SHP2 (IC50 = 70 nM)
Protein Tyrosine Phosphatase, Non-Receptor Type 11 (SHP2/PTPN11) (IC50 = 0.071 μM) [2]
Protein Tyrosine Phosphatase, Non-Receptor Type 11 (SHP2/PTPN11) (specifically targeting SHP2 E69K mutant associated with leukemia) [3]
Protein Tyrosine Phosphatase, Non-Receptor Type 11 (SHP2/PTPN11) (allosteric binding to the interface of N-terminal SH2, C-terminal SH2, and PTP domains) [1]

SHP099 exhibits dose-dependent inhibition of tumor growth in models of xenografts. The pharmacodynamic marker p-ERK is exposed and modulated in a dose-dependent manner in the xenografts following single doses of 30 and 100 mg/kg. 19% and 61%, respectively, of tumor growth inhibition can be obtained with a daily oral dose of 10 or 30 mg/kg. 100 mg/kg results in tumor stasis. The Novartis Guide for the Care and Use of Laboratory Animals was followed in all animal studies. Parental KYSE-520 cells were subcutaneously injected into female nude mice in Hank's balanced salt solution in a suspension containing 50% phenol red-free matrigel (BD Biosciences). Mice were given one dose of vehicle control or one by oral gavage for PK/PD studies after tumors had grown to a size of approximately 500 mm3. The mice were then put to sleep at pre-arranged intervals after receiving a single dosage of the compound. At that time, plasma and xenograft fragments were taken out to measure the concentration of 1 and the modulation of p-ERK, respectively. Mice were calipersed twice a week in two dimensions for efficacy studies. Mice were randomized to treatment groups when tumor sizes approached 200 mm3. Mice were given erlotinib (80 mg/kg qd) or vehicle (10, 30, or 100 mg/kg qd) by oral gavage for the efficacy study. The body weight of the mice and the tumor volume were measured twice a week. Using a commercially available kit (Meso Scale Discovery catalog number K15107D), total and phospho-ERK1/2 were measured to evaluate MAPK pathway modulation in xenograft protein lysates. With the exception of incubating the protein lysate overnight, the assay was carried out exactly as Meso Scale Discovery advised.

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1. SHP099 is efficacious in mouse tumour xenograft models, inhibiting the growth of tumours driven by receptor tyrosine kinases by suppressing RAS–ERK signalling [2]

The binding of bis-tyrosylphorphorylated peptides to SHP2's Src Homology 2 (SH2) domains causes allosteric activation of the protein. The release of SHP2's auto-inhibitory interface during the latter activation step makes the SHP2 PTP active and ready for substrate recognition and reaction catalysis. In a prompt fluorescence assay format, the catalytic activity of SHP2 was observed through the use of the surrogate substrate DiFMUP. More specifically, using a final reaction volume of 25 μL and the following assay buffer conditions: 60 mM HEPES, pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% P-20, and 5 mM DTT, the phosphatase reactions were carried out at room temperature in 384-well black polystyrene plate, flat bottom, low flange, non-binding surface (Corning, Cat# 3575). The assay employed to measure the inhibition of SHP2 by the tested compounds (with concentrations ranging from 0.003 to 100 μM) involved incubating 0.5 nM of SHP2 with 0.5 μM of the peptide IRS1_pY1172(dPEG8)pY1222 (sequence: H2NLN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide). Following a 30- to 60-minute incubation period at 25 oC, the reaction was supplemented with the surrogate substrate DiFMUP (Invitrogen, cat# D6567, 200 μM) and further incubated for 30 minutes at 25 oC (200 μM for 2-593, 100 μM for 1-525 construct). Subsequently, 5 μL of a 160 μM bpV(Phen) solution (Enzo Life Sciences cat# ALX-270-204) was added to quench the reaction. Using excitation and emission wavelengths of 340 nm and 450 nm, respectively, a microplate reader (Envision, Perki-Elmer) was used to monitor the fluorescence signal. Normalized IC50 regression curve fitting with control-based normalization was used to analyze the inhibitor dose response curves.

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1. A high throughput screen was conducted to identify progressable chemical matter for SHP2 inhibition; X-ray crystallography was employed to determine the binding location of SHP099 in the allosteric binding pocket of SHP2, and structure-based drug design was used to optimize the inhibitor by characterizing new protein-ligand interactions to enhance SHP2 inhibitory potency and selectivity [1]
2. Enzymatic activity assays were performed to determine the IC50 of SHP099 against SHP2, with the value measured as 0.071 μM; the assays confirmed that SHP099 inhibits SHP2 activity via an allosteric mechanism by stabilizing the auto-inhibited conformation of the protein, rather than direct binding to the active site [2]
3. Enzymatic assays were used to evaluate the inhibitory effect of SHP099 on wild-type and mutant (including E69K) SHP2 proteins, confirming its selective inhibition of the leukemia-associated SHP2 E69K mutant; the binding mode of SHP099 to SHP2 (PDB: 5EHR) was analyzed via co-crystal structure, with key residues (D61, E69, A72, E76) in the N-SH2 (wheat), C-SH2 (green), and PTP (blue) domains identified as relevant to the interaction [3]

p-ERK cellular assay with PerkinElmer's AlphaScreen® SureFireTM Phospho-ERK 1/2 Kit: After being grown in 96-well plate culture for the entire night, KYSE-520 cells (30,000 cells/well) were treated with SHP2 inhibitors for two hours at 37 °C at concentrations of 20, 6.6, 2.2, 0.74, 0.24, 0.08, and 0.027 μM. Thirty microliters of lysis buffer (PerkinElmer), which came with the SureFire phospho-extracellular signal-regulated kinase (p-ERK) assay kit (PerkinElmer), were added to end the incubations. Samples underwent processing in compliance with manufacturer's instructions. Two measurements of the p-ERK fluorescence signal were made using a Perkin Elmer Envision 2101 multilabel reader. The entire ERK signal was used to normalize the percentage of inhibition, and it was then contrasted with the DMSO vehicle control.

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1. For receptor-tyrosine-kinase-driven human cancer cell lines, cell proliferation assays were carried out to assess the anti-proliferative effect of SHP099; cells were exposed to varying concentrations of SHP099, and cell viability was measured to confirm the inhibition of cell proliferation mediated by the suppression of RAS–ERK signalling [2]
2. Stable TF-1 cell lines expressing four different SHP2 mutants (including E69K) were established; immunoblotting was used to analyze SHP2 expression in parental and mutant-expressing TF-1 cells (anti-SHP2 antibody detected both endogenous wildtype and exogenous mutant SHP2, anti-Flag antibody recognized exogenous Flag-tagged SHP2 mutants); the cells were cultured in GM-CSF free medium with different concentrations of SHP099, viable cells were counted on Day 5 to evaluate growth inhibition, and immunoblot analysis of cell lysates was performed after 2 days of SHP099 treatment to examine changes in SHP2-related signalling molecules [3]

10, 30, or 100 mg/kg qd by oral gavage.
Female nude mice were inoculated subcutaneously (3 x 106 cells) in a suspension containing 50% phenol red-free matrigel (BD Biosciences) in Hank’s balanced salt solution with parental KYSE-520 cells.

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1. Mouse tumour xenograft models were established using receptor-tyrosine-kinase-driven human cancer cells; SHP099 was administered orally (specific dosage and frequency not specified in the literature) to nude mice (both male and female were used), and tumour growth was monitored to evaluate the in vivo efficacy of the inhibitor, with the mechanism confirmed as suppression of RAS–ERK signalling in tumour tissues [2]

1. SHP099 has oral bioavailability, but no specific parameters (e.g., half-life, absorption rate, distribution, metabolism, excretion, or percentage of oral bioavailability) are provided [1]

[1]. J Med Chem . 2016 Sep 8;59(17):7773-82.

[2]. Nature . 2016 Jul 7;535(7610):148-52.

[3]. Leukemia . 2018 May;32(5):1246-1249.

1. SHP2 (encoded by PTPN11) is a non-receptor protein tyrosine phosphatase involved in cell growth, differentiation (via the MAPK signaling pathway) and programmed cell death pathway (PD-1/PD-L1); it is an oncoprotein associated with a variety of cancer-related diseases and a potential immunomodulator, so inhibiting SHP2 is a promising therapeutic strategy [1]
2. SHP2 was the first reported oncogenic tyrosine phosphatase; activation mutations of SHP2 are associated with developmental pathologies (e.g., Noonan syndrome) and various cancer types (leukemia, lung and breast cancer, neuroblastoma); SHP2 is widely expressed and mainly regulates cell survival and proliferation by activating the RAS-ERK signaling pathway, and also mediates the PD-1 and BTLA immune checkpoint pathways; reduced SHP2 activity can inhibit tumor cell growth, validating the effectiveness of SHP2 pharmacological inhibition as a cancer treatment method [2]
3. SHP2 E69K is a leukemia-associated mutant, and SHP099 has a selective inhibitory effect on this mutant, highlighting its potential application value in the treatment of SHP2 E69K mutation-driven leukemia [3]

C16H19CL2N5

352.26

351.101

C, 54.55; H, 5.44; Cl, 20.13; N, 19.88

1801747-42-1

SHP099 monohydrochloride;2200214-93-1;SHP099 hydrochloride;1801747-11-4

118238298

Light yellow to yellow solid powder

1.3±0.1 g/cm3

530.4±50.0 °C at 760 mmHg

274.5±30.1 °C

0.0±1.4 mmHg at 25°C

1.626

3.97

2

5

2

23

402

0

ClC1C(=C([H])C([H])=C([H])C=1C1C(N([H])[H])=NC(=C([H])N=1)N1C([H])([H])C([H])([H])C(C([H])([H])[H])(C([H])([H])C1([H])[H])N([H])[H])Cl

YGUFCDOEKKVKJK-UHFFFAOYSA-N

InChI=1S/C16H19Cl2N5/c1-16(20)5-7-23(8-6-16)12-9-21-14(15(19)22-12)10-3-2-4-11(17)13(10)18/h2-4,9H,5-8,20H2,1H3,(H2,19,22)

6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 1.2 mg/mL (3.41 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 12.0 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: ≥ 1.2 mg/mL (3.41 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 12.0 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: ≥ 1.2 mg/mL (3.41 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 12.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 0.67 mg/mL (1.90 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 0.67 mg/mL (1.90 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 6: ≥ 0.13 mg/mL (0.37 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 7: 10 mg/mL (28.39 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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