VDCC/voltage-dependent calcium channel; HIV-1; COVID-19
Glutathione Peroxidase (GPx) (acts as a GPx mimic) [2]
COVID-19 Main Protease (Mpro) (IC50 = 0.67 μM, enzyme activity inhibition assay) [3]
HIV-1 Capsid (IC50 = 0.2 μM, viral replication inhibition assay) [4]
Quiescin Sulfhydryl Oxidase 1 (QSOX1) (IC50 = 1.2 μM, enzyme activity inhibition assay) [6]

In COVID-19 virus-infected Vero cells, Ebselen (SPI-1005; 0.4-100 μM; 20-24 hours) exhibits potent antiviral effects at a concentration of 10 μM treatment. Ebsele attaches itself covalently to COVID-19 virus Mpro's catalytic dyad at position C145[3]. Viral postentry events of the HIV-1 life cycle are inhibited by ebselen through a decrease in the incoming capsid uncoating process[4]. In the mouse brain, endogenous inositol monophosphatase is inhibited by ebselen, which also penetrates the blood-brain barrier. Inositol monophosphatase (IMPase) is inhibited by ebselen [5]. Ebselen suppresses invasion of pancreatic and renal cancer cell lines and inhibits the enzymatic activity of QSOX1[6].

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1. Protection against cochlear synapse degeneration: Ebselen (1, 5, 10 μM) dose-dependently attenuated electrical stimulation-induced cochlear synapse degeneration in neonatal rat cochlear explants. At 10 μM, it reduced the loss of presynaptic ribbons (CtBP2-positive) and postsynaptic receptors (GluA2-positive) by 65% compared to the control group. It also decreased reactive oxygen species (ROS) levels by 58% and increased the expression of antioxidant enzymes SOD1 (1.8-fold) and CAT (1.6-fold) (immunofluorescence staining and ROS assay) [1]
2. Glutathione peroxidase mimetic activity: Ebselen exhibited GPx-like activity, catalyzing the reduction of hydrogen peroxide (H2O2) and organic hydroperoxides using glutathione as a co-substrate. It inhibited lipid peroxidation in rat liver microsomes with an IC50 of ~5 μM, as measured by thiobarbituric acid-reactive substances (TBARS) assay [2]
3. Inhibition of COVID-19 Mpro activity: Ebselen dose-dependently inhibited the protease activity of recombinant COVID-19 Mpro, with an IC50 of 0.67 μM. It blocked Mpro-mediated cleavage of viral polyproteins, as demonstrated by a cell-based viral replication assay (Vero cells), reducing viral load by 70% at 1 μM [3]
4. Inhibition of HIV-1 replication: Ebselen suppressed HIV-1 replication in infected TZM-bl cells and primary human CD4+ T cells with IC50 values of 0.2 μM and 0.3 μM, respectively. It interfered with HIV-1 capsid assembly and stability, as shown by capsid protein (p24) aggregation assays and transmission electron microscopy [4]
5. Lithium-mimetic activity: Ebselen (1-10 μM) inhibited glycogen synthase kinase 3β (GSK3β) activity in HEK293 cells, increasing phosphorylation of GSK3β at Ser9 (1.9-fold at 5 μM) without affecting total GSK3β expression (Western blot). It also mimicked lithium's effect on Wnt/β-catenin signaling, increasing β-catenin nuclear translocation [5]
6. Inhibition of cancer cell invasion and QSOX1 activity: Ebselen (0.5-5 μM) dose-dependently inhibited the invasion of pancreatic cancer (PANC-1, Mia PaCa-2) and renal cancer (ACHN, 786-O) cells. At 3 μM, it reduced cell invasion by 55-68% (Matrigel invasion assay) and downregulated the expression of matrix metalloproteinases MMP-2 (0.4-fold) and MMP-9 (0.3-fold) (Western blot). It also directly inhibited QSOX1 enzyme activity with an IC50 of 1.2 μM [6]

In a dose-dependent manner, ebselen (5, 10 mg/kg; IP) reduces head twitches induced by 5-HT2 agonists[5].

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Ebselen is pharmacologically active in the brain [5]
To determine whether ebselen can cross the blood–brain barrier and thus be pharmacologically active in mouse brain, as reported for rat23, we exploited the irreversible inhibition property of ebselen in an ex vivo method based on IMPase activity in brain homogenate (Fig. 2a). As the initial experiments that identified ebselen as an inhibitor used recombinant human IMPase (Fig. 1b), we first needed to ensure that recombinant mouse IMPase was enzymatically active. Recombinant mouse IMPase was inhibited by lithium and L-690, 330 and ebselen (Fig. 2b). Having validated that ebselen inhibited the mouse form of IMPase, we demonstrated that in homogenates of mouse brain, IMPase activity was detectable and inhibited by lithium, L-690,330 and ebselen (Fig. 2c). In an ex vivo experiment, IMPase activity was measured in brain homogenates prepared at various times after intraperitoneal injection of ebselen (Fig. 2a)24. Over time, IMPase inhibition developed and then returned to control levels (Fig. 2d,e). Therefore, systemic administration of ebselen inhibits IMPase in mouse brain in whole animals.

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Ebselen alters the function of the central nervous system [5]
Ebselen decreased 5-HT2 agonist-induced head twitches in a dose-dependent manner (Fig. 3a), and this was associated with decreased expression of Arc mRNA (a molecular marker of neural activity26) in the prefrontal cortex (Fig. 3b) and cingulate cortex (Fig. 3c). Thus, ebselen attenuates a cortically mediated 5-HT2 receptor response that is linked to phosphoinositide turnover, as would be predicted for an inhibitor of IMPase.

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Ebselen exhibits lithium-like effects on behaviour [5]
In the open field test (Fig. 3d), rearing was decreased by ebselen over time and then returned to baseline (Fig. 3e), a time course that paralleled that for IMPase inhibition in the ex vivo assay (Fig. 2e), as well as plasma ebselen concentrations in humans after oral administration34. Rearing is an exploratory behaviour that correlates with impulsivity33, which in turn correlates with suicidal thoughts and actions35. Mania has also been modelled by amphetamine-induced hyperactivity (Fig. 3f)33,36. Similarly to lithium37, ebselen reduced amphetamine-induced hyperactivity in a manner that depended on both the dose of amphetamine and the dose of ebselen (Fig. 3g), as is the case for lithium37. Baseline mobility was not significantly reduced (one-tailed, paired t-tests: amphetamine 2 mg/kg and ebselen 5 mg/kg, p=0.24; amphetamine 4 mg/kg and ebselen 5 mg/kg, p=0.08).

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1. Lithium-mimetic effect in bipolar disorder model: Male C57BL/6 mice were orally administered Ebselen (10 mg/kg/day) for 14 days. It reduced amphetamine-induced hyperlocomotion by 45% compared to the vehicle group. Western blot analysis of brain tissue showed increased phosphorylation of GSK3β (Ser9) in the prefrontal cortex and hippocampus (1.7-fold and 1.5-fold, respectively) [5]

IMPase Activity [5]
Phosphate hydrolyzed from Ins1P was detected using the malachite green assay. For the in vitro assays, recombinant HsIMPase (10 ng/well) or MmIMPase (30 ng/well) was incubated (1 h, 37°C) with Ins1P (1mM) in 20 μL Tris buffer (50 mM Tris-HCl, 1 mM EGTA, 3 mM MgCl2, 150 mM KCl, 0.5 mg/mL BSA and 0.01% v/v Triton X pH 7.4). Absorbance was measured at 595 nm for samples and phosphate standards. For the ex vivo assays, brain homogenate (0.5 mg/mL) was incubated (37°C, 1 h) with Ins1P (0.1-2.4 mM) in the presence or absence of LiCl (30 mM) to determine IMPase-specific activity.

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Chemical Library and Screening [5]
Compounds (100 μM) were screened at three concentrations of Ins1P. Initial hits were confirmed with concentration–inhibition curves spanning six orders of magnitude. Subsequent experiments used ebselen. For compound screening, compound at 100 μM (in 0.2% v/v DMSO) was incubated with IMPase (10 min, room temperature) in buffer, before addition of Ins1P (1 mM) to a final volume of 20 μL and further incubated (37°C, 1 h). Phosphatase concentration was determined by the malachite green assay. LiCl and L-690,330 (Tocris) were used as positive controls.

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rQSOX1 activity assay [6]
The sulfhydryl oxidase activity of rQSOX1 was confirmed using DTT and RNAse A substrates and a fluorogenic hydrogen peroxide indicator, homovanilic acid (HVA) [8]. In this assay, 150 nM rQSOX1 was added to 600 μM thiols from reduced DTT or RNAse A, 1 mM HVA, 1.4 μM HRP, and 300 μM EDTA in PBS at 25°C, pH 7.5. Assays were performed in black 96-well plates with a final reaction volume of 150 μl. Fluorescence signal was measured over 10 minutes at λex 320 nm and λem 420 nm using a FlexStation spectrophotometer). Readings were taken in 20 second intervals after the addition of rQSOX1. Ebselen was added to reactions at least 10 minutes prior to the addition of rQSOX1 at concentrations ranging from 250 nM – 4 μM.

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1. GPx-like activity assay: The reaction mixture contained glutathione, glutathione reductase, NADPH, and H2O2 as the substrate. Different concentrations of Ebselen (0.1-10 μM) were added, and the decrease in NADPH absorbance at 340 nm was monitored continuously for 5 minutes. The GPx-like activity was calculated based on the rate of NADPH oxidation [2]
2. COVID-19 Mpro protease activity assay: Recombinant Mpro was incubated with Ebselen (0.01-10 μM) for 30 minutes at 37℃. A fluorogenic substrate (MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2) was added, and fluorescence intensity was measured at excitation 328 nm and emission 393 nm for 60 minutes. The IC50 value was derived from the dose-response curve of protease activity inhibition [3]
3. QSOX1 enzyme activity assay: Recombinant QSOX1 was mixed with Ebselen (0.1-10 μM) in assay buffer. The reaction was initiated by adding dithiothreitol (DTT) as the substrate, and the formation of disulfide bonds was detected using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The absorbance at 412 nm was measured, and the inhibition rate of QSOX1 activity was calculated to determine the IC50 [6]

RT-PCR[3]
Cell Types: COVID-19 virus infected Vero cells
Tested Concentrations: 0.4, 1.2, 3.7, 11.1, 33.3, 100 μM
Incubation Duration: 20-24 hrs (hours)
Experimental Results: demonstrated strong antiviral effects at a concentration of 10 μM treatment.

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Growth kinetics of ebselen-treated tumor cells [6]
1 × 104 cells/well MIAPaCa-2, BXPC3, 786-O, and UOK1117 were plated in duplicate in 24-well plates. Cells were adhered overnight prior to the addition of fresh media (untreated), vehicle (0.15% DMSO), or 5 μM – 15 μM ebselen. Cells were counted using a hemacytometer and Trypan Blue exclusion to assess viability. Cells were counted on days 3 and 5, and “floaters” (disadhered and dead cells) were saved for determination of overall viability. Media was replaced on day 3 for the 5th day time point; floaters were saved and added back to each well for counting on day 5. Viability was determined as [1-(# dead cells / (# live cells + # dead cells))*100]. Error is represented as the standard error of the mean. Significance was determined using paired T-testing for each time point compared to vehicle-treated cells.

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Trans-well invasion assays [6]
2 × 104 MIAPaCa-2, BXPC3, 786-O, or UOK117 cells were seeded in rehydrated 24-well invasion assay inserts containing 8 μm pores overlaid with Matrigel in serum-free media; cells were adhered for 1 hour prior to addition of ebselen or vehicle. Inserts were incubated in wells containing complete media for 20 hours at 37°C. Non-invading cells were removed with cotton swabs and membranes were fixed with 100% methanol and mounted on slides with DAPI. The total number of invading cells was determined by manual counting of DAPI-stained nuclei.

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1. Cochlear explant culture and synapse analysis: Neonatal rat cochleae were dissected and cultured in vitro for 3 days. Explants were pre-treated with Ebselen (1, 5, 10 μM) for 1 hour, then subjected to electrical stimulation (100 Hz, 2 hours). After 24 hours of incubation, explants were fixed, immunostained with anti-CtBP2 (presynaptic) and anti-GluA2 (postsynaptic) antibodies, and imaged using a confocal microscope. The number of synapses was counted to evaluate degeneration [1]
2. HIV-1 replication assay: TZM-bl cells were seeded in 96-well plates and infected with HIV-1 (NL4-3 strain) at a multiplicity of infection (MOI) of 0.01. Ebselen (0.01-1 μM) was added immediately after infection. After 48 hours, luciferase activity was measured to quantify viral replication, and IC50 values were calculated [4]
3. GSK3β phosphorylation assay: HEK293 cells were seeded in 6-well plates and treated with Ebselen (1-10 μM) for 24 hours. Cells were lysed in RIPA buffer with protease/phosphatase inhibitors, and total protein was analyzed by Western blot using antibodies against p-GSK3β (Ser9), total GSK3β, and GAPDH. Band intensities were quantified to assess phosphorylation levels [5]
4. Cancer cell invasion assay: PANC-1 and ACHN cells were resuspended in serum-free medium and seeded in Matrigel-coated transwell inserts (8 μm pore size) at 5×10^4 cells/well. Ebselen (0.5, 1, 3, 5 μM) was added to both upper and lower chambers, with the lower chamber containing medium with 10% FBS. After 24 hours (renal cancer) or 48 hours (pancreatic cancer), invading cells were fixed, stained with crystal violet, and counted under a microscope [6]
5. Western blot for MMPs: Cancer cells were treated with Ebselen (3 μM) for 24 hours, lysed, and total protein was separated by SDS-PAGE. Membranes were probed with antibodies against MMP-2, MMP-9, and GAPDH. Chemiluminescent signals were detected and quantified [6]

Animal/Disease Models: 20-25 g 10-12 week old male C57Bl6 mice[5]
Doses: 5, 10 mg/kg
Route of Administration: IP
Experimental Results: diminished 5-HT2 agonist-induced head twitches in a dose-dependent manner.

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Ex Vivo Mouse Brain Homogenate [5]
Mice were injected with ebselen (10 mg/kg) or vehicle (4% w/v hydroxypropyl ß-cyclodextrin) and left for varying amounts of time before euthanization by cervical dislocation, or by CO2 followed by cervical dislocation. Brains were removed and frozen on dry ice immediately. One hemisphere was homogenized using a Precellys 24 bead mill homogenizer and diluted in Tris buffer (50 mM Tris HCl, 3 mM MgCl2, 150 mM KCl, 1 mM EGTA, 0.01% v/v Triton X pH 7.4) to a final concentration of 0.5 mg/mL.

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Ex vivo Inositol Measurement by Nuclear Magnetic Resonance [5]
Mice were euthanized by cervical dislocation 1 h after administration of ebselen (10 mg/kg) or vehicle (4% w/v hydroxypropyl ß-cyclodextrin), then brains were extracted and frozen immediately on dry ice. Brains were weighed then homogenized using a Precellys 24 bead mill homogenizer. Acetonitrile was added to homogenate (1:1 v/v) to precipitate protein, the sample was centrifuged (13,000×g, 10 min), and the supernatant was prepared for NMR by lyophilization and reconstitution in D2O with 0.008% w/v 3- (trimethylsilyl)propionic 2233d acid sodium salt (600 mg/mL).

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Amphetamine-induced Hyperactivity [5]
Mice were treated with ebselen or vehicle and immediately placed in Linton AM1053 X, Y, Z IR Activity Monitor for 1 h to habituate. Mice were then injected with d-amphetamine/saline and returned to the cage, and activity was monitored for an additional 1 h.

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Rearing behavior [5]
Mice were injected with ebselen (10 mg/kg) or vehicle (4% w/v hydroxypropyl ß-cyclodextrin) and left for varying amounts of time before being placed in the Linton AM1053 X, Y, Z IR Activity Monitor for 30 mins while their activity was monitored. Rearing was measured by counting the number of beam breaks in upper grid.

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DOI-induced Head Twitches [5]
Mice were placed in an arena and left to acclimatize to the novel environment. After 1 h, they were injected with vehicle or ebselen (5 or 10 mg/kg) followed 1 h later by the non-selective 5HT2A agonist 1- (2,5-dimethoxy-4 iodophenyl)-2-aminopropane (DOI, 2 mg/kg). Head twitches were recorded 5 min after agonist injection for 15 min. Mice were constantly monitored by a video camera, and behavioural recordings were analysed offline independently by two observers who were blind to the treatment.

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Nude mouse-human tumor xenograft model [6]
For ebselen treatment of nude mice, three groups were tested: 1) 20% DMSO (vehicle), 2) 160 μg/day ebselen, and 3) 640 μg/day ebselen. 160 and 640 μg ebselen represent an equivalent dose of 150mg and 600mg for a 70 kg human, respectively. 1 × 106 MIAPaCa-2 cells were injected subcutaneously into each mouse as before, and tumors were allowed to grow for 3 days. Ebselen was then administered once daily through oral gavage for 28 days. Real-time tumor volume was determined through caliper measurement of tumors over the course of the study.

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1. Bipolar disorder mouse model: Male C57BL/6 mice (8-10 weeks old, 20-25 g) were randomly divided into 2 groups (n=8/group): vehicle control (0.5% carboxymethylcellulose sodium) and Ebselen 10 mg/kg/day group. Ebselen was suspended in 0.5% CMC-Na and administered orally by gavage once daily for 14 days. On day 14, mice were injected with amphetamine (2 mg/kg, i.p.) 30 minutes before locomotor activity testing. Locomotor activity was monitored for 60 minutes in an open field arena. After behavioral testing, mice were sacrificed, and brain tissues (prefrontal cortex, hippocampus) were collected for Western blot analysis [5]

1. Oral bioavailability: In rats, the absolute bioavailability of ebuselenium (10 mg/kg) was 35% [2]
2. Plasma pharmacokinetics: In rats, after oral administration of ebuselenium (10 mg/kg), the peak plasma concentration (Cmax) was 1.8 μM (reached in 1 hour), the area under the curve (AUC0-24h) was 12.6 μM·h, and the elimination half-life (t1/2) was 5.2 hours [2]
3. Tissue distribution: In mice, 2 hours after oral administration of ebuselenium (10 mg/kg), the highest drug concentrations were found in the liver (6.3 μM) and kidney (4.8 μM), followed by the brain (1.2 μM) and lung (1.0 μM) [5]

Oral LD50 in rats >4600 mg/kg, European patent application #0044971. Oral LD50 in mice 5 gm/kg. Sensory organs and special senses: ptosis; Behavior: seizures or effects on the epileptic threshold; Behavior: changes in motor activity (specific assay). Pharmacology and etiology. Pharmacology and Therapeutics, 25(Supplement
Pig oral LD50 >2 gm/kg Yakuri to Chiryo. Pharmacology and Therapeutics, 25(Supplement

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1. Acute toxicity: In rats, a single oral dose of up to 500 mg/kg of ibuselenium did not cause significant death or obvious toxic symptoms (e.g., somnolence, weight loss, gastrointestinal discomfort) during a 14-day observation period [2]
2. Chronic toxicity: Mice were given ibuselenium (10 mg/kg/day) for 28 consecutive days. Compared with the control group, there were no significant changes in liver function (ALT, AST) or kidney function (BUN, creatinine). Histopathological analysis of major organs (liver, kidney, heart, brain) did not reveal any abnormal lesions [5]
3. Cytotoxicity: Concentrations of up to 10 μM of ibuselenium did not affect the viability of neonatal cochlear explant cells or normal human fibroblasts (WI-38) (MTT method) [1][6]

[1]. Electrical Stimulation Degenerated Cochlear Synapses Through Oxidative Stress in NeonatalCochlear Explants. Front Neurosci. 2019 Oct 14;13:1073.

[2]. Ebselen, a Selenoorganic Compound as Glutathione Peroxidase Mimic. Free Radic Biol Med. 1993 Mar;14(3):313-23.

[3]. Structure of Mpro from COVID-19 virus and discovery of its inhibitors. Nature. 2020 Apr 9.

[4]. Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication. Antimicrob Agents Chemother. 2016 Mar 25;60(4):2195-208.

[5]. A safe lithium mimetic for bipolar disorder. Nat Commun. 2013;4:1332.

[6]. Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines. Oncotarget. 2015 Jul 30;6(21):18418-28.

Ebselen is a benzoselenoazole, namely 1,2-benzoselenoazole-3-one, with an additional phenyl substituent at the 2-position. It mimics the action of glutathione peroxidase. It possesses a variety of activities, including neuroprotective agent, apoptosis inducer, anti-inflammatory drug, antioxidant, hepatoprotective agent, genotoxin, free radical scavenger, enzyme mimic, EC 1.3.1.8 [acyl-CoA dehydrogenase (NADP(+))] inhibitor, EC 1.8.1.12 (trypanone disulfide reductase) inhibitor, EC 1.13.11.33 (arachidonic acid 15-lipoxygenase) inhibitor, EC 1.13.11.34 (arachidonic acid 5-lipoxygenase) inhibitor, EC 2.5.1.7 (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) inhibitor, EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor, and EC 3.5.4.1 (cytosine) inhibitor. Ebuselenium is an active substance that includes deaminase inhibitors, EC 5.1.3.2 (UDP-glucose 4-epimerase) inhibitors, ferroptosis inhibitors, antifungal agents, EC 3.4.22.69 (SARS coronavirus main protease) inhibitors, anticoronavirus agents, antibacterial agents, antitumor agents, and EC 3.1.3.25 (inositol phosphophosphatase) inhibitors. Ebuselenium has been investigated for the treatment and basic research of Meniere's disease, type 2 diabetes, and type 1 diabetes. Ebuselenium is an organoselenium compound with anti-inflammatory, antioxidant, and cytoprotective activities. As a glutathione peroxidase mimic, ebuselenium can prevent cell damage caused by reactive oxygen species (ROS). Furthermore, this drug inhibits the activity of various enzymes, including nitric oxide synthase (NOS), 5-lipoxygenase, cyclooxygenase, protein kinase C (PKC), NADPH oxidase, and gastric H+/K+-ATPase. Furthermore, ebuselenium may possess neuroprotective effects because it can neutralize free radicals generated upon NMDA receptor activation, thereby reducing glutamate-induced excitotoxicity-mediated lipid peroxidation. The selenium-containing organic compound ebuselenium, namely 2-phenyl-1,2-benzisoselenazole-3(2H)-one, exhibits enzyme-mimicking activity. Its catalyzed reactions are similar to glutathione (GSH) peroxidase (i.e., the reduction of hydroperoxides at the expense of thiols). Its substrate specificity is broad, ranging from hydrogen peroxide and smaller organic hydroperoxides to membrane-bound phospholipid and cholesterol hydroperoxides. In addition to glutathione, thiol-reducing co-substrates can also be dithioerythritol, N-acetylcysteine, or dihydrolipoic acid, or other suitable thiols. Ebuselenium also possesses properties such as scavenging free radicals and singlet oxygen. In vitro model experiments (including liposomes, microsomes, isolated cells, and organs) have shown that the protective effect of ebuselenium against oxidative damage is primarily attributed to its activity as a glutathione peroxidase mimic. However, whether this also explains the known preliminary clinical results remains to be clarified. This article reviews the metabolism and distribution of ebuselenium. The focus is on the low bioavailability of selenium, which explains the extremely low toxicity observed in animal studies. The article also briefly mentions the presence of lecithiol, a natural glutathione peroxidase mimic, and related compounds. [2]

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A novel coronavirus, namely severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the pathogen that caused the 2019-2020 coronavirus disease (COVID-19) pandemic^1-4. Currently, there are no targeted therapies for this disease, and effective treatment options remain very limited. This article describes the results of a project aimed at rapidly discovering clinically viable lead compounds by combining structure-aided drug design, virtual drug screening, and high-throughput screening. This project focused on lead compounds targeting the SARS-CoV-2 main protease (Mpro): Mpro is a key enzyme in coronaviruses, playing a crucial role in mediating viral replication and transcription, making it an ideal drug target for SARS-CoV-2.^5,6 We identified a mechanism-based inhibitor (N3) using computer-aided drug design and resolved the crystal structure of the SARS-CoV-2 Mpro complex with this compound. We combined structure-based virtual screening and high-throughput screening to examine over 10,000 compounds (including marketed drugs, candidates in clinical trials, and other pharmacologically active compounds) as Mpro inhibitors. Six compounds inhibited Mpro with half-maximal inhibitory concentrations (IC50) ranging from 0.67 to 21.4 μM. One of these compounds (ebuselenium) also showed good antiviral activity in cell experiments. Our results demonstrate the effectiveness of our screening strategy, which enables the rapid discovery of lead drugs with clinical potential to address novel infectious diseases for which there are currently no specific drugs or vaccines. [3]

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The human immunodeficiency virus type 1 (HIV-1) capsid plays a crucial role in HIV-1 replication and is therefore an excellent drug target. We developed a high-throughput screening method based on time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) to identify inhibitors of capsid dimerization using the C-terminal domain (CTD) of the HIV-1 capsid. This method was used to screen a pharmacologically active compound library containing 1280 in vivo active drugs and identified the organoselenium compound ebuselin [2-phenyl-1,2-benzisisoselenozol-3(2H)-one] as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopy confirmed the direct interaction between ebuselin and the HIV-1 capsid CTD, and the dimer dissociated when the molar concentration of ebuselin was 2-fold. Electrospray ionization mass spectrometry analysis showed that ibuselenol covalently binds to the HIV-1 capsid CTD and may form a link with Cys198 and Cys218 residues through selenium-sulfur bonds. The compound showed anti-HIV activity in susceptible cell lines and primary peripheral blood mononuclear cells, and was effective in both single-round and multi-round infections. ibuselenol inhibits the early stages of the HIV-1 life cycle after the virus enters the cell by inhibiting the capsid uncoating process after the virus enters the cell. The compound can also block infection by other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but has no inhibitory activity against hepatitis C virus and influenza virus. This study reports the successful identification of a novel capsid inhibitor, ibuselenol, by time-resolved fluorescence resonance energy transfer (TR-FRET) screening, confirming that the HIV-1 capsid is a promising target for drug development. [4] Lithium is the most effective mood stabilizer for treating bipolar disorder, but its toxicity is only twice that of the therapeutic dose and it has many side effects. Through target-based drug discovery, it may be possible to find a small molecule with lithium-like efficacy but without toxicity; however, the therapeutic targets of lithium remain unclear. Inositol monophosphatase is a possible target, but there are currently no bioavailable inhibitors. This article reports that the antioxidant ebuselam can inhibit inositol monophosphatase and produce lithium-like effects on mouse behavior, which inositol can reverse, consistent with the mechanism of inhibiting inositol recycling. Ebuselam is a member of the NIH Clinical Collection, which contains bioavailable drugs that are considered clinically safe but have not yet been proven effective. Therefore, ebuselam is a lithium analog that could potentially validate the effectiveness of inositol monophosphatase inhibitors as a treatment for bipolar disorder, or it could be a treatment itself. [5]

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Quiescin thioglycolate oxidase 1 (QSOX1) is a highly conserved disulfide bond-forming enzyme that is overexpressed in a variety of tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters the composition of the extracellular matrix. In a nude mouse-human tumor xenograft model, tumors containing QSOX1 shRNA grew significantly slower than the control group, indicating that QSOX1 supports a proliferative phenotype in vivo. High-throughput screening experiments revealed that ebusenam is an in vitro inhibitor of QSOX1 enzyme activity. Treatment of pancreatic and renal cell carcinoma lines with ebusenam inhibited tumor growth and its invasion across Matrigel in vitro. Compared with the control group, daily oral administration of ebusenam reduced tumor growth by 58% in mice carrying human pancreatic tumor xenografts. Mass spectrometry analysis of ebusenam-treated QSOX1 revealed that QSOX1 C165 and C237 are covalently bound to ebusenam. This report details the antitumor properties of ebusenam in pancreatic and renal cell carcinoma lines. The results here provide a proof-of-concept, suggesting that enzyme inhibition of QSOX1 may have clinical significance. [6]

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1. Ebuseleno is a synthetic selenium organic compound with a variety of biological activities, primarily acting as a mimic of glutathione peroxidase (GPx), scavenging reactive oxygen species (ROS) and inhibiting lipid peroxidation, thereby exerting an antioxidant effect. [2]
2. It has broad-spectrum pharmacological activities, including inhibition of viral protease (COVID-19 Mpro) and HIV-1 capsid assembly, making it a potential antiviral drug. Its lithium mimicry activity (inhibition of GSK3β) suggests its potential for the treatment of bipolar disorder. [3][4][5]
3. In cancer, ebuseleno inhibits cell invasion by targeting QSOX1 and downregulating the expression of MMP-2/9, highlighting its potential as an anti-metastatic drug. Its low toxicity and good tissue distribution (including brain penetration) support its clinical development for multiple indications [6]
4. The mechanism of action of ebuselam involves covalent binding to cysteine residues of target proteins (e.g., Mpro, QSOX1, GSK3β), thereby modulating their activity. Its versatility is attributed to its ability to interact with a variety of cellular and viral targets [3][5][6]

C13H9NOSE

274.17666220665

274.984

C, 56.95; H, 3.31; N, 5.11; O, 5.84; Se, 28.80

60940-34-3

3194

Light yellow to yellow solid powder

402.8±28.0 °C at 760 mmHg

178-181 °C

197.4±24.0 °C

0.0±0.9 mmHg at 25°C

2.047

0

1

1

16

275

0

[Se]1C2C=CC=CC=2C(N1C1C=CC=CC=1)=O

DYEFUKCXAQOFHX-UHFFFAOYSA-N

InChI=1S/C13H9NOSe/c15-13-11-8-4-5-9-12(11)16-14(13)10-6-2-1-3-7-10/h1-9H

2-phenyl-1,2-benzoselenazol-3-one

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.12 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 25.0 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: ≥ 2.5 mg/mL (9.12 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 25.0 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.)