Ebselen and Diphenyl Diselenide Inhibit SARS-CoV-2 Replication at Non-Toxic Concentrations to Human Cell Lines

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the causative agent of the COVID-19 pandemic, a global public health problem. Despite the numerous studies for drug repurposing, there are only two FDA-approved antiviral agents (Remdesivir and Nirmatrelvir) for non-hospitalized patients with mild-to-moderate COVID-19 symptoms. Consequently, it is pivotal to search for new molecules with anti-SARS-CoV-2 activity and to study their effects in the human immune system. Ebselen (Eb) is an organoselenium compound that is safe for humans and has antioxidant, anti-inflammatory, and antimicrobial properties. Diphenyl diselenide ((PhSe)2) shares several pharmacological properties with Eb and is of low toxicity to mammals. Herein, we investigated Eb and (PhSe)2 anti-SARS-CoV-2 activity in a human pneumocytes cell model (Calu-3) and analyzed their toxic effects on human peripheral blood mononuclear cells (PBMCs). Both compounds significantly inhibited the SARS-CoV-2 replication in Calu-3 cells. The EC50 values for Eb and (PhSe)2 after 24 h post-infection (hpi) were 3.8 µM and 3.9 µM, respectively, and after 48 hpi were 2.6 µM and 3.4 µM. These concentrations are safe for non-infected cells, since the CC50 values found for Eb and (PhSe)2 on Calu-3 were greater than 200 µM. Importantly, the concentration rates tested on viral replication were not toxic to human PBMCs. Therefore, our findings reinforce the efficacy of Eb and demonstrate (PhSe)2 as a new candidate to be tested in future trials against SARS-CoV-2 infection/inflammation conditions.


Introduction
The first identified cases of infection by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were detected in December 2019 in Wuhan, China [1], and soon after, the virus was identified as the causative agent of coronavirus disease 2019 . In March 2020, COVID-19 was declared a pandemic by the World Health Organization, spreading rapidly across the world and becoming one of the worst and most deadly pandemics in the world [2]. Currently, it is estimated that more than 767 million the compounds did not induce any signs of toxicity in human PBMCs, as evaluated by parameters of viability loss, apoptosis, morphological changes, and cell cycle disruption. In the peroxidation assay, the compounds also did not exhibit pro-oxidant action at any concentration tested.

Eb and (PhSe) 2 Anti-SARS-CoV-2 Activity in Calu-3 Cells
Calu-3 (cell line from the submucosal gland that recapitulates type II pneumocytes) cells were infected with a SARS-CoV-2 isolate (lineage B.1, GenBank #MT710714, SisGen AC58AE2) at a multiplicity of infection (MOI) of 0.01 or 0.1 for 1 h at 37 • C. After that, the culture medium was removed and the treatments with Eb or (PhSe) 2 , ranging from 0.78 to 12.5 µM, were performed for 24 h and 48 h. The replicative ability of SARS-CoV-2, in the presence and absence of the compounds, was evaluated by counting the plaqueforming units (PFU/mL). For the PFU assay, Vero E6 (African green monkey kidney, ATCC CRL-1586) cells, previously seeded in 96-well plates containing 2 × 10 4 cells/well, were incubated with a serial dilution (1:200 to 1:12,800) of supernatants. After 1 h of infection, 10× Dulbecco's Modified Eagle Medium (DMEM, Gibco, Grand Island, NY, USA) containing 2% fetal bovine serum (FBS; Gibco TM , Fisher Scientific, Loughborough, UK) and 2.4% carboxymethylcellulose (Sigma-Aldrich, Burlington, MA, USA) was added to the cells and the incubation was maintained at 37 • C for 72 h. The fixation of the biological material was performed with 4% formalin solution for 3 h. After that, the cells were stained with 0.04% crystal violet solution for 1 h at room temperature. Then, the material was washed in water, and after drying the quantification of the PFU was performed. All procedures involving the handling of SARS-CoV-2 were performed in a biosafety level 3 (BSL3) environment, in accordance with the World Health Organization guidelines [23].

Human Subjects and Blood Collection
Peripheral blood was collected from healthy volunteers (4 men, 4 women, mean age of 30 ± 10). Exclusion criteria included alcohol abuse or drug dependence, diseases, and drug treatments in the last 2 weeks. The blood collection was performed following the Declaration of Helsinki. Volunteers were not exposed to any risk while participating in the study. The protocol was approved by the Ethics Committee for Research with Humans at the Federal University of Santa Maria (number 67825122.1.0000.5346).

Peripheral Blood Mononuclear Cells' (PBMCs) Isolation and Treatments
Peripheral blood mononuclear cells (PBMCs) were isolated following the methodology described by Ecker et al. [24], with some modifications. In this step, peripheral blood (20 mL) was collected from each volunteer under aseptic conditions into tubes coated with sodium heparin (Hipolabor Farmacêutica Ltda., Belo Horizonte, MG, Brazil). Whole blood was diluted 1:1 with PBS buffer (136 mM NaCL, 2.68 mM KCL, 1.47 mM KH 2 PO 4 , 8.1 mM Na 2 HPO 4 , pH 7.4) and layered over Ficoll-Paque Plus (GE Healthcare, Piscataway, NJ, USA) before the centrifugation at 577× g for 30 min to separate PBMCs. Cells were washed three times with PBS (followed by centrifugations at 400× g for 10 min, 256× g for 10 min, and 178× g for 5 min, respectively), and then cultured in RPMI media supplemented with 2% PHA, 10% BFS, and 1% antibiotic-antimycotic, containing Eb and (PhSe) 2 (both diluted in DMSO p.a as a vehicle). PBMCs were cultured in ELISA microplates maintained in culture for 24 h at 37 • C and 5% CO 2 . The number of cells used was 0.5 × 10 6 PBMCs per group. Two controls were used (with and without final DMSO of 0.25%). However, only the PBS buffer control is shown in the results since there was no statistical difference between these groups among all the evaluated parameters.

Cytotoxicity Assays
The possible toxicity of compounds in the cell models was determined by the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; Sigma-Aldrich) assay Vaccines 2023, 11, 1222 4 of 16 following the methodology described by Mosmann [25], with some modifications. Herein, PBMCs and Calu-3 were exposed to different concentration curves, ranging from 0.25 to 200 µM in PBMCs or 25 to 200 µM in Calu-3, of the tested compounds (dissolved in DMSO p.a, final: 0.1% v/v) for 24 or 72 h, respectively. After the treatment period, 0.16 mg/mL or 5 mg/mL of MTT, previously diluted in PBS, was added to the medium of PBMCs or Calu-3, respectively. Then, the samples were incubated for 2 h at 37 • C, in the dark. Subsequently, the samples were centrifuged, the supernatant was discarded, and the formazan crystals were solubilized with 10% SDS or DMSO p.a. After 2 h, the samples were read in an ELISA microplate reader at 540 or 570 nm (TP-Reader Thermo Plate), depending on the cell type, and the dehydrogenase enzyme activity was expressed as a percentage of the control, only exposed to DMSO at the same concentration used in the treated conditions.

Lipid Peroxidation by the Thiobarbituric Acid-Reactive Substances (TBARS) Method
Lipid peroxidation was assessed to evaluate the possible pro-oxidant activity of the compounds. The capacity of the compounds to induce oxidation of the glycerophospholipid phosphatidylcholine was tested by the TBARS method, as described by Ohkawa [26] and Prestes [27], with minor modifications. Briefly, the phosphatidylcholine (4.29 mg/mL) was incubated for 30 min at 37 • C with 10 mM of Tris buffer, pH 7.4, and fresh Fe 2 SO 4 (214 µM, pro-oxidant control), in the absence or presence of (PhSe) 2 or Eb (0.5, 1, 2, 5, 10, 25 µM). The reaction was stopped with SDS (final 1.35%). The color reaction was developed by incubating the medium with acetic acid/HCL buffer, pH 3.4, and 0.2% TBA, pH 6, for 60 min at 100 • C. The levels of TBARS were measured at 532 nm. The results were expressed as % of the control.

Morphological Parameters
Morphological changes of PBMCs (5 × 10 5 cells/sample) were analyzed after 24 h of exposure to (PhSe) 2 or Eb at 2.5 µM in a flow cytometry apparatus (BD Accury C6 Cytometer). In this test, the size and granularity were verified through digital signals of forward scatter (FSC) and side scatter (SSC), respectively. A total of 100,000 events were acquired for each sample, as described by Prestes et al. [28], with adaptations.

Viability, Apoptosis, and Necrosis Indexes
Cell viability, early apoptosis, advanced apoptosis, and necrosis rates were measured by the propidium iodide (PI) plus Annexin V kit (Alexa Fluor ® 488 Annexin V/Dead Cell Apoptosis Kit). After 24 h of exposure to (PhSe) 2 or Eb at 2.5 µM, PBMC samples (5 × 10 5 cells/sample) were processed as indicated by the manufacturer, and analyzed in a flow cytometer (BD Accury C6 Cytometer), where the rate of viable cells (not marked by Annexin V and PI), cells in early apoptosis (marked only by Annexin V), cells in advanced apoptosis (marked by both Annexin V and PI), or necrotic cells (marked only by PI) were quantified. To avoid signal overlap, fluorescence compensation was performed for the FL1 (Annexin) and FL3 channels (PI). Here, 100,000 events were recorded per sample, as described by Ecker et al. [29].

Cell Cycle Assay
The phases of the cell cycle were analyzed according to William-Faltaos et al. [29] and Ecker et al. [29], with minor modifications. Here, 0.5 × 10 6 cells/mL per group of PBMCs were seeded into 96-well plates (200 µL per sample) containing (PhSe) 2 or Eb at 2.5 µM for 24 h. Then, the cells were removed, washed with PBS buffer, and fixed in ethanol (70%) for 24 h, at −20 • C. Subsequently, PBMCs were centrifuged (500× g for 10 min), washed once with PBS, and then resuspended in a labeling solution (30 mM PI in PBS buffer containing 200 mg/mL Dnase-free Rnase and 0.1% Triton-X) during 30 min in the dark. Then, the G0-G1, S, and G2/M phases were analyzed by flow cytometry using the FL2 channel. Here, 100,000 events were acquired for each sample.

Statistical Analysis
One-way ANOVA followed by Tukey's or Dunnett's multiple tests, as appropriate, were used to perform statistical analyses. Results were expressed as mean ± SD or mean ± SEM. All experiments were independently carried out three to six times. Differences were considered statistically significant when p ≤ 0.05. The GraphPad Prism version 8.00 for Windows (GraphPad Software, La Jolla, CA, USA) was used to create the graphics.

MTT and TBARS Assays
The performance of diphenyl diselenide ((PhSe) 2 ) and ebselen (Eb) on the viability of distinct cellular lines was initially evaluated in this study. For a better understanding and a more satisfactory discussion, the chemical structures of these molecules are presented in Figure 1.

Statistical Analysis
One-way ANOVA followed by Tukey's or Dunnett's multiple tests, as were used to perform statistical analyses. Results were expressed as mean ± S SEM. All experiments were independently carried out three to six times. Diffe considered statistically significant when p ≤ 0.05. The GraphPad Prism vers Windows (GraphPad Software, La Jolla, CA, USA) was used to create the gra

MTT and TBARS Assays
The performance of diphenyl diselenide ((PhSe)2) and ebselen (Eb) on th of distinct cellular lines was initially evaluated in this study. For a better und and a more satisfactory discussion, the chemical structures of these molecul sented in Figure 1.   Via the MTT assay, we found that both (PhSe) 2 and Eb induced a significant decrease in the PBMCs' dehydrogenase activity from 50 µM. From the concentration curves, the IC 50 values calculated for (PhSe) 2 and Eb were seen to be 38.75 and 34.84 µM, respectively ( Figure 2).

Statistical Analysis
One-way ANOVA followed by Tukey's or Dunnett's multiple tests, as appropriat were used to perform statistical analyses. Results were expressed as mean ± SD or mean SEM. All experiments were independently carried out three to six times. Differences we considered statistically significant when p ≤ 0.05. The GraphPad Prism version 8.00 f Windows (GraphPad Software, La Jolla, CA, USA) was used to create the graphics.

MTT and TBARS Assays
The performance of diphenyl diselenide ((PhSe)2) and ebselen (Eb) on the viability of distinct cellular lines was initially evaluated in this study. For a better understanding and a more satisfactory discussion, the chemical structures of these molecules are pre sented in Figure 1. Via the MTT assay, we found that both (PhSe)2 and Eb induced a significant decrea in the PBMCs' dehydrogenase activity from 50 µM. From the concentration curves, the IC values calculated for (PhSe)2 and Eb were seen to be 38.75 and 34.84 µM, respectively (Fi ure 2). Cell viability of human PBMCs exposed to Eb and (PhSe)2. Here, 0.5 × 10 6 human cells we exposed to (PhSe)2 (A) or Eb (B) at 0.25, 0.5, 1, 2, 5, 10, 25, 50, 100, and 200 µM, for 24 h, at 37 °C in sterile environment containing 5% CO2. Data represent the mean ± SEM of four independent expe iments and were analyzed by one-way ANOVA followed by Tukey's multiple test. * Indicates si nificant difference when compared to the control (p ≤ 0.05). Cell viability of human PBMCs exposed to Eb and (PhSe) 2 . Here, 0.5 × 10 6 human cells were exposed to (PhSe) 2 (A) or Eb (B) at 0.25, 0.5, 1, 2, 5, 10, 25, 50, 100, and 200 µM, for 24 h, at 37 • C in a sterile environment containing 5% CO 2 . Data represent the mean ± SEM of four independent experiments and were analyzed by one-way ANOVA followed by Tukey's multiple test. * Indicates significant difference when compared to the control (p ≤ 0.05).
Toxicity was also evaluated in the SARS-CoV-2 cellular infection model used in this study. Thereby, the viability of non-infected cells was ensured in treatments with high concentrations of selenium compounds (25-200 µM), and the percentage of viable cells of each treatment was obtained in comparison to the control samples treated only with DMSO, Vaccines 2023, 11, 1222 6 of 16 at the same percentages as those to which the ebselen-and diphenyl diselenide-treated cells were subjected ( Figure 3). We found that both compounds were less toxic in Calu-3 than in PBMCs, which was reflected in the CC 50 of 200 µM ( Figure 3 and Table 1).
Toxicity was also evaluated in the SARS-CoV-2 cellular infection mod study. Thereby, the viability of non-infected cells was ensured in treatme concentrations of selenium compounds (25-200 µM), and the percentage of each treatment was obtained in comparison to the control samples treat DMSO, at the same percentages as those to which the ebselen-and diphen treated cells were subjected ( Figure 3). We found that both compounds wer Calu-3 than in PBMCs, which was reflected in the CC50 of 200 µM (Figure 3  In the TBARS assay, all tested concentrations of (PhSe)2 or Eb induced the glycerophospholipid phosphatidylcholine when compared to the con which was used as the pro-oxidant control ( Figure 4A,B, respectively). The treatments with FeSO4 also showed that (PhSe)2 and Eb, at the tested conce not prevent iron-induced lipid peroxidation ( Figure 4A,B, respectively). Effect of Eb and (PhSe) 2 on the viability of non-infected Calu-3 cells. Cells were exposed to Eb or (PhSe) 2 at concentrations from 25 to 200 µM, for 72 h, at 37 • C. Then, the viability was estimated by the MTT assay and the CC 50 values of Calu-3 were calculated from the concentration curves. Data represent the mean ± SD of three independent experiments, analyzed by one-way ANOVA, followed by Dunnett's multiple tests (n = 5). * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001. In the TBARS assay, all tested concentrations of (PhSe) 2 or Eb induced oxidation of the glycerophospholipid phosphatidylcholine when compared to the control or FeSO 4 , which was used as the pro-oxidant control ( Figure 4A,B, respectively). The results of co-treatments with FeSO 4 also showed that (PhSe) 2 and Eb, at the tested concentrations, did not prevent iron-induced lipid peroxidation ( Figure 4A,B, respectively).

Viral Replication in Calu-3 Cells Exposed to (PhSe) 2 and Eb
Human type II pneumocytes, target cells of the COVID-19 infectious agent, were infected and treated under different experimental conditions. In an infection model with MOI 0.01, levels ≥ 95% inhibition of SARS-CoV-2 replication were observed after treatment at a 12.5 µM concentration, regardless of the time proposed in this analysis ( Figure 5A,B). Importantly, 86% of viral inhibition was also observed after 24 h of treatment with both organoselenium compounds in Calu-3 cells (MOI 0.1) ( Figure 5C). Keeping the same virus/cell ratio, reductions of 74% and 81% in the replicative capacity of SARS-CoV-2 were also demonstrated after 48 h of treatment with (PhSe) 2 and Eb, respectively ( Figure 5D). In addition, it was observed that the EC 50 values of (PhSe) 2 and Eb in Calu-3 cells, previously infected with MOI 0.01, were 3.9 and 3.8 µM (24 h of treatment) and 3.4 and 2.6 µM (48 h of treatment), while in those infected with MOI 0.1, the values were 4.2 and 3.1 µM (24 h of treatment) and 3.5 and 3.9 µM (48 h of treatment), respectively. Despite the different viral particle numbers and treatment times proposed for this cell line, the concentration required to inhibit 50% of viral replication was similar in all conditions assumed in this study. The EC 50 values of (PhSe) 2 and Eb were demonstrated in Table 1.

Viral Replication in Calu-3 Cells Exposed to (PhSe)2 and Eb
Human type II pneumocytes, target cells of the COVID-19 infectious agent, were infected and treated under different experimental conditions. In an infection model with MOI 0.01, levels ≥ 95% inhibition of SARS-CoV-2 replication were observed after treatment at a 12.5 µM concentration, regardless of the time proposed in this analysis ( Figure 5A,B). Importantly, 86% of viral inhibition was also observed after 24 h of treatment with both organoselenium compounds in Calu-3 cells (MOI 0.1) ( Figure 5C). Keeping the same virus/cell ratio, reductions of 74% and 81% in the replicative capacity of SARS-CoV-2 were also demonstrated after 48 h of treatment with (PhSe)2 and Eb, respectively ( Figure 5D). In addition, it was observed that the EC50 values of (PhSe)2 and Eb in Calu-3 cells, previously infected with MOI 0.01, were 3.9 and 3.8 µM (24 h of treatment) and 3.4 and 2.6 µM (48 h of treatment), while in those infected with MOI 0.1, the values were 4.2 and 3.1 µM (24 h of treatment) and 3.5 and 3.9 µM (48 h of treatment), respectively. Despite the different viral particle numbers and treatment times proposed for this cell line, the concentration required to inhibit 50% of viral replication was similar in all conditions assumed in this study. The EC50 values of (PhSe)2 and Eb were demonstrated in Table 1.

Flow Cytometric Assays
For the flow cytometry assays, we chose the concentration of 2.5 µM to expose the PBMCs for 24 h. The analyzed toxicological parameters included: morphology and cell cycle changes, viability loss, apoptosis, and necrosis. A decreased size and increased granularity preceded DNA fragmentation and may be an early indicator of apoptosis. The results from Figure 6 relative to the morphology of the PBMCs showed that there were no alterations in the size of the PBMCs exposed to both selenium compounds when compared to the control cells ( Figure 6B). Likewise, 2.5 µM of (PhSe) 2 and Eb did not induce changes in the granularity of the cells ( Figure 6C).
The viability and indexes of early apoptosis, advanced apoptosis, and necrosis of PBMCs exposed to 2.5 µM of (PhSe) 2 and Eb were determined using Annexin V and PI reagents (Figure 7). In this assay, we did not find changes in the cell viability of PBMCs exposed to (PhSe) 2 and Eb ( Figure 7B). Exposure to 2.5 µM of (PhSe) 2 or Eb also did not induce signals of early apoptosis ( Figure 7C), advanced apoptosis ( Figure 7D), and/or necrosis ( Figure 7E) in the cells.
Data from Figure 8 showed that exposure of PBMCs to 2.5 µM of (PhSe) 2 and Eb also did not change the cell cycle phases, as indicated by the comparison of G0-G1, S, and G2/M ratios in relation to the control cells.

Flow Cytometric Assays
For the flow cytometry assays, we chose the concentration of 2.5 µM to expose the PBMCs for 24 h. The analyzed toxicological parameters included: morphology and cell cycle changes, viability loss, apoptosis, and necrosis. A decreased size and increased granularity preceded DNA fragmentation and may be an early indicator of apoptosis. The results from Figure 6 relative to the morphology of the PBMCs showed that there were no alterations in the size of the PBMCs exposed to both selenium compounds when compared to the control cells ( Figure 6B). Likewise, 2.5 µM of (PhSe)2 and Eb did not induce changes in the granularity of the cells ( Figure 6C). The viability and indexes of early apoptosis, advanced apoptosis, and necrosis of PBMCs exposed to 2.5 µM of (PhSe)2 and Eb were determined using Annexin V and PI reagents (Figure 7). In this assay, we did not find changes in the cell viability of PBMCs exposed to (PhSe)2 and Eb ( Figure 7B). Exposure to 2.5 µM of (PhSe)2 or Eb also did not induce signals of early apoptosis ( Figure 7C), advanced apoptosis ( Figure 7D), and/or necrosis ( Figure 7E) in the cells. Figure 6. Size and granularity of human lymphocytes exposed to Eb and (PhSe) 2 . Human PBMCs (0.5 × 10 6 cells/mL per sample) were exposed to (PhSe) 2 or Eb, at 2.5 µM for 24 h at 37 • C in a sterile environment containing 5% CO 2 . (A) Representative histogram of PBMCs by flow cytometry. FSC and SSC axes correspond to the size and granularity of the cells, respectively. (B,C) Arbitrary units of the size and granularity of the cells. Results represent the mean ± SEM of four independent experiments. Here, 50,000 events were acquired for each sample analysis. The one-way ANOVA followed by Tukey's multiple test was done. n = 6 for each group.
Vaccines 2023, 11, x FOR PEER REVIEW 10 of 17 Figure 7. Viability, early apoptosis, advanced apoptosis, and necrosis in lymphocytes exposed to Eb  Figure 7. Viability, early apoptosis, advanced apoptosis, and necrosis in lymphocytes exposed to Eb and (PhSe)2. Human PBMCs (0.5 × 10 6 cells/mL per sample) were exposed to (PhSe)2 or Eb, at 2.5 µM for 24 at 37 °C in a sterile environment containing 5% CO2. Then, the cells were treated with Annexin V/PI reagents, and the fluorescence was measured on a BD Accuri TM C6 flow cytometer. Here, 100,000 events were recorded per sample. No difference was observed between the groups by one-way ANOVA followed by Tukey's multiple tests.
Data from Figure 8 showed that exposure of PBMCs to 2.5 µM of (PhSe)2 and Eb also did not change the cell cycle phases, as indicated by the comparison of G0-G1, S, and G2/M ratios in relation to the control cells. Figure 7. Viability, early apoptosis, advanced apoptosis, and necrosis in lymphocytes exposed to Eb and (PhSe) 2 . Human PBMCs (0.5 × 10 6 cells/mL per sample) were exposed to (PhSe) 2 or Eb, at 2.5 µM for 24 at 37 • C in a sterile environment containing 5% CO 2 . Then, the cells were treated with Annexin V/PI reagents, and the fluorescence was measured on a BD Accuri TM C6 flow cytometer. Here, 100,000 events were recorded per sample. No difference was observed between the groups by one-way ANOVA followed by Tukey's multiple tests.

Discussion
Since the outbreak and spread of COVID-19, research has been carried out to under-

Discussion
Since the outbreak and spread of COVID-19, research has been carried out to understand the mechanisms underlying the infection for the further development of approaches capable of containing and/or reducing the spread of the virus and its complications. Although scientists around the world are studying SARS-CoV-2 and COVID-19, and much progress has been made in a short period of time with the vaccination development, there is still an urgent need to repurpose drugs against SARS-CoV-2 and the effects of COVID-19, which has a hallmark of an acute inflammatory response that is exacerbated in the elderly and individuals with chronic disorders, such as obesity and diabetes mellitus [30,31].
Here, we repurposed the use of two organic selenium compounds that have already had their toxicological and pharmacological properties extensively studied by our research group: ebselen (Eb) and diphenyl diselenide ((PhSe) 2 ) [24,[32][33][34][35][36]. We found very satisfactory and promising results, which showed the effectiveness of both compounds in significantly inhibiting SARS-CoV-2 replication in Calu-3 cells (that recapitulate type II pneumocytes), exhibiting EC 50 values at concentration ranges that were safe for human cell lines: alveolar lung cells and lymphocytes. Our research group has already demonstrated the activity of (PhSe) 2 against the SARS-CoV-2 main protease (Mpro) and its replication in Vero E6 cells [36]. In addition, other groups have already demonstrated the activity of Eb in this same enzyme through in silico and in vitro approaches [37][38][39][40][41]. The protease Mpro is highly conserved across the different SARS-CoV-2 "variants of concern" (VOCs) identified, for example, Alpha, Beta, Gamma, Delta, and Omicron [42][43][44]. Therefore, although we used a SARS-CoV-2 B.1 lineage isolate, it is strongly suspected that Eb and (PhSe) 2 would have similar effects on other SARS-CoV-2 variants. Vero E6 and Calu-3 cell types have particularities in the virus entry process, because the interactions of SARS-CoV-2 Spike proteins and cell receptors are different [45][46][47][48]. Using specially cultured lymphocytes in a battery of the toxicological tests, we found that Eb and (PhSe) 2 , even at concentrations higher than those that inhibited the viral replication, did not induce a loss of viability, apoptosis, or changes in the cell cycle and cell morphology. Indeed, the compounds did not present an effective pro-oxidant effect, as evaluated by a lipid peroxidation estimation. Selenium (Se) is a trace element, essential for numerous cellular functions in animals. In humans, Se is necessary for the synthesis of at least 25 selenoproteins, where the antioxidant enzymes such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) isoforms are highlighted. Of pharmacological importance, same organic selenium compounds, including (PhSe) 2 and Eb, can mimic GPx and/or be substrates for TrxR [34,49]. Along with the antioxidant activity, Eb and (PhSe) 2 have pharmacological implications against several experimental human pathologies via anti-inflammatory, anticarcinogenic, antidiabetogenic, and neuroprotective action. It is worth noting here that clinical trials have already been carried out with Eb to treat brain ischemia, noise-induced hearing loss, and bipolar disorder [34,[49][50][51].
On the other hand, high levels of Se can be toxic to living organisms. There is strong evidence that many inorganic/organic selenium compounds can oxidize low-and highmolecular-weight thiols, causing redox unbalance [52]. In this sense, the first toxicological works carried out by our research group toward organoselenium compounds demonstrated the pro-oxidant effect of several selenium-containing molecules, including (PhSe) 2 and Eb. These compounds can inhibit sulfhydryl-containing enzymes such as α-ALA-D and Na − K + in both in vitro and in vivo models [53][54][55]. More recently, our works have suggested that the compounds can act as weak electrophiles, a property that also seems to be involved in their antioxidant potential. In fact, by oxidizing -SH groups of keap-1, Eb and (PhSe) 2 promote the activation of the transcription factor NRF2, which triggers the antioxidant machinery of the cells [34,[49][50][51]. The oxidation of Cys from critical enzymes has also been recognized as the main mechanism involved in the antimicrobial activity of the compounds [34,[49][50][51]. There is evidence that Eb and (PhSe) 2 can oxidize critical thiolcontaining proteins from viruses, bacteria, and fungi [15]. In the virus context, Eb has already been indicated as a potent inhibitor of key thiol-containing enzymes of the human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus [15]. Regarding (PhSe) 2 , the literature reports its antiviral action against in vitro herpes simplex virus 2 (HSV-2), and in reducing the inflammation in HSV-2-infected mice [56]. (PhSe) 2 was also efficient against bovine alphaherpesvirus 2, both in vitro and in a sheep model [56].
Especially on SARS-CoV-2, Eb was one of the first molecules to be identified by virtual drug screening as a strong inhibitor of Mpro activity, where the compound displayed inhibition of Mpro activity with an IC 50 value of 0.67 µM, and viral replication in Vero cells with an EC 50 value of 4.67 µM [17,18]. Recently, Eb's efficacy was also observed on SARS-CoV-2 replication in Calu-3 cells, displaying an EC 50 value of 5 µM, similar to our results [56]. Then, Eb was used in this study as a control for (PhSe) 2 assays. Molecular and atomistic simulations found that Eb covalently binds to the Cys145 residue from the Mpro active site [35,37]. There is evidence that Eb is also an irreversible inhibitor of PLpro from SARS-CoV-2, with an IC 50 value of approximately 2 µM [57]. For this enzyme, molecular modeling showed an interaction of the compound with the residue Cys111 from the active site [40]. Herein, our results reinforced the promising findings previously found for Eb, as well as demonstrated its efficacy in human cell lines targeted by SARS-CoV-2 and COVID-19 [22]. Of toxicological significance, the EC 50 values of compounds on infected Calu-3 cells were very low, reaching concentrations that did not compromise the viability of human cell lines, in the case of Calu-3 and PBMCs. In addition, Eb did not induce deeper signs of cell damage, such as viability loss, morphological changes, lipid peroxidation, and cell death.
Our findings also shed light on the hopeful role of (PhSe) 2 , which exhibited a similar profile of antiviral behavior as Eb, at concentrations that did not cause apparent toxicity to human cell lines. In this sense, our work presented a new promising molecule for studying SARS-CoV-2 inhibition and COVID-19 complications. In terms of the action mechanism, a recent in silico study on the interaction between Mpro and PLpro from SARS-CoV-2 and selenium compounds compared the effects of (PhSe) 2 , Eb, and some derivatives. Although Eb showed higher negative binding energies on the enzyme active sites (Mpro: −6.5 kcal·mol −1 , Plpro: −6.1 kcal·mol −1 ), (PhSe) 2 exhibited an interesting effect, with a ∆G value of −6.2 kcal·mol −1 for Mpro and −4.1 kcal·mol −1 for PLpro [35].
Our research group has already demonstrated the activity of (PhSe) 2 against the SARS-CoV-2 main protease (Mpro) and its replication in Vero E6 cells [36]. In addition, other groups have already demonstrated the activity of Eb in this same enzyme through in silico and in vitro approaches [37][38][39][40][41]. The protease Mpro is highly conserved across the different SARS-CoV-2 "variants of concern" (VOCs) identified, for example, Alpha, Beta, Gamma, Delta, and Omicron [42][43][44]. Therefore, although we used a SARS-CoV-2 B.1 lineage (Alpha) isolate, it is strongly suspected that Eb and (PhSe) 2 would have similar effects on other SARS-CoV-2 variants.
We emphasize here that in addition to viral replication inhibition via thiol oxidation, Eb and (PhSe) 2 have been reported as potent anti-inflammatory agents, an effect that could simultaneously help in the inflammatory phase of COVID-19. In fact, Eb and (PhSe) 2 have been shown to be efficient in controlling the inflammation in a variety of in vitro and in vivo injury models, including ischemia and reperfusion cerebral, arthritis, food paw edema, pleurisy, and ulcerative colitis models [52]. Reduction of neutrophil infiltrate, inhibition of pro-inflammatory enzymes, reduction of nitric oxide (NO) and pro-inflammatory cytokines' production, and modulation of the purinergic system are among the phenomena underlying the anti-inflammatory potential of the compounds [15].
Studies have demonstrated that the Se status is low in blood samples from COVID-19 patients and that the in situ SARS-CoV-2 infection significantly reduces the expression of a number of selenoproteins, including SEL P, S, K, GPx, and Trx [58,59]. Se deficiency is also known to be prevalent during other viral infections, including in hepatitis and HIV [60,61]. Although several trials have already reported the beneficial effects of Se supplementation, cellular and molecular findings are still lacking to clarify the role of Se in the viral infection. However, clinical evidence from HIV infection has highlighted that Se supplementation can delay the CD4 decline in HIV-infected individuals, as well as affect the expression of selenoproteins in HIV-infected T cells in a hierarchical manner, with GPx1, GPx4, SEL H, SEL K, SEL S, and SEL T being the mostly sensitive selenoproteins [62][63][64]. In general, these events strengthen the additional benefit that the supplemental compounds containing selenium could have under viral infections. Especially regarding (PhSe) 2 , we have already demonstrated the chronic dietary increases in the levels of Se in the brain, along with the relative transcriptional levels of GPx isoforms and SEL P [65,66], effects that could be interesting under selenium-deficiency conditions.

Conclusions
Our findings showed the potential effects of Eb and (PhSe) 2 against SARS-CoV-2 replication at concentrations non-toxic to human cells. Although more efforts need to be made to elucidate the molecular and cellular action mechanisms of compounds on viral replication, we believe that one probable action is via inhibition of the viral proteases Mpro and PLpro, as previously indicated by molecular simulations. Therefore, our promising results in infected cells led us to suggest the use of these molecules in other trials, such as modulation of inflammation prompted by SARS-CoV-2 infection.