Articles pubs.acs.org/acschemicalbiology

Development of Accessible Peptidic Tool Compounds To Study the Phosphatase PTP1B in Intact Cells Christoph Meyer,† Birgit Hoeger,† Koen Temmerman,‡ Marianna Tatarek-Nossol,§ Vivian Pogenberg,‡ Jürgen Bernhagen,§ Matthias Wilmanns,‡ Aphrodite Kapurniotu,∥ and Maja Köhn*,† †

Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Hamburg Outstation, European Molecular Biology Laboratory, c/o DESY, Hamburg, Germany § Institute of Biochemistry and Molecular Cell Biology, RWTH Aachen University, Aachen, Germany ∥ Division of Peptide Biochemistry, Technische Universität München, Freising, Germany ‡

S Supporting Information *

ABSTRACT: Protein tyrosine phosphatases (PTPs) play crucial roles in health and disease. Chemical modulators of their activity are vital tools to study their function. An important aspect is the accessibility of these tools, which is usually limited or not existent due to the required, often complex synthesis of the molecules. We describe here a strategy for the development of cellular active inhibitors and in-cell detection tools for PTP1B as a model PTP, which plays important roles in diabetes, obesity, and cancer. The tool compounds are based on a peptide sequence from PTP1B’s substrate Src, and the resulting compounds are commercially accessible through standard peptide synthesis. The peptide inhibitor is remarkably selective against a panel of PTPs. We provide the co-crystal structure of PTP1B with the sequence from Src and the optimized peptide inhibitor, showing the molecular basis of the interaction of PTP1B with part of its natural substrate and explaining the crucial interactions to enhance binding affinity, which are made possible by simple optimization of the sequence. Our approach enables the broad accessibility of PTP1B tools to researchers and has the potential for the systematic development of accessible PTP modulators to enable the study of PTPs.

C

such chemical tools need to be easily accessible. In addition to inhibitors, tools to study the presence and/or subcellular localization of PTPs in different cell types are desirable. This can, for example, be achieved through molecular biology methods.7 Chemical methods are also available, such as the attachment of a fluorophore to the inhibitor, which requires careful selection of the attachment site on the inhibitor in order to maintain the bioactivity.7 Approaches that have been undertaken so far to develop PTP inhibitors cover a broad chemical space ranging from small molecules to peptides and peptidomimetic compounds.5,8 Often rationally derived active site targeting moieties that mimic the pY, like the O-malonyltyrosyl, fluoro-O-malonyltyrosyl, or difluorophosphonomethylphenylalanine (F2Pmp) group,

ells constantly receive, transmit, and integrate signals from external stimuli. Propagation of the signal inside the cell is achieved by a variety of different mechanisms.1 One of the crucial mechanisms involves highly dynamic, yet wellbalanced changes in the phosphorylation level of specific tyrosine residues. Thus, protein-tyrosine phosphatases (PTPs) that are capable of catalyzing the hydrolysis of phosphotyrosine (pY) residues play important roles in eukaryotic signal transduction in conjunction with their counteracting enzyme class, the protein-tyrosine kinases (PTKs).2 Misregulation of the otherwise tightly controlled phosphorylation equilibrium can contribute to severe diseases such as cancer or diabetes.3,4 Therefore, it is highly desirable to develop and apply inhibitors and other chemical tools to further understand and eventually treat impaired PTP activity. However, to date inhibitors have been developed only for 165 of the 88 active PTPs6, and very few specific inhibitors are commercially available. Still, in order to be useful for researchers interested in signaling processes, © 2014 American Chemical Society

Received: September 10, 2013 Accepted: January 5, 2014 Published: January 5, 2014 769

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our strategy more likely to be successful to other PTPs that have a decent affinity to peptide substrates.

define the core binder, whereas the surrounding parts of the molecules modulate strength and selectivity of their inhibitory activity.5,8 Peptides and peptidomimetics seem especially interesting lead structures for the development of PTP inhibitors since knowledge about the interactions of the enzymes with their natural substrates could give important suggestions for a successful and rational inhibitor development. In addition, peptides are accessible to all researchers through peptide synthesis companies and, as opposed to small molecules, also easily and flexibly modifiable with, for example, affinity or fluorescence tags, opening up a broad range of potential applications.9 A common misconception is that broader substrate specificity of a PTP often results in the lack of selectivity toward a peptidic inhibitor, but previous studies show that, similarly to small molecule inhibitors, finetuning to increase the affinity and specificity is possible.10,11 In addition, some PTPs indeed show quite remarkable substrate specificity,6 which can be a benefit for rational peptide-based inhibitor design. The major limitations using phosphopeptides have so far been that these come with intrinsic properties causing challenges such as lack of cell permeability or being prone to degradation inside cells.8 Therefore, unmodified phosphopeptides are generally not applied to study PTPs inside cells. Nevertheless, such pY-mimic containing peptides are useful for in vitro applications. For example, they were used to delineate substrate specificities of PTPs, in particular PTP1B. PTP1B was the first PTP identified and is the most studied to date.5,12,13 It is a validated drug target in type 2 diabetes and obesity.14,15 Furthermore, overexpression of PTP1B in mouse models was identified as a driving source of ErbB2 induced breast cancer,16,17 which is associated with increased c-Src kinase activity induced by the dephosphorylation of the inactivating pY530 on c-Src by PTP1B.18 An example for the application of pY-mimic containing peptides to study substrate preferences of PTP1B is the preparation of a library of peptides based on the hexapeptide DADEF2PmpL (single-letter amino acid code) derived from the dephophorylation site (amino acids 988−993) of the epidermal growth factor receptor (EGFR), a natural substrate of PTP1B. 10 In another project, a combinatorial library of malonyltyrosine containing peptides was screened for binding to PTP1B.12 More knowledge about PTP1B’s preferences for binding to groups surrounding the dephosphorylated pY has been acquired by X-ray crystallography19,20 or by determining the ability of PTP1B to dephosphorylate pY-peptide libraries in solution or with microarray techniques.21−26 The general conclusion of these extensive studies is that PTP1B accepts a wide range of pYpeptides as substrates, but that it prefers acidic stretches and that the affinities can be fine-tuned through exchanging amino acids.10,11,21−26 In this work, we sought to develop a strategy that would allow researchers studying PTPs or processes that involve PTPs to use the same type of chemical tools for inhibition and detection of PTPs inside cells. Importantly, the tools should be commercially accessible. We describe here the development of this principle based on nonhydrolizable phosphopeptides and applied it to the prototypical PTP PTP1B. We chose PTP1B because (i) it is the most well studied phosphatase with a relation to diseases, (ii) data are available to compare our chemical tools to existing ones, and (iii) it accepts many peptide substrates. The latter is important to show that simple fine-tuning of the affinity of peptides to even such an active phosphatase is possible, which would make the application of

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RESULTS AND DISCUSSION Identification of a Peptidic Active Site Binder for PTP1B. In the first step of the inhibitor development we had to determine a peptide sequence as starting point for our strategy. The high degree of structural and sequential similarity (72% sequence identity in the catalytic domain27) of PTP1B to its closest related enzyme T-cell PTP (TCPTP) makes the development of selective active site binders extremely challenging.5,8 Interestingly, their substrates in vivo are quite different. For example, dephosphorylation of the inhibitory pY530 of the c-Src kinase by PTP1B activates the enzyme,28 while dephosporylation of pY418 by TCPTP leads to an inactivation of the kinase.29 Therefore, we sought to investigate if the C-terminal stretch covering the pY530 site, EPQpYQPGENL, of the Src kinase, which includes the dephosphorylation site recognized by PTP1B, was a suitable starting point to develop such a chemical tool. This represents the first time this sequence is evaluated as PTP binder. Furthermore, the amino acids adjacent to pY in this sequence seem rather unusual for known PTP1B substrates and substrate preferences,25 leading to the question if this sequence could represent an equally strong binder of PTP1B as, for example, the one derived from the DADEpYL sequence.30 Therefore, the traditionally used PTP1B-binding peptide sequence DADEpYL was applied for comparison as benchmark for a good peptidic PTP1B inhibitor. To achieve high affinity binding, we employed the aforementioned pY-mimic F2Pmp,30 which is also commercially available.31 A peptide derived from the c-Src kinase (peptide Ac-1, Table 1, see also Supplementary Figure S1) and also the EGFR-based sequence (peptide Ac-2, Table 1) containing the F2Pmp group Table 1. Inhibition of Peptides toward PTP1B and TCPTPa IC50 [μM]b peptide

origin/name

Ac-1

Src

Ac-2 Ac-3

EGFR Src(Q+1A)

Ac-4

Src(P-2A)

Ac-5

random purchasedc

sequence

PTP1B

TCPTP

AcEPQXQPGENLNH2 Ac-DADEXL-NH2 AcEPQXAPGENLNH2 AcEAQXQPGENLNH2 AcASGAXAGGSANH2 CinnGEL

2.13 ± 0.62

2.34 ± 0.80

2.23 ± 0.27 7.26 ± 4.51

2.43 ± 0.28 8.28 ± 4.40

0.36 ± 0.12

0.58 ± 0.26

12.8 ± 1.90

12.0 ± 4.97

3.46 ± 0.90

7.45 ± 1.05

a

IC50 values of the F2Pmp containing sequences derived from Src (Ac1), EGFR (Ac-2), the Src variants Q+1A (Ac-3) and P-2A (Ac-4), and the random sequence (Ac-5) measured by the pNPP dephosphorylation assay (see the Supporting Information and Supplementary Figure S1 for graphs). The peptides are acetylated (Ac) at the Nterminus and carry an amide at the C-terminus. X = F2Pmp. The results are presented as the mean ± standard deviation (n = 3). bAll IC50’s were measured in the following buffer: 25 mM HEPES (pH = 7.2), 50 mM NaCl, 2.5 mM EDTA, 2 mM DTT; except CinnGEL, which was measured at the reported conditions:37 100 mM sodium acetate (pH = 6.0), 1 mM EDTA, 0.1% Triton X-100, 15 mM ßmercaptoethanol. cFrom Biomol, Germany. 770

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Table 2. Binding Constants of Peptides toward PTP1B and Other Proteins or Protein Domains KD [nM]a peptide

origin/name

CF-1 CF-2 CF-4 CF-5

Src EGFR Src(P-2A) random

PTP1B 113.4 137.1 55.0 998.8

± ± ± ±

5.9 9.1 1.5 387.6

TCPTP

RPTPα

VHR

Src-SH2

± ± ± ±

np np np np

np np np np

np np np np

118.9 157.9 56.2 984.4

16.8 7.4 5.7 216.4

a KD values measured by FP of peptides CF-1, -2, -4, and -5 carrying the fluorophore carboxyfluorescein (CF) instead of Ac at the N-terminus. np = no polarization. The results are presented as the mean ± standard deviation (n = 3).

efficacy of the inhibitor. Furthermore, contrary to a previous report,11 this showed that high potency of a peptidic inhibitor can be achieved without negatively charged amino acids directly neighboring the pY at positions −1 and −2. As a further control for the importance of the F2Pmp-surrounding amino acids on the inhibitory efficacy, we tested a random peptide containing uncharged polar and nonpolar amino acids (peptide Ac-5, Table 1). The more than 30-fold loss in potency compared to that of the Src(P-2A) variant Ac-4 demonstrates once more the importance of the surrounding amino acids in inhibiting PTP1B and TCPTP. The F2Pmp nevertheless contributes strongly to the binding, which is shown by an only 6-fold lower potency of the random sequence Ac-5 compared to Ac-1 and Ac-2. We also tested a commercially available inhibitor, CinnGEL,37 in this assay in order to benchmark our inhibitor. Under the reported assay conditions37 we obtained an IC50 of 3.5 μM that lies in the range of the reported one of 1.3 μM. To our knowledge, no data were previously available regarding the potency of CinnGEL with respect to TCPTP. According to our measurement, the IC50 is 7.4 μM. Therefore we concluded that peptide Ac-4 compared quite favorably to this commercially available active site inhibitor in vitro. In order to determine the binding affinity of the peptides toward the PTPs (decoupled from the catalytic activities), we measured their binding constants (KD’s) in a fluorescence polarization (FP) assay. To this end, the peptides were labeled with carboxyfluorescein (CF) at the N-terminus (Table 2). Both naturally derived sequences (CF-1 and CF-2) bound PTP1B in the 100−150 nM range. The Src(P-2A) variant CF-4 showed a 2-fold stronger affinity compared to CF-1 and CF-2, and the random sequence CF-5 was a more than 18-fold weaker binder compared to CF-4 and about 9-fold weaker against CF-1 and CF-2. These results show that the relationship between binding affinity and inhibition potency is not linear. Nevertheless, in comparison to the IC50 values an influence of the CF on the KD’s cannot be excluded, but this effect should be similar in all peptides as all carry a CF. In addition to the catalytically active phosphatases also the affinity of the peptides to proteins with pY binding domains, such as SH2 domains, can be measured using FP. This is of special interest in this case since efficient binding of the F2Pmp-containing peptide to cSrc’s SH2 domain could potentially activate the enzyme by outcompeting the inhibitory C-terminal part of c-Src.38 When using this sequence as an PTP1B inhibitor to suppress c-Src activation, this would counteract the aspired effect. Therefore, the recombinantly expressed SH2-domain of the Src kinase was measured against all four CF-peptides (Table 2), but no polarization could be observed in the concentration range tested (up to 10 μM), which showed that binding of these sequences is at least 2 orders of magnitude weaker to this SH2 domain than to PTP1B. We also tested TCPTP, the D1 domain of receptor-type PTP alpha (RPTPα) that also

instead of pY were synthesized by Fmoc solid phase peptide synthesis (see the Supporting Information). The required NFmoc-F2Pmp-OH building block was prepared as previously described.32 The IC50 values of these sequences against TCPTP and PTP1B were determined with a well-established phosphatase activity assay employing the unnatural substrate p-nitrophenylphosphate (pNPP).33,34 The IC50 values of both peptides were similar toward both PTPs in the low micromolar range (Table 1), confirming the applicability of the Src-derived sequence as PTP binder. Considering the different properties of amino acids and the lack of negatively charged amino acids adjacent to the F2Pmp that were previously thought to be crucial,11,19 this result was quite remarkable. However, neither of the peptides showed selectivity toward PTP1B compared to TCPTP, confirming the similar binding specificities of the two phosphatases in vitro8 and contrasting the differences in vivo.28,30 The IC50 of the EGFR sequence to PTP1B (Ac-2, Table 1) that we obtained via the pNPP activity assay did not correspond well to previously published IC50 data (100 nM).30 This discrepancy is likely caused by the different methods with which the values were determined, as previously the inhibitory constant was measured using purified insulin receptors labeled with radioactive phosphate.30 A similar discrepancy in the determination of IC50 values by the pNPP assay in the range of approximately 1 order of magnitude has been described before in the protein serine threonine phosphatase field when comparing the pNPP to a phosphorylase assay format, suggesting that this is a general issue with this type of assay.35 Optimization of the Peptidic PTP1B Inhibitor. In the next step, we sought to investigate if a simple fine-tuning strategy could improve affinity and selectivity of the Src-derived peptide. To this end, we synthesized a library of sequences in which all positions (except the F2Pmp) were exchanged iteratively to alanine (Supplementary Table S1a) and a library of truncated peptides (Supplementary Table S1b). Similar to a previous report using the DADEpYL sequence in Michaelis− Menten kinetic assays,36 the truncation scan did not lead to an improvement of efficacy. In the alanine scan, exchanging the glutamine at position +1 (peptide Ac-3, Table 1) with respect to the F2Pmp resulted in a more than 3-fold loss of potency with PTP1B and TCPTP. This result was somewhat unexpected as this position is in general considered less crucial for the binding of monophosphorylated peptide substrates to PTP1B.11,25,36 However, the Src-derived sequence is very different than previously tested ones, demonstrating that the experimental outcome is highly dependent on the nature of the sequence and hard to predict. Remarkably, replacing the proline at position −2 with alanine (Src(P-2A) variant, peptide Ac-4, Table 1) led to a 6-fold increase in potency for PTP1B (IC50 = 360 nM) and 4-fold for TCPTP (IC50 = 580 nM) (Table 1), resulting in a nanomolar inhibitor and demonstrating that the simple strategy indeed led to a remarkable improvement of the 771

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Figure 1. Zoom into the crystal structures of peptides Ac-1 and Ac-4 bound to PTP1B. (a) Ac-1:PTP1B (PDB entry 3ZMP); (b) Ac-4:PTP1B (PDB entry 3ZMQ) (see Supporting Information and Supplementary Figure 2 for full surface representation). The peptide binding region is shown, with PTP1B in gray surface representation and the docked peptide in wheat-colored stick representation. Peptide amino groups are in blue, and carboxylate groups are in red. In the Ac-1 structure the final Leu (position +6) is invisible; in the Ac-4 structure the sequence GENL (position +3 to +6) is lacking due to differences in X-ray data collection quality. The peptide as well as PTP1B residues involved in peptide interactions are in stick representation. Pink dotted lines represent polar contacts between residues. Contact differences between the two structures are highlighted by either full circles or dotted circles, for potential ineractions.

difference is that the flexible side chain of R47 contributes to the binding of the IR- and the EGFR-derived peptides and also of cyclic inhibitors,19,20,41 whereas it is unbound in the PTP1B:Ac-1 structure (Figure 1a), which can also be explained by the sterical constraint exerted by proline at position −2. The importance of glutamine at position +1, as shown in the alanine scan, is explained by a hydrogen bond formed by its side chain with the side chain of D48. Such interaction was not observed in the previous structures.19,20,40,41 The entire C-terminal part is highly flexible in both structures, and in the PTP1B:Ac-4 structure (Figure 1b) the C-terminal residues from position +3 to +6 are not visible (see also Supplementary Figure S2). Crucial new interactions are made possible by the P-2A mutation and the resulting increase in peptide flexibility distal from the F2Pmp. A new hydrogen bond between R45 and the backbone amide of the glutamic acid at position −3 of the peptide (circle in Figure 1b) is formed. The previously flexible, unbound side chain of D47 reorientates to a hydrogen-bound position with the side chain of the glutamine at position −1 (circle in Figure 1b). Additionally, the glutamic acid at position −3 could potentially form a salt bridge with R47; however, our current data is inconclusive (dotted circle in Figure 1b). All other interactions are maintained, explaining the gain of potency between Ac-1 and Ac-4. This analysis demonstrates that although the pY (-mimic) of peptides is bound by PTP1B in a similar manner, other interactions are flexible and can be optimized. Development of a Cell-Permeable Peptide Inhibitor. In the next step, we aimed to develop these potent inhibitory peptides further to be functional for applications in cells. The highly negatively charged nature of the essential F2Pmp in combination with the net negative charge of the peptides was

dephosphorylates pY530 on c-Src,39 and vaccinia H1-related phosphatase (VHR) as unrelated tyrosine-specific phosphatase.6 Whereas similar affinities of the peptides to TCPTP and PTP1B were measured, none of the peptides showed binding against RPTPα and VHR (Table 2). RPTPα was previously described to show low reactivity against peptides,25 which could explain the lack of binding affinity. Together, the peptides show good selectivity in binding PTP1B over the tested proteins, with the expected exception of TCPTP. Structural Basis of the Inhibitor−PTP1B Interaction. To understand the structural basis of the PTP1B−Src peptide interaction and the cause of the strong improvement of the potency when exchanging the proline at position −2 to alanine, we solved the crystal structures of PTP1B with both peptides (Ac-1 and Ac-4). The parent peptide Ac-1 containing the 2Dpalindromic sequence PQF2PmpQP bound in a single orientation to PTP1B (Figure 1a, the C-terminal leucine is not visible), which is the same as for other PTP1B-binding peptides in crystal structures.19,20,40,41 In the reported structures and also in the PTP1B:Ac-1 structure, the carboxylic moiety of D48 of PTP1B forms two hydrogen bonds with the backbone amide groups of the peptide at the pY (-mimic) and of the amino acid at position +1, and the pY (-mimic) is oriented similarly in all structures (Figure 1). Also in agreement with the other structures, a hydrogen bond is formed between the main chain amide group of R47 of PTP1B and a carbonyloxygen atom of Ac-1. However, in the structures with the peptides derived from the EGFR19 and insulin receptor20 (IR) the interaction happens at position −2 of the peptide, whereas with the Src sequence Ac-1 the bond is formed with the glutamic acid at position −3, which is likely due to the proline at position −2 restricting the flexibility of the peptide. Another 772

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expected to prohibit cell penetration of these molecules.42 Indeed, CF-1 was poorly taken up by cells (Figure 2a).

and eight (peptide CF-1-R8) C-terminally appended arginines indicated by a brighter and more equally distributed fluorescent signal throughout the cytosol (Figure 2, arrows). The signals were still not very strong (Supplementary Figure 3), but nevertheless clearly observable. All peptides also showed vesicular distribution inside cells to a similar extent, suggesting endocytic uptake. To test the mode of uptake, we measured cell penetration at 4 °C to reduce active transport and random endocytosis.9 The results showed that the peptides are taken up passively at 4 °C, indicating that the mechanism involves passive and endocytic transport at 37 °C (Supplementary Figure 4). Inversely to the increased uptake, the binding strength of these peptides against PTP1B decreased gradually with an increasing number of C-terminally added arginines (Table 3 and Supplementary Figure S5). This agrees with a previous report, where polycationic peptide sequences that were attached via a linker to the active compound were shown to counteract the binding of the F2Pmp group to PTP1B.42 Nevertheless, peptide CF-1-R6 with six C-terminal arginines showed improved cell penetration and an intermediate binding affinity. The binding strength was further improved by using the optimized Src(P-2A) sequence in the R6-containing peptide (CF-4-R6, Table 3), and this peptide was therefore used for the following studies. In order to make it useful for cellular applications, we needed to evaluate the selectivity of our cell permeable inhibitor further. To this end, phosphatase activity assays were carried out using Ac-4-R6 by a phosphatase profiling service with MKP5, PTP-MEG1, PTP-MEG2, PTPβ, RPTPμ, VHR, CD45, and SHP1 (see the Supporting Information and Supplementary Figure S6a). Most of these PTPs were not inhibited up to 300 μM Ac-4-R6, and only RPTPμ and PTP-MEG2 were inhibited to about 50% and 30% at 300 μM peptide, respectively. The IC50’s toward PTP1B and TCPTP using 6,8-difluoro-4methylumbelliferyl phosphate (DIFMUP) as a substrate (as used by the profiling service) were in the low micromolar range (5.53 ± 0.60 and 33.8 ± 3.95 μM, respectively) (Supplementary Figure S6b and Supplementary Table S2). Since this selectivity contrasted the one measured using pNPP and the non-cell penetrating version Ac-4 (Table 1), we determined the IC50 again using pNPP and Ac-4-R6 (Supplementary Table S2). The results showed that the selectivity is lost when using pNPP as substrate (IC50 = 10.6 ± 3.15 μM for PTP1B and 3.15 ± 1.17 μM for TCPTP). Thus the selectivity seems to depend here on the substrate, which is a phenomenon that was reported for different enzymes before.44 We also tested a buffer containing BSA (bovine serum albumin) and a detergent in order to test if protein aggregation could have an influence on the measured IC50’s. The results show that protein aggregation did not play a role here (Supplementary Table S2).

Figure 2. Comparison of cellular uptake and localization of CF-1 bearing increasing amounts of C-terminal arginine residues. HeLa Kyoto cells were incubated with the peptides (50 μM) for 12 h at 37 °C (see the Supporting Information). Scale bar = 20 μM. Shown is a representative experiment out of three. (a) Src parent peptide CF-1; (b) Src parent peptide plus four arginines (CF-1-R4), (c) plus six arginines (CF-1-R6), and (d) plus eight arginines (CF-1-R8). Arrows denote diffuse cytosolic localization. The strong, dotted signals could result from endocytosed peptides (see also Supplementary Figure S4 showing uptake at 4 °C) and are present in similar amounts in all experiments. The pictures shown here were optimized in contrast and brightness for better clarity; for the raw data see Supplementary Figure S3.

Application of a cell penetration strategy involving addition of positively charged arginines seemed an attractive way to solve this problem.9,42,43 To this end, we aimed to determine the optimal number of arginines promoting cellular uptake while retaining binding affinity to PTP1B (Figure 2b−d). Direct attachment to the C-terminus was chosen (i) to keep the chemistry as simple and reproducible as possible to ensure commercial accessibility of the resulting peptides and (ii) because the C-terminus showed to be flexible and less involved in binding in the crystal structure. Whereas the attachment of four arginines did not lead to an increase in cellular uptake, we observed a considerable gain in cellular uptake when testing the fluorescently labeled Src peptide with six (peptide CF-1-R6)

Table 3. Affinity Measurement of the Poly-arginine Peptides against PTP1B by Fluorescence Polarization (FP)a

a

peptide

sequence

name

CF-1-R4 CF-1-R6 CF-1-R8 CF-4-R6 CF-5-R6 CF-6-R6

CF-EPQXQPGENLRRRR-NH2 CF-EPQXQPGENLRRRRRR-NH2 CF-EPQXQPGENLRRRRRRRR-NH2 CF-EAQXQPGENLRRRRRR-NH2 CF-ASGAXAGGSARRRRRR-NH2 CF-EPQYQPGENLRRRRRR-NH2

Src-R4 Src-R6 Src-R8 Src(P-2A)-R6 random-R6 Src(Y)

KD PTP1B [nM] 198.2 ± 619.1 ± 811.5 ± 425.3 ± >20,000 np

12.7 73.3 112.2 21.5

See Supplementary Figure S5 for graphs). (X = F2Pmp; np = no polarization). The results are presented as the mean ± standard deviation (n = 3). 773

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Application of the Peptidic Inhibitor Inside Cells. Next, it was investigated whether the cell penetrating peptide CF-4R6 would co-localize with PTP1B in living cells in order to test its application as a detection tool for PTP1B. As control the cell-permeable but not PTP1B-binding CF-Src(Y) peptide (CF-6-R6) (Table 3) was used. Overexpression of a fusionprotein of full length PTP1B with an N-terminal mKate fluorescent protein showed that PTP1B localized at the perinuclear/endoplasmic reticulum (ER) region. This was expected since a C-terminal anchor sequence determines PTP1B’s natural cellular localization mainly to the ER membrane with the catalytic domain facing to the cytosol.45 For both peptides, uptake in vesicles and also a homogenously distributed fluorescent signal was detected inside cells (Figure 3), as observed previously with CF-1-R6 (Figure 2). Addition-

peptide Ac-6-R6. This result was consistent with the suggestion that Ac-4-R6 inhibits the dephosphorylation of this PTP1B substrate inside cells (Figure 4). To exclude peptide

Figure 4. Effects of Ac-4-R6, Ac-5-R6, and Ac-6-R6 on insulin-mediated tyrosine phosphorylation of insulin receptor β (IRβ) in human breast cancer cells. MDA-MB-468 cells were incubated with insulin or insulin mixtures with Ac-4-R6 (100, 200, and 400 μM), Ac-5-R6, and Ac-6-R6 (200 μM). Phosphorylated proteins were immunoprecipitated with anti-phosphotyrosine antibody (anti-PY20). Phosphorylated IRβ was revealed by WB with anti-insulin receptor β-subunit antibody (antiIRβ), and equal amounts of cellular proteins were used as shown by βactin WB (see the Supporting Information). (a) A representative WB showing the effect of insulin alone (100 nM) versus mixtures of insulin with Ac-4-R6 or Ac-6-R6 (both at 200 μM) as indicated. b) Densitometric quantification of the results of the above IR activation assay. Data of mixtures of insulin with Ac-4-R6 (200 μM) and Ac-5-R6 (200 μM) are means (±SEM) of 5−7 assays. Data for mixtures of insulin with Ac-6-R6 (200 μM), Ac-4-R6 (100 μM), and Ac-4-R6 (400 μM) are from 1 or 2 assays. Significant differences were found between the effects of the mixture of insulin with Ac-4-R6 (200 μM) versus insulin alone (p < 0.005) and the effects of mixtures of insulin with Ac4-R6 (200 μM) versus insulin with Ac-5-R6 (200 μM) (p < 0.05) by ANOVA.

Figure 3. mKate-PTP1B co-localizes with CF-4-R6, but not with a control peptide, in living cells. U2OS cells were transformed with the mKate-PTP1B vector and incubated with the peptides CF-4-R6 and CF-6-R6 (50 μM) or water vehicle (no peptide) for 12 h at 37 °C (see the Supporting Information). Scale bar = 10 μm. Shown is a representative experiment out of three. The pictures shown here were optimized in contrast and brightness for better clarity; for the raw data see Supplementary Figure S7. For more evidence see Supplementary Figure S8.

ally, we observed a nonvesicular concentration of CF-4-R6 at the perinuclear/ER region, which co-localized with the signal of mKate-PTP1B, whereas this was not the case in cells treated with the control peptide CF-6-R6 lacking F2Pmp. This indicated that CF-4-R6 entered the cells and bound to PTP1B, demonstrating that this peptide can be applied to detect PTP1B inside intact cells. We then investigated if Ac-4-R6 would inhibit PTP1B inside cells. To this end, we applied cultured MDA-MB-468 human breast cancer cells that express the insulin receptor,46 which is a substrate of PTP1B.20 After treatment for 1 h with peptides or vehicle alone and following a 10 min incubation with insulin, cells were lysed, and proteins phosphorylated on tyrosine were immunoprecipitated using an anti-phosphotyrosine antibody. Western blot (WB) analysis using anti-insulin receptor βsubunit antibody (anti-IRβ) revealed that treatment with 200 μM Ac-4-R6 resulted in a 2.5-fold increase in the amount of phosphorylated IRβ as compared to vehicle alone or the control

degradation as cause between the different performance of the Src-derived peptides Ac-4-R6 and Ac-6-R6, we determined the cell lysate stability of the two peptides as CF-forms in the MDA-MB-468 cells over a time frame of 8 h, which covered the conditions of the cellular assay. The results showed that the F2Pmp-containing parent Src peptide CF-4-R6 was stable over 8 h in this setting (Supplementary Figure S9), thus confirming that this peptide is stable enough to be applied in cells. CF-6-R6 appeared slightly less stable starting between 4 and 8 h, but due to the variability of the signal this was not significant. We concluded thus that the Src sequence itself is quite stable under the applied conditions, making it a very suitable sequence for peptidic tool design for applications inside cells and excluding degradation as cause for the different activity in the cellular assay. Finally, cell-penetrating polybasic stretches can cause 774

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cytotoxicity.8 In order to ensure that the inhibitory activity of Ac-4-R6 observed in the cellular assay was not due to cytotoxic effects of the peptide, we performed the commonly used MTT cell proliferation assay applying the MDA-MB-468 cells and a similar concentration range as in the previous cellular assay (Supplementary Figure S10). The results showed that Ac-4-R6 is not cytotoxic under these conditions, supporting that the intracellular activity of this peptide is not due to cytotoxic effects. In summary, we describe here a straightforward strategy to derive peptidic tools to inhibit and detect PTP1B in intact cells. To our knowledge this is the first commercially available (through standard peptide synthesis) cell penetrating phosphono-peptide that is active inside cells, remarkably selective over other phosphatases, and proteolytically stable under the experimental conditions. In addition, our peptidic approach ensures that the probes to detect PTP1B in intact cells are still commercially available through standard peptide synthesis. Other potent small molecule active site inhibitors have been developed but are not commercially available and often require complex syntheses,5,10 and this is also true for fluorescently modified small molecules to detect PTP1B inside cells,8 limiting their accessibility to researchers significantly. We also provide crystal structures of both inhibiting peptides with PTP1B, one of which is the first structure of PTP1B with a sequence of its natural substrate Src. In addition to giving insight into PTP1B’s binding mode to its natural substrate, these structures can be used in the future for further fine-tuning of the binding affinity and selectivity of the peptides for example through computational methods.47 PTP1B is a highly active phosphatase that accepts multiple peptide substrates in vitro.10,11 Therefore, our results suggest that this strategy could be applicable to other PTPs with decent affinities toward peptide substrates, which would enable creating systematically accessible peptidic tool compounds for the investigation of PTPs.

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using beamlines ID29 and ID23-2, respectively. We acknowledge the support of the EMBL Protein Expression and Purification Core Facility, the EMBL Chemical Biology Core Facility, and the EMBL Advanced Light Microscopy Facility. The authors thank Peter Sehr for support with the FP measurements.

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.K. thanks the German Science Foundation for support within the Emmy-Noether program (KO 4013/1-1). C.M. thanks the EMBL International PhD Programme for a fellowship. B.H. thanks the Austrian Academy of Sciences for a DOC fellowship. This work was supported by DFG grant IRTG1508 to J.B. K.T. is supported by an EMBL Interdisciplinary Postdoc (EIPOD) fellowship under Marie Curie Actions (COFUND). This study was technically supported by the High Throughput Crystallization Laboratory Facility at EMBL Hamburg. The X-ray experiments were performed at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. We are grateful to Christoph Müller-Dieckmann and Silvia Russi for providing assistance in 775

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