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Research Communications |
a Département de Pharmacochimie Moléculaire et Structurale, INSERM U266-CNRS UMR 8600, UFR des Sciences Pharmaceutiques et Biologiques, 75270 Paris Cedex 06, France
b INSERM U248, Institut CURIE, 75248 Paris Cedex 05, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSREFERENCES |
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Key Words: proline-rich peptidimers • Grb2 SH3 domains inhibition • Sos inhibition
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSREFERENCES |
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In this work, we propose another approach, which consists of inhibiting the interaction between Sos and the two SH3 domains of Grb2. To interact with both SH3 domains of Grb2, peptidimers have been developed by coupling two proline-rich sequences from Sos through an optimized linker. Such peptides have high affinity for Grb2 and are able to inhibit Grb2–Sos interaction in vitro. Since these compounds were not able to enter cells, one was coupled with a carrier consisting of the homeodomain of the transcription factor of Antennapedia and tested for its ability to inhibit PC12 cell differentiation induced by nerve growth factor (NGF) and to inhibit the cloning efficiency of NIH3T3/HER2-transfected cells. In both cases the vectorized peptidimer was found to be active at micromolar concentrations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSREFERENCES |
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Chemistry
Assembly of the protected peptide chains was carried out using the stepwise solid-phase method of Merrifield (16) on an Applied Biosystems (ABI) 431A automated peptide synthesizer with ABI small-scale Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on an HMP resin and DCC/HOBt coupling method. Fmoc groups were removed by piperidine (20% in dichloromethane) (17).
Peptidimer 2 was synthesized by coupling Fmoc-Lys(Fmoc)-OH to the resin; after Fmoc deprotection, the two VPPPVPPRRR motifs were coupled simultaneously to both the side chain and backbone amino groups of the lysine. Peptidimer 2 was obtained after total amino acid deprotection and cleavage from the resin with TFA.
Antennapedia peptidimer conjugate 4 was obtained in a similar way: each amino acid of Antennapedia was first coupled as Fmoc protected amino groups and with nonbasic sensitive protections of the lateral chains (Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH) on the HMP resin; Ahx and Lys with Fmoc protection were then coupled. After deprotection of both NH2 groups of the latter lysine residue, dimeric coupling of each amino acid of the proline-rich sequence was performed as for peptidimer 2.
Finally, peptides 2 and 4 were purified by high-performance liquid chromatography on a C18 column (250X10 mm - Vydac) and a linear gradient of B (where A is trifluoroacetic acid 0.1% and B is CH3CN 70%, trifluoroacetic acid 0.09%) at a flow rate of 2 ml/min, with detection at 220 nm. The identity of the peptides was checked by electrospray mass spectroscopy (peptidimer 2: MS=2451.6; peptidimer conjugate 4: MS=4792.7).
Affinity measurement
Fluorescence measurements were performed on a Perkin Elmer fluorimeter in a 10 x 10 mm cuvette at 25°C, with stirring as described by Cussac et al. (18). Briefly, the excitation was at 292 nm (band width 2.5 nm) and emission was recorded at 345 nm (band width 14 nm). The buffer was Hepes 50 mM, pH 7.5, DTT 1 mM. Grb2 was a gift from Dr. P. Chardin (Nice, France). Preparation of purified Grb2 has been described previously (18).
Cell culture
ER 22 cells were grown and lysed as described by Vidal et al. (19). PC12 were typically maintained in RPMI medium supplemented with 10% horse serum and 5% fetal calf serum (all from GIBCO, Chagrin Falls, Ohio), and grown on collagen-coated tissues culture plates (Falcon, Oxnard, Calif.). For NGF and epidermal growth factor (EGF) treatment, PC12 cells were serum starved overnight and stimulated with NGF (10 ng/ml) or EGF (100 ng/ml) (all from Sigma, St. Louis, Mo.) for different times at 37°C. The lysis buffer was the same as the one used for ER 22 cell lysis. NIH3T3 cells transfected with HER2 (a kind gift from Dr. A. Ullrich, Germany) were typically maintained in RPMI medium supplemented with 10% fetal calf serum (all from GIBCO).
Transformation assays
The efficiency of colony formation in soft agar was determined by plating 25,000 cells in 3 ml of 0.2% agar (GIBCO-BRL, Paisley, U.K.) in the presence of different concentrations of Antennapedia peptidimer conjugate 4. As described by Hudziak et al. (20), after 2–4 wk, colonies of about 100 cells or more were counted.
Peptide coupling to Sepharose beads
Ten milligrams of the peptidimer 2 were coupled to 1 ml of CNBr-activated Sepharose 4B (Pharmacia, Piscataway, N.J.), according to Hermanson et al. (21). 30 µl of peptide beads were then incubated overnight at 4°C in 500 µl of cell lysate (2 mg of protein). Beads were washed four times with phosphate-buffered saline (PBS). Affinity-precipitated proteins were eluted by boiling sodium dodecyl sulfate (SDS) sample buffer for 5 min. After SDS-polyacrylamide gel electrophoresis (PAGE) separation and transfer, protein was detected by an anti-Sos or anti-Grb2 (control) Western blot.
To avoid steric hindrance at the level of the two VPPPVPPRRR motifs, peptidimers (2 mg) was coupled by its lysine COOH-terminal group to EAH Sepharose 4B beads (500 µl) according to the described protocol (Pharmacia). Peptide beads (30 µl) were then incubated overnight at 4°C in 500 µL of cell lysate (2 mg of protein). Beads were washed four times with PBS. Affinity-precipitated proteins were eluted by boiling in SDS sample buffer for 5 min. After SDS-PAGE separation and transfer, precipitated proteins were detected by an anti-Grb2, anti-Nck, or anti-PI3K Western blot.
Antibodies
Anti-active MAP kinase antibody (anti-ERK1 and ERK2 pAb) were purchased from Promega (Madison, Wis.). Anti-Grb2 (IgG1 mAb: immunoglobulin G1 monoclonal antibody), anti-Nck (IgG2b mAb), and anti-PI3K (IgG1 mAb) were from Transduction Laboratories (Lexington, Ky.). Anti-Sos (anti-Sos1 pAb) was from Upstate Biotechnology (Lake Placid, N.Y.); anti-mouse and anti-rabbit were from Sigma.
Immunoblotting experiments
As already described by Vidal et al. (19), precipitated proteins were separated and transferred onto polyvinylidene difluoride membrane. After blocking, the membrane was incubated with mAb antibody in blocking buffer (Super Block Blocking Buffer-Blotting in TBS, Pierce, Rockford, Ill.) for 2 h. After successive washes in PBS, 0.05% Tween 20, bound proteins were detected by incubation with peroxidase-conjugated anti-mouse antibody and revealed by the enhanced chemiluminescence method. For the MAP kinase phosphorylation assay, 10 µg of total cellular extract was used for the immunoblotting experiment.
RESULTS
Design and affinity of the peptidimers for Grb2
The design of Grb2-Sos inhibitors was executed by taking into account the observation that transfection of a Grb2 mutant, with its amino-terminal SH3 domain deleted, into transformed NIH3T3 cells expressing the activated HER2/neu receptor, induced inhibition of Shc-Grb2–Sos interaction, and phenotypic reversion of the cells (22). This was reinforced by results showing that expression of a Sos mutant restricted to the COOH-terminal region, which contains the proline-rich sequences necessary for SH3 domain binding, was able to hamper the formation of functional Shc–Grb2–Sos complexes and subsequent MAP kinase phosphorylation (23). Moreover, the approach, which consists of inhibiting Grb2–Sos interaction, was expected to be very selective but was difficult to carry out due to the relatively weak affinity (~20–100 µM) of Sos-derived, proline-rich peptides for Grb2 (18). To circumvent this problem, we took advantage of the presence of two spatially close SH3 domains in Grb2 (13) to which proline-rich peptides from Sos, such as VPPPVPPRRR, were shown to bind after a specific orientation in which Pro2 and Val5 side chains interacted with aromatic residues constituting a hydrophobic binding site and Arg8 with an acidic cluster of the domain (14, 24–27). Based on these structural data, we therefore have designed peptidimers in which two VPPPVPPRRR peptides were joined together by their COOH-terminal part with flexible linkers in order to allow their simultaneous binding to Grb2. Molecular modeling studies were used to select spacers, allowing dual interactions of the VPPPVPPRRR sequences with Grb2 (to be published elsewhere). A linker as short as a lysine appeared to fulfill this criteria. Accordingly, after minimization, the two polyproline type II helices of the peptidimer were found to interact with their respective recognition platform present in each SH3 domain (
Fig. 1),
as observed in nuclear magnetic resonance (NMR) studies (14). Peptidimers were therefore synthesized by solid phase on an HMP resin, using both amino groups of a lysine to anchor proline-rich sequences by their COOH-terminal parts ( Fig. 1).
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The affinities of the dimers and the corresponding monomer 1 for Grb2 were determined using a fluorescence assay (18). The expected affinities of such dimeric structures are theoretically equal to the product of the binding constant of each monomer, plus a term accounting for their linker (28). Peptidimer 2, containing twice the VPPPVPPRRR sequence, has an affinity two and three orders of magnitude higher for the entire Grb2 protein (Kd=40 nM) than the monomer 1 for the amino-terminal (Kd=2.6 µM) and COOH-terminal SH3 (Kd=40 µM) domains of Grb2, respectively. These results suggest a dual interaction of peptidimer 2 with both Grb2 SH3 domains. This was confirmed by the much lower affinities observed between Grb2 and control peptides, where the second proline-rich motif was either deleted (monomer 1; Kd=18 µM) or replaced by a scramble proline-rich sequence (peptidimer 3; Kd=16 µM), devoid of the consensus PX
PXR motif (=hydrophobic amino acid; X=any amino acid) required for SH3 domain type II recognition (29–31) (see 
Fig. 1). Together, these results strongly support the simultaneous interaction of both proline-rich sequences of peptidimer 2 with the two Grb2 SH3 domains or, alternatively, suggest that the peptidimer recognizes these domains through a `flip-flop' mechanism (32, 33). However, as already observed with other dimers, the affinity of peptidimer 2 for Grb2 is somewhat lower than expected from the product of the monomer affinities for SH3 domains. This is probably due to unfavorable entropic factors.
Since these SH3-directed peptides did not enter cells, they were tested in vitro to evaluate their effect on Grb2–Sos complexes.
Effect of the peptidimer in vitro on ER22 cell extracts
Increasing concentrations of peptidimer 2 or monomer 1 as a control were added to a cell lysate of exponentially growing ER22 cells. Thereafter, the Grb2–Sos complexes were pulled down using Sepharose beads bearing a phosphopeptide (PSY(PO3H2)VNVPD) derived from Shc, which is able to recognize the SH2 domain of Grb2 (18). As expected, Fig. 2
shows that 50 nM of peptidimer 2 is sufficient to almost completely inhibit the Grb2–Sos complexation, whereas 5 µM of monomer 1 as a control is necessary to obtain a similar effect ( Fig. 2). The capacity of peptidimer 2 to block the Grb2–Sos interaction at a low concentration suggests that such dimers might constitute potential inhibitors of cell proliferation and/or differentiation mediated by the Ras signaling pathway. Finally, it was essential to verify their specificity toward Grb2, since several signaling proteins bear SH3 domains.
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Specificity of the peptidimer 2
The specificity toward Grb2–Sos complexation was studied by immobilizing peptidimer 2 on Sepharose beads through its lysine carboxylic group. Affinity-precipitated proteins from an ER22 cell homogenate were separated by SDS-PAGE and revealed by immunoblotting experiments with antibodies directed toward the p85 subunit of PI3K, Grb2, and Nck ( Fig. 3),
three proteins containing one, two, and three SH3 domains, respectively. Only the anti-Grb2 antibody provided a positive response, demonstrating that peptidimer 2 specifically recognizes the structure of Grb2.
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In vivo effect on ER22 cells: coupling of peptidimer 2 to a carrier
Due to its high affinity and specificity for Grb2 as well as its capacity to inhibit Grb2–Sos interaction in vitro, peptidimer 2 could be a good candidate to inhibit Ras signaling-derived pathway in vivo provided it could penetrate the cells. This was rendered possible by coupling the peptidimer, by the free carboxylic group of its lysine residue, to a 16 amino acid-long peptide: RQIKIWFQNRRMKWKK ( Fig. 1A). This peptide, corresponding to the third helix of the homeodomain of Antennapedia, had been shown to deliver biologically active peptides inside living cells by a nonreceptor-derived process (34, 35). Incubation of ER22 cells for 15 min with the modified peptidimer conjugate 4, at doses as low as 10 µM, inhibited Grb2–Sos interaction (100%) and MAP kinase phosphorylation (60%) evoked by a 5 min EGF stimulation (data not shown), without modifying cellular growth. Thus, to test the capacity of the peptidimer conjugate to inhibit overexpressed Grb2–Sos interactions in a cellular model, it was tested successively on NGF-induced differentiation of PC12 cells and on the cloning efficiency of NIH3T3 cells transfected with HER2.
In vivo effect of peptidimer conjugate 4 on PC12 cells
The neural crest-derived pheocytochroma PC12 cells constitute an interesting model with which to test the biological activity of peptidimers, since Ras signaling activation appears to be essential for their neuronal differentiation by NGF (36–40). Microinjection of oncogenic Ras protein in PC12 cells produces neurite outgrowth (41), whereas microinjection of anti-p21Ras antibodies blocks NGF-induced differentiation (42). Moreover PC12 cell apoptosis is observed after withdrawal of NGF (43). In these cells, the Ras signaling pathway can also be activated by EGF, which is involved only in cellular growth. Although PC12 cells have approximately equal numbers of EGF and NGF surface receptors, and most of the early signal transduction pathways elicited by both factors are qualitatively similar, NGF causes a much more sustained level of MAP kinase activation in relation to its capacity to induce cell differentiation (36–40). Therefore, PC12 cells were treated with 10 µM of peptidimer conjugate 4 and stimulated with either NGF or EGF. As previously reported, PC12 cells stimulated with NGF are able to differentiate into neurite-bearing cells and EGF-stimulated cells do not (36). Cells treated with peptidimer conjugate 4 no longer have the ability to form neurites after a 24 h culture with NGF ( Fig. 4).
With longer culture times, cells progressively lose their adherence properties and die in a manner similar to that observed after NGF withdrawal (43). Under the same conditions, neurite outgrowth induced by NGF was not affected by the Antennapedia carrier alone (0.5 mM). In addition, treatment with peptidimer conjugate 4 of PC12 cells after EGF stimulation does not inhibit their growth (data not shown).
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Effect on MAP kinase activation of peptidimer conjugate 4
It has already been shown that the proline-rich part (carboxyl-terminal region) of Sos is able to inhibit MAP kinase activation in the Ras signaling pathway (23). Thus, to analyze the role of peptidimer conjugate 4 on PC12 cell signaling, its intracellular effects were analyzed by measuring MAP kinase phosphorylation in cellular homogenates 5 min after either EGF or NGF stimulation. As discussed, the level of MAP kinase activation is higher when cells are stimulated with NGF than with EGF ( Fig. 5),
highlighting a critical difference in Ras signaling between the pathways eliciting PC12 cell differentiation or growth. In agreement with its inhibitory effect on neurite formation discussed above, peptidimer conjugate 4 (at 10 µM) reduced MAP kinase phosphorylation more efficiently when using PC12 cells stimulated with NGF (60%) rather than cells stimulated with EGF (30%). Antennapedia carrier alone had no inhibitory effects on MAP kinase activation in the same concentration range (data not shown). It is interesting that the high and sustained level of MAP kinase activation in PC12 cells treated with NGF resembles the constitutive activation of MAP kinase observed in transformed fibroblasts expressing the oncogene HER2 (44, 45).
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Antiproliferative activity of 4 on NIH3T3 cells transfected with HER2
NIH3T3 cells transfected with HER2, the oncogenic form of EGF receptor, provide a good model to test the potential antitumor activity of the peptidimer conjugate 4, since the latter targets Grb2–Sos complexation, located downstream from HER2 in the signaling pathway. Thus, peptidimer conjugate 4 was tested for its ability to inhibit the colony formation of NIH3T3/HER2 cells in soft agar (20). As expected and as depicted in Fig. 6,
compound 4 is able to inhibit NIH3T3/HER2 cells cloning, with an IC50 of around 1 µM. This result shows that by inhibiting Grb2–Sos complex in cells overexpressing HER2, peptidimer conjugate 4 displays cellular antiproliferative activity.
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DISCUSSION
Here we describe the effect of a rationally designed inhibitior of the Grb2–Sos interaction. Briefly, starting from X-ray and NMR structural derived data, peptidimers made of two proline-rich sequences from Sos were designed with the aid of molecular modeling. These peptidimers interact with Grb2 with the highest affinity ever reported and inhibit Grb2–Sos interaction in vitro in cellular extracts.
Several approaches have previously been proposed to increase the affinity of proline-rich peptides for SH3 domains. The SH3 domain of Src has been the most extensively studied for new ligand discovery. Schumacher et al. (46) have proposed a strategy to search for nondegradable compounds, which consists of identifying D-peptide ligands through mirror image phage display. Using this approach, nonnatural peptides with affinities of the same order of magnitude as natural ligands (~60µM) have been obtained. Combinatorial chemistry approaches directed toward the same SH3 domain, using split-pool synthesis, have slightly improved the affinity of the parent peptide (47, 48); however, all the compounds defined by this approach retain a proline-rich sequence (PLPPLPP). Solution structures of two of these ligands complexed with the Src SH3 domain (49) have provided insights into their mode of interaction and are the basis of current efforts to improve the affinity and specificity of nonnatural SH3 ligands. Recently, screening another library containing nonpeptide elements likely to interact with the SH3 domains of Src and Hck (two highly homologuous proteins), specific ligands for these SH3 domains have been discovered (50, 51). The compounds obtained by this approach have a 10-fold better affinity than the natural peptide ligands. Thus, it might be interesting to use combinatorial chemistry to find peptidomimetic or non-entire peptide structures with higher affinities for each Grb2 SH3 domain; then, using the same strategy as that described here for peptidimers, connect these ligands with an appropriate linker that allows their interaction with both SH3 domains.
The rational design of biologically active compounds is sometimes balked by the inability of these compounds to penetrate cells and find their targets. Different approaches have been developed to overcome these problems. Biophysical techniques such as electroporation (52) constitute an important stress for the cells. Liposomes, with or without coupling to a ligand, able to induce receptor-mediated endocytosis (53, 54) or bioreversible protecting groups increasing the molecule lipophilicity and intracellular delivery through enzymatic mechanisms (55) have been tested. Another approach consists of coupling the drug to a peptidic carrier, allowing its penetration into cells (56). Among these vectors, several peptides have been proposed, such as the Antennapedia homeodomain (34, 35), signal peptides (57), or the truncated HIV-1 Tat protein basic domain (58). Here, a peptide from Antennapedia homeodomain was chosen since it was easily introduced by peptide synthesis and was expected to increase the water solubility of the peptidimer. This approach had already been used with success to target a tyrosine-phosphorylated Grb2-binding peptide corresponding to the P85 subunit of the PI3 kinase (59) or the FGFR high-affinity binding site for PLC
(60). In our case, the coupling of the peptidimer 2 to a fragment of the third helix of the Antennapedia homeodomain yielded compound 4, which displayed efficient cell penetration. Several peptides have been described to target molecules specifically into cellular organelles (61). The use of such tools to target SH3 inhibitors to different subcellular localizations could thus provide new and exciting tools for deciphering the signal transduction mechanisms.
In line with in vitro activity of 2, the peptidimer conjugate was able to inhibit NGF-induced differentiation in PC12 cells. In the presence of 10 µM peptidimer, cells lose their ability to form neurites when cultured with NGF. This blockade of NGF-induced cell differentiation is confirmed by biochemical analysis, since MAP kinase phosphorylation is highly inhibited by peptidimer conjugate 4 (43). Moreover, this approach allowed us to assertain that the blockade of Grb2 SH3-mediated interactions are essential for the Ras signaling pathway, as previously described in the case of the P49L mutant of Grb2 (8) or by the overexpression of the carboxyl terminus part of Sos (23). Finally, the antiproliferative activity of the peptidimer conjugate was demonstrated on the colony formation of NIH3T3 cells transfected with HER2 (IC50 around 1 µM), an oncogenic form of the EGF receptor, truncated in its extracellular domain and constitutively activated (20). In agreement with the specificity of our targeted approach, the peptidimer conjugate has no effect on the cloning efficiency of NIH3T3 fibroblasts transfected with oncogenic Ras (personal communication from Dr. J. F. Riou). Indeed, in these cells, uncontrolled overexpression of oncogenic Ras is located downstream of the Grb2–Sos interaction. Together, these results confirm the crucial role of Grb2 as a molecular switch for Ras signal transduction induced by tyrosine kinase receptor activation. The peptidimer conjugate, exhibiting no toxicity on ER22 cells, thus constitutes a new lead for the conception of antitumor agents and an interesting probe to decipher the interconnexions of the different signaling pathways, including seven-pass membrane spanning receptors, in which Grb2 seems to play an important role (62).
Since many signaling proteins have well-defined neighboring domains with known tridimensional structures, the design of dimers able to target two domains can be an effective method for ligand discovery. It has already been used to inhibit ZAP 70 interactions, targeting its two SH2 domains with peptides in which two tyrosine-phosphorylated ITAM motifs had been linked (63). Moreover, this approach could be extended to the targeting of more than two domains. In the case of DNA, intercalating trimers were designed with affinities of around 10-12 M, starting from monomers with affinities of 10-5 M (64).
In conclusion, our data demonstrate that highly selective compounds able to recognize with high affinity and to inhibit with great efficiency protein–protein interactions can be designed by a dimerization or oligomerization approach.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: EGF, epidermal growth factor; Ig, immunoglobulin; mAb, monoclonal antibody; MAP, mitogen-activated protein; NGF, nerve growth factor; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; PBS, phosphate-buffered saline; RTK, receptors with tyrosine kinase activity; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Received for publication April 28, 1998. Accepted for publication September 29, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSREFERENCES |
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