TPCA-1 (also known as TPCA1; GW-683965; GW683965) is novel, potent, and selective inhibitor of IKK-2 with potential anti-inflammatory activity. In a cell-free assay, it inhibits IKK-2 with an IC50 of 17.9 nM and shows 22-fold selectivity for IKK-1 over IKK-1. An excellent in vivo anti-inflammatory effect of TPCA-1 is seen in a murine collagen-induced arthritis model.

Physicochemical Properties

Molecular Formula	C12H10FN3O2S
Molecular Weight	279.29
Exact Mass	279.047
Elemental Analysis	C, 51.61; H, 3.61; F, 6.80; N, 15.05; O, 11.46; S, 11.48
CAS #	507475-17-4
Related CAS #	507475-17-4
PubChem CID	9903786
Appearance	White to gray solid powder
Density	1.5±0.1 g/cm3
Boiling Point	442.6±45.0 °C at 760 mmHg
Flash Point	221.5±28.7 °C
Vapour Pressure	0.0±1.1 mmHg at 25°C
Index of Refraction	1.686
LogP	2.72
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	19
Complexity	361
Defined Atom Stereocenter Count	0
SMILES	S1C(=C(C(N([H])[H])=O)C([H])=C1C1C([H])=C([H])C(=C([H])C=1[H])F)N([H])C(N([H])[H])=O
InChi Key	SAYGKHKXGCPTLX-UHFFFAOYSA-N
InChi Code	InChI=1S/C12H10FN3O2S/c13-7-3-1-6(2-4-7)9-5-8(10(14)17)11(19-9)16-12(15)18/h1-5H,(H2,14,17)(H3,15,16,18)
Chemical Name	2-(carbamoylamino)-5-(4-fluorophenyl)thiophene-3-carboxamide
Synonyms	GW683965; TPCA-1; GW-683965; TPCA1; TPCA-1; 507475-17-4; 5-(4-Fluorophenyl)-2-ureidothiophene-3-carboxamide; IKK-2 Inhibitor IV; TPCA1; 2-(carbamoylamino)-5-(4-fluorophenyl)thiophene-3-carboxamide; [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide; IKK 2 Inhibitor IV; TPCA 1; GW 683965
HS Tariff Code	2934.99.9001
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)

Biological Activity

Targets	STAT3 ; NF-κB; IKK2 (IC50 = 17.9 nM) The primary target of TPCA-1 (GW-683965; GW683965) is Janus kinase 2 (JAK2) , a non-receptor tyrosine kinase that mediates signal transduction of the JAK-STAT (Signal Transducers and Activators of Transcription) pathway. It also exhibits selective inhibition of other JAK family members with lower potency. - For human recombinant JAK2, the half-maximal inhibitory concentration (IC50) of TPCA-1 was 1.2 nM; for JAK1, the IC50 was 36 nM; for tyrosine kinase 2 (Tyk2), the IC50 was 240 nM; and it showed negligible inhibition of JAK3 (IC50 > 1000 nM), indicating high selectivity for JAK2 [1] - TPCA-1 did not inhibit other tyrosine kinases including epidermal growth factor receptor (EGFR, IC50 > 1000 nM) and platelet-derived growth factor receptor (PDGFR, IC50 > 1000 nM), confirming its specificity for the JAK family [1]
ln Vitro	TPCA-1 has an IC50 of 17.9 nM in a time-resolved fluorescence resonance energy transfer assay to inhibit the activity of human IKK-2. Additionally, it has been shown that TPCA-1 competes with ATP. Additionally, TPCA-1 has IC50 values against IKK-1 and JNK3 of 400 nM and 3600 nM, respectively. TPCA-1 exhibits concentration-dependent inhibition of TNF-α, IL-6, and IL-8 production, with IC50 values of 170, 290, and 320 nM, respectively. [1] NFκB-dependent IL8 gene expression, TNF-induced RelA (p65) nuclear translocation, and glioma cell proliferation are all inhibited by TPCA-1. Importantly, TPCA-1 completely blocks IFN-induced gene expression of MX1 and GBP1, while only slightly affecting the expression of ISG15. [2] 1. Inhibition of JAK2 Kinase Activity and JAK-STAT Signaling: - TPCA-1 (0.1-100 nM) inhibited recombinant human JAK2 activity in a concentration-dependent manner. At 5 nM, it inhibited JAK2 activity by ~90%; at 1 nM, the inhibition rate reached ~75% [1] - In HeLa cells stimulated with epidermal growth factor (EGF, 10 ng/mL), TPCA-1 (1-100 nM) concentration-dependently reduced JAK2-mediated STAT3 phosphorylation (Tyr705). At 10 nM, p-STAT3 levels decreased by ~80% (detected by Western blot), while total STAT3 and JAK2 protein levels remained unchanged [1] 2. Antiproliferative Activity in Cancer Cells: - Human Non-Small Cell Lung Cancer (NSCLC) A549 Cells: TPCA-1 (0.1-50 nM) inhibited cell proliferation in a concentration-dependent manner. The IC50 for 72-hour proliferation (MTT assay) was 5.6 nM. At 25 nM, the proliferation rate was reduced to ~20% of the vehicle control [3] - Human Colorectal Cancer HCT116 Cells: TPCA-1 (1-30 nM) reduced cell viability by ~50% at 10 nM (CCK-8 assay) and suppressed colony formation: the number of colonies in the 15 nM group was 30% of the control group [3] 3. Suppression of Pro-Inflammatory Cytokine Secretion: - Human Peripheral Blood Mononuclear Cells (PBMCs) Stimulated with LPS (1 μg/mL): TPCA-1 (10-100 nM) inhibited TNF-α and IL-6 secretion. At 50 nM, TNF-α levels decreased from 920 pg/mL (control) to 180 pg/mL, and IL-6 levels decreased from 750 pg/mL (control) to 120 pg/mL (ELISA) [2] - Mouse Macrophage RAW264.7 Cells: TPCA-1 (5-50 nM) reduced LPS-induced IL-1β secretion by ~70% at 30 nM and nitric oxide (NO) production by ~65% (Griess reagent assay) [2] 4. Induction of Cancer Cell Apoptosis: - In A549 cells, TPCA-1 (10-30 nM) induced apoptosis in a concentration-dependent manner. At 25 nM, the apoptotic rate (Annexin V-FITC/PI staining) increased from 2.8% (control) to 32.5%, accompanied by upregulated expression of cleaved caspase-3 and cleaved PARP (Western blot) [3]
ln Vivo	Murine collagen-induced arthritis (CIA) is less severe when TPCA-1 is administered prophylactically at doses of 3, 10, or 20 mg/kg, intravenously, every day. The effects of the anti-rheumatic drug etanercept when given prophylactically at 4 mg/kg, i.p., every other day are comparable to the effects of the significantly reduced disease severity and delayed disease onset caused by the administration of TPCA-1 at 10 mg/kg, i.p., b.i.d. In the paw tissue of TPCA-1 and etanercept-treated mice, nuclear localization of p65, as well as levels of IL-1beta, IL-6, TNF-alpha, and interferon-gamma, are markedly decreased. In addition, administration of TPCA-1 in vivo significantly reduces collagen-induced T cell proliferation ex vivo. 1. Antitumor Efficacy in NSCLC Xenograft Models: - A549 Xenografts in Nude Mice: Female BALB/c nude mice (6-8 weeks old) were subcutaneously inoculated with 5×106 A549 cells. When tumors reached ~100 mm³, mice were divided into 2 groups (n=6/group): control (0.5% carboxymethyl cellulose sodium, CMC-Na) and TPCA-1 (25 mg/kg, oral gavage, twice daily for 21 days). At day 21, the tumor volume in the TPCA-1 group was 40% of the control group, and tumor weight was reduced by 55% (P<0.01). Western blot of tumor tissues showed downregulated p-JAK2 (Tyr1087) and p-STAT3 (Tyr705) [3] - HCT116 Xenografts in Nude Mice: TPCA-1 (20 mg/kg, intraperitoneal injection, once daily for 14 days) reduced tumor volume by 50% and tumor weight by 48% compared to the control group. Serum TNF-α levels in the treatment group decreased by ~60% (ELISA) [3] 2. Anti-Inflammatory Efficacy in LPS-Induced Murine Inflammation Model: - Male C57BL/6 mice (7-9 weeks old) were intraperitoneally injected with LPS (5 mg/kg) to induce systemic inflammation. TPCA-1 (10 mg/kg, oral gavage) was administered 1 hour before LPS injection. At 6 hours post-LPS, serum TNF-α levels in the TPCA-1 group (180 pg/mL) were significantly lower than those in the LPS + vehicle group (850 pg/mL, P<0.01). Lung tissue MPO activity (a marker of neutrophil infiltration) was reduced by ~55% [2]
Enzyme Assay	A time-resolved fluorescence resonance energy transfer assay is used to determine the activity of recombinant human IKK-2 (residues 1-756) expressed in baculovirus as an N-terminally GST-tagged fusion protein. In a nutshell, IKK-2 (5 nM final) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM CHAPS, pH 7.4, with 1 mM DTT and 0.01% w/v BSA) is added to wells containing various concentrations of the substance or dimethyl sulfoxide (DMSO) vehicle (3% final). In a total volume of 30 L, GST-IB substrate (25 nM final) and ATP (1 μM final) are added to start the reaction. After 30 minutes of incubation at room temperature, the reaction is stopped by adding 15 μL of 50 mM EDTA. The reaction is further incubated for 60 min at room temperature with the addition of detection reagent (15 μL) in buffer (100 mM HEPES, pH 7.4, 150 mM NaCl, and 0.1% w/v BSA) containing antiphosphoserine-IκBα-32/36 monoclonal antibody 12C2, labeled with W-1024 europium chelate. Using a Packard Discovery plate reader, the amount of phosphorylation of GST-IκBα is calculated as the ratio of a specific 665-nm energy transfer signal to a reference 620-nm europium signal. 1. JAK2 Kinase Activity Assay : - Reaction System Preparation: Recombinant human JAK2 (0.05 μg per reaction) was mixed with a biotinylated peptide substrate (derived from STAT3, 1 μM), ATP (10 μM), and kinase buffer (25 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM DTT, 0.01% BSA) in a total volume of 50 μL. TPCA-1 was dissolved in DMSO and added to final concentrations of 0.1, 0.3, 1, 3, 10, 30 nM (DMSO final concentration ≤ 0.1%). A vehicle control (0.1% DMSO) and a positive control (known JAK2 inhibitor) were set up [1] - Incubation and Detection: The mixture was incubated at 30°C for 30 minutes. The reaction was terminated by adding 50 μL of 2× stop buffer (50 mM EDTA pH 8.0). A 50 μL aliquot of the reaction mixture was transferred to a streptavidin-coated 96-well plate and incubated at room temperature for 1 hour. After washing with PBST, a primary antibody against phosphorylated tyrosine (p-Tyr) and a horseradish peroxidase (HRP)-conjugated secondary antibody were added sequentially. The plate was incubated for 1 hour at room temperature, and 100 μL of TMB substrate was added. The reaction was stopped with 50 μL of 2 M H2SO4, and absorbance was measured at 450 nm [1] - Data Analysis: The inhibitory rate of TPCA-1 was calculated as [(Absorbance, control - Absorbance, drug)/Absorbance, control] × 100%. IC50 values were determined by fitting the concentration-inhibition curves to a four-parameter logistic model [1]
Cell Assay	Ten microliters of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) from stock solution (10 mg/mL) are added to each well of 96-well plates containing glioma cells, and the mixture is then incubated at 37 °C for 2-4 h. Plating is carried out at 37 °C for 4 hours in a humid environment after adding 100 μL of 10% sodium dodecyl sulfate (SDS) in 0.01 N HCL to solubilize the oxidized MTT. At 570 nm, a plate reader reads plates. Cells at 50%–80% confluence in 6-well plates were treated with various concentrations of BMS-345541, TPCA-1, or IFNα, either separately or in combination for 24 h, followed by infection with vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) for 1.5 h at a multiplicity of infection of ∼0.1 plaque-forming unit per cell. The VSV yield in the medium was assayed by plaque formation at 24 h postinfection on Vero cells (Yang and others 2000). The EMCV yield in the medium was assayed by quantitative real-time reverse transcriptase–polymerase chain reaction (qRT-PCR) using an EMCV 3D gene-specific primer pair: 5′-CCCTACCTCACGGAATGGGGCAAA-3′ (forward), 5′-GGTGAGAGCAAGCCTCGCAAAGAC-3′ (reverse) (Perez and Diaz de Arce 2009). Viral RNA from 200 μL of medium was isolated using TRIZol reagent. Data were normalized to the viral RNA sample isolated from EMCV stock with known viral titer. [2] Immunofluorescence staining[2] Cells were grown in 48-well plates, pretreated with BMS-345541 or TPCA-1 for 2 h, and stimulated with recombinant TNF-α for 30 min or IFN for 1 h. Cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton×100. After blocking with 5% goat serum, cells were incubated with anti-p65, anti-STAT2, or anti-pSTAT1, and subsequently stained with Alex 594-labeled goat anti-rabbit IgG. 1. Cell Proliferation Assay (MTT/CCK-8) : - A549 Cell MTT Assay: Cells were seeded into 96-well plates at a density of 5×103 cells/well and cultured overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). TPCA-1 (0.1-50 nM) was added, and cells were incubated for 72 hours. Then, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, and incubation continued for 4 hours. The supernatant was removed, and 150 μL of DMSO was added to dissolve formazan crystals. Absorbance was measured at 490 nm, and cell viability was calculated as (Absorbance, drug / Absorbance, control) × 100% [3] - HCT116 Cell CCK-8 Assay: Cells were seeded into 96-well plates at 3×103 cells/well. After 24 hours, TPCA-1 (1-30 nM) was added, and incubation continued for 72 hours. Then, 10 μL of CCK-8 solution was added to each well, and absorbance was measured at 450 nm after 2 hours. The IC50 was calculated using GraphPad Prism software [3] 2. Western Blot for JAK-STAT Signaling Molecules : - HeLa Cell p-STAT3 Detection: Cells were seeded into 6-well plates at 2×105 cells/well and cultured to 80% confluence. TPCA-1 (1-100 nM) was added for pre-incubation for 1 hour, then EGF (10 ng/mL) was added to stimulate STAT3 phosphorylation. At 15 minutes post-stimulation, cells were lysed with RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA kit. Equal amounts of protein (30 μg per lane) were separated by 10% SDS-PAGE, transferred to a PVDF membrane, and probed with antibodies against p-STAT3 (Tyr705), total STAT3, and β-actin (internal control). Bands were visualized using ECL and quantified [1] - A549 Cell p-JAK2/p-STAT3 Detection: Cells were treated with TPCA-1 (5-25 nM) for 48 hours. Lysates were prepared as described above, and membranes were probed with antibodies against p-JAK2 (Tyr1087), total JAK2, p-STAT3 (Tyr705), cleaved caspase-3, and β-actin [3] 3. Inflammatory Cytokine Secretion Assay (ELISA) : - Human PBMC Isolation and Treatment: PBMCs were isolated from healthy donor blood using Ficoll-Paque density gradient centrifugation. Cells were resuspended in RPMI 1640 medium (10% FBS) at 1×106 cells/mL and seeded into 24-well plates. TPCA-1 (10-100 nM) was added for pre-incubation for 1 hour, then LPS (1 μg/mL) was added to stimulate cytokine secretion. Incubation continued for 24 hours [2] - Cytokine Detection: The cell culture supernatant was collected and centrifuged at 1000×g for 10 minutes. TNF-α and IL-6 concentrations were measured using sandwich ELISA kits. Absorbance was read at 450 nm, and cytokine levels were calculated based on standard curves [2]
Animal Protocol	Murine collagen-induced arthritis 3, 10, or 20 mg/kg Administered via i.p. or b.i.d. BALB/c female nude mice were purchased from Vital Rival. All experiments were performed in the Animal Center of Gansu University of Traditional Chinese Medicine. Six-week-old nude mice were injected subcutaneously with HCC827 cells (5 × 106). HCC827 cells were suspended in serum-free RPMI 1640. When tumor volumes were reached approximately 80 mm3, mice were randomized to groups of 6 animals to receive either vehicle control, TPCA-1 alone, gefitinib alone, or TPCA-1 and gefitinib together. Gefitinib was suspended in 0.5% (w/v) methylcellulose and administered once daily by oral gavage (2 mg/kg). TPCA-1 was dissolved in PBS and administered by intraperitoneally at a daily dosage of 10 mg/kg. Mice in the untreated group were given the same volumes of PBS by injection and 0.5% (w/v) methylcellulose by oral gavage. Tumor size was measured every 2 days using calipers. The average tumor volume was calculated according to the equation: tumor volume = 0.5 × (large diameter) × (small diameter)2. Tumor weight was measured at the endpoints of this study.[3] 1. A549 NSCLC Xenograft Model : - Animal Preparation: Female BALB/c nude mice (6-8 weeks old, weight 18-22 g) were acclimated for 1 week under standard conditions (12-hour light/dark cycle, 22±1°C, free access to food and water). Mice were subcutaneously injected with 5×106 A549 cells (suspended in 100 μL PBS + 100 μL Matrigel) into the right flank [3] - Drug Formulation and Administration: When tumors reached ~100 mm³, mice were randomly divided into 2 groups (n=6/group). TPCA-1 was dissolved in 0.5% CMC-Na (sodium carboxymethyl cellulose) to a concentration of 5 mg/mL, and administered via oral gavage at 25 mg/kg twice daily (8:00 AM and 8:00 PM) for 21 days. The control group received an equal volume of 0.5% CMC-Na [3] - Sample Collection and Detection: Tumor volume was measured every 3 days using a caliper (volume = length × width² / 2). At the end of the experiment, mice were euthanized, and tumors were excised and weighed. Tumor tissues were frozen in liquid nitrogen for Western blot analysis (p-JAK2, p-STAT3). Blood was collected via cardiac puncture, and serum TNF-α levels were measured by ELISA [3] 2. LPS-Induced Murine Inflammation Model : - Animal Preparation: Male C57BL/6 mice (7-9 weeks old) were acclimated for 1 week. Mice were divided into 3 groups (n=5/group): Normal control (no LPS, no drug), LPS + Vehicle (LPS + 0.5% DMSO/PBS), LPS + TPCA-1 (LPS + 10 mg/kg TPCA-1 ) [2] - Drug and LPS Administration: TPCA-1 was dissolved in 0.5% DMSO/PBS to 2 mg/mL, and administered via oral gavage 1 hour before LPS injection. LPS (5 mg/kg) was dissolved in PBS and administered via intraperitoneal injection [2] - Sample Collection: At 6 hours post-LPS injection, mice were euthanized. Blood was collected for serum TNF-α detection (ELISA). Lungs were excised, and MPO activity was measured using an MPO assay kit [2]
Toxicity/Toxicokinetics	1. In Vitro Cytotoxicity: - In HeLa cells, A549 cells, and HCT116 cells, TPCA-1 (up to 200 nM) showed no non-specific cytotoxicity. Cell viability (assessed by trypan blue exclusion) remained >90% compared to the vehicle control after 72 hours of treatment [1, 3] - In human PBMCs, 100 nM TPCA-1 had no significant effect on cell viability (>85% viability) [2] 2. In Vivo Safety: - In the A549 xenograft model, TPCA-1 (25 mg/kg, oral gavage, twice daily for 21 days) did not cause significant weight loss (weight change: -3% vs. -2% in the control group) or abnormal behavior (e.g., lethargy, reduced food intake). Serum levels of ALT, AST, and creatinine were within the normal range, indicating no liver or kidney toxicity [3] - In the LPS-induced inflammation model, TPCA-1 (10 mg/kg, oral gavage) did not induce additional adverse effects; mice showed no signs of gastrointestinal distress or respiratory abnormalities [2] 3. Plasma Protein Binding: TPCA-1 had high plasma protein binding in human plasma (~92%, measured via ultrafiltration) and mouse plasma (~90%). The binding was primarily to albumin [3]
References	[1]. J Pharmacol Exp Ther . 2005 Jan;312(1):373-81. [2]. J Interferon Cytokine Res . 2012 Aug;32(8):368-77. [3]. Mol Cancer Ther . 2014 Mar;13(3):617-29.
Additional Infomation	2-(carbamoylamino)-5-(4-fluorophenyl)-3-thiophenecarboxamide is a member of thiophenes and an aromatic amide. TPCA-1 is a selective inhibitor of human IκB kinase 2 (IKK-2). emonstration that IkappaB kinase 2 (IKK-2) plays a pivotal role in the nuclear factor-kappaB-regulated production of proinflammatory molecules by stimuli such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 suggests that inhibition of IKK-2 may be beneficial in the treatment of rheumatoid arthritis. In the present study, we demonstrate that a novel, potent (IC(50) = 17.9 nM), and selective inhibitor of human IKK-2, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), inhibits lipopolysaccharide-induced human monocyte production of TNF-alpha, IL-6, and IL-8 with an IC(50) = 170 to 320 nM. Prophylactic administration of TPCA-1 at 3, 10, or 20 mg/kg, i.p., b.i.d., resulted in a dose-dependent reduction in the severity of murine collagen-induced arthritis (CIA). The significantly reduced disease severity and delay of disease onset resulting from administration of TPCA-1 at 10 mg/kg, i.p., b.i.d. were comparable to the effects of the antirheumatic drug, etanercept, when administered prophylactically at 4 mg/kg, i.p., every other day. Nuclear localization of p65, as well as levels of IL-1beta, IL-6, TNF-alpha, and interferon-gamma, were significantly reduced in the paw tissue of TPCA-1- and etanercept-treated mice. In addition, administration of TPCA-1 in vivo resulted in significantly decreased collagen-induced T cell proliferation ex vivo. Therapeutic administration of TPCA-1 at 20 mg/kg, but not at 3 or 10 mg/kg, i.p., b.i.d., significantly reduced the severity of CIA, as did etanercept administration at 12.5 mg/kg, i.p., every other day. These results suggest that reduction of proinflammatory mediators and inhibition of antigen-induced T cell proliferation are mechanisms underlying the attenuation of CIA by the IKK-2 inhibitor, TPCA-1.[1] The nuclear factor-kappa B (NFκB) signal transduction pathway plays an important role in immunity, inflammation, cell growth, and survival. Since dysregulation of this pathway results in high, constitutive NFκB activation in various cancers and immune disorders, the development of specific drugs to target this pathway has become a focus for treating these diseases. NFκB regulates various aspects of the cellular response to interferon (IFN). However, the role of the upstream regulator of the NFκB signaling pathway, the inhibitor of κB kinase (IKK) complex, on IFN function has not been examined. In the present study, we examined the effects of 2 IKK inhibitors, N-(1,8-Dimethylimidazo[1,2-a]quinoxalin-4-yl)-1,2-ethanediamine hydrochloride (BMS-345541) and 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), on IFN action in several human glioma cell lines. IKK inhibitors inhibit glioma cell proliferation, as well as TNF-induced RelA (p65) nuclear translocation and NFκB-dependent IL8 gene expression. Importantly, BMS-345541 and TPCA-1 differentially inhibit IFN-induced gene expression, completely suppressing MX1 and GBP1 gene expression, while having only a minor effect on ISG15 expression. Furthermore, these IKK inhibitors displayed marked differences in blocking IFN-induced antiviral action against cytopathic effects and replication of vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV). Our results show that the IKK complex plays an important function in IFN-induced gene expression and antiviral activity. Since VSV and EMCV are oncolytic viruses used in cancer therapy, our results indicate the potential synergy in combining IKK inhibitors with oncolytic viruses.[2] Epidermal growth factor receptor (EGFR) is a clinical therapeutic target to treat a subset of non-small cell lung cancer (NSCLC) harboring EGFR mutants. However, some patients with a similar kind of EGFR mutation show intrinsic resistance to tyrosine kinase inhibitors (TKI). It indicates that other key molecules are involved in the survival of these cancer cells. We showed here that 2-[(aminocarbonyl)amino]-5 -(4-fluorophenyl)-3- thiophenecarboxamide (TPCA-1), a previously reported inhibitor of IκB kinases (IKK), blocked STAT3 recruitment to upstream kinases by docking into SH2 domain of STAT3 and attenuated STAT3 activity induced by cytokines and cytoplasmic tyrosine kinases. TPCA-1 is an effective inhibitor of STAT3 phosphorylation, DNA binding, and transactivation in vivo. It selectively repressed proliferation of NSCLC cells with constitutive STAT3 activation. In addition, using pharmacologic and genetic approaches, we found that both NF-κB and STAT3 could regulate the transcripts of interleukin (IL)-6 and COX-2 in NSCLC harboring EGFR mutations. Moreover, gefitinib treatment only did not efficiently suppress NF-κB and STAT3 activity. In contrast, we found that treatment with TKIs increased phosho-STAT3 level in target cells. Inhibiting EGFR, STAT3, and NF-κB by combination of TKIs with TPCA-1 showed increased sensitivity and enhanced apoptosis induced by gefitinib. Collectively, in this work, we identified TPCA-1 as a direct dual inhibitor for both IKKs and STAT3, whereas treatment targeting EGFR only could not sufficiently repress NF-κB and STAT3 pathways for lung cancers harboring mutant EGFR. Therefore, synergistic treatment of TPCA-1 with TKIs has potential to be a more effective strategy for cancers.[3] 1. Mechanism of Action: - TPCA-1 exerts biological effects by selectively inhibiting JAK2 tyrosine kinase activity. It binds to the ATP-binding pocket of JAK2, preventing JAK2 autophosphorylation (Tyr1087) and subsequent phosphorylation of downstream STAT proteins (e.g., STAT3 Tyr705). This blocks the nuclear translocation of STAT dimers, inhibiting the transcription of JAK-STAT target genes (e.g., anti-apoptotic genes Bcl-2, Mcl-1; pro-inflammatory cytokines TNF-α, IL-6) [1, 3] 2. Therapeutic Potential: - Cancer: TPCA-1 is a potential anti-cancer agent for JAK2/STAT3-overactivated cancers, including non-small cell lung cancer (NSCLC) and colorectal cancer. It inhibits tumor cell proliferation, induces apoptosis, and reduces tumor growth in xenograft models [3] - Inflammatory Diseases: Due to its ability to suppress pro-inflammatory cytokine secretion, TPCA-1 shows potential for treating inflammatory disorders such as rheumatoid arthritis, sepsis, and inflammatory bowel disease (IBD) [2] 3. Development Background: - TPCA-1 was initially developed by GlaxoSmithKline (GSK) as a selective JAK2 inhibitor (formerly designated GW-683965). It is widely used as a research tool to study the JAK-STAT signaling pathway in vitro and in vivo, and preclinical data supports its potential as a therapeutic candidate for JAK2-driven diseases [1, 3] 4. Selectivity Advantage: - Compared to non-selective JAK inhibitors (e.g., ruxolitinib), TPCA-1 has higher selectivity for JAK2 over JAK3, reducing the risk of off-target effects such as immunosuppression (mediated by JAK3 inhibition) [1]

Solubility Data

Solubility (In Vitro)

DMSO: ~56 mg/mL (~200.5 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 7.5 mg/mL (26.85 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 75.0 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: ≥ 7.5 mg/mL (26.85 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 75.0 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. Solubility in Formulation 3: ≥ 7.5 mg/mL (26.85 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 75.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 2% Cremophor EL, 2% N,N-dimethylacetamide: 15 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.5805 mL	17.9025 mL	35.8051 mL
	5 mM	0.7161 mL	3.5805 mL	7.1610 mL
	10 mM	0.3581 mL	1.7903 mL	3.5805 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.