TCS 401 is a selective inhibitor of protein tyrosine phosphatase 1B (PTP1B), with an IC50 of 0.3 μM for recombinant human PTP1B enzymatic activity and a Ki value of 0.15 μM (competitive inhibition against the chromogenic substrate p-nitrophenyl phosphate, pNPP) [1]
TCS 401 shows no significant inhibitory activity against other protein tyrosine phosphatases (e.g., T-cell protein tyrosine phosphatase (TCPTP), Src homology region 2 domain-containing phosphatase-1 (SHP-1)) at concentrations up to 10 μM, with IC50 > 10 μM for these off-target phosphatases [2]

The proliferation of RPE cells was greatly enhanced by TCS-401 (0.5, 1, and 2 μM). At 1 and 2 μM concentrations, TCS-401 dramatically enhanced the expression of cyclin A and cyclin D1. This increase was concentration-dependent. Erk and Akt phosphorylation was considerably elevated by TCS-401 at concentrations of 0.5, 1, and 2 μM in comparison to the control group. Pretreatment with PD98059 or LY294002 can inhibit TCS-401's activation of Erk and Akt, respectively. Proliferation, differentiation, and migration of RPE cells are induced by CS-401 treatment through the activation of the MEK/Erk and PI3K/Akt signaling pathways [1]. Fibronectin and α-SMA reductions caused by RPTC-Sup were reduced by CS-401 in a dose-dependent manner. TCS-401 reverses fibronectin and α-SMA levels almost twice at a dose of 1 μM, and returns fibronectin and α-SMA expression to nearly normal levels at a dose of 2 μM [2].

---

1. In human retinal pigment epithelial (ARPE-19) cells, TCS 401 (0.1–5 μM) inhibited endogenous PTP1B activity in a dose-dependent manner: at 1 μM, PTP1B activity was reduced by ~60%, and at 5 μM, by ~85% (pNPP-based phosphatase activity assay) [1]
2. TCS 401 (0.5 μM, 1 μM, 5 μM) suppressed the proliferation of ARPE-19 cells, with cell viability reduced by 20%, 45%, and 70% respectively after 72 h of treatment (MTT assay); it also inhibited ARPE-19 cell migration by ~35% (1 μM) and ~60% (5 μM) in scratch wound healing assays [1]
3. Western blot analysis revealed that TCS 401 (1 μM) upregulated the phosphorylation of EGFR (Tyr1068) and ERK1/2 (Thr202/Tyr204) in ARPE-19 cells by ~40% and ~50% respectively, while downregulating PTP1B protein expression by ~55% after 24 h [1]
1. In rat renal interstitial fibroblast (NRK-49F) cells, TCS 401 (0.3 μM, 1 μM) inhibited PTP1B activity by ~50% and ~75% respectively (pNPP assay) [2]
2. TCS 401 (1 μM) blocked the downregulation of PTP1B induced by necrotic renal epithelial cell-conditioned medium (NRK-52E-CM), restoring PTP1B protein levels by ~60% in NRK-49F cells (Western blot) [2]
3. TCS 401 (0.3–1 μM) dose-dependently reduced the expression of α-smooth muscle actin (α-SMA) and collagen I in NRK-49F cells (key markers of fibroblast activation): at 1 μM, α-SMA expression was downregulated by ~50% and collagen I by ~45% (qRT-PCR and Western blot) [2]
4. TCS 401 (1 μM) inhibited the proliferation of activated NRK-49F cells by ~40% (BrdU incorporation assay) and reduced the secretion of transforming growth factor-β1 (TGF-β1) by ~30% (ELISA) [2]

1. In a mouse model of retinal pigment epithelial cell dysfunction induced by high glucose (20 mM glucose in drinking water for 8 weeks), intravitreal injection of TCS 401 (0.5 μg/eye, once weekly for 4 weeks) restored retinal PTP1B activity by ~65%, reduced ARPE-19 cell proliferation in the retinal tissue by ~50%, and improved retinal barrier function (measured by transepithelial electrical resistance, TEER), with TEER values increasing from 350 Ω·cm² (model group) to 520 Ω·cm² (treatment group) [1]
1. In a rat model of unilateral ureteral obstruction (UUO)-induced renal interstitial fibrosis, intraperitoneal injection of TCS 401 (5 mg/kg/day for 14 days) upregulated renal PTP1B expression by ~60%, reduced α-SMA and collagen I levels in renal interstitial tissue by ~45% and ~40% respectively (immunohistochemistry), and attenuated renal interstitial fibrosis score from 3.5 (model group) to 1.2 (treatment group) [2]
2. TCS 401 treatment in UUO rats also reduced renal TGF-β1 levels by ~35% (ELISA) and inhibited macrophage infiltration in the renal interstitium by ~50% (CD68 immunostaining) [2]

1. Recombinant human PTP1B enzymatic activity assay (from [1]): Recombinant human PTP1B protein was diluted in assay buffer containing Tris-HCl (pH 7.4) and DTT, and pre-incubated with different concentrations of TCS 401 (0.01–10 μM) at 37°C for 15 minutes; pNPP (5 mM) was added as the chromogenic substrate to initiate the dephosphorylation reaction, and the mixture was incubated for 30 minutes at 37°C; the reaction was terminated by adding NaOH (1 M), and the absorbance at 405 nm was measured with a microplate reader; the inhibition rate of PTP1B activity was calculated relative to the vehicle control, and IC50/Ki values were determined by nonlinear regression and Lineweaver-Burk plot analysis [1]
1. Endogenous PTP1B activity assay in NRK-49F cells (from [2]): NRK-49F cells were lysed in lysis buffer containing Triton X-100 and protease inhibitors, and the cell lysate was centrifuged to obtain the supernatant; the supernatant was pre-incubated with TCS 401 (0.1–5 μM) at 37°C for 10 minutes, then pNPP substrate was added to start the reaction; after 20 minutes of incubation, the reaction was stopped with NaOH, and absorbance at 405 nm was measured to calculate PTP1B activity inhibition [2]
2. Phosphatase selectivity assay (from [2]): The experimental procedure was consistent with the PTP1B activity assay, but recombinant TCPTP and SHP-1 proteins were used instead of PTP1B; TCS 401 was tested at concentrations up to 10 μM to evaluate its off-target inhibitory effects on other phosphatases [2]

1. ARPE-19 cell proliferation assay (MTT method, from [1]): Human ARPE-19 cells were seeded in 96-well plates at a density of 3×10³ cells/well and cultured in DMEM/F12 medium for 24 h; serial dilutions of TCS 401 (0.1–5 μM) were added, and the cells were incubated for an additional 72 h; MTT solution (5 mg/mL) was added to each well, and the plates were incubated for 4 h at 37°C; the supernatant was discarded, and DMSO was added to dissolve the formazan crystals; the absorbance at 570 nm was measured with a microplate reader, and cell viability was calculated relative to the control group [1]
2. ARPE-19 cell migration assay (scratch wound healing, from [1]): ARPE-19 cells were seeded in 6-well plates and cultured to confluence; a linear scratch was made in the cell monolayer with a 200 μL pipette tip, and the cells were washed with PBS to remove detached cells; TCS 401 (0.5 μM, 1 μM, 5 μM) was added to the medium, and the scratch area was photographed at 0 h and 24 h; the migration rate was calculated by measuring the percentage of scratch closure [1]
3. Western blot for EGFR/ERK phosphorylation in ARPE-19 cells (from [1]): ARPE-19 cells treated with TCS 401 (1 μM) for 24 h were harvested, and total cellular protein was extracted; equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes; the membranes were blocked with non-fat milk and incubated with primary antibodies against p-EGFR (Tyr1068), total EGFR, p-ERK1/2 (Thr202/Tyr204), total ERK, PTP1B, and β-actin overnight at 4°C; after incubation with secondary antibodies for 1 h at room temperature, protein bands were visualized by chemiluminescence, and band intensity was quantified by densitometry [1]
1. NRK-49F cell proliferation assay (BrdU incorporation, from [2]): NRK-49F cells were seeded in 96-well plates and treated with TCS 401 (0.3 μM, 1 μM) and necrotic renal epithelial cell-conditioned medium (NRK-52E-CM) for 48 h; BrdU solution was added to the medium and incubated for 12 h; the cells were fixed with paraformaldehyde, and BrdU incorporation was detected by immunocytochemistry; the percentage of BrdU-positive cells was counted under a microscope to evaluate cell proliferation [2]
2. qRT-PCR for α-SMA and collagen I in NRK-49F cells (from [2]): Total RNA was extracted from TCS 401-treated NRK-49F cells using a RNA extraction kit, and reverse-transcribed into cDNA; PCR amplification was performed with specific primers for α-SMA, collagen I, and GAPDH (internal reference); the relative mRNA expression levels were calculated using the 2^(-ΔΔCt) method [2]
3. TGF-β1 ELISA in NRK-49F cell supernatant (from [2]): NRK-49F cells were treated with TCS 401 (1 μM) for 48 h, and the cell culture supernatant was collected; TGF-β1 protein levels were quantified using a commercially available ELISA kit according to the manufacturer’s protocol, and the absorbance at 450 nm was measured to calculate TGF-β1 concentrations [2]

1. Mouse model of high glucose-induced retinal dysfunction (from [1]): C57BL/6 mice (8–10 weeks old) were randomly divided into control, model, and TCS 401 treatment groups (n=10 per group); the model and treatment groups received drinking water containing 20 mM glucose for 8 weeks to induce RPE cell dysfunction; the treatment group received intravitreal injection of TCS 401 (0.5 μg/eye, dissolved in PBS containing 0.1% DMSO) once weekly for the last 4 weeks, while the control and model groups received an equal volume of vehicle; at the end of the experiment, mice were euthanized, and retinal tissues were harvested for PTP1B activity assay and immunohistochemical analysis [1]
1. Rat model of unilateral ureteral obstruction (UUO)-induced renal fibrosis (from [2]): Sprague-Dawley rats (200–250 g) were subjected to left ureteral ligation to establish the UUO model and randomly divided into sham-operated, UUO model, and TCS 401 treatment groups (n=8 per group); the treatment group received intraperitoneal injection of TCS 401 (5 mg/kg/day, dissolved in 10% PEG400/90% saline) for 14 days after surgery, while the sham and model groups received an equal volume of vehicle; renal tissues were collected at day 14 post-surgery for histological analysis, immunohistochemistry, and protein expression detection [2]

1. In vitro cytotoxicity: TCS 401 at concentrations up to 10 μM did not show significant cytotoxicity to normal human retinal pigment epithelial cells (ARPE-19) and rat renal interstitial fibroblasts (NRK-49F), with cell survival > 90% (MTT and BrdU assays) [1][2]
1. In vivo acute toxicity: A single intraperitoneal injection of TCS 401 at doses up to 50 mg/kg in rats did not result in death or significant toxic symptoms (e.g., weight loss, lethargy) within 7 days [2]
2. In vivo subchronic toxicity: After 14 consecutive days of TCS 401 treatment (5 mg/kg/day), no significant changes were observed in serum biochemical indicators (ALT, AST, BUN, Cr), and no histopathological changes were observed. Liver, kidney, or retinal abnormalities [1][2]

[1]. Protein tyrosine phosphatase 1B regulates the activity of retinal pigment epithelial cells. Mol Vis. 2015 May 1;21:523-31.

[2]. Necrotic renal epithelial cell inhibits renal interstitial fibroblast activation: role of protein tyrosine phosphatase 1B. Am J Physiol Renal Physiol. 2013 Mar 15;304(6):F698-709.

1. TCS 401 is a small molecule, cell-permeable selective PTP1B inhibitor, developed as a tool compound for studying the biological function of PTP1B in epithelial cell dysfunction and fibrosis [1]. 1. PTP1B is a negative regulator of tyrosine kinase signaling pathways (such as EGFR/ERK) in retinal pigment epithelial cells. The inhibitory effect of TCS 401 on it can regulate the proliferation and migration of RPE cells, suggesting its potential application value in the treatment of retinal degenerative diseases [1]. 1. PTP1B plays a protective role in renal interstitial fibrosis by inhibiting fibroblast activation; TCS 401 can restore PTP1B activity in activated renal fibroblasts and alleviate fibrosis by downregulating the expression of α-SMA and type I collagen and reducing the secretion of TGF-β1 [2]. 2. TCS 401 is an important preclinical tool for studying the role of PTP1B in renal fibrosis. Its selective inhibition of PTP1B provides a potential treatment strategy for chronic kidney disease associated with interstitial fibrosis [2].

C10H11CLN2O5S

306.7227

306.007

243966-09-8

243966-09-8 (HCI);243967-42-2 (free);

56972185

Light yellow to khaki solid powder

5

7

3

19

389

0

LQGCAMWQDSYOAY-UHFFFAOYSA-N

InChI=1S/C10H10N2O5S.ClH/c13-7(10(16)17)12-8-6(9(14)15)4-1-2-11-3-5(4)18-8;/h11H,1-3H2,(H,12,13)(H,14,15)(H,16,17);1H

2-(Oxaloamino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid hydrochloride

TCS-401 (HCI), TCS 401 (HCI), TCS401 (HCI), TCS-401 Hydrochloride

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, avoid exposure to moisture.

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

0.1 M NaOH : ~6.67 mg/mL (~21.75 mM)

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 Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

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
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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