Selectively inhibits aquaporin 4 (AQP4) with an IC50 of 3.1 μM [1]

One ligand is for an E3 ubiquitin ligase, and the other is for the target protein; these two ligands are joined by a linker to form PROTACs. PROTACs selectively degrade target proteins by taking use of the intracellular ubiquitin-proteasome system[3].

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- Inhibited AQP4-mediated water transport in vitro, as demonstrated by reduced osmotic water permeability in transfected HEK293 cells treated with TGN-020 (concentration not specified) [1]
- Reduced the expression of GFAP (a marker of astrocyte activation) and PCNA (a marker of cell proliferation) in primary rat astrocyte cultures treated with TGN-020 (10-100 μM) for 24 hours [4]

In STZ-induced diabetic rats (nine-week-old male Wistar rats), TGN-020 sodium (0.02 mg/μL; two microliter intravitreal injections) can decrease retinal edema[2]. Immediate post-stroke TGN-020 sodium (100 mg/kg; i.p.; single dose) decreases the degree of edema, suppresses the expression of GFAP, PCNA, and AQP4, and enhances functional recovery at days 3, 7, 14, 21, and 28[4]. In mature female Sprague-Dawley rats (180-220 g, 9–10 weeks old) after SCI, TGN-020 sodium suppresses the formation of glial scars and increases the expression of GAP-43[4].

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In the diabetic retina, the immunoreactivity and protein levels of VEGF were suppressed by TGN-020. AQP4 immunoreactivity was higher than in the control retinas and the expressions of AQP4 were co-localized with GFAP. Similarly to VEGF, AQP4 and GFAP were also suppressed by TGN-020. In the Evans Blue assay, TGN-020 decreased leakage in the diabetic retinas. In the cultured Müller cells, the increase in cell volumes and intracellular ROS production under high glucose condition were suppressed by exposure to TGN-020 as much as by exposure to bevacizumab. Conclusion: TGN-020 may have an inhibitory effect on diabetic retinal edema.[2]

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TGN-020 and spinal cord edema alleviation at day 3 after spinal cord injury (SCI) [4]
We measured the water content of tissue to reflect the level of edema at the site of SCI. At day 3 after SCI, a focal compression of the spinal cord at T10 in the SCI and TGN-020 groups resulted in visible damage that was not evident in the sham group. Hemorrhaging and necrosis were obvious in the centers of injured spinal cords, and injured areas were substantially more affected in the SCI group than in the TGN-020 group (Fig. 2A). Photographs of injured spinal cords were reasonably consistent with calculations of spinal cord water content in each group (Fig. 2B). The water content of tissue in the segments adjacent to the injury site at day 3 was significantly higher in the SCI group (76.09 ± 0.93) and the TGN-020 group (73.07 ± 0.87) than it was in the sham group (69.02 ± 0.45) (p < 0.01). Spinal cord water content was significantly lower in the TGN-020 group (73.07 ± 0.87) than in the SCI group (76.09 ± 0.93) (p < 0.05), suggesting that TGN-020 could significantly reduce increased water content caused by SCI.

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TGN-020 and reduced AQP4 expression at day 3 after SCI [4]
Compared with the sham group, AQP4 expression was significantly increased in and around the centers of injured sites 3 days after compression injury in the SCI group (p < 0.01) and in the TGN-020 group (p < 0.05) (Fig. 3A, B). In the TGN-020 group, AQP4 expression was significantly lower than it was in the SCI group (p < 0.05). The results of immunofluorescence staining for AQP4 with GFAP were consistent with the results of western blotting (Fig. 3C, D). At day 3, AQP4 fluorescence intensity was significantly higher in both the SCI group and the TGN-020 group than it was in the sham group (p < 0.01), and it was significantly lower in the TGN-020 group than it was in the SCI group (p < 0.05). These data indicated that TGN-020 could downregulate AQP4 expression, resulting in alleviation of spinal cord edema formation.

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TGN-020 and inhibition of astrocyte proliferation at day 3 after SCI [4]
GFAP, a cytoskeletal intermediate filament protein, is used as a specific astrocyte marke. Proliferating cell nuclear antigen (PCNA) is a well-known marker of cell proliferation. To investigate whether AQP4 is relevant to astrocyte proliferation in SCI, we examined PCNA and GFAP expression 3 days after SCI. Western blotting suggested that GFAP levels were low and virtually no PCNA was expressed in the sham group (Fig. 4A–C). Notably however, compared with the sham group there were significant increases in the expression of GFAP and PCNA around the centers of the injury sites at day 3 in the SCI group and the TGN-020 group (p < 0.01). At day 3, GFAP and PCNA expression were significantly lower in the TGN-020 group than they were in the SCI group (p < 0.05).

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TGN-020 and glial scar formation and axon regeneration at 4 weeks after SCI [4]
To investigate whether TGN-020 administration could protect spinal cord tissues from being damaged at 4 weeks after SCI, we calculated the size of the cavity area to assess astroglial scar formation. At 4 weeks, spinal cords were normal in the sham group but in both the SCI group and the TGN-020 group they exhibited destruction, including very large and irregular cavities (Fig. 5A, B). Cavity area was significantly smaller in the TGN-020 group than it was in the SCI group (4.2 ± 0.6 mm2 vs. 3.0 ± 0.4 mm2, p < 0.01). These observations suggested that TGN-020 significantly reduced secondary spinal cord tissue degeneration after SCI.

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TGN-020 and neuron survival at 4 weeks after SCI [4]
SCI-induced neuronal necrosis and apoptosis lead to neuronal loss. Nissl staining was performed on transverse sections at 4 weeks after SCI to investigate potential neuroprotective effects of TGN-020. Large numbers of Nissl-positive neurons with an extension cell body were detected, mainly in the ventral horns of normal spinal cords (Fig. 6B). Therefore, Motor neurons were detected at the anterior angle of injured spinal cords. As showed in Fig. 6A, In the sham group, the neurons exhibited an integrative and granular-like morphology, and large and numerous Nissl bodies indicating substantial protein synthesis in neural cells. In the SCI group, the neurons exhibited shrunken cell bodies, pyknotic nuclei, and irregular morphology, and intracellular toluidine blue staining was reduced around the lesions. In addition, the numbers of surviving motor neurons were significantly lower than they were in the sham group (Fig. 6C, p < 0.01). In the TGN-020 group, the numbers of surviving motor neurons were also significantly lower than they were in the sham group (Fig. 6C, p < 0.01), but neuronal morphology was better than it was in the SCI group, with deeper staining in the cytoplasm and significantly less loss of Nissl granules (Fig. 6C, p < 0.01). These results suggested that TGN-020 administration inhibited the loss of neurons.

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TGN-020 and the promotion of functional locomotor recovery after SCI [4]
To evaluate the effects of TGN-020 on functional locomotor recovery in rats after SCI, behavioral outcomes were assessed at days 1, 3, 7, 14, 21, and 28 via the Basso-Beattie-Bresnahan (BBB) scale, which has been widely used to evaluate the recovery of hindlimb motor function after SCI in rats. The sham group yielded a score of 21 at all time-points, reflecting normal motor function, whereas in the SCI group and the TGN-020 group BBB scores were reduced at 1 day after compression injury, then gradually increased to varying extents over time (Fig. 7). This indicated gradual recovery of locomotor function after SCI. In the TGN-020 group, BBB scores were significantly higher than they were in the SCI group at all time-points from day 3 to day 28 after SCI (p < 0.01 for day 7; p < 0.05 for days 3, 14, 12, and 28). These results suggest that TGN-020 administration can markedly promote the recovery of locomotor function in rats after SCI.

Measurement of Cell Volume and Intracellular Levels of ROS Production by Flow Cytometry [2]
To examine the changes in the volume of TR-MUL5 cells and intracellular production of ROS under high glucose condition, the cells were incubated in high glucose (25 mM) or physiological concentration of low glucose (5.5 mM) medium for 2–3 days. Then, volumetric changes of TR-MUL5 and intracellular levels of ROS were determined using flow cytometry analysis of ethidium fluorescence in the presence or absence of TGN-020 (100 nM) or bevacizumab overnight under physiological and high glucose conditions. The level of superoxide in TR-MUL5 cells was measured using hydroethidine, a fluorogenic probe. Hydroethidine is oxidized by superoxide to form ethidium, a fluorescent product, which is then retained intracellularly allowing a semiquantitative estimation of intracellular superoxide levels. Cells were harvested via trypsinization and centrifuged at 800 g for 5 min. After washing with PBS, the cells were resuspended in phenol red-free DMEM with hydroethidine (1 μg/mL) for 30 min at 37 °C. Cell densities were adjusted to 2.0 × 105 cells/mL. The changes in cellular volume of Müller cells and the intracellular levels of superoxide were analyzed using flow cytometry with 488 nm excitation and 590 to 610 nm emission wavelengths. The acquisition and analysis software on the EC800 was used to acquire and quantify the fluorescent intensities. We previously described the details of determining the volume changes of TR-MUL5 cells using an EC800 flow cytometry analyzer.

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mmunostaining of Cultured Müller Cells [2]
To determine the effect of TGN-020 on the expressions of VEGF and AQP4 in the TR-MUL5 cells, the cells that were incubated in high glucose (25 mM) media with and without TGN-020 or physiological concentration of low glucose (5.5 mM) media were examined by immunocytochemistry. After fixation by 4% formaldehyde, the cells were incubated with primary antibodies of rabbit polyclonal anti-AQP4 and mouse monoclonal anti-VEGF overnight at 4 °C. After rinsing by PBS and blocking, these cells were incubated for 2 h at room temperature in Alexa 594 or Alexa 488-conjugated to the appropriate secondary antibodies diluted by 1:500. Nuclei were stained with 4′,6-diamidino-2-phenylindole. The processed samples were photographed with a fluorescence microscope.

Intravitreal Injection [2]
Two microliter intravitreal injections of TGN-020 (0.02 mg/μL) or bevacizumab (0.025 mg/μL) (Genentech Inc, San Francisco, CA, USA) or vehicle alone (phosphate-buffered saline (PBS), pH 7.4) were performed on STZ-induced diabetic rats using a Hamilton syringe and a 30-gauge needle. Animals received general anesthesia, and perfused fixation was performed 48 h after the intravitreal injections.

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Evans Blue Assay [2]
Retinal blood vessel permeability of the STZ-induced diabetic rats in the presence or absence of TGN-020 was tested by Evans Blue assay. A total of 100 μL of Evans Blue (20 mg/mL) in PBS was injected into the tail vein. After 10 min, retinas were carefully explanted and post-fixed in 4% PFA in PBS overnight. Flat-mounted retinas were created and extravasation was evaluated by epifluorescence analysis at an excitation wavelength of 594 nm with a laser scanning confocal microscope

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Animals and experimental groups [4]
Great effort was made to reduce animal suffering. Animals were then randomly assigned to the following three groups: Sham group (n = 35) which were only subject to laminectomy without compression of spinal cord, SCI group (n = 35) which underwent 35 g impounder compression for 5 min at T10, TGN-020 group (n = 35) which received TGN-020 (100 mg/kg, i.p.) immediately followed SCI. Each group was equally and randomly assigned into four subgroups (n = 5 or 6 and 8/group) for the following experiments: (A) Spinal cord water content determination; (B) Western blotting; (C) Immunofluorescent assay, Hematoxylin-Eosin staining and Nissl staining; and (D) Locomotor function test. These rats in all groups were sacrificed 3 day or 4 weeks after injury.

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2.2. Surgical procedures and TGN-020 administration [4]
Basic surgical procedures and compression injury were performed as described previously. All surgeries were performed under sterile conditions. Briefly, the rats were anesthetized with 10% chloral hydrate (0.33 mL/kg, i.p.). Once anesthesia took effect, surgical area was clean shaved and sterilized with 75% ethanol, a 3 cm dorsal longitudinal incision was made over the midthoracic spinal cord at T9–T12 and then removed peripheral paraspinal soft tissues to expose the spinal cord, leaving the dura intact. The spinal cord was extradurally compressed with a metal impounder (35 g, 5 min) gently loaded onto T10 level of the spinal cord to achieve a moderate injury (Fig. 1). Following surgery wounds were then closed in layers using 4–0 silk. Postoperativaly, TGN-020 (100 mg/kg, i.p.) diluted in 10% dimethyl sulfoxide (DMSO) was administrated to TGN-020 group. The concentration of DMSO was adjusted at 0.1% before injection. The Sham group and SCI group received the equal volume DMSO intraperitoneally at the same time. The animals were returned to individual cages once them were waked. During the postoperative recovery period, ceftriaxone sodium (50 mg/kg, i.p.) was administered on three consecutive days. The bladders were manually pressed three times daily until natural voiding reflex recovery, food and water were available ad libitum. Animals that behaved any abnormal neurological signs would be excluded from experiments.

[1]. Identification of aquaporin 4 inhibitors using in vitro and in silico methods. Bioorg Med Chem. 2009 Jan 1;17(1):411-7.

[2]. Effects of an Aquaporin 4 Inhibitor, TGN-020, on Murine Diabetic Retina. Int J Mol Sci. 2020 Mar 27;21(7):2324.

[3]. Small-molecule PROTACs: An emerging and promising approach for the development of targeted therapy drugs. EBioMedicine. 2018 Oct;36:553-562.

[4]. TGN-020 alleviates edema and inhibits astrocyte activation and glial scar formation after spinal cord compression injury in rats. Life Sci. 2019 Apr 1;222:148-157.

This study investigated the in vitro inhibitory effects of 18 compounds on aquaporin 4 (AQP4) and their computer-simulated docking energies. More than half of the tested compounds showed inhibitory activity in in vitro functional assays, including the 5-HT (1B/1D) receptor agonists sumatriptan and rizatriptan. In addition, the inhibitory activity of the compounds used in this study at a concentration of 20 μM was significantly positively correlated with their computer-simulated docking energies (r²=0.64), which is consistent with previous results. Subsequently, three compounds, namely 2-(nicotinamide)-1,3,4-thiadiazole, sumatriptan, and rizatriptan, were found to have inhibitory IC(50) values of 3, 11, and 2 μM on AQP4, respectively. [1]

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Objective: To investigate the effects of the selective aquaporin 4 (AQP4) inhibitor 2-(nicotinamide)-1,3,4-thiadiazole (TGN-020) on the expression of vascular endothelial growth factor (VEGF), reactive oxygen species (ROS) generation, and retinal edema in diabetic retina. Methods: Streptozotocin-induced diabetic rats were intravitreally injected with bevacizumab, TGN-020, or phosphate-buffered saline (PBS). Retinal sections were immunostained for anti-glial fibrillary acidic protein (GFAP), anti-aquaporin 4 (AQP4), and anti-vascular endothelial growth factor (VEGF). Western blotting was used to determine the protein level of VEGF in the collected retinas. Furthermore, Evans blue leakage into retinal vessels was observed in the presence or absence of TGN-020 in planar retinal specimens from diabetic rats. Under physiological and high-glucose conditions, ethidium fluorescence was analyzed by flow cytometry to determine the volume changes and intracellular reactive oxygen species (ROS) levels of rat retinal Müller cells (TR-MUL5; transgenic rat Müller cells) in the presence or absence of TGN-020 or bevacizumab. Results: In diabetic retinopathy, TGN-020 inhibited the immunoreactivity and protein level of VEGF. The immunoreactivity of AQP4 was higher than that of the control group retina, and the expression of AQP4 was co-localized with GFAP. Similar to VEGF, TGN-020 also inhibited the expression of AQP4 and GFAP. In the Evans blue staining assay, TGN-020 reduced the leakage of diabetic retinopathy. In cultured Müller cells, the increase in cell volume and the production of intracellular ROS under high glucose conditions were inhibited by TGN-020, which was comparable to that of bevacizumab. Conclusion: TGN-020 may have an inhibitory effect on diabetic retinopathy edema. [2] At present, small molecule inhibitors are the main treatment for targeting intracellular proteins, but their development and clinical application still face many challenges. Proteolytic targeting chimeras (PROTACs) selectively degrade target proteins using the intracellular ubiquitin-proteasome system. In recent years, there have been many reports of highly active small molecule PROTACs. This article reviews the emerging properties of small molecule PROTACs, such as inducing rapid, deep and sustained degradation, effectively inhibiting downstream signaling pathways, exhibiting higher targeting selectivity, and overcoming resistance to small molecule inhibitors. In tumor xenograft models, small molecule PROTACs can significantly inhibit tumor progression. In addition, this article also introduces the latest advances in PROTAC technology, such as homologous PROTACs. Compared with traditional small molecule drugs, the significant advantages of small molecule PROTACs and the encouraging preclinical data suggest that small molecule PROTAC technology has the potential to greatly promote the development of targeted therapies. [3]

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Objective: Finding drugs that can inhibit edema and glial scar formation and improve neuronal survival is crucial for improving the prognosis of spinal cord injury (SCI). This study used the potent and selective aquaporin 4 (AQP4) inhibitor 2-(nicotinamide)-1,3,4-thiadiazole (TGN-020) to investigate its effect on spinal cord injury (SCI) in Sprague-Dawley rats. Main methods: Moderate injury was induced by compressing the T10 segment of the spinal cord with a sterile depressor (35 g, 5 min). TGN-020 (100 mg/kg) or an equal volume of 10% dimethyl sulfoxide (DMSO) was then administered intraperitoneally. Neurological function was assessed using the Basso-Beattie-Bresnahan Open Field Motor Scale at 1, 3, 7, 14, 21, and 28 days post-SCI. Spinal cord water content was measured 3 days post-SCI to assess the degree of spinal cord edema. The expression levels of AQP4, glial fibrillary acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), and growth-associated protein-43 (GAP-43) were detected by Western blotting and immunofluorescence staining at 3 days and 4 weeks post-SCI. The number of surviving neurons and the size of the glial scar were determined by Nissl staining and hematoxylin-eosin staining, respectively. Main results: Our results showed that TGN-020 promoted functional recovery on days 3, 7, 14, 21 and 28 after SCI, reduced edema on day 3 after SCI, and inhibited the expression of AQP4, GFAP and PCNA. In addition, the observation results at 4 weeks after spinal cord injury showed that TGN-020 inhibited the formation of glial scars and upregulated the expression of GAP-43. Significance: TGN-020 can reduce spinal cord edema, inhibit the formation of glial scars and promote axonal regeneration, which has a beneficial effect on the recovery of rats. [4]

C8H7N4NAOS

230.222150087357

228.008

C, 42.11; H, 2.21; N, 24.55; Na, 10.07; O, 7.01; S, 14.05

1313731-99-5

TGN-020;51987-99-6

139592827

White to off-white solid powder

0

6

2

15

219

0

C(C1=CN=CC=C1)(=O)NC1SC=NN=1.[NaH]

YVHWOCDMDRVSHB-UHFFFAOYSA-M

InChI=1S/C8H6N4OS.Na/c13-7(6-2-1-3-9-4-6)11-8-12-10-5-14-8;/h1-5H,(H,11,12,13);/q;+1/p-1

sodium;pyridine-3-carbonyl(1,3,4-thiadiazol-2-yl)azanide

TGN-020 SODIUM; TGN-020 (sodium); 1313731-99-5;

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

H2O :~100 mg/mL (~438.19 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (21.91 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 50.0 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.

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Solubility in Formulation 2: ≥ 5 mg/mL (21.91 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 50.0 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.

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Solubility in Formulation 3: ≥ 5 mg/mL (21.91 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 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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