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Purity: ≥98%
SHP099 (SHP-099), identified through HTS and structure-based drug design, is a potent, selective, orally bioavailable, and highly efficacious allosteric inhibitor of SHP2 [Src homology-2 domain containing protein tyrosine phosphatase-2] with anticancer and anti-osteoarthritis activity. It inhibits SHP2 with an IC50 of 70 nM. The PTPN11 gene encodes SHP2, a nonreceptor protein tyrosine phosphatase (PTP) that is involved in cell growth and differentiation through the MAPK signaling pathway. Additionally, SHP2 is said to be crucial to the PD-1/PD-L1 pathway, which causes programmed cell death. SHP099 functions as an allosteric modulator (inhibitor) of SHP2, stabilizing the autoinhibited conformation. The binding location in a previously unidentified allosteric binding pocket was identified by X-ray crystallography.
| Targets |
SHP2 (IC50 = 70 nM)
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| ln Vitro |
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| ln Vivo |
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| Enzyme Assay |
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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| Cell Assay |
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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| References |
| Molecular Formula |
C16H19CL2N5
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| Molecular Weight |
352.26
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| Exact Mass |
351.10
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| Elemental Analysis |
C, 54.55; H, 5.44; Cl, 20.13; N, 19.88
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| CAS # |
1801747-42-1
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| Related CAS # |
SHP099 monohydrochloride;2200214-93-1;SHP099 hydrochloride;1801747-11-4
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| Appearance |
Solid powder
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| SMILES |
CC1(CCN(CC1)C2=CN=C(C(=N2)N)C3=C(C(=CC=C3)Cl)Cl)N
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| InChi Key |
YGUFCDOEKKVKJK-UHFFFAOYSA-N
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| InChi Code |
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)
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| Chemical Name |
6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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. 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. View More
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. 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. 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. 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. 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. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8388 mL | 14.1941 mL | 28.3881 mL | |
| 5 mM | 0.5678 mL | 2.8388 mL | 5.6776 mL | |
| 10 mM | 0.2839 mL | 1.4194 mL | 2.8388 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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