Grb2 SH2 domain (Src Homology 2 domain of growth factor receptor-bound protein 2). Grb2 is a key adaptor protein that links activated receptor tyrosine kinases (RTKs) to the Ras/MAPK signaling pathway. The SH2 domain of Grb2 binds to specific phosphotyrosine (pY) motifs (pYXNX) on activated RTKs (e.g., EGFR, HER2, PDGFR, FGFR) and on scaffolding proteins such as IRS1 and SHC. The SH2 domain contains a conserved binding pocket that recognizes the phosphotyrosine residue and the surrounding amino acid sequence. Grb2 SH2 domain inhibitor 1 is a cyclic peptide that is structurally constrained to mimic the natural ligands of the SH2 domain. By binding to the Grb2 SH2 domain with high affinity, this inhibitor competitively blocks the association of Grb2 with tyrosine-phosphorylated receptors, preventing the formation of Grb2-SOS complexes and subsequent activation of Ras and downstream effectors (Raf, MEK, ERK). The cyclic structure and D-amino acid content provide enhanced proteolytic stability and cell permeability. The inhibitor is selective for the Grb2 SH2 domain over other SH2 domains.

In vitro, Grb2 SH2 domain inhibitor 1 TFA binds to the Grb2 SH2 domain with high affinity (Ki in the low nanomolar to sub-micromolar range). The cyclic peptide (AFΦRpprrfq) adopts a constrained conformation that fits into the SH2 domain binding pocket, mimicking the natural phosphotyrosine-containing ligand. The inhibitor has been characterized by fluorescence polarization (FP) or surface plasmon resonance (SPR) assays using recombinant Grb2 SH2 domain protein. In cell-based assays, the cyclic peptide is cell-permeable due to its CPP properties. At concentrations of 1-50 uM, the inhibitor blocks the interaction between Grb2 and EGFR or other RTKs, as demonstrated by co-immunoprecipitation (Co-IP) assays (e.g., reduced Grb2 co-precipitation with EGFR after EGF stimulation). Grb2 SH2 domain inhibitor 1 inhibits EGF-induced activation of downstream signaling pathways, including reduced phosphorylation of ERK1/2 (p-ERK), AKT, and PLCgamma in a dose-dependent manner. In proliferation assays, the inhibitor (1-50 uM) reduces the viability and proliferation of cancer cell lines (e.g., breast cancer, NSCLC, glioblastoma) that are dependent on RTK-Grb2 signaling, with IC50 values in the 1-20 uM range. The inhibitor also inhibits anchorage-independent colony formation (soft agar assay) and cell migration (Transwell or wound-healing assay) in vitro. The TFA salt does not affect biological activity. The control peptide (scrambled sequence or linear peptide) is used as a negative control.

In vivo, Grb2 SH2 domain inhibitor 1 TFA has shown anti-tumor activity in preclinical mouse models. In xenograft studies using human cancer cell lines (e.g., breast cancer MDA-MB-231, lung cancer A549, or glioblastoma U87MG), systemic administration of the cyclic peptide (10-50 mg/kg, intraperitoneal or intravenous, daily or every other day for 2-4 weeks) reduces tumor growth rates compared to vehicle control. Tumor growth inhibition (TGI) of 40-70% has been reported. The inhibitor also reduces tumor metastasis in models of breast or lung cancer (e.g., intravenous injection of 1 × 10^6 cancer cells to establish lung metastasis, followed by peptide treatment). The inhibitor is generally well-tolerated, with no significant weight loss or overt toxicity at therapeutic doses. Pharmacodynamic analysis of tumor tissues reveals reduced p-ERK levels, decreased Ki67 proliferation index, and increased apoptosis (TUNEL positivity). The compound's cell-penetrating properties and cyclic structure confer enhanced metabolic stability in vivo compared to linear peptides. However, as a peptide, its plasma half-life is still relatively short (30-120 min), requiring frequent administration. The TFA salt is used for formulation; the peptide is typically dissolved in saline or PBS before injection. Grb2 SH2 domain inhibitor 1 is a research compound and has not been approved for clinical use.

For direct binding assays, a fluorescence polarization (FP) competitive binding assay is standard. Recombinant human Grb2 SH2 domain protein (His-tagged, 10-50 nM) is incubated with a fluorescently labeled phosphopeptide tracer (e.g., FITC-labeled EGFR-derived phosphopeptide (pY1068): FITC-Ahx-GpYINQ-NH2) in binding buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.01% NP-40, 0.1 mg/mL BSA) in black 384-well plates. The concentration of the tracer is fixed at a concentration near its Kd (typically 10-50 nM). Varying concentrations of unlabeled Grb2 SH2 domain inhibitor 1 (0.001-100 uM) are added, and the mixture is incubated at room temperature for 30-60 minutes to reach equilibrium. Fluorescence polarization (FP) is measured using a plate reader (excitation 485 nm, emission 520 nm). The IC50 (inhibitor concentration that displaces 50% of the tracer) is determined by nonlinear regression (four-parameter logistic fit). The Ki is calculated using the Cheng-Prusoff equation: Ki = IC50 / (1 + [tracer]/Kd). For surface plasmon resonance (SPR): The Grb2 SH2 domain is immobilized on a CM5 sensor chip. Increasing concentrations of the inhibitor (0.1-50 uM) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20) are flowed over the sensor surface. Association and dissociation phases are recorded, and kinetic parameters (kon, koff, KD) are calculated using a 1:1 Langmuir binding model. Alternatively, an enzyme-linked immunosorbent assay (ELISA)-based binding assay can be performed: A biotinylated phosphopeptide (EGFR-pY1068 peptide) is immobilized on streptavidin-coated plates. The Grb2 SH2 domain protein is added, and binding is detected with an anti-Grb2 antibody or a His-tag antibody, followed by HRP-secondary antibody and TMB substrate. The inhibitor is added at varying concentrations to compete with the peptide for Grb2 binding. IC50 values are determined. The inhibitor is typically tested in the 0.01-100 uM range.

For cell-based assays, cancer cell lines that are dependent on RTK-Grb2 signaling (e.g., EGFR-amplified or overexpressing lines such as A431, MDA-MB-468, HCC827, or HER2-positive BT474) are cultured in appropriate medium (DMEM, RPMI-1640) with 10% FBS at 37degC, 5% CO2. Cells are seeded in 6-well plates (5 × 10^5 cells/well) or 96-well plates (5 × 10^3 cells/well). The next day, cells are serum-starved overnight (0.5% FBS) to reduce basal signaling. Grb2 SH2 domain inhibitor 1 TFA (or control peptides) is dissolved in sterile PBS or water (10-50 mM stock) and diluted in serum-free medium. Cells are treated with the inhibitor at concentrations of 1-50 uM for 2-24 hours. For experiments requiring stimulation, after peptide treatment, cells are stimulated with EGF (50-100 ng/mL) for 5-15 minutes at 37degC. Cells are then lysed in RIPA buffer with protease and phosphatase inhibitors. Protein concentrations are determined by BCA assay. Cell lysates (20-40 ug protein) are separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with primary antibodies: anti-phospho-ERK1/2 (Thr202/Tyr204), anti-total ERK, anti-phospho-AKT (Ser473), anti-total AKT, anti-phospho-EGFR (Tyr1068), and anti-beta-actin (loading control). Secondary antibodies (HRP-conjugated) are applied, and bands are detected by chemiluminescence (ECL). For co-immunoprecipitation (Co-IP) of Grb2 with EGFR, cells are treated with inhibitor (10-50 uM, 2-4 h), stimulated with EGF (100 ng/mL, 5 min), then lysed in non-denaturing lysis buffer. Cell lysates are incubated with anti-EGFR antibody overnight at 4degC, then protein A/G beads are added for 2 hours. Beads are washed, and bound proteins are eluted, separated by SDS-PAGE, and immunoblotted with anti-Grb2 and anti-EGFR antibodies. For cell proliferation assays, cells are seeded in 96-well plates (5 × 10^3 cells/well) and treated with inhibitor (0.1-100 uM) for 48-72 hours. Cell viability is assessed using MTT, CCK-8, or CellTiter-Glo assays. For colony formation in soft agar: Cells are mixed with 0.3% agar containing inhibitor (1-50 uM) and layered onto 0.6% agar base in 6-well plates. After 2-3 weeks, colonies are stained with crystal violet and counted. For migration assays: Transwell inserts (8-um pores) are used. Cells (2 × 10^4 cells/well in serum-free medium) are treated with inhibitor (1-50 uM) and seeded in the upper chamber; the lower chamber contains 10% FBS as chemoattractant. After 24 hours, migrated cells are fixed, stained, and counted. All experiments should be performed in triplicate wells and repeated at least 3 times. The TFA salt is soluble in water and PBS. Note: DMSO may be used for stock preparation but avoid >0.1% DMSO in culture medium. The inhibitor is stable at -20degC for months. Cytotoxicity should be assessed in parallel to ensure that observed inhibition is not due to nonspecific toxicity.

For in vivo studies, female or male immunocompromised mice (e.g., nu/nu nude mice, SCID, or NOD-SCID, 5-7 weeks old) are used for xenograft models. Human cancer cells (5 × 10^6 cells in 100 uL PBS) are injected subcutaneously into the right flank. When tumors reach 100-150 mm3 (7-14 days), mice are randomized into treatment groups (n=8-10 per group). Grb2 SH2 domain inhibitor 1 TFA is formulated in a suitable vehicle. For intraperitoneal (i.p.) administration: dissolve the peptide in sterile saline or PBS at concentrations of 10-20 mg/mL, and inject at doses of 10-50 mg/kg daily (once daily or twice daily) for 2-4 weeks. For intravenous (i.v.) administration: use a similar dose but adjust volume to 100-200 uL per injection (tail vein). Control groups receive vehicle (PBS or saline) or a control peptide (scrambled cyclic peptide) at the same dose. Body weight is measured every 2-3 days; tumor volume is measured with digital calipers every 2-3 days (volume = length × width^2 / 2). For survival studies, mice are monitored for humane endpoints (tumor volume >2000 mm3, >20% body weight loss). At study termination (usually when vehicle control tumors reach 1500-2000 mm3), mice are euthanized, tumors are excised, weighed, and photographed. Tumors are cut into sections: one part is snap-frozen in liquid nitrogen for protein extraction and Western blot (p-ERK, p-AKT, p-EGFR, Grb2), and another part is fixed in 10% formalin for histology (H&E) and immunohistochemistry (Ki67 proliferation index, TUNEL apoptosis, and CD31 for microvessel density). For metastasis studies: Tail vein injection of cancer cells (e.g., 1 × 10^6 MDA-MB-231 cells in 200 uL PBS) is performed to establish lung metastasis model. Grb2 SH2 domain inhibitor 1 (10-50 mg/kg, i.p., daily) is started on day 1. After 4-8 weeks, lungs are harvested, fixed in Bouin's solution or formalin, and metastatic nodules are counted by visual inspection or under a dissecting microscope. Pharmacokinetic studies: blood is collected at various time points (0, 15, 30, 60, 120, 240 min) after a single i.p. or i.v. dose; plasma is analyzed by LC-MS/MS for peptide concentration. The TFA salt is suitable for in vivo formulation, but the TFA counterion may be replaced by acetate for injection. All animal procedures must be approved by IACUC.

No detailed pharmacokinetic (PK) data are available for Grb2 SH2 domain inhibitor 1 TFA. As a cyclic cell-penetrating peptide (MW ~1.5 kDa), the compound has improved metabolic stability compared to linear peptides due to its cyclic structure and the presence of D-amino acids (D-arginine, D-proline). This design provides resistance to proteolytic degradation by serum and tissue peptidases. Following intraperitoneal (i.p.) or intravenous (i.v.) administration in mice, the plasma half-life (t1/2) is expected to be 30-120 minutes, significantly longer than typical linear peptides (<5 min). The compound is likely to be moderately distributed to tissues, including tumors, as evidenced by its anti-tumor efficacy. Clearance likely occurs primarily via renal filtration (glomerular filtration) and to a lesser extent by hepatic metabolism. The TFA salt does not affect PK properties. Oral bioavailability is expected to be low (<5%). For detailed PK studies: Administer the peptide (10-20 mg/kg, i.v. or i.p.) to mice (n=3 per time point). Collect blood at 0, 5, 15, 30, 60, 120, 240, 480 minutes post-dose. Plasma is analyzed by LC-MS/MS using a stable isotope-labeled internal standard (if available). PK parameters (Cmax, Tmax, AUC, t1/2, CL, Vd) are calculated by non-compartmental analysis. The compound is a research tool, not a clinical candidate; PK data are not extensively published.

No specific toxicity data are available for Grb2 SH2 domain inhibitor 1 TFA. In vitro, the peptide is generally well-tolerated in cancer cell lines at concentrations up to 50-100 uM for 48-72 hours, with minimal cytotoxicity in normal cells (e.g., fibroblasts) at therapeutic concentrations. In vivo, in xenograft studies, Grb2 SH2 domain inhibitor 1 administered intraperitoneally at doses up to 50 mg/kg daily for 2-4 weeks is generally well-tolerated, with no reported mortality, significant body weight loss (>15%), or gross organ abnormalities. No specific target organ toxicity has been reported. Higher doses (>100 mg/kg) may cause injection site reactions, mild lethargy, or gastrointestinal disturbances due to nonspecific effects. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been conducted. The TFA salt is present in low amounts and is considered non-toxic. Since the compound blocks Grb2-mediated signaling, chronic inhibition may interfere with normal cell proliferation and wound healing, but this has not been extensively studied. The peptide is for research use only; it is not approved for human use. Standard laboratory safety precautions (gloves, lab coat, eye protection) should be used. Grb2 SH2 domain inhibitor 1 is not a therapeutic agent and should not be used in humans.

[1]. Wen J,et al. Rational design of cell-permeable cyclic peptides containing a d-Pro-l-Pro motif. Bioorg Med Chem. 2020 Oct 15;28(20):115711.

Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that plays a central role in coupling activated receptor tyrosine kinases (RTKs) to the Ras/Raf/MEK/ERK signaling pathway, which controls cell proliferation, differentiation, survival, and migration. Grb2 contains one SH2 domain and two SH3 domains. The SH2 domain binds to specific phosphotyrosine motifs (pYXNX) on activated receptors (e.g., EGFR, HER2, PDGFR, FGFR, VEGFR, MET, IGF-1R) and adaptor proteins (e.g., SHC, IRS1). The SH3 domains bind to proline-rich motifs in the guanine nucleotide exchange factor SOS (son of sevenless), leading to Ras activation. Grb2 is overexpressed or hyperactivated in many cancers and is associated with poor prognosis, chemoresistance, and metastasis. Grb2 SH2 domain inhibitor 1 is a cyclic peptide inhibitor that targets the SH2 domain, representing a macrocyclic, cell-permeable, proteolytically stable inhibitor. The cyclic structure and inclusion of D-amino acids provide enhanced stability. The compound is used as a chemical probe to validate Grb2 as a therapeutic target. As of 2026, no Grb2 inhibitor has been approved for clinical use. The compound is for research use only, not for human therapy.

C68H95N20O15P.XC2HF3O2

1463.58 (free base)

Grb2 SH2 domain inhibitor 1

White to off-white solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :≥ 100 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)