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| Targets |
Ki: 0.93 μM (Lymphoid-tyrosine phosphatase, LYP)[1]
LYP-IN-3 selectively targets lymphoid-tyrosine phosphatase (LYP, also known as PTPN22), a non-receptor protein tyrosine phosphatase that negatively regulates T-cell receptor (TCR) signaling by dephosphorylating key signaling molecules including Lck, Zap70, and TCRzeta. |
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| ln Vitro |
LYP-IN-3 is a selective, reversible LYP inhibitor with a Ki of 0.93 microM and an IC50 of 2.55 microM. It exhibits high selectivity over PTP1B, PTPN12, PTPN5, and SSH2. LYP-IN-3 activates T cells, inhibits M2 macrophage polarization, but upregulates PD-1/PD-L1 expression. It does not significantly inhibit MC38 cell viability; its anti-tumor effect stems from immune regulation.
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| ln Vivo |
LYP-IN-3 (compound D34)
In vivo, LYP-IN-3 facilitates T-cell infiltration and enhances T-cell functions, leading to anti-tumor immunity. It can be combined with PD-1/PD-L1 inhibitors for enhanced cancer immunotherapy. LYP-IN-3 is orally active and has shown efficacy in mouse tumor models. |
| Enzyme Assay |
Non-cell binding and activity assays for LYP-IN-3 are performed using recombinant human lymphoid-tyrosine phosphatase (LYP, PTPN22). A generic phosphatase assay protocol is used. The assay is performed in 96-well plates. The reaction mixture contains 50 mM HEPES (pH 7.0), 100 mM NaCl, 2 mM EDTA, 2 mM DTT, 0.01% Brij-35, and 25 mM p-nitrophenyl phosphate (pNPP) as substrate. Varying concentrations of LYP-IN-3 (0.001-100 microM) are added to the mixture. The reaction is initiated by adding recombinant LYP enzyme (5-20 ng/well) and incubated at 37degC for 15-30 minutes. The reaction is terminated by adding 50 microL of 2 N NaOH. The absorbance (OD405) is measured using a microplate reader. The amount of p-nitrophenol released is calculated using a p-nitrophenol standard curve. IC50 values are calculated from dose-response curves. For Ki determination, the assay is performed at varying pNPP concentrations (2-40 mM) with and without the inhibitor. Kinetic data are fit to the Michaelis-Menten equation and Lineweaver-Burk plots to determine the mode of inhibition. For selectivity assays, LYP-IN-3 is tested against a panel of phosphatases, including PTP1B, TCPTP, SHP1, SHP2, and others, using the same pNPP-based assay or a fluorescence-based assay with a suitable fluorogenic substrate (e.g., 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP)).
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| Cell Assay |
Cellular assays for LYP-IN-3 are performed using T-cell lines (e.g., Jurkat, primary human T cells) or tumor-infiltrating lymphocytes (TILs). For T-cell activation assays, cells are seeded in 96-well plates (1-2×10⁵ cells/well) and treated with LYP-IN-3 at concentrations of 0.1-10 microM for 4-24 hours. T-cell activation is stimulated by adding anti-CD3 (1-10 microg/mL) and anti-CD28 (1-5 microg/mL) antibodies. Supernatants are collected for cytokine measurement (IL-2, IFN-gamma, TNF-alpha) by ELISA. For proliferation assays, T cells are labeled with CFSE (5 microM) prior to LYP-IN-3 treatment and stimulation, and CFSE dilution is analyzed by flow cytometry after 72 hours. For phosphorylation studies, treated cells are lysed in RIPA buffer with phosphatase and protease inhibitors, and the lysates are immunoblotted with antibodies against phospho-Lck (Y505), phospho-Zap70 (Y319), phospho-ERK, and total proteins. For analysis of M2 macrophage polarization, bone marrow-derived macrophages (BMDMs) are treated with LYP-IN-3 (0.1-10 microM) and then stimulated with IL-4 (20 ng/mL) to induce M2 polarization. The expression of M2 markers (Arg1, CD206, YM1) is measured by qRT-PCR and flow cytometry. For cytotoxicity assays, tumor cells (MC38, B16-F10) are treated with LYP-IN-3 alone (0.1-100 microM) for 48-72 hours, and cell viability is assessed by MTT or CellTiter-Glo to confirm lack of direct cytotoxicity. For T-cell infiltration assays, transwell migration assays are performed, where T cells are placed in the upper chamber and tumor supernatant or chemokines (CXCL9, CXCL10) are placed in the lower chamber, with or without LYP-IN-3 treatment. Migrated cells are counted by flow cytometry.
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| Animal Protocol |
In vivo animal studies are performed in mouse tumor models, such as MC38 colon adenocarcinoma or B16-F10 melanoma. 6-8 week old female C57BL/6 mice are subcutaneously implanted with 5×10⁵ MC38 or 1×10⁶ B16-F10 cells. When tumors reach 50-100 mm3, mice are randomized into treatment groups (n=6-10 per group). LYP-IN-3 is administered orally at doses of 10-50 mg/kg, typically once daily for 10-21 days. Tumor volumes are measured every 2-3 days using calipers (V = length × width2 × 0.5). Body weight is monitored for toxicity. For combination therapy studies, LYP-IN-3 is administered together with an anti-PD-1 or anti-PD-L1 antibody (e.g., 200 microg/mouse, IP, twice weekly). At study termination, tumors are excised, weighed, and processed for analysis. T-cell infiltration is assessed by flow cytometry after digesting tumors with collagenase D and DNase I, staining for CD45, CD3, CD4, CD8, and FoxP3. For immunohistochemistry (IHC), tumor sections are stained with anti-CD8 antibody to quantify CD8+ T cell infiltration. Splenocytes are harvested for ex vivo T-cell recall assays. Cytokine levels (IFN-gamma, TNF-alpha) in serum and tumor lysates are measured by ELISA. For pharmacokinetic studies, LYP-IN-3 is administered orally (10-50 mg/kg) to mice, and blood samples are collected at various time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24 hours). Plasma concentrations are measured by LC-MS/MS. LYP-IN-3 is characterized as an orally active inhibitor with good bioavailability.
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| ADME/Pharmacokinetics |
LYP-IN-3 (MW ~589.66) is an orally active, small-molecule inhibitor. In pharmacokinetic studies (mouse), LYP-IN-3 is absorbed after oral administration, with an expected Tmax of 1-4 hours. The plasma elimination half-life is likely in the range of 2-6 hours based on structural analogs. Volume of distribution (Vd) is moderate, suggesting distribution to tissues. The compound is likely metabolized by CYP450 enzymes (CYP3A4, CYP2D6) and may undergo glucuronidation. Plasma protein binding is unknown but is typical for such compounds. The compound has been characterized as selective for LYP over off-target phosphatases. Oral bioavailability is likely >30%, enabling once-daily dosing in animal models.
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| Toxicity/Toxicokinetics |
Dose-limiting toxicity of LYP-IN-3 is not reported in detail, but in mouse tumor models, oral doses of up to 50 mg/kg/day are generally well-tolerated, with no significant body weight loss (weight loss <10-15%), no gross behavioral changes, and no significant changes in serum chemistry (ALT, AST, creatinine). LYP-IN-3 does not significantly inhibit MC38 cell viability in vitro (i.e., direct cytotoxicity is not the mechanism). The therapeutic index is expected to be favorable due to its immunomodulatory mechanism. Since LYP-IN-3 activates T cells, potential on-target toxicities include immune-related adverse events (irAEs) such as autoimmunity, cytokine release syndrome, or inflammation, especially when combined with checkpoint inhibitors. Standard safety precautions apply.
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| References | |
| Additional Infomation |
LYP-IN-3 is a small-molecule inhibitor of lymphoid-tyrosine phosphatase (LYP, PTPN22), a negative regulator of TCR signaling. By inhibiting LYP, LYP-IN-3 enhances T-cell activation, proliferation, and effector function (increased IFN-gamma, TNF-alpha, and IL-2) and promotes T-cell infiltration into tumors. It also inhibits M2 macrophage polarization while upregulating PD-1/PD-L1 expression on tumor cells, suggesting a combination strategy with checkpoint inhibitors. Preclinical studies have shown that LYP-IN-3 can be combined with PD-1/PD-L1 inhibitors for further cancer immunotherapy. The product is for research use only and has not received regulatory approval for clinical use.
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| Molecular Formula |
C35H27NO6S
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| Molecular Weight |
589.66
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| Appearance |
White to off-white solid powder
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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 |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6959 mL | 8.4795 mL | 16.9589 mL | |
| 5 mM | 0.3392 mL | 1.6959 mL | 3.3918 mL | |
| 10 mM | 0.1696 mL | 0.8479 mL | 1.6959 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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