PEPTIDES TARGETING SHP2 AND USES THEREOF

Abstract

The present invention relates to a peptide having the sequence from N-terminus to C-terminus X.sub.-.sub.2X.sub.-1ZX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5 wherein Z is tyrosine, phosphotyrosine or a non-natural analogue of phosphotyrosine, such as phosphonodifluoromethyl phenylalanine (F.sub.2Pmp) X.sub.-2 is a hydrophobic amino acid, such as Leu, Ile, Val, Phe, Tyr, Trp and Met X.sub.-1 is any amino acid X.sub.1 is a hydrophobic amino acid, such as Ile, Leu, Val, Phe, Tyr, Trp and Met X.sub.3 is a hydrophobic amino acid, such as Leu, Ile, Val, Phe, Tyr, Trp and Met X.sub.5 is a hydrophobic amino acid, such as Trp, Ile, Val, Phe, Tyr, and Met X.sub.2 and X.sub.4 are anionic amino acids, preferably each independently is Asp or Glu. The peptide inhibits protein-protein interactions of the Src homology 2 domain-containing phosphatase 2 (SHP2), for the treatment of cancer and RASopathies and as a biomedical research tool.

Claims

1. A peptide comprising the sequence from N-terminus to C-terminus: X.sub.-.sub.2X.sub.-1ZX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5 (SEQ ID NO: 1) wherein Z is a non-natural analogue of phosphotyrosine; X-.sub.2 is a hydrophobic amino acid; X.sub.-1 is any amino acid; X.sub.1 is a hydrophobic amino acid; X.sub.3 is a hydrophobic amino acid; X.sub.5 is a hydrophobic amino acid; and X.sub.2 and X.sub.4 are anionic amino acids; with the proviso that the hydrophobic amino in X.sub.5 acid is not Leu.

2. A peptide according to claim 1 comprising the sequence from N-terminus to C-terminus: X.sub.-.sub.2X.sub.-1ZX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5 (SEQ ID NO:1) wherein Z is a non-natural analogue of phosphotyrosine; X-.sub.2 is a hydrophobic amino acid; X.sub.-1 is any amino acid; X.sub.1 is a hydrophobic amino acid; X.sub.3 is a hydrophobic amino acid; X.sub.5 is a hydrophobic amino acid selected from the group consisting of: Trp, Phe and Tyr; and X.sub.2 and X.sub.4 are anionic amino acids wherein the amino acids may be each independently a natural or non-natural amino acid .

3. A peptide according to claim 1 comprising the sequence from N- terminus to C-terminus: X.sub.-.sub.2X.sub.-1ZX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5 (SEQ ID NO: 1) wherein Z is a non-natural analogue of phosphotyrosine X-.sub.2 is a hydrophobic amino acid, X.sub.-1 is any amino acid; X.sub.1 is a hydrophobic amino acid; X.sub.3 is a hydrophobic amino acid; X.sub.5 is Trp; and X.sub.2 and X.sub.4 are anionic amino acids wherein the amino acids may be each independently a natural or non-natural amino acid .

4. The peptide according to claim 1 wherein: X-.sub.2 is Leu and/or -X.sub.-1 is Asn and/or X.sub.1 is Ile and/or X.sub.2 is Asp and/or X.sub.3 is Leu and/or X.sub.4 is Asp and/or X.sub.5 is Trp or Phe .

5. The peptide according to claim 1 wherein Z is a non-natural analogue of phosphotyrosine, .

6. The peptide according to claim 5 comprising the sequence from N-terminus to C-terminus: LN-(F.sub.2PmP)-IDLDW (SEQ ID NO:4) or GLN-(F.sub.2PmP)-IDLDW (SEQ ID NO:5).

7. The peptide according to claim 1 wherein the N-terminus and/or the C-terminus of the peptide are modified.

8. The peptide according to claim 6 wherein the peptide N-terminus is acetylated, and its C-terminus is amidated.

9. A peptide according to claim 1 further comprising an aminoacidic sequence which favors penetration inside cells.

10. A non-covalent complex comprising the peptide according to claim 1 and an aminoacidic sequence which favors penetration inside cells.

11. A pharmaceutical composition comprising a peptide according to claim 1, at least one pharmaceutically acceptable carrier, excipient and/or diluent, and optionally further comprising at least one therapeutic agent.

12. The peptide according to claim 1 for use as a medicament, preferably for use: in the treatment of childhood myeloproliferative disorders, preferably juvenile myelomonocytic leukemia (JMML), childhood myelodysplastic syndromes (e.g, RAEB), childhood leukemia (e.g, acute monocytic leukemia (AMoL, FAB M5) and acute lymphoblastic leukemia (ALL), “common” subtype), adult myelodysplastic syndromes, myelogenous and lymphoblastic leukemia, pediatric/adult solid tumors associated with an aberrant activity of SHP2 due to the occurrence of somatic PTPN11 gain-of-function mutations, e.g. neuroblastoma, glioma, embryonal rhabdomyosarcoma, lung cancer, colon cancer, and melanoma), in the treatment of tumors associated with hyperactivation of the signal transduction pathways regulated by SHP2 (RAS-MAPK and PBK-AKT-mTOR), e.g. colon, cervix, endometrium, pancreas, large and small intestine, skin, prostate, head and neck, and lung tumors, in the treatment of post natal clinical manifestations of RASopathies caused by germline mutations of PTPN11 (Noonan syndrome and Noonan syndrome with multiple lentigines, also called LEOPARD syndrome), such as hypertrophic cardiomyopathy, short stature and predisposition to certain malignancies, particularly JMML, in cancer immunotherapy to avoid the tumor immune evasion mediated by SHP2’s activation of immune checkpoint pathways, such as the Programmed Cell Death 1 (PD-1) or signal-regulatory protein alpha (SIRPa)/CD47, thus modulating the immune response in cancer, as a cytotoxin-associated gene A (CagA) competitor in H. /.sub.j7 /7-rnediated gastric carcinoma.

13. (canceled)

14. A peptide according to claim 1 comprising the sequence from N-terminus to C-terminus: X.sub.-.sub.2X.sub.-1ZX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5 (SEQ ID NO: 1) wherein Z is phosphonodifluoromethyl phenylalanine (F.sub.2Pmp), tyrosine or phosphotyrosine (pY); X-.sub.2 is a hydrophobic amino acid selected from the group consisting of: Leu, Ile, Val, Phe, Tyr, Trp and Met; X.sub.-1 is any amino acid; X.sub.1 is a hydrophobic amino acid selected from the group consisting of: Ile, Leu, Val, Phe, Tyr, Trp and Met; X.sub.3 is a hydrophobic amino acid selected from the group consisting of: Leu, Ile, Val, Phe, Tyr, Trp and Met; X.sub.5 is a hydrophobic amino acid selected from the group consisting of: Trp, Ile, Val, Phe, Tyr, and Met; and X.sub.2 and X.sub.4 are each independently Asp or Glu wherein the amino acids may be each independently a natural or non-natural amino acid, such as Ca or N methylated, peptoids, beta amino acids or D amino acids.

15. A peptide according to claim 1 comprising the sequence from N-terminus to C-terminus: X.sub.-.sub.2X.sub.-1ZX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5 (SEQ ID NO:1) wherein Z is phosphonodifluoromethyl phenylalanine (F.sub.2Pmp), tyrosine or phosphotyrosine (pY); X-.sub.2 is a hydrophobic amino acid selected from the group consisting of: Leu, Ile, Val, Phe, Tyr, Trp and Met; - X-X.sub.1 is any amino acid; X.sub.1 is a hydrophobic amino acid selected from the group consisting of: Ile, Leu, Val, Phe, Tyr, Trp and Met; X.sub.3 is a hydrophobic amino acid selected from the group consisting of: Leu, Ile, Val, Phe, Tyr, Trp and Met; X.sub.5 is a hydrophobic amino acid selected from the group consisting of: Trp, Phe and Tyr; and X.sub.2 and X.sub.4 are each independently Asp or Glu wherein the amino acids may be each independently a natural or non-natural amino acid.

16. A peptide according to claim 1 comprising the sequence from N- terminus to C-terminus: X.sub.-.sub.2X.sub.-1ZX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5 (SEQ ID NO: 1) wherein Z is phosphonodifluoromethyl phenylalanine (F.sub.2Pmp), tyrosine or phosphotyrosine (pY) ; X-.sub.2 is selected from the group consisting of: Leu, Ile, Val, Phe, Tyr, Trp and Met; X.sub.-1 is any amino acid; X.sub.1 is a hydrophobic amino acid selected from the group consisting of: Ile, Leu, Val, Phe, Tyr, Trp and Met; X.sub.3 is a hydrophobic amino acid selected from the group consisting of: Leu, Ile, Val, Phe, Tyr, Trp and Met; X.sub.5 is Trp; and X.sub.2 and X.sub.4 are each independently Asp or Glu wherein the amino acids may be each independently a natural or non-natural amino acid.

17. The peptide according to claim 1 wherein: X-.sub.2 is Leu and/or X.sub.-1 is Asn and/or X.sub.1 is Ile and/or X.sub.2 is Asp and/or X.sub.3 is Leu and/or X.sub.4 is Asp and/or X.sub.5 is Trp.

18. The peptide according to claim 1 wherein Z is F.sub.2Pmp.

19. The peptide according to claim 9 wherein the aminoacidic sequence which favors penetration inside cells is TAT, penetratin, oligo-Arg, transportan 10, said amino acidic sequence being linked to the N-terminus and/or the C-terminus of the peptide.

20. The peptide according to claim 7, wherein the N-terminus is acetylated or labeled with CF and/or the C- terminus is amidated.

21. The non-covalent complex according to claim 10 wherein the aminoacidic sequence which favors penetration inside cells is Pepl.

Description

[0138] The present invention is therefore illustrated by means of non-limiting examples in reference to the following figures.

[0139] FIG. 1: SHP2 structure and scheme of the activation process.

[0140] Top: crystallographic structures for the closed, auto-inhibited and the open, active states of SHP2 (left and right, respectively). The N-SH2, C-SH2 and PTP domain are colored in light grey, black and white, respectively. The N-SH2 blocking loop (DE loop) and the PTP active site are highlighted in black. PDB codes of the two structures are 2SHP and 6CRF. Segments missing in the experimental structures were modeled as previously described [Bocchinfuso 2007].

[0141] Bottom: schematic model of the allosteric regulation mechanism.

[0142] FIG. 2: Binding curves for the phosphorylated and unphosphorylated IRS-1 pY1172 peptides.

[0143] [CF-P9]=1.0 nM (full symbols and solid line), [CF-P9Y0]=10 nM (empty symbols and dashed line); Replicate experiments are reported with different symbols and were fit collectively.

[0144] FIG. 3: effect of sequence length on the binding affinity.

[0145] Displacement experiments for analogs of different length. [CF-P9]=1.0 nM; [N-SH2]= 40 nM.

[0146] FIG. 4: in silico free energy calculations for different modified sequences

[0147] The free energy profile is reported as a function of the distance between the centers of mass of the N-SH2 domain and of the phosphopeptide. The simulations predict a loss in affinity of P8 with dephosphorylation of the pY (P8Y0), and a gain with substitution of the Leu in position +5 with Trp (P8W5), but not with Phe P8F). The additional substitution of Asp in +4 with Glu (P8E4W5) does not provide any further increase in affinity. Shaded areas correspond to standard deviations in the PMF profile.

[0148] FIG. 5: effect of substitutions at position +5 on the binding affinity

[0149] Left panel: direct binding experiments; [CF-P9W5]=0.10 nM, [CF-P9E4W5]=0.10 nM, [CF-P9]=1.0 nM. Data for CF-P9 are repeated here for comparison.

[0150] Right panel: displacement assay, [CF-P9W5]=0.10 nM, [N-SH2]=3.35 Nm

[0151] FIG. 6: binding selectivity of CF-P9W5 for the two SH2 domains of SHP2

[0152] Comparison of the association curves of CF-P9W5 to the N-SH2 and C-SH2 domains of SHP2. Experimental conditions for the N-SH2 binding experiments: see FIG. 5; for the C-SH2 binding experiments: [CF-P9W5]=1.0 nM.

[0153] FIG. 7: binding selectivity of Cy3-P9W5 for an array of SH2 domains.

[0154] The left panels report the fluorescence of the bound peptide, at a concentration of 0.5 nM (top), 5.0 nM (center) and 50 nM (bottom). Each SH2 domain is present in duplicate, and negative control spots (with GST only) are also present. The bright spots correspond to the N-SH2 domain of SHP2 and to the SH2 domain of the SH2 and PH domain-containing adapter protein APS (also called SHP2B2). The intensity of all other spots is comparable to that of the negative controls.

[0155] The top right panel shows the position of each SH2 domain in the array, while the top bottom panel is a control of the protein loading in each spot, performed with an anti-GST antibody.

[0156] FIG. 8: binding of the non-dephosphorylatable peptide CF-P9ND0W5 (or OP) to the N-SH2 domain.

[0157] For comparison, the curve for CF-P9W5 is also shown. [CF-P9ND0W5]=1.0 nM, [CF-P9W5]=0.10 nM

[0158] FIG. 9. OP resistance to proteolytic degradation in human serum and in DMEM

[0159] HPLC profiles of OP, after incubation with human serum (left) or DMEM (right). Profiles are reported in order of increasing incubation time, from bottom to top.

[0160] FIG. 10 Binding of the CF-OP peptide to the whole SHP2 protein (WT and pathogenic mutants)

[0161] Top panel: fraction of CF-OP peptide bound to the WT protein and selected mutants, obtained from fluorescence anisotropy experiments. The peptide bound fractions were obtained by following the variation of the peptide fluorescence anisotropy during the titration with increasing amounts of protein. [CF-OP]=1.0 nM.

[0162] Center panel: catalytic activity of the WT protein and selected mutants, under basal conditions (black bars) and after stimulation with 10 .Math.M BTAM peptide (grey bars).

[0163] Bottom panel: correlation between basal activity and affinity (association constant).

[0164] FIG. 11: dephosphorylation of P8W5 and other phosphopeptides by the SHP2 PTP domain.

[0165] The following phosphopeptides were used for comparison, in addition to P8 and P8W5, in a malachite green assay.

TABLE-US-00005 GAB1 Y657 (DKQVEpYLDLDL (SEQ ID NO:6))

TABLE-US-00006 p190A/RhoGAP Y1105 (EEENIpYSVPHD(SEQ ID NO:7))

TABLE-US-00007 EGFR Y1016 (VDADEpYLIPQQ(SEQ ID NO:8))

BTAM, or biphosphorylated SHSP-1 TAM1

TABLE-US-00008 (GGGGDIT(pY)ADLNLPKGKKPAPQAAEPNNHTE(pY)ASIQTS (SEQ ID NO:9),

with 4 N-terminal G residues)

[0166] A SHP2 construct lacking the N-SH2 domain (i.e. the first 104 residues) was used at a 95 nM concentration. Phosphopeptides were added at a 100 .Math.M concentration and the phosphate released was measured at different times. From the linear region of the phosphate versus time curve, the variation in absorbance at 655 nm in 1 min, due to phosphate released, was calculated and plotted.

[0167] FIG. 12. CF-OP association to the PTP domain.

[0168] [CF-P9ND0W5]=1.0 nM

[0169] FIG. 13: SHP2 activation by the OP

[0170] Basal activity is reported in light grey, while activities in the presence of 10 .Math.M BTAM or 10 .Math.M OP are shown in dark grey and black, respectively.

[0171] FIG. 14: cell uptake of the phosphopeptides

[0172] Left panel: FACS quantification of spontaneous and CPP-induced cell uptake in NIH3T3 cells after 120 minutes of incubation.

[0173] Right panel: time course of the cell uptake process, at a 10 .Math.M fluorescent peptide concentration.

[0174] FIG. 15: optimization of Pep1 concentration

[0175] FACS quantification of Pep1-induced cell uptake in NIH3T3 cells after 120 minutes of incubation. CF-P9W5 concentration was 0, 0.5 .Math.M, 1.0 .Math.M or 2.0 .Math.M.

[0176] FIG. 16: peptide toxicity in the cell uptake experiments

[0177] Cell viability in the FACS experiment was determined by using Sytox fluorescence.

[0178] FIG. 17: the OP partially rescues Shp2a-D61G-induced gastrulation defects and mortality in a dose dependent manner in zebrafish embryos. Embryos were injected at the one-cell stage with mRNA encoding GFP-2A-Shp2-D61G or GFP465 Shp2-wt with or without peptide at 0.3 .Math.M, 3 .Math.M and 5 .Math.M concentration. Non-injected embryos (ni) were evaluated as a control. A: ovality of embryos at 11 hpf, as indicated by the ratio of the long and the short axis. Tukey’s honest significant difference test was done to assess significance. In the box plot, the horizontal line indicates the median, box limits indicate the 25th and 75th percentiles (interquartile range), whiskers (error bars) extend to the maximum and minimum values, or to 1.5 times the interquartile range from the 25th and 75th percentiles, if some data points fall outside this range. In the latter case, outliers are indicated as single data points. B: embryo lethality. Surviving embryos were counted at 1 dpf and 4 dpf. Survival was plotted and Log-rank test was done to access differences between groups. Non significant (ns) p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001. The number of embryos that were analyzed are indicated in parentheses.

EXAMPLES

Materials and Methods

Materials and Methods

Materials

[0179] Fmoc fluorenylmethyloxycarbonyl)-amino acids were obtained from Novabiochem (Merck Biosciences, La Jolla, CA). Rink amide MBHA resin (0.65 mmol/g, 100-200 mesh) was purchased from Novabiochem. All other protected amino acids, reagents and solvents for peptide synthesis were supplied by Sigma-Aldrich (St. Louis, MO). The LB medium components, all the reagents used to prepare the buffers and the Bradford reagent were purchased from Sigma Aldrich. tris(2-carboxyethyl)phosphine (TCEP) was obtained from Soltec Ventures, Beverly, MA, USA. Spectroscopic grade organic solvents were purchased from Carlo Erba Reagenti (Milano, Italy). Cell culture media growth factors and antibodies were purchased from VWR International PBI (Milan, Italy), EuroClone (Milan, Italy), Promega (Madison, WI, USA), Invitrogen (Carlsbad, CA, USA), Cell Signaling (Danvers, MA, USA), Sigma-Aldrich (Saint Louis, MO, USA), and Santa Cruz Biotechnology (Dallas, TX, USA).

Peptide Synthesis

[0180] Assembly of peptides on the Syro Wave (Biotage, Uppsale, Sweden) peptide synthesizer was carried out on a 0.1 mmol scale by the FastMoc methodology, beginning with the Rink Amide MBHA resin (Merck Biosciences, La Jolla, CA) (155 mg, loading 0.65 mmol/g). The peptide was cleaved from the resin, filtered and collected. The solution was concentrated under a flow of nitrogen, and the crude peptide precipitated by addiction on diethyl ether. The crude peptides were purified by flash chromatography on Isolera Prime chromatographer (Biotage, Uppsale, Sweden) using a SNAP Cartridge KP-C18-HS 12 g or preparative RP-HPLC on a Phenomenex C18 column (22.1x250 mm, 10 .Math.m, 300 Å) using an Akta Pure GE Healthcare (Little Chalfont, UK) LC system equipped with an UV-detector (flow rate 15 mL/min) and a binary elution system: A, H2O; B, CH3CN/H2O (9: 1 v/v); gradient 25-55% B in 30 min. The purified fractions were characterized by analytical HPLC-MS on a Phenomenex Kinetex XB-C18 column(4.6 x 100 mm, 3.5 .Math.m, 100 Å) with an Agilent Technologies (Santa Clara, CA) 1260 Infinity II HPLC system and a 6130 quadrupole LC/MS. The binary elution system used was as follows: A, 0.05% TFA (trifluoroacetic acid) in H2O; B, 0.05% TFA in CH3CN; flow rate 1 mL/min. Retention times (Rt) for the synthetic peptides obtained from RP-HPLC (the elution conditions used for different peptides are listed in brackets) and molecular weights for the synthetic peptides experimentally determined by ESI-MS spectrometry are reported in Table 1.

[0181] Peptides were dissolved in DMSO to obtain stock solutions between 1 and 1.5 mM. The exact concentration was obtained by UV measurements, exploiting the signal of carboxyfluorescein for the labeled peptides and of pTyr, Tyr and Trp for the unlabeled peptides. To this end, CF-labeled peptides were diluted from the stocks (1:100) in buffer (pH 9), and their concentration was calculated from the CF signal at 490 nm using a molar extinction coefficient of 78000 M-1 cm-1 [Esbjörner 2007]. Unlabeled peptides were diluted 1:100 in a pH 7.4 buffer; molar extinction coefficients of Tyr, Phe and Trp were taken from Pace et al. [1995], while molar extinction coefficient of pY was taken from Bradshaw et al. [1999].

Protein Expression and Purification

[0182] The human esaHis-tagged PTPN11 (residues 1-528) cDNA was cloned in a pET-26b vector (Novagen, MA, USA). Nucleotide substitutions associated with NS or leukemia were introduced by site-directed mutagenesis (QuikChange site-directed mutagenesis kit; Stratagene, CA, USA). A construct containing the cDNA encoding the isolated PTP domain preceded by the C-SH2 domain (residues 105-528) was generated by PCR amplification of the full-length wild-type cDNA and subcloned into the pET-26b vector (SHP2Δ104). A similar procedure was followed for the constructs of the N-SH2 (residues 2-111), C-SH2 (109-217) and PTP (212-528) domains, and of the N-SH2/C-SH2 tandem (2-217). Primer sequences are available upon request.

[0183] Recombinant proteins were expressed as previously described [Martinelli 2012, Pannone 2017], using E.coli (DE3) Rosetta2 competent cells (Novagen). Briefly, following isopropil-β-D-1-tiogalattopiranoside (Roche) induction (2 hr at 30° C., or overnight at 18° C.), bacteria were centrifuged at 5,000 rpm, 4° C. for 15 minuts, resuspended in a lysozyme-containing lysis buffer (TRIS-HCl 50 mM , pH=8.5, NaCl 0.5 M, imidazole 20 mM, tris(2-carboxyethyl)phosphine (TCEP) ImM, lysozyme 100 mg/ml, 1 tablet of complete protease inhibition cocktail) and sonicated. The lysate was centrifuged at 16,000 rpm, 4° C. for 30 minutes. The supernatant was collected and the protein of interest was purified by affinity chromatography on a Ni-NTA column (Qiagen, Hilden, Germany), using a TRIS-HCl 50 mM, NaCl 0.5 M, TCEP 1 mM buffer containing 100 mM or 250 mM imidazole, for washing and elution, respectively.

[0184] To remove imidazole, the samples were then dialyzed in a 20 mM TRIS-HCl (pH 8.5) buffer, containing 1 mM TCEP and 1 mM EDTA and 50 mM NaCl (or 150 mM NaCl if no further purification steps followed). Full length proteins and the SHP2Δ104 construct were then further purified by sequential chromatography, using an Äkta FPLC system (Äkta Purifier 900, Amersham Pharmacia Biotech, Little Chanfont, UK). The samples were first eluted within an anion exchange Hi-Trap QP 1 ml-column (GE Helathcare, Pittsburgh, PA, USA); the elution was carried out using TRIS-HCl 20 mM (pH 8.5) in a NaCl gradient from 50 to 500 mM. The most concentrated fractions were then eluted in a gel filtration Superose column using TRIS-HCl 20 mM buffer containing NaCl (150 mM) as mobile phase . Sample purity was checked by SDS PAGE with Coomassie Blue staining and resulted to be always above 90%.

[0185] Proteins were quantitated by both the Bradford assay [Bradford 1976] and the UV absorbance of aromatic residues, calculating extinction coefficients according to Pace. [Pace 1995]. In general, the two methods were in agreement, but the values derived from UV absorbance were more precise and are reported in the Figures and Tables. The protein samples were used immediately after purification or stored at -20° C. and used within the following week. In this case, after thawing TCEP 2.5 mM was added, the samples were centrifuged at 13,000 rpm for 20 minutes, and the new concentration was re-evaluated. In the few cases where residual apparent absorbance due to light scattering was present in the UV spectra, it was subtracted according to Castanho et al., 1997.

Phosphatase Activity Assays

[0186] Catalytic activity was evaluated in vitro using 20 pmol of purified recombinant proteins in a 200-uL reaction buffer supplemented with 20 mM p-nitrophenyl phosphate (Sigma) as substrate, either basally or following stimulation with the protein tyrosine phosphatase nonreceptor type substrate 1 (PTPNS1) bisphosphotyrosyl-containing motif (BTAM peptide) (GGGGDIT(pY)ADLNLPKGKKPAPQAAEPNNHTE(pY)ASIQTS(SEQ ID NO:9)) (Primm, Milan, Italy), as previously described [Martinelli 2008]. Proteins were incubated for 15 min (SHP2Δ104) or 30 min (SHP2) at 30° C. Phosphate release was determined by measuring absorbance at 405 nm.

[0187] DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) assay was carried out as previously described [Garcia Fortanet 2016], with minor changes. Briefly, reactions were performed at room temperature in 96-well flat bottom, low flange, non-binding surface, black polystyrene plates (Corning, cat. no. 3991), using a final volume of 100 .Math.l and the following assay buffer: 60 mM HEPES, pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% Tween-20, 5 mM DTT. Catalytic activity was checked using 1 nM SHP2 and different concentrations of activating peptides. After 45 min at 25° C., 200 .Math.M of surrogate substrate DiFMUP (Invitrogen, cat. no. D6567) was added to the mix, and incubated at 25° C. for 30 min. The reaction was stopped by addition of 20 .Math.l of 160 .Math.M bpV(Phen) (Potassium bisperoxo(1,10-phenanthroline)oxovanadate (V) hydrate) (Enzo Life Sciences cat. no. SML0889-25MG). The fluorescence was monitored using a microplate reader (Envision, Perki-Elmer) using excitation and emission wavelengths of 340 nm and 455 nm, respectively.

[0188] The ability of SHP2 to dephosphorylate the phosphopeptides was evaluated through a malachite green phosphatase assay (PTP assay kit 1 Millipore, MA, USA). The BTAM peptide and the following monophosphorylated peptides derived from known SHP2 substrates were used for comparison: DKQVEpYLDLDL (SEQ ID NO:6) (GAB1Y657), EEENIpYSVPHD (SEQ ID NO:7) (p190A/RhoGAPY1105), and VDADEpYLIPQQ (SEQ ID NO:8) (EGFRY1016) (Primm) [Sun et al., 2009; Ren et al., 2011]. SHP2Δ104 or PTP (2.38 pmol) were incubated with 100 .Math.M of each phosphopeptide (total volume 25 .Math.l) for different times. The reaction was stopped by adding 100 .Math.l of malachite green solution. After 15 min, absorbance was read at 655 nm using a microplate reader, and compared with a phosphate standard curve to determine the release of phosphate. Data obtained in the linear region of the curve were normalized on the reaction time (1 min).

Fluorescence Anisotropy Binding Assay

[0189] Anisotropy measurements were carried out using a Horiba Fluoromax 4 spectrofluorimeter.

[0190] For the binding assays, the requested peptide amount (1 nM or 0.1 nM) was diluted in buffer (HEPES 10 mM, NaCl 150 mM, EDTA 1 mM, TCEP 1 mM, fluorescence buffer henceforth) and its anisotropy signal was recorded. The peptide was then titrated with increasing protein amounts, until the anisotropy signal reached a plateau at its maximum value, or up to a protein concentration where protein aggregation and consequent light scattering affected the anisotropy values (usually above 1 .Math.M). The measurements of CF-labeled peptides were carried out using an excitation wavelength of 470 nm and collecting the anisotropy values at an emission wavelength of 520 nm. A 495 nm emission filter was used. For the Cy3-labeled peptides, excitation and emission wavelengths of 520 and 560 nm were used. The lowest peptide concentration needed to have a sufficient fluorescent signal (0.1 nM) was used in the binding experiments. Higher concentrations (1 or 10 nM) were used for peptides with lower affinities, and therefore higher K.sub.d values.

[0191] The displacement assays were carried out with the same experimental settings. In this case, the labeled peptide-protein complex was titrated with increasing amounts of the unlabeled peptide, following the decrease in anisotropy. Measurements were carried out at the same CF-peptide concentration used for the corresponding binding experiments. Regarding the protein concentration, a compromise between two requirements is needed [Huang 2003]. On one hand, it is desirable to have a significant fraction of the CF-peptide bound to the protein, to maximize the dynamic range in the anisotropy signal, which decreases during the displacement experiment.

[0192] On the other hand, the protein concentration should be comparable or lower than the dissociation constant of the unlabeled peptide (K.sub.i), to allow a quantitative and reliable determination of its binding affinity. Since several unlabeled peptides had a higher affinity than their fluorescent counterparts, in the displacement assays authors used a protein concentration [P].sub.T~K.sub.d, or in some cases even ~K.sub.d/2.

[0193] The K.sub.d values were obtained fitting the data with the following equation, described in Van der Weert 2011, which avoids the need for the commonly used (but often unjustified) approximation of the concentration of unbound protein with the total concentration:

[00001]$\frac{r-r_0}{r_{max}-r_0}=\frac{{\left[P\right]}_T+{\left[L\right]}_T+K_d-\sqrt{{\left({\left[P\right]}_T+{\left[L\right]}_T+K_d\right)}^2-4{\left[P\right]}_T{\left[L\right]}_T}}{2{\left[L\right]}_T}$

Here, [P].sub.T and [L].sub.T are the total protein and ligand concentrations, while r, ro and r.sub.max are the anisotropy values at a given protein concentration, in the absence of protein and when the peptide is completely bound, respectively.

[0194] The affinity of unlabeled peptides was determined by competition experiments, in which a sample with fixed total protein and ligand concentrations ([P].sub.T and [L].sub.T) was titrated with the inhibitor, causing displacement of the fluorescent peptide and a decrease in anisotropy. From these data, the IC.sub.50 (i.e. the total concentration of unlabeled peptide that displaces half of the bound fluorescent analog) was determined, interpolating the displacement curve using a phenomenological Hill equation [Barlow 1989]:

[00002]$\frac{r-r_0}{r_{fin}-r_0}=\frac{{\left\{\raisebox{1ex}{${\left[I\right]}_T$}\!\left/ \!\raisebox{-1ex}{$I{C}_{50}$}\right.\right\}}^n}{1+{\left\{\raisebox{1ex}{${\left[I\right]}_T$}\!\left/ \!\raisebox{-1ex}{$I{C}_{50}$}\right.\right\}}^n}$

where [I].sub.T is the total concentration of the peptide causing the displacement, and r.sub.fin is the anisotropy corresponding to total displacement, while in this case r.sub.0 is the starting anisotropy, in the absence of displacing peptide.

[0195] Successively, the dissociation constant of the unlabeled peptide (K.sub.i) was calculated from the know values of IC.sub.50, K.sub.d, [P].sub.T and [L].sub.T, as described here below.

[00003]$K_i=\frac{I{C}_{50}-{\left[P\right]}_T+\frac{{\left[PL\right]}_0}{2}\left(1+\frac{K_d}{{\left[L\right]}_T-\frac{{\left[PL\right]}_0}{2}}\right)}{\left(\frac{2{\left[P\right]}_T}{{\left[PL\right]}_0}-1\right)\frac{{\left[L\right]}_T-\frac{{\left[PL\right]}_0}{2}}{K_d}-1}$

Here, [PL].sub.0 can be substituted with the following expression, analogous to Eq. (1):

[00004]${\left[PL\right]}_0=\frac{{\left[P\right]}_T+{\left[L\right]}_T+K_d-\sqrt{{\left({\left[P\right]}_T+{\left[L\right]}_T+K_d\right)}^2-4{\left[P\right]}_T{\left[L\right]}_T}}{2}$

In this way, K.sub.i is expressed as a function of the known quantities IC.sub.50, K.sub.d, [P].sub.T and [L].sub.T, without any approximation.

SH2 Domain Microarray

[0196] The microarray experiment was conducted by the Protein Array and Analysis Core at the MD Anderson Cancer Center (University of Texas, USA), as previously described [Roth 2019]. Briefly, a library of SH2 domains [Huang 2008] was expressed as GST fusion in E. coli and purified on glutathione-sepharose beads. The domains were spotted onto nitrocellulose-coated glass slides (Oncyte Avid slides, Grace Bio-Labs) using a pin arrayer [Espejo 2002]. Each domain was spotted in duplicate. After incubation with a Cy3-P9W5 solution (0.5, 5.0 nM, or 50 nM), fluorescence signals were detected using a GeneTACTM LSIV scanner (Genomic Solutions).

In Silico Studies

System Preparation

[0197] The initial structure of the N—SH2 complexed with phosphopeptide P8 (Table 1) was obtained by amino acid substitutions (and deletion) in the crystallographic structure of the protein complexed with the GAB1 peptide (sequence GDKQVE-pY-LDLDLD (SEQ ID NO:3)) (PDB code 4QSY). Side-chain configurations for mutated residues were chosen as the most probable in a backbone-dependent rotamer library [Dunbrack 2002]. The obtained complex was then used as the starting structure for subsequent amino acid substitutions in the bound peptide.

System Equilibration

[0198] MD simulations were performed using the GROMACS simulation package [Abraham 2015] and a variant of AMBER99SB force field with parameters for phosphorylated residues [Homeyer 2006]. Water molecules were described by the TIP3P model [Jorgersen 1983]. All the simulated systems were inserted in a pre-equilibrated triclinic periodic box containing about 24000 water molecules and counterions to neutralize system total charge. They were relaxed first by doing a minimization with 5000 steepest descent cycles, by keeping protein positions fixed and allowing water and ions to adjust freely, followed by a heating protocol in which temperature was progressively increased from 100 K to 300 K. The system was then equilibrated for 100 ps in the NVT ensemble at 300 K, using velocity rescaling with a stochastic term (relaxation time 1 ps) [Bussi 2007] and then for 500 ps at constant pressure (1 atm) using the Parrinello-Rhaman barostat (relaxation time 5 ps) [Parrinello 1981]. Long-range electrostatic interactions were calculated using the particle mesh Ewald method [Darden 1993] and the cut-off distance for the non-bonded interaction was set equal to 12.0 Å. The LINCS constraint to all the hydrogen atoms and a 2 fs time-step were used [Hess 1997].

Sampling of the Initial Configurations for Umbrella Sampling

[0199] For each system, a set of initial configurations was prepared by performing a center-of-mass (COM) pulling simulation. The distance between the peptide and N-SH2 domain COMs was constrained with a harmonic force (K=1000 kJ mol-1 nm-2). Pulling was performed by gradually increasing the value of the equilibrium distance with a constant-rate of 0.0025 nm/ps. The length of each simulation was about 2.5 ns. During the whole simulation, a positional restraint (1000 kJ/(mol .Math. nm) was applied to all heavy atoms in the N-SH2 domain except for atoms in loops around the binding region (residues 30-45, 52-75, 80-100). The choice of the optimal unbinding pathway is critical for a reliable estimation of the peptide binding free energy [Chovancova 2012, Vuong 2015]. In this work, three different directions were tested, corresponding to: i) the vector from the phosphate to the alpha carbon in pY, in the equilibrated complex; ii) the vector defined by the initial positions of the two COMs; iii) the vector perpendicular to the surface of the cavity flanked by the EF and BG loops, passing through the N-SH2 domain center of mass. Among the three different pathways, the third direction encountered less steric occlusion by the EF and BG loops, and was thus selected for further analyses.

Umbrella Sampling Simulations

[0200] A set of starting configurations was extracted from the pull-dynamics trajectory saving the peptide-protein center-of-mass distances every 2 Å in the range from 9 to about 40 Å, thus obtaining about 20 windows along the COM distance. The system in each window was preliminarily equilibrated for 1 ns with a strong positional restraint (1000 kJ/(mol .Math. nm) to all carbon alpha atoms except for those in loops flanking the binding region (as in the pull simulation), followed by a production run of 150 ns with the restraints. During this stage, an harmonic potential (K=1000 kJ/mol .Math. nm2 ) was applied on the distance between the two COMs. Additional sampling windows were added every 1 Å along the distance between the two COMs up to a distance of 15 Å. The resulting asymmetric distribution of sampling windows was used to calculate PMF on the production run trajectories. The Weighted Histogram Analysis Method (WHAM) [Kumar 1992] was used, with default settings (50 bins and tolerance of 10-6 kJ mol-1), using the gmx wham GROMACS tool [Hub 2010]. The analysis of the simulation was carried out on the 150 ns production dynamics, during which configurations were stored every 0.1 ns. The statistical uncertainty of the obtained PMF was estimated by bootstrapping analysis [Hub 2010].

Peptide Stability in Serum and in DMEM

[0201] The peptides were dissolved in DMSO (C=5 mg/mL). In eppendorf tubes, 1 mL of HEPES buffer (C = 25 mM, pH = 7.6) was temperature equilibrated at 37° C. before adding 250 .Math.L of human serum and 20 .Math.L of peptide solution; the reaction was followed for 90 minutes. At fixed intervals, 100 .Math.L of the solution were withdrawn and added to 200 .Math.L of absolute ethanol. These samples were kept on ice for 15 minutes, then centrifuged at 13,000 rpm for 5 minutes; the supernatant solutions were analyzed by HPLC and HPLC-MS with 20-60% B gradient in 20 minutes to follow the reaction. In parallel, samples containing peptide, buffer and ethanol only were also analyzed, too. A degradation resistance test was also conducted in DMEM (Dulbecco’s Modified Eagle Medium). The experimental conditions are similar to those described above; the reaction was followed for 72 hours. The enzymatic degradation resistance tests were followed by HPLC using a 5-50% B gradient in 20 minutes.

Cell Uptake Experiments

[0202] Cell uptake experiments were performed on NIH3T3 cells. About 200,000 cells were seeded on six-well plates and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1 % penicillin-streptomycin, at 37° C. with 5% CO.sub.2.

[0203] Two different strategies were followed. In a first set of experiments, the fluorescent peptide has been incubated with the cell-penetrating peptide (CPP) Pep-1. A Pep-1 stock solution was prepared by dissolving the peptide in ultrapure water. The fluorescent peptide was diluted in PBS from the DMSO stock solution. The peptides were mixed and incubated for 30 minutes at room temperature in a final volume of 400 .Math.l. The conditions for cell delivery were optimized by investigating different cargo to CPP ratios. The mixture was then added to the cells, washed with PBS, together with 600 .Math.l of serum-free medium (volumes for one well are reported). After one hour, 10% of fetal bovine serum (FBS) was added.

[0204] Cells were incubated for different times and then washed, collected and stained with SYTOX blue (1 mM, Invitrogen™) to identify the viable population and the green fluorescence of CFP9W5 was recordered to measure peptide uptake. Data were analysed with Kaluza Analysis Software (Beckman Coulter).

[0205] A second set of measurement was carried out using the TAT-conjugated peptide analog. In this case, cells were directly incubated with different peptide amounts.

In Vivo Zebrafish Rescue Experiments

[0206] One cell stage zebrafish embryos were injected with a mixture of 120 ng/.Math.l of mRNA encoding either GFP-2A-Shp2-D61G or GFP-2A-Shp2-wt (as a control), with or without OP, at 0.3 .Math.M, 3 .Math.M and 5 .Math.M concentration. Embryos were selected based on proper GFP expression and imaged at 11 hours post fertilization (hpf) in their lateral position using the Leica M165 FC stereomicroscope. Images were analyzed using ImageJ [Schneider 2012], by measuring the ratio of the major and minor axis from a minimum of 31 embryos. Statistical analysis was performed in GraphPad Prism, using the analysis of variance (ANOVA) complemented by Tukey’s honest significant difference test (Tukey’s HSD). To measure the survival of injected embryos, a minimum of 48 embryos per group were grown up to 4 days post fertilization (dpf) and counted at 1 dpf and 4 dpf. Survival curves were plotted using GraphPad Prism, and the differences between samples were determined using the Log-rank (Mantel-Cox) test.

Results

1) Characterization of IRS-1 pY1172/N-SH2 Binding

1.1) The IRS-1 pY1172 Peptide Binds the N-SH2 Domain With a Low Nanomolar Affinity

[0207] In a direct binding experiment, the fluorescently labeled peptide IRS-1 pY1172 analog CFP9 (Table 1) was titrated with increasing concentrations of the N-SH2 domain. The fraction of protein-bound peptide was determined from the increase in fluorescence anisotropy (FIG. 2), and a KD of 53±2 nM was obtained (Table 2).

1.2) Phosphorylation Contributes Only 30% of the Standard Binding Free Energy

[0208] Association of SHP2 domains with the partner proteins is regulated by phosphorylation, and therefore the phosphate group is necessarily responsible for a large fraction of the binding affinity. On the other hand, in order to have a good selectivity, the rest of the sequence must also contribute significantly to the peptide/protein interaction. To quantify this aspect, authors performed a binding experiment (FIG. 2) with an unphosphorylated analog of the labeled IRS-1 pY1172 peptide, CF-P9Y0 (Table 1). The affinity was approximately 100 times lower, with a KD of 6.6±0.6 .Math.M, compared with 53±2 nM for the phosphorylated peptide. The corresponding values for the standard free energy of binding (assuming a 1 M standard state) are -29.6±0.2 kJ/mol and -41.6±0.1 kJ/mol, respectively. Assuming additivity of contributions, the phosphate group results to be responsible for the difference of -12.0±0.2 kJ/mol, i.e. for less than 30% of the total standard binding free energy of the phosphorylated peptide. This result indicates that the contribution of the rest of the peptide predominates in the binding interactions.

2) Sequence Optimization

2.1) The Sequence Can Be Reduced to 8 Amino Acids Without Loss in Affinity

[0209] Literature data are partially contradictory regarding the effect of shortening the IRS-1 pY1172 sequence on the binding affinity. Kay [1998] reported that the sequence could be shortened at the C-terminus down to the +5 residue without any loss in affinity. By contrast, Case [1994] observed a significant reduction in affinity residues in determining the N-SH2 domain binding affinity, authors performed displacement studies (FIG. 3) with unlabeled peptide P9, and with the shortened analogues P8 and P7 (Table 1), where residues -3 or -2 and -3 were removed, respectively. No significant loss in affinity was observed by reducing the sequence to 8 residues, while elimination of the amino acid at position -2 caused a drastic perturbation of complex stability (FIG. 3). The -2 to +5 IRS-1 sequence is the minimal peptide with a low nM dissociation constant.

2.2) Single Amino Acids Substitutions Improve the K.SUB.d to the Low nM Range.

[0210] Based on the structures of phosphopeptide/N-SH2 complexes [Lee 1994, Hayashi 2017] the IRS-1 pY1172 sequence is expected to have several favorable interactions, since it has apolar residues at positions +1, +3 and +5, which point towards the hydrophobic groove in the N—SH2 sequence, and anionic amino acids at positions +2 and +4, which can interact with K residues in the BG loop.

[0211] In an effort to further optimize the binding affinity, authors have analyzed in silico the effect of different apolar amino acids in position +5. Free energy calculations indicated that substitution of L with the bulkier W (but not with F) could be favorable (FIG. 4). The additional substitution of D in +4 with the longer E was evaluated as well, in view of a possible strengthening of the electrostatic interactions. In this case, no further increase in binding affinity was predicted (FIG. 4). Analogs with F or W at position +5 (P8F5 and P8W5), as well as a labeled analog with the L to W substitution (CF-P9W5, Table 1) were synthesized and studied experimentally (FIG. 5). As predicted, introduction of W in +5 was highly favorable, leading to reduction in the dissociation constant by an order of magnitude, both for the labeled and the unlabeled analog (Tables 2 and 3). As a consequence, the K.sub.d for the P8W5 analog was 1.6±0.4 nM. By contrast, the additional D to E substitution resulted in a slight loss in binding affinity (FIG. 5 and Table 2 and 3).

[0212] Based on these results, further studies concentrated on the peptide with W in position +5.

3) Binding Selectivity

3.1) The Modified Sequence Is Highly Selective for the N-SH2 Domain of SHP2.

[0213] The selectivity of binding of CF-P9W5 was first assessed with respect to the C-SH2 domain of SHP2, again with the fluorescence anisotropy assay (FIG. 6). As reported in Table 2, the affinity for the C-SH2 domain was almost 1000 times less than that for the N-SH2 domain.

[0214] A more complete analysis of the binding selectivity was performed on a protein array of 97 human SH2 domains (FIG. 7). An analogue of CF-P9W5 was employed in this assay, where CF was substituted with the Cy3 dye, suitable for detection in the array reader. Control binding experiments showed that the change in fluorophore did affect peptide binding affinity only marginally (Table 2).

[0215] Strikingly, significant binding was observed only with the N-SH2 domain of SHP2, and, to a lesser extent, to the SH2 domain of the adapter protein APS (also called SHP2B2). It is worth noting that binding to the N-SH2 domain of SHP1, which has the highest identity with that of SHP2 [Liu 2006], was negligible, too.

4) Engineering Resistance to Degradation

4.1) Introduction of a Non-hydrolysable pY Mimic Is Compatible With Low nM Binding Affinity

[0216] In view of intracellular or in vivo applications of the peptide, it is essential to make it resistant to degradation. The most labile moiety is the phosphate group of the pY residue, which can be hydrolyzed by protein tyrosine phosphatase, possibly also including SHP2, of which IRS-1 pY 1172 has been shown to be a substrate [Noguchi 1994]. Authors substituted the pY with the non-hydrolysable mimetic phosphonodifluoromethyl phenylalanine (F.sub.2Pmp), which is isosteric with pY and has a total negative charge comparable to that of pY under physiologic pH conditions [Burke 2006].

[0217] Binding experiments demonstrated that the substituted analogue (CF-P9ND0W5, where ND stands for non-dephosphorylatable, Table 1) has a dissociation constant for the N-SH2 domain which is just an order of magnitude lower with respect to that of CF-P9W5 (68 ± 5 nM with respect to 4.6 ± 0.4 nM) (FIG. 8 and Table 2). Similarly, the dissociation constant for the unlabeled peptide P9ND0W5 was 15 ± 0.4 nM (with respect to 1.6 ± 0.4 nM for P8W5) and thus remained in the nM range (Table 3).

[0218] For the sake of brevity, in the following text, CF-P9ND0W5 and its unlabeled analogue P9ND0W5 will be also referred to as the optimized peptides, or CF-OP and OP, respectively.

4.2) The Optimized Peptide OP Is Resistant to Proteolytic Degradation

[0219] To test the resistance to proteases, the optimized peptide OP was incubated in human serum for up to 90 minutes, or in DMEM for three days, and then analyzed by HPLC. No significant degradation was observed in these time frames (FIG. 9). By contrast, the octadecapeptide HPA3NT3 [Park 2008], which authors used as a positive control, was completely degraded already after 5 minutes (data not shown). This result is important for in vivo applications of the peptide.

5) Binding to and Activation of the Whole SHP2 Protein

5.1) OP Binds to Pathogenic Mutants With Much Higher Affinity Than to the WT

[0220] As discussed in the introduction, authors and others have hypothesized that in the autoinhibited state the conformation of the N-SH2 domain prevents efficient association to binding partners, while the binding affinity to phosphorylated sequences is maximized in the open, active state [Keilhack 2005; Bocchinfuso 2007, Martinelli 2008, LaRochelle 2018]. This model has many relevant consequences, because it implies that pathogenic mutants have a twofold effect: they increase the activity of the phosphatase, but also its affinity towards binding partners. In principle, both effects could be the origin of the hyperactivation of the signal transduction pathways involved in the pathologies caused by PTPN11 mutations.

[0221] Notwithstanding the relevance of this aspect, to the best of our knowledge, no direct phosphopeptide binding experiments to the whole SHP2 protein have ever been performed, possibly due to the fact that pY can be dephosphorylated by the PTP domain. Now, peptide OP and its fluorescent analogue CF-OP allow us to directly assess the hypothesis described above.

[0222] FIG. 10 and Table 2 report the results of binding experiments performed with CF-OP and WT SHP2 or the pathogenic mutants A72S, E76K, D61H, F71L and E76V. E76K is among the most common lesions associated with leukemia, and has never been observed in individuals with Noonan syndrome (NS). This mutation is strongly activating, with a basal activity of the corresponding mutant being at least 10 times higher than that of the WT protein. Conversely, A72S specifically recurs among subjects with NS. In this case, basal activation is only twofold [Bocchinfuso 2007]. Mutations D61H, F71L and E76V have been identified as somatic events in haematopoietic and lymphoid neoplasms [Tartaglia 2003, Tartaglia 2005]. Interestingly, authors observed that the affinity for CF-OP nicely parallels the basal activity of these mutants (FIG. 10). This finding provides a first direct confirmation that the closed, autoinhibited state has a lower affinity for the binding partners, compared to the open, active conformation.

[0223] The present data have also another important consequence: in a cellular environment, the peptide would act as an effective inhibitor of the protein-protein interactions of mutant, hyperactivated SHP2, while it would leave essentially unperturbed the WT protein. This behavior is the exact opposite of what has been observed for allosteric inhibitors, such as SHP099, which have a significantly impaired activity in pathogenic variants of SHP2 [Sun 2018; LaRochelle 2018].

5.2) OP Is Also an Inhibitor of the PTP Domain.

[0224] Based on previous reports of the dephosphorylation of IRS-1 pY 1172 by SHP2 [Noguchi 1994], authors verified if P8 and P8W5 are also a substrate of this protein. As reported in FIG. 11, dephosphorylation was indeed observed, although to a lower extent than for other phosphopeptides.

[0225] Using the non-dephosphorylatable peptide CD-OP, authors measured directly binding to the PTP domain of SHP2 (FIG. 12). Significant association was observed, although with a much lower affinity than with the N-SH2 domain (K.sub.d=10.0 ± 0.8 .Math.M). This finding indicates that in principle, the non-dephosphorylatable OP could act as a double hit SHP2 inhibitor, acting on both protein-protein interactions and catalytic activity.

5.3) OP Activates SHP2 Only Weakly

[0226] SHP2 activation is caused by binding of mono- or bi-phosphorylated peptides. Authors tested the effect of OP on the WT protein, or on the A72S mutant (this experiment is not possible with E76K, as in that case the protein is essentially fully activated also in the absence of phosphopeptides). As shown in FIG. 13, activation was very weak, compared with that induced by the biphosphorylated BTAM peptide, and a significant effect was observed only with the mutant protein. Interestingly, under the experimental conditions used, both the WT and the A72S proteins should be nearly saturated by the OP, according to the binding experiments reported in FIG. 10. A possible explanation of this result is provided by the combination of two effects: opening of the protein, by peptide association to the N-SH2 domain, and partial inhibition of the catalytic activity by association to the phosphatase domain. This finding is interesting, as SHP2 activation by the OP might have been an undesirable side effect. The Authors’ findings indicate that the peptide can inhibit SHP2 protein-protein interactions, without causing a significant increase in catalytic activity.

6) Cell Uptake

6.1) Cell Penetrating Peptides Deliver the Phosphopeptides Intracellularly, Efficiently and Without Toxicity.

[0227] SHP2 is an intracellular target, but spontaneous cell uptake of the highly charged peptides developed here is highly unlikely. To solve this issue, authors developed two different strategies, involving cell-penetrating peptide (CPP) sequences. These peptides are able to spontaneously cross the cell membranes, carrying with them macromolecular cargoes to which they have been associated covalently or non covalently. For the non covalent strategy, authors simply mixed the peptides with the Pep1 CPP [Bobone 2011]. Authors also covalently linked the sequence of fragment 48-57 of the human immunodeficiency virus type 1 TAT to the C-terminus of the peptides (Table 1) [Brooks 2005].

[0228] Fluorescence-activated cell sorting (FACS) experiments on NIH3T3 cells with the CF-P9W5 peptide demonstrated that indeed spontaneous uptake is minimal. By contrast, cell uptake could be efficiently obtained with both the covalent and the non-covalent strategy (FIG. 14). In both cases, a significant, dose dependent intracellular concentration of labeled peptide was detected. No saturation effects were observed in the concentration range investigated. Time-course experiments showed that, even after 2 hours of incubation, cell uptake was not complete (FIG. 14).

[0229] The concentration of Pep1 to be used in the delivery studies was determined based on preliminary experiments, showing that cell uptake was maximal at 10 .Math.M CPP concentration, irrespective of the cargo concentration (FIG. 15). Under these conditions, Pep1 toxicity was negligible. CF-P9W5 TAT did not cause any cell killing at all the concentrations tested (FIG. 16).

7) OP Effectively Reverses the Effects of D61G Mutation in Vivo

[0230] The zebrafish model system was used to explore the in vivo effect of the peptide. Zebrafish SHP2a is highly homologous to human SHP2 (91.2% protein sequence identity); in particular, the sequence of the N-SH2 domain and of the N-SH2/PTP interface are identical in the human and fish proteins. RASopathies-associated mutants, including activating mutants of Shp2a, greatly impact zebrafish development. In humans, the D61G substitution has been found in both NS and leukemia [Kratz 2005] and in animal models it induces both NS-like features and myeloproliferative disease [Araki 2004]. Microinjection of synthetic mRNA encoding NS-associated mutants of Shp2 at the one-cell stage induces NS-like traits [Jopling 2007]. During gastrulation, convergence and extension movement are affected, resulting in oval-shaped embryos, with increased major/minor axis length ratio at 11 hpf [Jopling 2007]. Inventors coinjected Shp2a-D61G mRNA with OP in zebrafish embryos, to investigate whether OP rescues the defective cell movements during gastrulation [Bobone 2020]. As shown in FIG. 7, a dose-dependent decrease in Shp2a-D61G induced major/minor axis ratios was observed, with a rescue of the phenotype that was significant at 5 .Math.M peptide concentration. On the other hand, embryos injected with Shp2a-WT were almost perfect spheres at 11 hpf, and co-injection with 3 .Math.M peptide had no impact on their shape. As expected, a large portion of Shp2a-D61G injected embryos were severely affected and they died during embryonic development, whereas injection of WT Shp2a did not induce significant lethality. Following the survival of Shp2a-D61G injected embryos, a significant and dose dependent improvement in the survival of embryos was observed upon co-injection with 0.3 .Math.M, 3 .Math.M and 5 .Math.M OP. By contrast, lethality of WT Shp2a embryos was not affected by co-injection of 3 .Math.M OP. Altogether, these results indicate that co-injection of the OP rescued the developmental defects induced by a pathogenic, basally activated Shp2a variant, while it had no effect on WT embryos.

Tables

[0231] TABLE-US-00009 Peptide sequences investigated in the invention Abbreviation Sequence R.sub.t (min) [M+H].sup.+ Purity SEQ ID NO: P9 GLN-pY-IDLDL 21.2 (10-40%B in 30′) 1156.5 95% 2 P9Y0 GLN- Y-IDLDL 10.9 (20-50%B in 30′) 1076.6 99% 12 P8 LN-pY-IDLDL 24.3 (10-40 %B in 30′) 1099.4 92% 13 P7 N-pY-IDLDL 16.9 (10-40 %B in 30′) 986.3 92% 14 P8W5 LN-pY-IDLDW 12.5 (20-60%B in 20′) 1172.5 95% 15 P8F5 LN-pY-IDLDF 16.5 (10-95%B in 30′) 1133.5 93% 16 P8E4W5 LN-pY-IDLEW 12.4 (20-60%B in 20′) 1186.5 98% 17 P9ND0W5 (or OP) GLN-F.sub.2Pmp-IDLDW 15.7 (10-50%B in 20′) 1263.4 94% 5 CF-P9 CF-GLN-pY-IDLDL 14.9 (20-50%B in 30′) 1473.5 93% 18 CF-P9Y0 CF-GLN- Y-IDLDL 18.7 (20-50%B in 30′) 1392.6 99% 19 CF-P9W5 CF-GLN-pY-IDLDW 14.0 (20-60%B in 20′) 1545.5 96% 20 Cy3-P9W5 Cy3-GLN-pY-IDLDW 19.6 (20-60%B in 20′) 1626.7 95% 21 CF-P9E4W5 CF-GLN-pY-IDLEW 13.9 (20-60%B in 20′) 1559.6 97% 22 CF-P9ND0W5 (or CF-OP) CF-GLN-F.sub.2Pmp-IDLDW 16.1 (5-65%B in 20′) 1580.4 93% 23 P8W5-TAT LN-pY-IDLDW-GRKKRRQRRR 16.1 (5-65%B in 30′) 2550.4 95% 24 CF-P9W5-TAT CF-GLN-pY-IDLDW-GRKKRRQRRR 18.2 (5-65%B in 30′) 2924.6 90% 10

[0232] All peptides were amidated at the C-terminus. Unlabeled peptides were acetylated at the N-terminus. CF is 5,6 carboxyfluorescein, Cy3 is Cyanine 3 carboxylic acid and F.sub.2Pmp is the non-dephosphorylatable pY mimic phosphonodifluoromethyl phenylalanine . Retention times (Rt) for the synthetic peptides obtained from RP-HPLC (the different elution conditions used for the various peptides are shown in brackets) and molecular weights for the synthetic peptides experimentally determined by ESI-MS spectrometry, and purities are reported.

TABLE-US-00010 Dissociation constants obtained from the fluorescence anisotropy binding experiments. Peptide Domain/Protein K.sub.D (nM) CF-P9 N-SH2 53 ± 2 CF-P9Y0 N-SH2 6600 ± 600 CF-P9W5 N-SH2 4.6 ± 0.4 CF-P9W5 C-SH2 4200 ± 300 Cy3-P9W5 N-SH2 23 ± 2 CF-P9E4W5 N-SH2 8.2 ± 0.7 CF-P9ND0W5 (CF-OP) N-SH2 68 ± 5 CF-P9ND0W5 (CF-OP) PTP 10000 ± 800 CF-P9ND0W5 (CF-OP) WT 930 ± 70 CF-P9ND0W5 (CF-OP) A72S 400 ± 40 CF-P9ND0W5 (CF-OP) E76V 330 ± 10 CF-P9ND0W5 (CF-OP) D61H 170 ± 10 CF-P9ND0W5 (CF-OP) F71L 140 ± 10 CF-P9ND0W5 (CF-OP) E76K 48 ± 2

TABLE-US-00011 Inhibition constants obtained from the displacement experiments. All the measurements were performed on the N-SH2 domain of SHP2. Experiments were performed at [N-SH2]= 3.4 nM and [CF-P9W5]=0.5 nM (for P8 and P9ND0W5) or 0.1 nM (for the other peptides) Peptide IC.sub.50 (nM) K.sub.i (nM) P8 47 ± 4 25 ± 4 P8F5 16 ± 1 9 ± 2 P8W5 5.4 ± 0.3 1.6 ± 0.4 P8E4W5 11 ± 1 5 ± 2 P9ND0W5 32 ± 5 15 ± 5

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