7−SH2/peptide 5 interaction, we solved the X-ray crystal structure of the complex to 2.1 Å (Table 3). Grb7−SH2 adopted the canonical SH2 domain arrangement with peptide 5 positioned unambiguously across the βD strand (Figure S1). The core Grb7−SH2 binding motif (YXN) of peptide 5 maintained similar hydrogen bonding interactions as have been observed previously for Grb7−SH2/ peptide interactions.17,18,23 However, the structure revealed an unexpected positioning of the thioether linkage to the surface of the Grb7−SH2 domain and concomitant positioning of the lactam linker away from the surface (Figure 3A,B). In this position, the C-terminal amide is able to interact with Gln499 Table 3. X-ray Data Statistics Grb7−SH2/peptide 5 Data Collection wavelength (Å) space group unit cell dimensions a, b, c (Å) α, β, γ (deg) resolution (Å) Rmergea (%) Wilson B factor CC (1/2) (%) I/σI unique reflns measd completeness (%) multiplicity Refinement Rwork (%) Rfree (%) no. of atoms macromolecules ligands solvent mean B-factors (Å2) macromolecules ligands solvent RMSDs bond lengths (Å) bond angles (deg) Ramachandran plot (%) favored regions allowed regions

Grb7−SH2/peptide 6

Grb7−SH2/peptide 7

0.954 C2

0.954 P21

0.947 P21

122.18, 47.09, 94.61 90, 97.49, 90 40.49−2.10 (2.16−2.10) 5.8 (40.6) 99.7 (78.8) 9.7 (2.0) 30704 (2519) 97.7 (98.1) 2.5 (2.6)

47.98, 109.74, 48.33 90, 103.44, 90 35.74−2.10 (2.16−2.10) 13.8 (75.5) 22.96 99.6 (58.8) 5.1 (1.7) 28374 (2300) 99.9 (99.8) 3.6 (3.6)

45.07, 107.61, 48.01 90, 101.38, 90 47.07−2.15 (2.22−2.15) 12.0 (48.4) 24.21 85.2 (63.8) 4.9 (1.8) 24095 (2044) 98.7 (98.0) 2.8 (2.9)

19.66 (28.62) 24.95 (35.98)

21.79 (29.55) 26.09 (34.48)

20.97 (25.35) 22.05 (28.26)

3511 44 135

3595 101 191

3433 97 122

41.25 45.46 41.77

27.02 23.02 27.93

27.27 23.73 26.46

0.005 0.80

0.005 0.69

0.009 1.12

98.4 1.6

98.1 1.9

97.3 2.7

Rmerge = ∑hkl∑i|Ii(hkl) − ⟨I(hkl)⟩|/∑hkl∑iIi(hkl), where Ii(hkl) is the ith intensity measurement of reflection hkl and ⟨I(hkl)⟩ is its average. Values given in parentheses are for the high resolution shell.

a

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domain. Peptides 3 and 4 also showed negligible binding to the Grb10−SH2 and Grb2−SH2 showing that the linkage chemistry used between residues 1 and 8 does not impact on peptide binding specificity (Figure S2). A structural overlay of peptide 5 onto the Grb10−SH2 and Grb2−SH2 structures (solved in the absence of peptide ligand) provides possible insight into the specificity defining features of Grb7. It has previously been determined that the βD6 residue, Leu481, can dictate the ligand binding preference of the Grb7− SH2 domain.26 Structural analysis of the Grb7−SH2/peptide 5 complex clearly identifies the way in which Leu481 influences binding, with F2 and Y5 from peptide 5 straddling this hydrophobic residue (Figure S3). In contrast, the glutamine at the equivalent position in Grb10−SH2 would likely prevent binding due to steric clashes (Figure S3). This is also likely to occur for the Grb2−SH2 domain with a bulky lysine side chain potentially clashing with F2 positioning, as well as the Grb2−SH2 having an extended BG loop that could interfere with bicyclic peptide binding (Figure S3). Incorporating pY Mimetics Leads to Engagement of the Grb7−SH2 BC Loop and Removes the Requirement for Phosphate. Based on the structural insights from the Grb7−SH2/peptide 5 X-ray crystal structure, we hypothesized that replacing Y5 with a pY mimetic would enhance binding affinity and remove the requirement for high concentrations of phosphate. We have previously described the use of carboxymethylphenylalanine (cmF) or carboxyphenylalanine (cF) as pY mimetics;17 therefore, we incorporated these same non-natural amino acids into the peptide 5 bicyclic backbone (Figure 4A). The new pY containing bicyclic peptides, 6 (containing cmF) and 7 (containing cF), were tested for binding to the Grb7−SH2 in the presence of a physiological concentration of phosphate. Both peptides displayed enhanced affinity for the Grb7−SH2 with equilibrium dissociation constants of KD = 220 nM and 130 nM, respectively, representing an approximately 80-fold and 140-fold improvement over with the lead inhibitor peptide 1 (KD = 18.1 μM under the same conditions) (Figure 4B; Figure S4; Table 4). Both peptides displayed the slowed dissociation rate that was described for peptide 5 binding to the Grb7−SH2 in a high phosphate buffer. To deduce the effectiveness of the cmF and cF as pY mimetics, we also measured the binding affinity of the two peptides for the Grb7−SH2 in the high phosphate buffer (50 mM). As expected, the affinity decreased 14-fold and 6-fold, respectively, consistent with the phosphate outcompeting the peptides for Grb7−SH2 binding (Figure 4B; Figure S4; Table 4). Thus, by mimicking the positioning of phosphate, the incorporation of the cmF and cF into the bicyclic backbone has resulted in potent Grb7−SH2 binding peptides under physiologically relevant buffer conditions. The cF Phosphotyrosine Mimetic Effectively Closes off the pY Binding Pocket. When the two pY mimetics were designed, it was anticipated that the cmF containing peptide (6) would have enhanced affinity for the Grb7−SH2 compared with the cF bearing peptide (7). The cF group was predicted to have a less ideal geometry than the cmF, with the absence of the methylene group that would spatially mimic the oxygen in a phosphotyrosine. However, our binding experiments indicated that peptide 7 was the higher affinity ligand. To understand the molecular basis for this unexpected finding, we determined the X-ray crystal structures of the Grb7−SH2/peptide 7 and Grb7−SH2/peptide 6 complexes (Table 3).

Figure 3. X-ray crystal structure of the Grb7−SH2/peptide 5 complex and specificity analysis. (A, B) Surface representations of the Grb7−SH2 (gray) with either bound peptide 2 (blue sticks) or peptide 5 (green sticks), (C, D) Close-up of the Grb7−SH2 pY binding pocket with key amino acids shown in stick format and hydrogen bonds displayed as dashed lines (PDB IDs 5EEL and 5U1Q). (E, F) SPR equilibrium binding curves for peptide 5 binding to the Grb7−SH2 (gray), Grb2−SH2 (wheat), and Grb10−SH2 (purple) in either high phosphate buffer (50 mM; +PO43−) or low phosphate buffer (1 mM; −PO43−). Representative sensorgrams are provided as Supporting Information.

conformation and one in which there is no electronegative ion and the BC loop exists in a more “open” conformation. This likely reflects the molecular basis for the enhanced peptide binding observed in the presence of higher phosphate concentration−with the highly electronegative phosphate ion effecting coordination between peptide and protein and promoting the “closed” conformation of the BC loop. The Lactam Peptide 5 Selectively Binds Grb7−SH2. As well as developing high affinity binding peptides, it was also important to assess whether the designed molecules maintained specificity for the Grb7−SH2 target. Establishing inhibitor specificity against the Grb7−SH2 is critical, with over 120 human proteins bearing SH2 domains. We chose to test binding against two closely related SH2 domains, Grb2 and Grb10, with Grb2 sharing the same preferential pYXN binding motif as Grb7, and Grb10 representing a member of the Grb7 family.24,25 Using SPR, we determined that peptide 5 showed negligible binding to both Grb2−SH2 and Grb10−SH2 with binding responses that were too weak for equilibrium dissociation constants to be determined (Figure 3E,F; Figure S2). The preferential binding to Grb7−SH2 was shown to occur regardless of the phosphate concentration, reinforcing that bicyclic peptide 5 is robustly preferential for the Grb7−SH2 9353



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Figure 4. Redesigning pY mimetic containing bicyclic peptides. (A) Chemical structure of peptides 6 and 7 with the pY mimetics (cmF and cF) highlighted by the pink and purple boxes, respectively. (B) SPR equilibrium binding curves of peptide 6 (top) and peptide 7 (bottom) binding to the Grb7−SH2 in both high (50 mM; +PO43−) and low (1 mM; −PO43−) phosphate containing buffer conditions. Representative sensorgrams are provided as Supporting Information. (C) Close-up of the Grb7−SH2 pY binding pocket (gray) highlighting peptide 6 (top) and peptide 7 (bottom) interacting residues (stick format). Waters are displayed as red spheres and hydrogen bonds as dashed lines. (D) Surface representation of the Grb7−SH2 (colored according to atom type: N = blue, O = red, S = yellow, C = gray), with either peptide 6 (top) or peptide 7 (bottom). Grb7−SH2/peptide 6 PDB ID 5U06, Grb7−SH2/peptide 7 PDB ID 5TYI.

was described for the Grb7−SH2/peptide 5 structure via chloride and water ion interactions. In contrast, the cF moiety of peptide 7 does not extend deeply into the pY pocket and instead forms hydrogen bonds with the Asn463 and Ser460 side chain oxygens that are closer to the entry of the binding pocket (Figure 4C). A coordinating water hydrogen bonds with the cF carboxyl leading to engagement of Gln461 and Arg458. This positioning places the aromatic ring of cF in a more planar arrangement against the Grb7−SH2 binding surface and allows Arg462 to extend over the pY pocket via interactions with the G4 carbonyl (Figure 4D). Thus, although both pY mimetics engage the BC loop and hold it in the optimal closed conformation, the shortened pY mimetic appears to facilitate the most stabilized complex with the Grb7−SH2 domain through additional favorable interactions. The Optimized Inhibitors Successfully Block Grb7 Mediated Interactions in Breast Cancer Cells. We next sought to determine whether the optimized inhibitors were effective at disrupting Grb7 interactions with HER2, SHC, and FAK in a HER2+ breast cancer cell line.5,11 To ensure cell entry, the peptides were conjugated to the cell penetrating peptide (CPP) penetratin (denoted by P). We also tested first generation monocyclic peptide 1 and its cF containing equivalent (peptide 8) as a comparison.17 Whole cells were treated with peptide (to test their internalization) and washed, and then Grb7 immunoprecipitated from the cell lysate and associated partners were identified by Western blot (Figure 5A). All of the penetratin conjugated peptides (1P, 5P, 7P, and 8P) disrupted Grb7 interactions with SHC, HER2, and FAK to a similar extent (The full blots are provided in Figure S6).

Table 4. Binding Parameters of Peptides 6 and 7 for the Grb7−SH2 Measured in a High (50 mM; +PO43−) or Low (1 mM; −PO43−) Phosphate Concentration −PO43−

+PO43−

peptide

KD (μM)

Bmax/ theoretical Bmaxb (%)

6 7

0.22 ± 0.0004 0.13 ± 0.0004

30 32

a

a

KD (μM)

Bmax/ theoretical Bmaxb (%)

2.94 ± 0.008 0.83 ± 0.001

52 53

Equilibrium dissociation constants calculated from fits by a single-site binding model. The errors shown are the standard error arising from the fits. bThe range of Bmax values that were derived from fits by a single-site binding model are displayed as a % of the calculated theoretical Bmax. a

The Grb7−SH2 domain in both structures adopted the classical fold, with peptides 6 and 7 unambiguously positioned across the βD strand as described for the Grb7−SH2/peptide 5 complex structure (Figure S5). The peptide 6 and peptide 7 backbones maintained similar positioning to peptide 5 with an α-carbon RMSD of 0.2 and 0.3 Å respectively, and formed the same interactions with Grb7−SH2 amino acids (hydrogen bonding with Arg438, His479, Leu481, and Met495) (Figure S5). As expected, the only difference between the three complex structures was in the engagement of the BC loop and interactions with the pY binding pocket. In the case of peptide 6, the cmF group extends into the back of the pY binding pocket with the carboxylate forming bidentate salt bridges with Arg458 at the base of the pY binding pocket, as well as with the Arg438 guanidino group and hydrogen bonds with the Ser460 side-chain oxygen (Figure 4C). Through these interactions, the BC loop is held in the optimal closed conformation that 9354



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Figure 5. Optimized inhibitors block Grb7 interactions with cellular partners. (A) SKBR3 cells were serum starved overnight and treated for 1 h with Grb7 targeting peptides (20 μM) prior to stimulation for 10 min with fibronectin (10 μg·mL−1). Washed cells were lysed and immunoprecipitated using Grb7 antibodies before being probed with various antibodies for the detection of Grb7 binding partners: SHC, top panel; HER2, second panel; FAK, third panel; Grb7 on the bottom panel served as a loading control. FN = fibronectin, P = penetratin control peptide. (B) As per panel A, with the exception that the Grb7 targeting peptides were added following cell lysis. Schematics of the penetratin coupled peptides tested are shown on the right of the image with the peptide sequence conveyed as single letter code and code displayed in color and the remainder of the schematic conveying the peptide chemical structure.

18-fold increase to KD = 1 μM (in physiologically relevant phosphate concentrations). Fascinatingly, this improvement was not seen for peptide 4 that only differed in the orientation of the lysine and glutamic acid lactam precursors within the amino acid sequence. To understand the structural basis for the improved interaction, we solved the X-ray crystal structure of the Grb7−SH2/peptide 5 complex. This structure revealed a novel binding mode adopted by the bicyclic peptide, with the thioether linkage forming part of the binding surface and the lactam linker not engaging the Grb7−SH2 as predicted. Thus, the basis for the peptide binding was revealed, but not the reason for the favored lactam orientation. Peptide stabilization through lactam linkers has been used previously in peptide design, mainly to constrain peptides in an α-helical conformation.30 Examples of the use of lactam constraints in other structural motifs are rarer but include successful application in the development of SH2 domain inhibitors. Inhibitors of Src−SH2 cyclized through a lactam linkage displayed >60-fold improved affinity compared with the linear peptides,31 and Grb2−SH2 targeting lactam-linked bicyclic inhibitors improved the IC50 17-fold compared to the monocyclic peptide.32 Interestingly, in the case of these Grb2− SH2 targeted inhibitors, selectivity depending upon the lactam amino acid orientation was also described suggesting an important structural difference that can occur when the lactam orientation is reversed. Our binding experiments revealed that a high concentration of phosphate in the buffer was required for high affinity binding between Grb7−SH2 and the tyrosine containing bicyclic peptides, consistent with our previous studies of Grb7 targeting peptides.18,20 The phosphate is likely to play a role in the pY binding pocket analogous to that played by the pY phosphate of a cellular ligand, contributing greatly to the overall affinity of binding through interactions with the surrounding residues including the BC loop. The Grb7−SH2/peptide 5 complex

To test whether there was any discrimination between the ability of the peptides to disrupt Grb7 interactions with binding partners more directly we also tested their ability to block Grb7 mediated interactions when added directly to cell lysate (Figure 5B). The optimized inhibitors (5P, 7P and 8P) all blocked Grb7 mediated interactions to a greater extent than the original lead 1P inhibitor. Thus, the incorporation of the lactam linker and/or phosphotyrosine mimetic has effectively translated into improved inhibitors of Grb7 interactions.

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DISCUSSION The development of biological agents that target disease related PPIs is a burgeoning area in the field of therapeutic design.27 SH2 domains are an obvious target for the development of therapeutics with the negatively charged pY binding pocket, a distinctive feature for small molecules to bind. However, ensuring selectivity over other SH2 domains has led to larger ligands, such as peptides, being utilized in SH2 inhibitor design, with surfaces adjacent to the pY binding pocket helping to confer substrate specificity.28 Here, we have described the development of peptides that potently and selectively target the SH2 domain of the breast cancer target Grb7. Based on a combination of structural and binding information, we developed bicyclic peptide inhibitors that bind to the Grb7−SH2 with nanomolar affinity, representing an overall 140-fold improvement on the first-generation monocyclic inhibitor peptide 1.20 We previously discovered a bicyclic peptide tethered by an olefin linkage that showed enhanced affinity over peptide 1, likely due to a reduced entropic loss effect.29 Here we tested the effects of alternative linkage chemistries on the Grb7−SH2/peptide binding affinity using SPR. These were designed to maintain peptide rigidity but also include potential electron accepting functionalities that could form additional contacts with the Grb7−SH2 domain. This strategy identified that the lactam linker in peptide 5 facilitates an 9355



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We also show that these peptides are deliverable to cells. They therefore not only represent tools to elucidate the specific function of Grb7 in cells but also represent a significant step toward the development of novel Grb7-targeted therapeutics for the treatment of breast and other cancers.

structure revealed the presence of a chloride ion that could effect the equivalent interactions alongside peptide 5. In light of this structural information, we incorporated either a carboxymethylphenylalanine (cmF) or carboxyphenylalanine (cF) into the peptide 5 bicyclic backbone resulting in the affinity improving a further 8-fold to KD = 130 nM (in a physiological concentration of phosphate). Both non-natural amino acids successfully acted as phosphotyrosine mimetics with the SPR analysis revealing that phosphate competes for Grb7−SH2 binding, and the X-ray crystal structures of Grb7−SH2 in complex with peptides 6 or 7 revealing engagement of the BC loop and pY binding pocket residues. Phosphotyrosine mimetics are typically designed to spatially mimic a phosphotyrosine. In this study, we discovered that the shorter cF pY mimetic (lacking the oxygen mimicking methylene of cmF) increased the binding affinity to a greater extent than the more traditional cmF pY mimetic and was less affected by the presence of high concentrations of phosphate (6-fold lower affinity compared with 14-fold difference). This is consistent with our studies of monocyclic peptides;17 however, we have now revealed the structural basis for the improvement in the interaction with crystal structures of Grb7−SH2 domain in complex with peptides 6 and 7. While the cmF group extends to the base of the pY binding pocket, the shorter cF group binds BC loop residues at the entry to the pocket. Through these interactions, the binding pocket is effectively closed off, forming a tighter interaction surface. This finding suggests that shortened pY mimetics such as carboxyphenylalanine could also be applicable in the design of peptides targeted to other SH2 domains to produce high affinity interactions. We have also determined that the peptide 5 scaffold is specific for Grb7−SH2 over the closely related SH2 domains of Grb2 and Grb10. Our structural investigations show the way in which this bicyclic scaffold complements the surface of Grb7−SH2, particularly across the βD6 amino acid Leu481 over which F2 and Y5 are straddled. Despite the very close homology and similar ligand binding preference of Grb10−SH2 and Grb2−SH2 respectively, the equivalent interaction is predicted to be impeded for these SH2 domains. Thus, the optimized peptides not only possess higher affinity but also maintain specificity for Grb7. Lastly, we explored whether the optimized inhibitors could disrupt Grb7 mediated interactions when attached to the cell penetrating peptide, penetratin. This confirmed that the peptide inhibitors could successfully enter the cell and block Grb7 interactions with HER2, SHC, and FAK. Improved inhibition of Grb7 interactions by the bicyclic peptides over the first-generation monocyclic peptides was apparent, but only when the peptides were delivered directly to the cell lysate. Thus, we postulate that there could be differences in the effective delivery of the bicyclic peptides across the cell membrane. It has been demonstrated that the incorporation of different cargoes can alter the cellular uptake efficiencies by CPPs.33−35 It is possible that incorporating the lactam-linker has altered the ability of penetratin to deliver the peptides to the cytosol, and a different CPP is required for optimal uptake.

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EXPERIMENTAL SECTION

Statement of Purity. All proteins were purified ≥95% purity using size-exclusion chromatography as assessed using SDS-PAGE. All peptides were purified to ≥95% purity using reversed-phase HPLC and mass spectrometry for verification (as shown in Supporting Information). Peptide Synthesis. Peptide Synthesis Materials. The allyloxycarbamate protected N-Fmoc lysine and the N-Fmoc-L-glutamic acid α-allyl ester, to synthesize the lactam peptides (4−7), were purchased from Purar chemicals (Melbourne, Victoria, Australia). The triazole linker containing peptide (3) was synthesized from N-Fmoc-5azidopentanoic acid, purchased from AAPPTec LLC (Louisville, Kentucky, USA) and N-Fmoc-L-propargyl serine, prepared from N-Fmoc-L-serine. To further incorporate a phosphotyrosine mimetic within the peptides required the substitution of the tyrosine residue within peptides (6 and 7) with either N-Fmoc-(4-tert-butyloxycarbonyl)-L-phenylalanine, purchased from Santa Cruz Biotechnology Inc. (Dallas, Texas, USA) or N-Fmoc-(4-methoxycarbonylmethyl)-Lphenylalanine, purchased from (Epichem Pty Ltd., Bentley, Western Australia, Australia). All other N-Fmoc protected α amino acids incorporated within the peptide sequences were purchased from GL Biochem Shanghai Ltd. (Shanghai, China). At the end of the peptide elongation cycle, the resin was thoroughly washed with CH2Cl2 and Et2O and air-dried before further manipulation (see below). Peptide Synthesis (Peptides 3−7). Peptides 3−7 were synthesized via solid phase peptide synthesis on a 0.25 mmol scale using standard Fmoc-chemistry on Rink amide resin (0.7 mmol/g), similarly to previously reported syntheses.17,18 Details of the procedure for click chemistry, lactamization, thioether formation, and peptide verification are provided below. Click Chemistry (Peptide 3). Click chemistry was per that performed, on resin, on a 0.25 mmol scale. 2,6-lutidine (70 μL, 0.6 mmol), L-ascorbic acid (70 mg, 0.4 mmol), and NaHCO3 (33 mg, 0.4 mmol) in H2O (1.5 mL) were added to a solution of CuI (29 mg, 0.15 mmol) in CH3CN (12 mL) and DMSO (3 mL). The resulting solution was degassed by bubbling a stream of argon for 10 min and then added to the peptide loaded resin followed by gentle agitation for 16 h at room temperature. The resin was filtered and washed sequentially with CH3CN, H2O, DMF, and CH2Cl2, and the resin was soaked in DMF before continuing to the N-terminus deprotection step. Lactamization (Peptides 4−7). The glutamic acid and lysine residue protecting groups (allyl ester and allyloxycarbonyl, respectively) were removed simultaneously, on resin, on a 0.25 mmol scale. CHCl3 (15 mL) was degassed by bubbling a stream of argon for 10 min. A portion of the CHCl3 (2 mL) was used to preswell the resin, while PhSiH3 (740 μL, 6 mmol) and Pd(PPh3)4 (580 mg, 0.5 mmol) were added to the remainder of the sparged CHCl3. The mixture was vigorously shaken until homogeneity was achieved. The resin was subsequently soaked in the Pd(PPh3)4 solution for 2 h with gentle agitation. The resin was filtered and washed with CH2Cl2 and DMF to remove the catalyst. Lactamization of the lysine and glutamic acid residues was achieved on a 0.25 mmol scale. The resin was washed with DMF, and then a solution of HBTU (3 equiv to resin loading), HOBt (3 equiv to resin loading), and DIPEA (4.5 equiv to resin loading), dissolved in DMF (4 mL), was added to the resin and gently agitated for 45 min. The resin was subsequently washed with DMF before continuing to the N-terminus deprotection step. N-Terminus Deprotection, Thioether Formation, and Peptide Analysis (Peptides 3−7). The terminal Fmoc protecting group was removed with 2× wash (5 mL each) of 20% (v/v) piperidine in DMF (with 0.1 M HOBt) for 15 min each, and the resin was treated with chloroacetic anhydride (171 mg, 1 mmol) and DIPEA (100 μL) in

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CONCLUSIONS This study thus describes a major advance in the development of potent and specific Grb7 targeting peptides. We propose that the bicyclic scaffold described herein not only confers rigidity to the peptide helping to enhance binding affinity but would also be anticipated to protect the peptide against proteases in vivo. 9356



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injected over all four flow cells at 30 μL·min−1 for 60−80 s. The +PO43− buffer consisted of 50 mM Na3PO4, 150−300 mM NaCl, and 1 mM DTT (pH 7.4), whereas the −PO43− buffer consisted of 1 mM Na3PO4 (reflecting a physiological concentration), 150 mM NaCl, 20 mM Tris, 1 mM DTT (pH 7.4). Peptide 3 required regeneration with 2 M NaCl before injection of the next peptide cycle. Data were analyzed using Scrubber2.0 (BioLogic Software, Campbell, ACT, Australia) and Prism 6.0 (GraphPad Software, California, USA). Structure Determination of Grb7−SH2/Peptide Complexes. The Grb7−SH2/peptide 5 complex formed diffracting crystals using 7.5 mg·mL−1 protein in a 1:1.5 M ratio of protein/peptide in a condition containing 18% (w/v) PEG 6000, 0.1 M MES (pH 5.5), and 0.2 M CaCl2. The crystals were cryoprotected in mother liquor supplemented with 15% (v/v) glycerol and flash-cooled in liquid nitrogen. Diffraction images were collected at the Australian Synchrotron MX2 high-throughput protein crystallography beamline with an ADSC Quantum 315r CCD detector using the BLU-ICE acquisition software.38 Diffraction images were indexed using IMOSFLM39 and scaled using AIMLESS40 from the CCP4 suite.41 Phases were generated via molecular replacement (using PHASER) with one chain of apo Grb7−SH2 as the search model (PDB ID 2QMS).42 Subsequent model refinement was conducted using PHENIX43 and model building was performed with COOT.44 The Grb7−SH2/peptide 6 complex crystals formed using 10 mg·mL−1 protein in a 1:1.5 M ratio of protein/peptide in a condition containing 25% (w/v) PEG3350, 0.2 M KSCN, and 0.1 M BTP (pH 8.5). The crystals were cryoprotected in mother liquor supplemented with 10% (v/v) glycerol and flash-cooled in liquid nitrogen. Diffraction data was collected at the Australian Synchrotron as per that of the Grb7−SH2/ peptide 5 complex and subsequently indexed and using IMOSFLM39 and scaled using AIMLESS40 from the CCP4 suite.41 Molecular replacement, model building, and refinement occurred as per that of the Grb7−SH2/peptide 5 complex. The Grb7−SH2/peptide 7 complex formed diffracting crystals using 10 mg·mL−1 protein in a 1:1.5 M ratio of protein/peptide in a condition containing 20% (w/v) PEG3350 and 0.2 M NaSCN. The crystals were cryoprotected in mother liquor saturated with sucrose and flash-cooled in liquid nitrogen. Diffraction images were collected at the Australian Synchrotron as described above. The images were indexed using XDS45 and scaled using AIMLESS.40 Molecular replacement, model building, and refinement occurred as per that of the Grb7−SH2/peptide 5 complex. The restraint files for all peptides were generated using eLBOW from SMILES strings.46 MOLPROBITY was used to assess the quality of the final models.47 The data collection and processing statistics are displayed in Table 3. Cell Culture and Coimmunoprecipitation Experiments. The SKBR3 HER2+ breast cancer cell line was obtained from the American Type Culture Collection (ATCC, USA) and maintained according to ATCC guidelines. Overnight serum starved cells in a 10 cm dish were stimulated for 10 min with fibronectin (10 μg.mL−1, 37 °C), washed 2 times with ice cold PBS and lysed with 1 mL of RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 10 mM sodium pyrophosphate, 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 μg·mL−1 aprotinin, and 10 μg·mL−1 leupeptin). Supernatant protein concentration was determined using the BradfordUltra reagent from Expedeon (catalog no. BFU1L).48 Rabbit Grb7 (N-20) antibodies (Santa Cruz Biotechnology) were used to immunoprecipiate Grb7 and associating proteins, and this was collected with protein A sepharose beads (Zymed Laboratories). The Grb7 targeting peptides (20 μM) were either added to 1 mg of cell lysate or, prior to fibronectin stimulation, live cells were treated with peptide for 1 h. Immunoprecipitate samples were resolved by SDS-PAGE (8−12%) and subject to Western blotting using the appropriate antibodies. Grb7 and its bound proteins were visualized by chemiluminescence from the corresponding secondary antibodies. Mouse FAK (catalog no. F15020) and mouse SHC (catalog no. 610879) were obtained from BD Transduction Laboratories. Rabbit HER2 (catalog no. 06-562) was obtained from Merck Millipore. Donkey anti-mouse HRP (catalog no.

DMF (3 mL) for 45 min to afford a choloroacetyl-capped N-terminus. The resin was subsequently washed with DMF, CH2Cl2, and Et2O and air-dried. Cleavage was performed on 0.25 mmol of the resin, by treating the resin with a cleavage solution (15 mL) comprising 2.5% (v/v) distilled water (375 μL), 2.5% (v/v) triisopropylsilane (375 μL), and 0.5% (v/v) ethanedithiol (75 μL) in TFA for 3 h. The TFA was evaporated under a stream of N2, the peptide was precipitated by addition of Et2O (40 mL), and the precipitate was filtered and redissolved in 50% aqueous CH3CN for lyophilization. Thioether formation occurred by dissolving the crude peptides in 100 mM aqueous NH4HCO3 solution made up in 50% aqueous CH3CN for 2 h at room temp. The cyclized peptides were purified using reversedphase HPLC. Hydrolysis of cmF Methyl Ester (Peptide 6). The para-carboxymethyl phenylalanine methyl ester was hydrolyzed by dissolving the peptide (∼5 mg·mL−1) in aqueous LiOH solution for 2 h at room temp. The success of hydrolysis was confirmed using mass spectrometry, and all peptides were purified to ≥95% purity using reversed-phase HPLC (as shown in Supporting Information). All peptide identities were verified by mass spectrometry. Peptide 3: Mexpected (C49H61N14O17S)− = 1149.4; Mmeasured (C49H61N14O17S)− = 1149.3; M e x p e c t e d (C 4 9 H 6 0 N 1 4 O 1 7 S) 2 − = 574.2; M m e a s u r e d (C49H60N14O17S)2− = 574.2. Peptide 4: Mexpected (C49H63N12O17S)− = 1123.4; M m e a s u r e d (C 4 9 H 6 3 N 1 2 O 1 7 S) − = 1123.1; M e x p e c t e d (C49H62N12O17S)2− = 561.2; Mmeasured (C49H62N12O17S)2− = 561.1. Peptide 5: M expected (C 49 H 63 N 12 O 17 S) − = 1123.4; M measured (C49H63N12O17S)− = 1123.4; Mexpected (C49H62N12O17S)2− = 561.2; M measured (C 49 H 62 N 12 O 17 S) 2− = 561.1. Peptide 6: M expected (C51H65N12O18S)− = 1165.4; Mmeasured (C51H65N12O18S)− = 1165.1; Mexpected (C51H64N12O18S)2− = 582.2; Mmeasured (C51H64N12O18S)2− = 582.0. Peptide 7: Mexpected (C50H63N12O18S)− = 1151.4; Mmeasured (C50H63N12O18S)− = 1151.0; Mexpected (C50H62N12O18S)2− = 575.2; Mmeasured (C50H62N12O18S)2− = 575.1. Penetratin Coupled Peptides. Penetratin coupled peptide 1 (1P) was prepared as previously described.36 Mexpected (C171H246N48O38S2)6+ = 608.3; Mmeasured (C171H246N48O38S2)6+ = 608.6. Penetratin coupled peptides 5, 7, and 8 (5P, 7P, and 8P), and a propargyl penetratin control peptide were synthesized by Purar Chemicals (Australia) to >95% purity based on LC-MS (as shown in Supporting Information). The synthesis of peptide 8 (without penetratin) has been previously described.17 Peptide 5P: Mexpected (C153H230N46O36S2)4+ = 839.5; Mmeasured(C153H230N46O36S2)4+ = 839.8; Mexpected (C153H230N46O36S2)5+ = 671.8; Mmeasured(C153H230N46O36S2)5+ = 672.0. Peptide 7P: Mexpected (C155H231N45O37S2)4+ = 846.5; Mmeasured(C155H231N45O37S2)4+ = 847.0; Mexpected (C155H231N45O37S2)5+ = 677.4; Mmeasured(C155H231N45O37S2)5+ = 677.8. Peptide 8P: M expected (C 172 H246 N48 O39 S 2 ) 4+ = 919.6; M measured(C172H246N48O39S2)4+ = 919.6; M expected (C172H246N48O39S2)5+ = 735.9; Mmeasured(C172H246N48O39S2)5+ = 735.9. Penetratin control peptide: Mexpected (C111H180N36O21)4+ = 596.6; Mmeasured (C111H180N36O21)4+ = 596.5; Mexpected (C111H181N36O21)5+ = 477.5; Mmeasured (C111H181N36O21)5+ = 477.4; Mexpected (C111H182N36O21)6+ = 398.1; Mmeasured (C111H182N36O21)6+ = 398.0. Peptide Concentration Determination. Solution peptide concentrations were determined spectrophotometrically based on absorbance at 280 nm using extinction coefficients derived from amino acid content.37 If the absorbance could not be measured at 280 nm, the DirectDetect (Millipore) system was used. Protein Expression and Purification. GST tagged Grb7−SH2, Grb2−SH2, and Grb10−SH2, GST alone, and untagged Grb7−SH2 were expressed and purified as previously described.17 Surface Plasmon Resonance. SPR experiments were conducted at 25 °C as previously described on a BIAcore T100 using BIAcore CM5 series S sensor chips (GE Life Sciences).17,18 Briefly, polyclonal anti-GST antibody (Abcam, Cambridge) was immobilized on all flow cells via amine coupling with capture levels between 2280 RU and 6700 RU (GE Life Sciences GST capture kit). As a control, GST alone was captured on the reference flow cell (between 520 RU and 1280 RU) and the GST tagged proteins were immobilized on the active flow cells (between 690 RU and 1990 RU). Lyophilized peptides (in triplicate) were resuspended in analysis buffer and 9357



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1706515) and goat anti-rabbit HRP antibodies were obtained from Bio-RadAbcam.

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(2) Pawson, T. Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 2004, 116, 191−203. (3) Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 2011, 144, 646−674. (4) Gao, M.; Cheng, K.; Yin, H. Targeting protein-protein interfaces using macrocyclic peptides. Biopolymers 2015, 104, 310−316. (5) Stein, D.; Wu, J.; Fuqua, S. A.; Roonprapunt, C.; Yajnik, V.; D’Eustachio, P.; Moskow, J. J.; Buchberg, A. M.; Osborne, C. K.; Margolis, B. The SH2 domain protein GRB-7 is co-amplified, overexpressed and in a tight complex with HER2 in breast cancer. EMBO J. 1994, 13, 1331−1340. (6) Tanaka, S.; Mori, M.; Akiyoshi, T.; Tanaka, Y.; Mafune, K.; Wands, J. R.; Sugimachi, K. Coexpression of Grb7 with epidermal growth factor receptor or Her2/erbB2 in human advanced esophageal carcinoma. Cancer Res. 1997, 57, 28−31. (7) Tanaka, S.; Pero, S. C.; Taguchi, K.; Shimada, M.; Mori, M.; Krag, D. N.; Arii, S. Specific peptide ligand for Grb7 signal transduction protein and pancreatic cancer metastasis. J. Natl. Cancer Inst. 2006, 98, 491−498. (8) Wang, Y.; Chan, D. W.; Liu, V. W.; Chiu, P.; Ngan, H. Y. Differential functions of growth factor receptor-bound protein 7 (GRB7) and its variant GRB7v in ovarian carcinogenesis. Clin. Cancer Res. 2010, 16, 2529−2539. (9) Slamon, D. J.; Clark, G. M.; Wong, S. G.; Levin, W. J.; Ullrich, A.; McGuire, W. L. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987, 235, 177−182. (10) Pradip, D.; Bouzyk, M.; Dey, N.; Leyland-Jones, B. Dissecting GRB7-mediated signals for proliferation and migration in HER2 overexpressing breast tumor cells: GTP-ase rules. Am. J. Cancer Res. 2013, 3, 173−195. (11) Han, D. C.; Guan, J. L. Association of focal adhesion kinase with Grb7 and its role in cell migration. J. Biol. Chem. 1999, 274, 24425− 24430. (12) Ramsey, B.; Bai, T.; Hanlon Newell, A.; Troxell, M.; Park, B.; Olson, S.; Keenan, E.; Luoh, S. W. GRB7 protein over-expression and clinical outcome in breast cancer. Breast Cancer Res. Treat. 2011, 127, 659−669. (13) Chu, P. Y.; Li, T. K.; Ding, S. T.; Lai, I. R.; Shen, T. L. EGFinduced Grb7 recruits and promotes Ras activity essential for the tumorigenicity of Sk-Br3 breast cancer cells. J. Biol. Chem. 2010, 285, 29279−29285. (14) Giricz, O.; Calvo, V.; Pero, S. C.; Krag, D. N.; Sparano, J. A.; Kenny, P. A. GRB7 is required for triple-negative breast cancer cell invasion and survival. Breast Cancer Res. Treat. 2012, 133, 607−615. (15) Lim, R. C.; Price, J. T.; Wilce, J. A. Context-dependent role of Grb7 in HER2+ve and triple-negative breast cancer cell lines. Breast Cancer Res. Treat. 2014, 143, 593−603. (16) Pero, S. C.; Oligino, L.; Daly, R. J.; Soden, A. L.; Liu, C.; Roller, P. P.; Li, P.; Krag, D. N. Identification of novel non-phosphorylated ligands, which bind selectively to the SH2 domain of Grb7. J. Biol. Chem. 2002, 277, 11918−11926. (17) Watson, G. M.; Gunzburg, M. J.; Ambaye, N. D.; Lucas, W. A.; Traore, D. A.; Kulkarni, K.; Cergol, K. M.; Payne, R. J.; Panjikar, S.; Pero, S. C.; Perlmutter, P.; Wilce, M. C.; Wilce, J. A. Cyclic peptides incorporating phosphotyrosine mimetics as potent and specific inhibitors of the Grb7 breast cancer target. J. Med. Chem. 2015, 58, 7707−7718. (18) Gunzburg, M. J.; Kulkarni, K.; Watson, G. M.; Ambaye, N. D.; Del Borgo, M. P.; Brandt, R.; Pero, S. C.; Perlmutter, P.; Wilce, M. C.; Wilce, J. A. Unexpected involvement of staple leads to redesign of selective bicyclic peptide inhibitor of Grb7. Sci. Rep. 2016, 6, 27060. (19) Pero, S. C.; Shukla, G. S.; Cookson, M. M.; Flemer, S., Jr.; Krag, D. N. Combination treatment with Grb7 peptide and Doxorubicin or Trastuzumab (Herceptin) results in cooperative cell growth inhibition in breast cancer cells. Br. J. Cancer 2007, 96, 1520−1525. (20) Gunzburg, M. J.; Ambaye, N. D.; Del Borgo, M. P.; Pero, S. C.; Krag, D. N.; Wilce, M. C.; Wilce, J. A. Interaction of the non-

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01320. X-ray crystal structure of the Grb7−SH2/peptide 5 complex, SPR specificity experiments for peptides 3−5 binding to Grb7−SH2 domain, surface representation of peptide 5 bound to SH2 domains, SPR binding experiments for peptides 6 and 7 binding to Grb7−SH2, X-ray crystal structures of the Grb7−SH2/peptide 6 complex and the Grb7−SH2/peptide 7 complex, full Western blots and LC chromatograms and MS for peptides (PDF) Molecular formula strings (CSV) Chemical structure data (CDX) Accession Codes

Atomic coordinates and structure factors have been deposited in the RCSB protein data bank under the accession numbers 5U1Q (Grb7−SH2/peptide 5 complex), 5U06 (Grb7−SH2/ peptide 6 complex), and 5TYI (Grb7−SH2/peptide 7 complex).

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AUTHOR INFORMATION

Corresponding Author

*Jacqueline Wilce. E-mail: [email protected]. Phone: + 613 9902 9226. Fax: + 613 9902 9500. ORCID

Jacqueline A. Wilce: 0000-0002-8344-2626 Funding

This work was supported by a grant from the National Health and Medical Research Council awarded to J.A.W. (APP1045309) and a National Health and Medical Research Senior Research Fellowship awarded to M.C.J.W. (FP1079611), as well as the Victorian Government’s Operational Infrastructure Support Program. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Danielle Cini for assistance with setting up crystallization experiments, Roger Daly for input towards the cellular experiments, and all the staff at the MX2 beamline at the Australian Synchrotron, Victoria, Australia.

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ABBREVIATIONS Grb7, growth receptor bound protein; SH2 domain, Src homology domain; PPI, protein−protein interaction; HER2, human epidermal growth factor receptor 2; FAK, focal adhesion kinase; SHC, SH2-containing protein; cmF, carboxymethylphenylalanine; cF, carboxyphenylalanine; SPR, surface plasmon resonance; pY, phosphotyrosine; CPP, cell penetrating peptide

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REFERENCES

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phosphorylated peptide G7−18NATE with Grb7-SH2 domain requires phosphate for enhanced affinity and specificity. J. Mol. Recognit. 2012, 25, 57−67. (21) Eck, M. J.; Shoelson, S. E.; Harrison, S. C. Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck. Nature 1993, 362, 87−91. (22) Bevington, A.; Mundy, K. I.; Yates, A. J.; Kanis, J. A.; Russell, R. G.; Taylor, D. J.; Rajagopalan, B.; Radda, G. K. A study of intracellular orthophosphate concentration in human muscle and erythrocytes by 31P nuclear magnetic resonance spectroscopy and selective chemical assay. Clin. Sci. 1986, 71, 729−735. (23) Ambaye, N. D.; Pero, S. C.; Gunzburg, M. J.; Yap, M.; Clayton, D. J.; Del Borgo, M. P.; Perlmutter, P.; Aguilar, M. I.; Shukla, G. S.; Peletskaya, E.; Cookson, M. M.; Krag, D. N.; Wilce, M. C.; Wilce, J. A. Structural basis of binding by cyclic nonphosphorylated peptide antagonists of Grb7 implicated in breast cancer progression. J. Mol. Biol. 2011, 412, 397−411. (24) Pero, S. C.; Daly, R. J.; Krag, D. N. Grb7-based molecular therapeutics in cancer. Expert Rev. Mol. Med. 2003, 5, 1−11. (25) Songyang, Z.; Shoelson, S. E.; McGlade, J.; Olivier, P.; Pawson, T.; Bustelo, X. R.; Barbacid, M.; Sabe, H.; Hanafusa, H.; Yi, T. Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. Mol. Cell. Biol. 1994, 14, 2777−2785. (26) Janes, P. W.; Lackmann, M.; Church, W. B.; Sanderson, G. M.; Sutherland, R. L.; Daly, R. J. Structural determinants of the interaction between the erbB2 receptor and the Src homology 2 domain of Grb7. J. Biol. Chem. 1997, 272, 8490−8497. (27) Scott, D. E.; Bayly, A. R.; Abell, C.; Skidmore, J. Small molecules, big targets: drug discovery faces the protein-protein interaction challenge. Nat. Rev. Drug Discovery 2016, 15, 533−550. (28) Kraskouskaya, D.; Duodu, E.; Arpin, C. C.; Gunning, P. T. Progress towards the development of SH2 domain inhibitors. Chem. Soc. Rev. 2013, 42, 3337−3370. (29) Gunzburg, M. J.; Ambaye, N. D.; Del Borgo, M. P.; Perlmutter, P.; Wilce, J. A. Design and testing of bicyclic inhibitors of Grb7are two cycles better than one? Biopolymers 2013, 100, 543−549. (30) Lau, Y. H.; de Andrade, P.; Wu, Y.; Spring, D. R. Peptide stapling techniques based on different macrocyclisation chemistries. Chem. Soc. Rev. 2015, 44, 91−102. (31) Nam, N.; Ye, G.; Sun, G.; Parang, K. Conformationally constrained peptide analogues of pTyr-Glu-Glu-Ile as inhibitors of the Src SH2 domain binding. J. Med. Chem. 2004, 47, 3131−3141. (32) Quartararo, J. S.; Wu, P.; Kritzer, J. A. Peptide bicycles that inhibit the Grb2 SH2 domain. ChemBioChem 2012, 13, 1490−1496. (33) Fischer, R.; Waizenegger, T.; Kohler, K.; Brock, R. A quantitative validation of fluorophore-labelled cell-permeable peptide conjugates: fluorophore and cargo dependence of import. Biochim. Biophys. Acta, Biomembr. 2002, 1564, 365−374. (34) Maiolo, J. R.; Ferrer, M.; Ottinger, E. A. Effects of cargo molecules on the cellular uptake of arginine-rich cell-penetrating peptides. Biochim. Biophys. Acta, Biomembr. 2005, 1712, 161−172. (35) El-Andaloussi, S.; Jarver, P.; Johansson, H. J.; Langel, U. Cargodependent cytotoxicity and delivery efficacy of cell-penetrating peptides: a comparative study. Biochem. J. 2007, 407, 285−292. (36) Ambaye, N. D.; Lim, R. C.; Clayton, D. J.; Gunzburg, M. J.; Price, J. T.; Pero, S. C.; Krag, D. N.; Wilce, M. C.; Aguilar, M. I.; Perlmutter, P.; Wilce, J. A. Uptake of a cell permeable G7−18NATE contruct into cells and binding with the Grb-7-SH2 domain. Biopolymers 2011, 96, 181−188. (37) Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995, 4, 2411−2423. (38) McPhillips, T. M.; McPhillips, S. E.; Chiu, H. J.; Cohen, A. E.; Deacon, A. M.; Ellis, P. J.; Garman, E.; Gonzalez, A.; Sauter, N. K.; Phizackerley, R. P.; Soltis, S. M.; Kuhn, P. Blu-Ice and the Distributed Control System: software for data acquisition and instrument control at macromolecular crystallography beamlines. J. Synchrotron Radiat. 2002, 9, 401−406.

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