Aurintricarboxylic acid inhibits influenza virus neuraminidase (NA) activity (IC50 values: 7.2 μg/mL for PR8, 16.6 μg/mL for NC, 16.4 μg/mL for NY, 6.3 μg/mL for influenza B virus, 0.52 μg/mL for recombinant N1 protein, and 1.0 μg/mL for recombinant N4 protein) [2].
Aurintricarboxylic acid inhibits TWEAK-Fn14-NF-κB signaling (IC50 of ~0.6 μM in HEK293 NF-κB-Luc/Fn14 cells) [4].

P2X2/3Rs, P2X2Rs, P2X4Rs, or P2X7Rs are all slightly inhibited by aurine tricarboxylic acid[1]. In a concentration-dependent manner, auricin tricarboxylic acid suppresses the current of ATP activation [1]. via controlling the action of calcineurin [2]. Aurin tricarboxylic acid suppresses TWEAK-Fn14 in a weak manner. In GBM cells, aurin tricarboxylic acid (10 μM; 0.5-2 hours) suppresses NF-κB, Akt, and Src phosphorylation mediated by TWEAK-Fn14 [4]. Aurin tricarboxylic acid inhibits TWEAK and stimulates gold tricarboxylic acid (compound 8), which can prevent exogenous siRNA from being loaded by RISC but cannot control endogenous let-7 loading on AGO2 in cultivated cells [4].

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Aurintricarboxylic acid (ATA) protected MDCK cells from infection with influenza A (H1N1 strains PR8, NC; H3N2 strain NY) and B viruses, as measured by neutral red uptake assay (p < 0.0001). ATA caused 50% cytotoxicity (CC50) in MDCK cells at 577 μg/mL and protected 50% of cells (EC50) from PR8 infection at 6.5 μg/mL, resulting in a selective index of 88.8. Pre-exposing cells to ATA for 24h prior to infection did not protect from CPE [2].
ATA reduced influenza A PR8 viral nucleoprotein (NP) RNA levels in MDCK cells in a concentration-dependent manner, as determined by RT-PCR [2].
ATA (50 and 100 μg/mL) dramatically reduced virus yield in supernatants of infected MDCK cells by 93% and 95% for PR8, 86% and 94% for NC, and 72% and 81% for NY, respectively, as quantified by ELISA [2].
ATA treatment (100 μg/mL) significantly reduced both cell-associated (to 5×10^4 PFU/mL) and extracellular (to 1.3×10^4 PFU/mL) virus yields compared to untreated control (1×10^7 PFU/mL for both) in PR8-infected MDCK cells, as determined by plaque assay [2].
Simultaneous treatment with ATA and amantadine hydrochloride (AH) showed additive protective effects against influenza A virus-induced CPE and nearly abolished virus production, as shown by neutral red assay and ELISA [2].
Pre-incubation of influenza A and B viruses with ATA (50, 100 μg/mL) reduced the number of plaques, indicating direct viral inactivation [2].
Electron microscopy revealed that ATA (100 μg/mL) treatment of PR8-infected MDCK cells induced viral aggregation on the cell surface [2].
ATA inhibited the enzymatic activity of NA derived from PR8, NC, NY, influenza B, WSN, and oseltamivir-resistant H274Y viruses, as well as recombinant N1 and N4 proteins, in a cell-free chemiluminescent assay [2].
ATA inhibited replication of both wild-type WSN and oseltamivir-resistant H274Y viruses in a dose-responsive manner in MDCK cells, with IC50 values of 2 μg/mL and 18 μg/mL, respectively, for NA inhibition [2].
In glioma cell lines (T98G, A172) and a patient-derived xenograft line (GBM44), ATA treatment (10 μM) did not alter Fn14 protein levels but abrogated TWEAK-induced phosphorylation of NF-κB (p65), Akt, and Src [4].
ATA (10 μM) suppressed TWEAK-induced activation of Rac1 in A172 and T98G glioma cells [4].
ATA (10 μM) suppressed TWEAK-induced binding of TRAF2 to Fn14 in A172 and GBM44 cells, as shown by co-immunoprecipitation [4].
ATA (10 μM) significantly repressed TWEAK-induced glioma cell chemotactic migration (Transwell assay) and invasion (Matrigel assay) in T98G, A172, and GBM44 cells without altering cell viability (CellTiterGlo assay) [4].
ATA (10 μM) abrogated the survival phenotype conferred by TWEAK in glioma cells, as evidenced by increased PARP cleavage and significantly impaired colony formation following treatment with temozolomide (TMZ) or radiation (2 Gy) [4].
Aurintricarboxylic acid (10 μM) increased luciferase signals by approximately fivefold in a luciferase-based cellular assay for miR-21 function, indicating inhibition of miR-21. It did not affect miR-30 or miR-93. ATA did not alter mature or primary miR-21 expression levels, suggesting it inhibits miR-21 function, not its expression [6].
In A498 renal carcinoma cells, ATA (10 μM) decreased the IC50 of topotecan from 1 μM to 90 nM in a dose-dependent cell viability assay and from 2 μM to 74 nM in a 2-week clonogenic assay, indicating restored chemosensitivity [6].

In an intracranial xenograft mouse model, depletion of Fn14 (target of ATA) in GBM44 glioma cells, in conjunction with TMZ treatment (50 mg/kg for 5 days via oral gavage), significantly enhanced survival compared to TMZ treatment alone (p < 0.0008). Tumors with decreased Fn14 expression showed significantly higher levels of apoptosis markers γH2AX and cleaved-caspase 3 [4].

Influenza Neuraminidase Inhibition Assay (Cell-Free): A chemiluminescent assay was used to measure NA inhibition. Serial dilutions of ATA (ranging from 2000 μg/mL to 0.002 μg/mL) were prepared in assay buffer (26 mM MES, pH 6.0, 4 mM CaCl2). 25 μL of each drug dilution was combined with 25 μL of diluted viruses or recombinant NA proteins in assay buffer and incubated at 37°C for 30 min. 10 μL of NA-Star substrate (10 μM final concentration) was added and incubated at 37°C for 30 min. 60 μL of NA-Star accelerator was then added, and the chemiluminescent signal was measured. IC50 values were calculated from the percentage of NA activity relative to controls [2].
Rac1 Activation Assay: Glioma cells were pre-incubated with 10 μM ATA or vehicle for 1 hour prior to stimulation with TWEAK (100 ng/mL). Cell lysates were harvested, and equal protein concentrations were assessed for Rac1 activity using a commercial pulldown assay kit according to the manufacturer's protocol [4].
Co-Immunoprecipitation (Co-IP) for TRAF2-Fn14 Binding: A172 and GBM44 cells were pre-treated with 10 μM ATA or vehicle for 1 hour before stimulation with 100 ng/mL TWEAK for 2 minutes. Cells were lysed, and 1 mg of protein was immunoprecipitated using a TRAF2 antibody or a control isotype-matched antibody. The immunoprecipitates were washed, resolved by SDS-PAGE, transferred to nitrocellulose, and probed with Fn14 and TRAF2 antibodies [4].

Western Blot analysis [4]
Cell Types: T98G, A172, GBM44 glioma cells
Tested Concentrations: 10 μM
Incubation Duration: 0.5 hrs (hours), 1 hour, 2 hrs (hours)
Experimental Results: Abolition of the activation of TWEAK downstream signals, including phospholipids of the NF-κB family 6]. members p65, Akt, and Src in all three GBM cell lines.

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Neutral Red Assay for Cytotoxicity and Antiviral Activity: MDCK cell monolayers in 24-well plates were exposed to ATA for 48h to assess cytotoxicity. For antiviral activity, after viral adsorption, cells were incubated with ATA for 48h. Neutral red dye (0.033%) was added for 2h, cells were fixed, and the incorporated dye was solubilized. Absorbance was measured at 540 nm. Cytotoxicity was presented as percentage of untreated controls; protection was presented as percentage of uninfected controls [2].
Reverse Transcription-PCR (RT-PCR): Total RNA was extracted from cells 48h post-infection and ATA treatment. DNase I-treated RNA (200 ng) was used in a one-step RT-PCR with primers for influenza PR8 nucleoprotein (NP) (forward: 5’-ACTCACATGATGATCTGG-3’, reverse: 5’-CTGCATTGTCTCCGAAGA-3’). RT was at 50°C for 30 min, followed by 10 cycles (94°C 30s, 58°C 30s, 68°C 45s) and then 18 cycles (with 5s added to elongation each cycle), and a final extension at 68°C for 7 min. Products were visualized on a 1% agarose gel [2].
ELISA for Viral Antigen: Supernatants from infected and treated cells were clarified and added to wells coated with an anti-influenza A nucleoprotein monoclonal antibody. After incubation and washing, a biotinylated rabbit polyclonal anti-influenza virus antibody was added. Following further washes, streptavidin conjugate was added, then a substrate solution. The reaction was stopped with H2SO4, and absorbance was read at 450 nm. Viral abundance was calculated as HA units from a standard curve [2].
Virus Yield Reduction Assay (Plaque Assay): Confluent MDCK cells in 6-well plates were infected with PR8 (MOI 0.001) and treated with compounds for 48h. Cells and supernatants were collected separately. Cells were lysed by freeze-thaw cycles. Supernatants and lysates were incubated with MDCK monolayers, then overlaid with maintenance medium containing agarose. After 3 days, plaques were stained with neutral red or crystal violet and counted [2].
Direct Inactivation Assay (Plaque Assay): Viruses (~100 pfu) were incubated with ATA at 37°C for 30 min. The mixture was then transferred to MDCK cell monolayers, incubated for 2h, and subjected to plaque assay as described above [2].
Western Blot Analysis for Signaling Studies: Glioma cells (T98G, A172, GBM44) were stimulated with TWEAK (100 ng/mL) in the presence or absence of ATA (10 μM). Cells were lysed in SDS sample buffer. Proteins (30 μg) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies against Fn14, phospho-NF-κB p65, total NF-κB p65, phospho-AKT, total AKT, phospho-SRC, total SRC, cleaved PARP, and α-tubulin [4].
Chemotactic Migration and Matrigel Invasion Assays: Glioma cells were seeded in the top well of a collagen-coated transwell chamber (for migration) or in growth factor-reduced Matrigel (for invasion). TWEAK (100 ng/mL) with or without ATA (10 μM) was added to the lower wells. After 24h, non-invaded/migrated cells were removed, and cells that had migrated/invaded were fixed, stained with DAPI, and counted in five high-power fields [4].
Clonogenic Assay: Glioma cells were treated with TMZ (500 μM) or radiation (2 Gy), with or without TWEAK (100 ng/mL) and/or ATA (10 μM). After 24h, 250 cells were plated in triplicate in 35 mm dishes. Colonies were allowed to grow for 6-7 days, then fixed, stained with crystal violet, and counted. Surviving fractions were determined relative to non-treated controls [4].
Apoptosis Study (PARP Cleavage): Cells were treated with TMZ (250 μM), TWEAK (100 ng/mL), and ATA (10 μM) for 48h. Whole cell lysates were analyzed for cleaved PARP by Western blot [4].
Cell Viability Assay (CellTiterGlo): T98G, A172, and GBM44 cells were seeded at 3000 cells/well in 96-well plates. Increasing concentrations of ATA were added for 72h. CellTiterGlo reagent was added, and luminescence was measured. Raw values were normalized to vehicle (100% viability) and MG132 (0% viability) controls [4].

Intracranial Xenograft Model and TMZ Treatment: Athymic nude mice were intracranially implanted with 3×10^5 GBM44 glioma cells expressing control shRNA or Fn14 shRNA. Mice with established tumors were randomized into treatment groups (n=10 per group): vehicle control and TMZ (50 mg/kg). Treatment was given for 5 days through oral gavage. Mice were observed daily and euthanized upon reaching a moribund state. Brains were removed, fixed in 10% neutral buffered formalin for 48 hours, and paraffin-embedded [4].

In MDCK cells, the 50% cytotoxic concentration (CC50) of Aurintricarboxylic acid was 577 μg/mL, and the selective index (SI) was 88.8, suggesting relatively low toxicity in tissue culture [2].
Aurintricarboxylic acid did not demonstrate any cytotoxic effects on HEK293 NF-κB-Luc or NF-κB-Luc/Fn14 cells at concentrations up to 100 μM, as measured by CellTiterGlo assay [4].
Aurintricarboxylic acid treatment (up to 10 μM) did not alter cell viability in T98G, A172, or GBM44 glioma cells, as measured by CellTiterGlo assay [4].
Previous research in hamsters showed that infusion of ATA at a dose of up to 1 mg/kg/hour for 2 weeks was well tolerated [2].

[1]. Identification of aurintricarboxylic acid as a potent allosteric antagonist of P2X1 and P2X3 receptors. Neuropharmacology. 2019 Nov 1;158:107749.

[2]. Aurintricarboxylic acid is a potent inhibitor of influenza A and B virus neuraminidases. PLoS One. 2009 Dec 17;4(12):e8350.

[3]. Aurintricarboxylic acid, a putative inhibitor of apoptosis, is a potent inhibitor of DNA topoisomerase II in vitro and in Chinese hamster fibrosarcoma cells. Biochem Pharmacol. 1995 Jan 31;49(3):305-13.

[4]. Identification of aurintricarboxylic acid as a selective inhibitor of the TWEAK-Fn14 signaling pathway in glioblastoma cells. Oncotarget. 2017 Feb 14; 8(7): 12234–12246.

[5]. Discovery of a Bioactive Inhibitor with a New Scaffold for Cystathionine γ-Lyase. J Med Chem. 2019 Feb 14;62(3):1677-1683.

[6]. Small molecules with big roles in microRNA chemical biology and microRNA-targeted therapeutics. RNA Biol. 2019 Jun; 16(6): 707-718.

Gold-containing tricarboxylic acid (GC) belongs to the quinone methane class of compounds. Its chemical name is 3-methylene-6-oxocyclohexyl-1,4-diene-1-carboxylic acid, where the methylene hydrogen is replaced by a 4-carboxyl-3-hydroxyphenyl group. Its trisodium salt is the biological staining agent "Chromium Violet CG," and its triammonium salt is "ammonium aluminate." It can be used as a histological dye, an insulin-like growth factor receptor 1 antagonist, and a fluorescent dye. It is a monohydroxybenzoic acid, belonging to the quinone methane class of compounds, and is also a tricarboxylic acid. It is the conjugate acid of gold-containing tricarboxylic acid salt. It is a dye that inhibits protein synthesis in the early stages of protein biosynthesis. Its ammonium salt (ammonium aluminate) is a reagent used for colorimetric determination of aluminum content in water, food, and tissues.

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Aurintricarboxylic acid is a polyaromatic carboxylic acid derivative that has been shown to inhibit nucleases and nucleic acid processing enzymes. It has previously demonstrated antiviral activity against HIV, vesicular stomatitis virus, SARS-CoV, and vaccinia virus [2].
The anti-influenza mechanism of ATA is attributed to direct inhibition of viral neuraminidase, leading to viral aggregation on the cell surface [2].
Aurintricarboxylic acid was identified as a specific inhibitor of the TWEAK-Fn14 signaling axis in a high-throughput screen of the LOPAC1280 library using HEK293 cells engineered to express Fn14 and an NF-κB-driven luciferase reporter. It did not inhibit TNFα-induced NF-κB activation [4].
Aurintricarboxylic acid has been used as a chemical probe to study cellular processes. For example, it was used to discover a novel regulatory pathway involving miR-221/222, myoD, and myomiRs in muscle cells [6].
Aurintricarboxylic acid has been reported to have various biological activities, including as an inhibitor of the JAK-STAT pathway and as an anti-apoptotic factor [2].

C22H14O9

422.35

422.063

4431-00-9

13186-45-3 (tri-hydrochloride salt);569-58-4 (tri-ammonium salt);63451-31-0 (calcium (2:3) salt);93480-02-5 (calcium (1:3) salt)

2259

Brown to black solid powder

1.6±0.1 g/cm3

759.6±60.0 °C at 760 mmHg

300 °C(lit.)

427.1±29.4 °C

0.0±2.7 mmHg at 25°C

1.751

4.09

5

9

5

31

841

0

GIXWDMTZECRIJT-UHFFFAOYSA-N

InChI=1S/C22H14O9/c23-16-4-1-10(7-13(16)20(26)27)19(11-2-5-17(24)14(8-11)21(28)29)12-3-6-18(25)15(9-12)22(30)31/h1-9,23-24H,(H,26,27)(H,28,29)(H,30,31)

5-[(3-Carboxy-4-hydroxyphenyl)-(3-carboxy-4-oxocyclohexa-2,5-dien-1-ylidene)methyl]-2-hydroxybenzoic acid

Aurintricarboxylic acid ATA NSC 4056 NSC4056 NSC-4056

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)

DMSO : ~125 mg/mL (~295.97 mM)
NH4OH : 10 mg/mL (~23.68 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.92 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 20.8 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: ≥ 2.08 mg/mL (4.92 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 20.8 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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 (Please use freshly prepared in vivo formulations for optimal results.)