Next Article in Journal
Influenza Vaccination Uptake and Prognostic Factors among Health Professionals in Italy: Results from the Nationwide Surveillance PASSI 2015–2018
Previous Article in Journal
Improving Protection to Prevent Bacterial Infections: Preliminary Applications of Reverse Vaccinology against the Main Cystic Fibrosis Pathogens
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Vaccines
Volume 11
Issue 7
10.3390/vaccines11071222
vaccines-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Guilherme Wildner
1,†,
Amanda Resende Tucci
2,3,†,
Alessandro de Souza Prestes
1,
Talise Muller
1,
Alice dos Santos Rosa
2,3,
Nathalia Roberto R. Borba
2,
Vivian Neuza Ferreira
2,
João Batista Teixeira Rocha
1,
Milene Dias Miranda
2,3,*,‡ and
Nilda Vargas Barbosa
1,*,‡
1
Programa de Pós-Graduação em Bioquímica Toxicológica, Universidade Federal de Santa Maria, Santa Maria 97105-900, RS, Brazil
2
Laboratório de Morfologia e Morfogênese Viral, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro 21041-250, RJ, Brazil
3
Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro 21041-250, RJ, Brazil
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
‡
These authors also contributed equally to this work.
Vaccines 2023, 11(7), 1222; https://0-doi-org.brum.beds.ac.uk/10.3390/vaccines11071222
Submission received: 27 April 2023 / Revised: 18 June 2023 / Accepted: 25 June 2023 / Published: 10 July 2023
(This article belongs to the Special Issue Progress on Antiviral Drugs Research in Epidemics)
Browse Figures
Review Reports Versions Notes

Abstract

:
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.

1. 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 (COVID-19). 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 cases of infection have been recorded since the beginning of the pandemic, and that more than 6.9 million deaths have occurred worldwide [3].
SARS-CoV-2 is a β-coronavirus, composed of a capsid containing a single-strand RNA (ssRNA) and four structural proteins: the nucleocapsid protein (N), the membrane protein (M), the envelope protein (E), and the spike protein (S) [4]. The virion envelope is made up of the proteins S, M, and E, where the S protein is responsible for the entry of the virus into the host cells. The S protein attaches to the host cells through the interaction with a specific cellular receptor, angiotensin-converting enzyme 2 (ACE2), together with a host factor, the surface serine protease (TMPRSS2) [5].
Upon entry into the host cells, the viral replication and transcription complex is formed with non-structural proteins (nsps), as a result of processing two polyproteins (pp1a and pp1ab) translated from the virus genomic RNA. The main protease (Mpro, or 3C-like protease—3CLpro) and papain-like protease (PLpro) are critical sulfhydryl enzymes for the viral replication and transcription due to their post-translational action on pp1a and pp1ab processing [6]. Together with the S protein, the SARS-CoV-2 proteases became important targets of antiviral approaches and research on the mechanisms underlying the infection [7,8,9].
After continuous multiplication, the virus reaches the lungs, where it causes alveoli inflammation, leading to pneumonia. This physiological state can rapidly progress to acute respiratory distress syndrome (ARDS) [10,11]. In this phase, the hyperactivation of immune cells and excessive cytokine production are responsible for the severe inflammatory process characteristic of COVID-19, known as cytokine storm [12]. So far, the number of new SARS-CoV-2 infections and deaths from COVID-19 pathogenesis highlight the importance of new therapies, since those that currently exist are limited [13]. The U.S. Food and Drug Administration (FDA) approved the Emergency Use Authorization (EUA) for some drugs to treat mild-to-moderate COVID-19 patients likely to progress to a more severe illness. Although Remdesivir and Paxlovid (nirmatrelvir and ritonavir) are the only antiviral drugs approved by the FDA for the treatment of adults with mild-to-moderate COVID-19 who are at high risk for severe disease progression, which can lead to hospitalization and death, the antiviral Lagevrio (Molnupiravir) has also been used as a treatment under an Emergency Use Authorization, as proposed by the National Institutes of Health (NIH) [14]. However, there is still an urgent need to repurpose medication against the SARS-CoV-2 infection and acute inflammatory response of COVID-19.
Drug repurposing, molecular simulations in search of viral protease inhibitors, and screenings for antiviral/anti-inflammatory molecules have been strategies widely used in the global research against the SARS-CoV-2 virus and the effects of COVID-19. In this context, here, we highlight the study of two organic selenium compounds, ebselen (Eb) and diphenyl diselenide ((PhSe)2). In addition to the well-known antioxidant and anti-inflammatory effects in several experimental models of human pathologies, the compounds have been reported as antimicrobial agents by oxidizing Cys residues from critical enzymes in different microorganisms [15,16]. Importantly, one of the first computational experimental screenings toward SARS-CoV-2 identified Eb as one of the most promising inhibitors of the protease Mpro, as well as a potent inhibitor of the enzyme in vitro and of SARS-CoV-2 replication [17,18]. The antiviral activity of (PhSe)2 is less explored, but there is evidence of its efficacy against other viruses, including the herpes simplex virus 2 (HSV-2) and the bovine alphaherpesvirus 2 (BoHV-2) [19,20,21]. Moreover, our group has already demonstrated that (PhSe)2 is capable of covalently interacting with the SARS-CoV-2 Mpro in silico and inhibiting virus replication in a Vero E6 cell model.
Herein, we investigated the effects of Eb and (PhSe)2 on SARS-CoV-2 replication using Calu-3 cells as the virus-targeted cells. To ensure the safety of the antiviral concentrations tested, we also exposed human PBMCs to the Eb and (PhSe)2 to ascertain some endpoints of toxicity. We found that both compounds significantly inhibited the replication of SARS-CoV-2 in human pneumocytes, presenting low EC50 values, comparable to others already described in the literature for anti-SARS-CoV-2 molecules [22]. Of toxicological significance, 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.

2. Materials and Methods

2.1. 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 plaque-forming 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 × 104 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; GibcoTM, 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].

2.2. 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).

2.3. 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 KH2PO4, 8.1 mM Na2HPO4, 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% CO2. The number of cells used was 0.5 × 106 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.

2.4. 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 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.

2.5. 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 Fe2SO4 (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.

2.6. Morphological Parameters

Morphological changes of PBMCs (5 × 105 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.

2.7. 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 × 105 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].

2.8. 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 × 106 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.

2.9. 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.

3. Results

3.1. 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.
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 IC50 values calculated for (PhSe)2 and Eb were seen to be 38.75 and 34.84 µM, respectively (Figure 2).
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, 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 CC50 of 200 µM (Figure 3 and Table 1).
In the TBARS assay, all tested concentrations of (PhSe)2 or Eb induced oxidation of the glycerophospholipid phosphatidylcholine when compared to the control or FeSO4, which was used as the pro-oxidant control (Figure 4A,B, respectively). The results of co-treatments with FeSO4 also showed that (PhSe)2 and Eb, at the tested concentrations, did not prevent iron-induced lipid peroxidation (Figure 4A,B, respectively).

3.2. 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.

3.3. 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.

4. 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 EC50 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 high-molecular-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 thiol-containing 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 IC50 value of 0.67 μM, and viral replication in Vero cells with an EC50 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 EC50 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 IC50 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 EC50 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 ΔG 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.

5. 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.

Author Contributions

Conceptualization, J.B.T.R., M.D.M. and N.V.B.; data curation, J.B.T.R., M.D.M. and N.V.B.; formal analysis, M.D.M.; funding acquisition, J.B.T.R. and M.D.M.; investigation, G.W., A.R.T., A.d.S.P., A.d.S.R., N.R.R.B., V.N.F. and M.D.M.; methodology, G.W., A.R.T., A.d.S.P., T.M., A.d.S.R., N.R.R.B., V.N.F. and M.D.M.; project administration, J.B.T.R.; resources, M.D.M.; supervision, M.D.M. and N.V.B.; validation, A.R.T., A.d.S.R., V.N.F. and M.D.M.; writing—original draft, A.R.T. and M.D.M.; writing—review and editing, M.D.M. and N.V.B. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). A.R.T., A.S.R., V.N.S.F. and M.D.M. were supported by Laboratório de Morfologia e Morfogênese Viral, Instituto Oswaldo Cruz (IOC), Fiocruz, FIOTEC (grant number IOC-023-FIO-18-2-58), CAPES (scholarships and grant numbers: 88887.648435/2021-00, 88887.717861/2022-00, 88887.694990/2022-00), and FAPERJ (E-26/201.426/2022, E-26/201.574/2021, E-26/210.151/2020). The APC was funded by Instituto Oswaldo Cruz. N.V.B. and J.T.R. are the recipients of CNPq fellowships. CAPES (grant number: CAPES EPIDEMIAS 09 PROC.88887.505377/2020).

Institutional Review Board Statement

The protocol was approved by the Ethics Committee for Research with Humans at the Federal University of Santa Maria (number 67825122.1.0000.5346).

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

Thanks are due to Oswaldo Cruz Institute for access to the Biosafety Level 3 (BSL-3) facility at the Pavilhão Leonidas Deane, Fiocruz, RJ, and Andre Sampaio from Farmanguinhos, platform RPT11M, for kindly donating the Calu-3 cells.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Zhang, J.J.; Dong, X.; Cao, Y.Y.; Yuan, Y.D.; Yang, Y.B.; Yan, Y.Q.; Akdis, C.A.; Gao, Y.D. Clinical characteristics of 140 patients infected with SARS-CoV-2 in Wuhan, China. Allergy 2020, 75, 1730–1741. [Google Scholar] [CrossRef] [PubMed]
  2. WHO. WHO Director-General’s Opening Remarks at the Media Briefing on COVID-19-11 March 2020. Available online: https://www.who.int/director-general/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19---11-march-2020 (accessed on 10 April 2023).
  3. WHO. WHO Coronavirus (COVID19) Dashboard. Available online: https://covid19.who.int/ (accessed on 16 June 2023).
  4. Brian, D.A.; Baric, R.S. Coronavirus genome structure and replication. In Coronavirus Replication and Reverse Genetics; Current Topics in Microbiology and Immunology book series; Springer: Berlin/Heidelberg, Germany, 2005; Volume 287, pp. 1–30. [Google Scholar] [CrossRef] [Green Version]
  5. Hoffmann, M.; Kleine-Weber, H.; Schroeder, S.; Kruger, N.; Herrler, T.; Erichsen, S.; Schiergens, T.S.; Herrler, G.; Wu, N.H.; Nitsche, A.; et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 2020, 181, 271–280.e8. [Google Scholar] [CrossRef] [PubMed]
  6. Jeong, G.U.; Song, H.; Yoon, G.Y.; Kim, D.; Kwon, Y.C. Therapeutic Strategies against COVID-19 and Structural Characterization of SARS-CoV-2: A Review. Front. Microbiol. 2020, 11, 1723. [Google Scholar] [CrossRef] [PubMed]
  7. Agost-Beltran, L.; de la Hoz-Rodriguez, S.; Bou-Iserte, L.; Rodriguez, S.; Fernandez-de-la-Pradilla, A.; Gonzalez, F.V. Advances in the Development of SARS-CoV-2 Mpro Inhibitors. Molecules 2022, 27, 2523. [Google Scholar] [CrossRef] [PubMed]
  8. Shang, J.; Wan, Y.; Luo, C.; Ye, G.; Geng, Q.; Auerbach, A.; Li, F. Cell entry mechanisms of SARS-CoV-2. Proc. Natl. Acad. Sci. USA 2020, 117, 11727–11734. [Google Scholar] [CrossRef]
  9. Steuten, K.; Kim, H.; Widen, J.C.; Babin, B.M.; Onguka, O.; Lovell, S.; Bolgi, O.; Cerikan, B.; Neufeldt, C.J.; Cortese, M.; et al. Challenges for Targeting SARS-CoV-2 Proteases as a Therapeutic Strategy for COVID-19. ACS Infect. Dis. 2021, 7, 1457–1468. [Google Scholar] [CrossRef]
  10. Camporota, L.; Cronin, J.N.; Busana, M.; Gattinoni, L.; Formenti, F. Pathophysiology of coronavirus-19 disease acute lung injury. Curr. Opin. Crit. Care 2022, 28, 9–16. [Google Scholar] [CrossRef]
  11. Sherren, P.B.; Ostermann, M.; Agarwal, S.; Meadows, C.I.S.; Ioannou, N.; Camporota, L. COVID-19-related organ dysfunction and management strategies on the intensive care unit: A narrative review. Br. J. Anaesth. 2020, 125, 912–925. [Google Scholar] [CrossRef]
  12. Paludan, S.R.; Mogensen, T.H. Innate immunological pathways in COVID-19 pathogenesis. Sci. Immunol. 2022, 7, eabm5505. [Google Scholar] [CrossRef]
  13. Solomon, M.; Liang, C. Human coronaviruses: The emergence of SARS-CoV-2 and management of COVID-19. Virus Res. 2022, 319, 198882. [Google Scholar] [CrossRef]
  14. FDA. Coronavirus (COVID-19)|Drugs. Available online: https://www.fda.gov/drugs/emergency-preparedness-drugs/coronavirus-covid-19-drugs (accessed on 16 June 2023).
  15. Nogueira, C.W.; Barbosa, N.V.; Rocha, J.B.T. Toxicology and pharmacology of synthetic organoselenium compounds: An update. Arch. Toxicol. 2021, 95, 1179–1226. [Google Scholar] [CrossRef] [PubMed]
  16. Benelli, J.L.; Poester, V.R.; Munhoz, L.S.; Klafke, G.B.; Stevens, D.A.; Xavier, M.O. In vitro anti-Cryptococcus activity of diphenyl diselenide alone and in combination with amphotericin B and fluconazole. Braz. J. Microbiol. 2021, 52, 1719–1723. [Google Scholar] [CrossRef] [PubMed]
  17. Jin, Z.; Du, X.; Xu, Y.; Deng, Y.; Liu, M.; Zhao, Y.; Zhang, B.; Li, X.; Zhang, L.; Peng, C.; et al. Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors. Nature 2020, 582, 289–293. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Sies, H.; Parnham, M.J. Potential therapeutic use of ebselen for COVID-19 and other respiratory viral infections. Free Radic. Biol. Med. 2020, 156, 107–112. [Google Scholar] [CrossRef]
  19. Sartori, G.; Jardim, N.S.; Marcondes Sari, M.H.; Dobrachinski, F.; Pesarico, A.P.; Rodrigues, L.C., Jr.; Cargnelutti, J.; Flores, E.F.; Prigol, M.; Nogueira, C.W. Antiviral Action of Diphenyl Diselenide on Herpes Simplex Virus 2 Infection in Female BALB/c Mice. J. Cell. Biochem. 2016, 117, 1638–1648. [Google Scholar] [CrossRef]
  20. Sartori, G.; Jardim, N.S.; Sari, M.H.; Flores, E.F.; Prigol, M.; Nogueira, C.W. Diphenyl Diselenide Reduces Oxidative Stress and Toxicity Caused by HSV-2 Infection in Mice. J. Cell. Biochem. 2017, 118, 1028–1037. [Google Scholar] [CrossRef]
  21. Amaral, B.P.; Cargnelutti, J.F.; Mortari, A.P.G.; Merchioratto, I.; Feio, L.M.; Nogueira, C.W.; Weiblen, R.; Flores, E. Diphenyl diselenide and cidofovir present anti-viral activity against Bovine Alphaherpesvirus 2 in vitro and in a sheep model. Res. Vet. Sci. 2021, 134, 78–85. [Google Scholar] [CrossRef]
  22. Citarella, A.; Scala, A.; Piperno, A.; Micale, N. SARS-CoV-2 M(pro): A Potential Target for Peptidomimetics and Small-Molecule Inhibitors. Biomolecules 2021, 11, 607. [Google Scholar] [CrossRef]
  23. WHO. Laboratory Biosafety Guidance Related to Coronavirus Disease 2019 (COVID-19); WHO: Geneva, Switzerland, 2020. [Google Scholar]
  24. Ecker, A.; Ledur, P.C.; da Silva, R.S.; Leal, D.B.R.; Rodrigues, O.E.D.; Ardisson-Araujo, D.; Waczuk, E.P.; da Rocha, J.B.T.; Barbosa, N.V. Chalcogenozidovudine Derivatives with Antitumor Activity: Comparative Toxicities in Cultured Human Mononuclear Cells. Toxicol. Sci. 2017, 160, 30–46. [Google Scholar] [CrossRef]
  25. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [Google Scholar] [CrossRef]
  26. Ohkawa, H.; Ohishi, N.; Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 1979, 95, 351–358. [Google Scholar] [CrossRef] [PubMed]
  27. De Souza Prestes, A.; Stefanello, S.T.; Salman, S.M.; Pazini, A.M.; Schwab, R.S.; Braga, A.L.; de Vargas Barbosa, N.B.; Rocha, J.B. Antioxidant activity of beta-selenoamines and their capacity to mimic different enzymes. Mol. Cell. Biochem. 2012, 365, 85–92. [Google Scholar] [CrossRef] [PubMed]
  28. De Souza Prestes, A.; Dos Santos, M.M.; Ecker, A.; de Macedo, G.T.; Fachinetto, R.; Bressan, G.N.; da Rocha, J.B.T.; Barbosa, N.V. Methylglyoxal disturbs the expression of antioxidant, apoptotic and glycation responsive genes and triggers programmed cell death in human leukocytes. Toxicol. Vitr. 2019, 55, 33–42. [Google Scholar] [CrossRef] [PubMed]
  29. William-Faltaos, S.; Rouillard, D.; Lechat, P.; Bastian, G. Cell cycle arrest and apoptosis induced by oxaliplatin (L-OHP) on four human cancer cell lines. Anticancer Res. 2006, 26, 2093–2099. [Google Scholar] [PubMed]
  30. Al-Sabah, S.; Al-Haddad, M.; Al-Youha, S.; Jamal, M.; Almazeedi, S. COVID-19: Impact of obesity and diabetes on disease severity. Clin. Obes. 2020, 10, e12414. [Google Scholar] [CrossRef] [PubMed]
  31. Bae, S.; Kim, S.R.; Kim, M.N.; Shim, W.J.; Park, S.M. Impact of cardiovascular disease and risk factors on fatal outcomes in patients with COVID-19 according to age: A systematic review and meta-analysis. Heart 2021, 107, 373–380. [Google Scholar] [CrossRef] [PubMed]
  32. Dos Santos, M.M.; de Macedo, G.T.; Prestes, A.S.; Loro, V.L.; Heidrich, G.M.; Picoloto, R.S.; Rosemberg, D.B.; Barbosa, N.V. Hyperglycemia elicits anxiety-like behaviors in zebrafish: Protective role of dietary diphenyl diselenide. Prog. Neuropsychopharmacol. Biol. Psychiatry 2018, 85, 128–135. [Google Scholar] [CrossRef] [PubMed]
  33. Ecker, A.; da Silva, R.S.; Dos Santos, M.M.; Ardisson-Araujo, D.; Rodrigues, O.E.D.; da Rocha, J.B.T.; Barbosa, N.V. Safety profile of AZT derivatives: Organoselenium moieties confer different cytotoxic responses in fresh human erythrocytes during in vitro exposures. J. Trace Elem. Med. Biol. 2018, 50, 240–248. [Google Scholar] [CrossRef]
  34. Barbosa, N.V.; Nogueira, C.W.; Nogara, P.A.; de Bem, A.F.; Aschner, M.; Rocha, J.B.T. Organoselenium compounds as mimics of selenoproteins and thiol modifier agents. Metallomics 2017, 9, 1703–1734. [Google Scholar] [CrossRef]
  35. Nogara, P.A.; Omage, F.B.; Bolzan, G.R.; Delgado, C.P.; Aschner, M.; Orian, L.; Teixeira Rocha, J.B. In silico Studies on the Interaction between Mpro and PLpro from SARS-CoV-2 and Ebselen, its Metabolites and Derivatives. Mol. Inf. 2021, 40, e2100028. [Google Scholar] [CrossRef]
  36. Omage, F.B.; Madabeni, A.; Tucci, A.R.; Nogara, P.A.; Bortoli, M.; Rosa, A.D.S.; Neuza Dos Santos Ferreira, V.; Teixeira Rocha, J.B.; Miranda, M.D.; Orian, L. Diphenyl Diselenide and SARS-CoV-2: In silico Exploration of the Mechanisms of Inhibition of Main Protease (M(pro)) and Papain-like Protease (PL(pro)). J. Chem. Inf. Model. 2023, 63, 2226–2239. [Google Scholar] [CrossRef]
  37. Menendez, C.A.; Bylehn, F.; Perez-Lemus, G.R.; Alvarado, W.; de Pablo, J.J. Molecular characterization of ebselen binding activity to SARS-CoV-2 main protease. Sci. Adv. 2020, 6, eabd0345. [Google Scholar] [CrossRef] [PubMed]
  38. Qiao, Z.; Wei, N.; Jin, L.; Zhang, H.; Luo, J.; Zhang, Y.; Wang, K. The Mpro structure-based modifications of ebselen derivatives for improved antiviral activity against SARS-CoV-2 virus. Bioorg. Chem. 2021, 117, 105455. [Google Scholar] [CrossRef]
  39. Amporndanai, K.; Meng, X.; Shang, W.; Jin, Z.; Rogers, M.; Zhao, Y.; Rao, Z.; Liu, Z.J.; Yang, H.; Zhang, L.; et al. Inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives. Nat. Commun. 2021, 12, 3061. [Google Scholar] [CrossRef] [PubMed]
  40. Sahoo, P.; Lenka, D.R.; Batabyal, M.; Pain, P.K.; Kumar, S.; Manna, D.; Kumar, A. Detailed Insights into the Inhibitory Mechanism of New Ebselen Derivatives against Main Protease (M(pro)) of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). ACS Pharm. Transl. Sci. 2022, 6, 171–180. [Google Scholar] [CrossRef] [PubMed]
  41. Huff, S.; Kummetha, I.R.; Tiwari, S.K.; Huante, M.B.; Clark, A.E.; Wang, S.; Bray, W.; Smith, D.; Carlin, A.F.; Endsley, M.; et al. Discovery and Mechanism of SARS-CoV-2 Main Protease Inhibitors. J. Med. Chem. 2022, 65, 2866–2879. [Google Scholar] [CrossRef] [PubMed]
  42. Lu, R.; Zhao, X.; Li, J.; Niu, P.; Yang, B.; Wu, H.; Wang, W.; Song, H.; Huang, B.; Zhu, N.; et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: Implications for virus origins and receptor binding. Lancet 2020, 395, 565–574. [Google Scholar] [CrossRef] [Green Version]
  43. Lee, J.T.; Yang, Q.; Gribenko, A.; Perrin, B.S., Jr.; Zhu, Y.; Cardin, R.; Liberator, P.A.; Anderson, A.S.; Hao, L. Genetic Surveillance of SARS-CoV-2 M(pro) Reveals High Sequence and Structural Conservation Prior to the Introduction of Protease Inhibitor Paxlovid. mBio 2022, 13, e0086922. [Google Scholar] [CrossRef]
  44. Vangeel, L.; Chiu, W.; De Jonghe, S.; Maes, P.; Slechten, B.; Raymenants, J.; Andre, E.; Leyssen, P.; Neyts, J.; Jochmans, D. Remdesivir, Molnupiravir and Nirmatrelvir remain active against SARS-CoV-2 Omicron and other variants of concern. Antivir. Res. 2022, 198, 105252. [Google Scholar] [CrossRef]
  45. Kanimozhi, G.; Pradhapsingh, B.; Singh Pawar, C.; Khan, H.A.; Alrokayan, S.H.; Prasad, N.R. SARS-CoV-2: Pathogenesis, Molecular Targets and Experimental Models. Front. Pharm. 2021, 12, 638334. [Google Scholar] [CrossRef]
  46. Jackson, C.B.; Farzan, M.; Chen, B.; Choe, H. Mechanisms of SARS-CoV-2 entry into cells. Nat. Rev. Mol. Cell. Biol. 2022, 23, 3–20. [Google Scholar] [CrossRef] [PubMed]
  47. Essalmani, R.; Jain, J.; Susan-Resiga, D.; Andreo, U.; Evagelidis, A.; Derbali, R.M.; Huynh, D.N.; Dallaire, F.; Laporte, M.; Delpal, A.; et al. Erratum for Essalmani et al. “Distinctive Roles of Furin and TMPRSS2 in SARS-CoV-2 Infectivity”. J. Virol. 2022, 96, e0074522. [Google Scholar] [CrossRef] [PubMed]
  48. Lee, S.Y.; Kim, D.W.; Jung, Y.T. Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation. Virus Genes. 2022, 58, 172–179. [Google Scholar] [CrossRef] [PubMed]
  49. Cardoso, B.R.; Roberts, B.R.; Bush, A.I.; Hare, D.J. Selenium, selenoproteins and neurodegenerative diseases. Metallomics 2015, 7, 1213–1228. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  50. Kil, J.; Harruff, E.E.; Longenecker, R.J. Development of ebselen for the treatment of sensorineural hearing loss and tinnitus. Hear. Res. 2022, 413, 108209. [Google Scholar] [CrossRef]
  51. Singh, N.; Halliday, A.C.; Thomas, J.M.; Kuznetsova, O.V.; Baldwin, R.; Woon, E.C.; Aley, P.K.; Antoniadou, I.; Sharp, T.; Vasudevan, S.R.; et al. A safe lithium mimetic for bipolar disorder. Nat. Commun. 2013, 4, 1332. [Google Scholar] [CrossRef] [Green Version]
  52. Nogueira, C.W.; Rocha, J.B. Toxicology and pharmacology of selenium: Emphasis on synthetic organoselenium compounds. Arch. Toxicol. 2011, 85, 1313–1359. [Google Scholar] [CrossRef]
  53. Barbosa, N.B.; Rocha, J.B.; Zeni, G.; Emanuelli, T.; Beque, M.C.; Braga, A.L. Effect of organic forms of selenium on delta-aminolevulinate dehydratase from liver, kidney, and brain of adult rats. Toxicol. Appl. Pharm. 1998, 149, 243–253. [Google Scholar] [CrossRef]
  54. Meotti, F.C.; Borges, V.C.; Zeni, G.; Rocha, J.B.; Nogueira, C.W. Potential renal and hepatic toxicity of diphenyl diselenide, diphenyl ditelluride and Ebselen for rats and mice. Toxicol. Lett. 2003, 143, 9–16. [Google Scholar] [CrossRef] [PubMed]
  55. Borges, V.C.; Rocha, J.B.; Nogueira, C.W. Effect of diphenyl diselenide, diphenyl ditelluride and ebselen on cerebral Na(+), K(+)-ATPase activity in rats. Toxicology 2005, 215, 191–197. [Google Scholar] [CrossRef]
  56. Centers for Disease Control and Prevention. Oseltamivir-resistant novel influenza A (H1N1) virus infection in two immunosuppressed patients-Seattle, Washington, 2009. MMWR Morb. Mortal. Wkly. Rep. 2009, 58, 893–896. [Google Scholar]
  57. Weglarz-Tomczak, E.; Tomczak, J.M.; Talma, M.; Burda-Grabowska, M.; Giurg, M.; Brul, S. Identification of ebselen and its analogues as potent covalent inhibitors of papain-like protease from SARS-CoV-2. Sci. Rep. 2021, 11, 3640. [Google Scholar] [CrossRef] [PubMed]
  58. Skesters, A.; Kustovs, D.; Lece, A.; Moreino, E.; Petrosina, E.; Rainsford, K.D. Selenium, selenoprotein P, and oxidative stress levels in SARS-CoV-2 patients during illness and recovery. Inflammopharmacology 2022, 30, 499–503. [Google Scholar] [CrossRef] [PubMed]
  59. Rayman, M.P. The importance of selenium to human health. Lancet 2000, 356, 233–241. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  60. Lin, H.S.; Lin, X.H.; Wang, J.W.; Wen, D.N.; Xiang, J.; Fan, Y.Q.; Li, H.D.; Wu, J.; Lin, Y.; Lin, Y.L.; et al. Exhausting T Cells During HIV Infection May Improve the Prognosis of Patients with COVID-19. Front. Cell. Infect. Microbiol. 2021, 11, 564938. [Google Scholar] [CrossRef] [PubMed]
  61. Shreenath, A.P.; Ameer, M.A.; Dooley, J. Selenium Deficiency. In StatPearls [Internet]; StatPearls Publishing: Treasure Island, FL, USA, 2023. Available online: https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/books/NBK482260 (accessed on 25 July 2022).
  62. Muzembo, B.A.; Ngatu, N.R.; Januka, K.; Huang, H.L.; Nattadech, C.; Suzuki, T.; Wada, K.; Ikeda, S. Selenium supplementation in HIV-infected individuals: A systematic review of randomized controlled trials. Clin. Nutr. ESPEN 2019, 34, 1–7. [Google Scholar] [CrossRef]
  63. Guillin, O.M.; Vindry, C.; Ohlmann, T.; Chavatte, L. Interplay between Selenium, Selenoproteins and HIV-1 Replication in Human CD4 T-Lymphocytes. Int. J. Mol. Sci. 2022, 23, 1394. [Google Scholar] [CrossRef] [PubMed]
  64. Martinez, S.S.; Huang, Y.; Acuna, L.; Laverde, E.; Trujillo, D.; Barbieri, M.A.; Tamargo, J.; Campa, A.; Baum, M.K. Role of Selenium in Viral Infections with a Major Focus on SARS-CoV-2. Int. J. Mol. Sci. 2021, 23, 280. [Google Scholar] [CrossRef]
  65. Antunes Dos Santos, A.; Ferrer, B.; Marques Goncalves, F.; Tsatsakis, A.M.; Renieri, E.A.; Skalny, A.V.; Farina, M.; Rocha, J.B.T.; Aschner, M. Oxidative Stress in Methylmercury-Induced Cell Toxicity. Toxics 2018, 6, 47. [Google Scholar] [CrossRef] [Green Version]
  66. Dos Santos, M.M.; de Macedo, G.T.; Prestes, A.S.; Ecker, A.; Muller, T.E.; Leitemperger, J.; Fontana, B.D.; Ardisson-Araujo, D.M.P.; Rosemberg, D.B.; Barbosa, N.V. Modulation of redox and insulin signaling underlie the anti-hyperglycemic and antioxidant effects of diphenyl diselenide in zebrafish. Free Radic. Biol. Med. 2020, 158, 20–31. [Google Scholar] [CrossRef]
Vaccines 11 01222 g001 550
Figure 1. Chemical structures of diphenyl diselenide ((PhSe)2) (A) and ebselen (Eb) (B).
Figure 1. Chemical structures of diphenyl diselenide ((PhSe)2) (A) and ebselen (Eb) (B).
Vaccines 11 01222 g001
Vaccines 11 01222 g002 550
Figure 2. Cell viability of human PBMCs exposed to Eb and (PhSe)2. Here, 0.5 × 106 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% CO2. 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).
Figure 2. Cell viability of human PBMCs exposed to Eb and (PhSe)2. Here, 0.5 × 106 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% CO2. 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).
Vaccines 11 01222 g002
Vaccines 11 01222 g003 550
Figure 3. 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 CC50 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.
Figure 3. 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 CC50 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.
Vaccines 11 01222 g003
Vaccines 11 01222 g004 550
Figure 4. Effects of Eb and (PhSe)2 on lipid peroxidation by thiobarbituric acid-reactive substances (TBARS). Graphs illustrate TBARS production by 4.26 mg/mL of phosphatidylcholine exposed, or not, to Eb and (PhSe)2 in the presence or absence of 214 µM of FeSO4, as previously described. (A) (PhSe)2 and (B) Eb at 0.5, 1, 2, 5, 10, and 25 µM. Data represent the mean ± SEM of four independent experiments, analyzed by one-way ANOVA, followed by Tukey’s multiple tests (n = 3). Letters “a” and “b” indicate statistical equivalence.
Figure 4. Effects of Eb and (PhSe)2 on lipid peroxidation by thiobarbituric acid-reactive substances (TBARS). Graphs illustrate TBARS production by 4.26 mg/mL of phosphatidylcholine exposed, or not, to Eb and (PhSe)2 in the presence or absence of 214 µM of FeSO4, as previously described. (A) (PhSe)2 and (B) Eb at 0.5, 1, 2, 5, 10, and 25 µM. Data represent the mean ± SEM of four independent experiments, analyzed by one-way ANOVA, followed by Tukey’s multiple tests (n = 3). Letters “a” and “b” indicate statistical equivalence.
Vaccines 11 01222 g004
Vaccines 11 01222 g005 550
Figure 5. Effect of (PhSe)2 and Eb on SARS-CoV-2 replication in Calu-3 cells. Percentage of SARS-CoV-2 replication in Calu-3 cells exposed to Eb or (PhSe)2 under different experimental conditions: MOI 0.01 (A,B) or MOI 0.1 (C,D), and 24 h (A,C) or 48 h (B,D) of treatment time. Statistical analysis was obtained in comparison to the infected control group. Data represent the mean ± SD of five independent experiments, analyzed by one-way ANOVA, followed by Dunnett’s multiple tests. ** p ≤ 0.01 and *** p ≤ 0.001.
Figure 5. Effect of (PhSe)2 and Eb on SARS-CoV-2 replication in Calu-3 cells. Percentage of SARS-CoV-2 replication in Calu-3 cells exposed to Eb or (PhSe)2 under different experimental conditions: MOI 0.01 (A,B) or MOI 0.1 (C,D), and 24 h (A,C) or 48 h (B,D) of treatment time. Statistical analysis was obtained in comparison to the infected control group. Data represent the mean ± SD of five independent experiments, analyzed by one-way ANOVA, followed by Dunnett’s multiple tests. ** p ≤ 0.01 and *** p ≤ 0.001.
Vaccines 11 01222 g005
Vaccines 11 01222 g006 550
Figure 6. Size and granularity of human lymphocytes exposed to Eb and (PhSe)2. Human PBMCs (0.5 × 106 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% CO2. (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.
Figure 6. Size and granularity of human lymphocytes exposed to Eb and (PhSe)2. Human PBMCs (0.5 × 106 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% CO2. (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 11 01222 g006
Vaccines 11 01222 g007 550
Figure 7. Viability, early apoptosis, advanced apoptosis, and necrosis in lymphocytes exposed to Eb and (PhSe)2. Human PBMCs (0.5 × 106 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 AccuriTM C6 flow cytometer. (A) Histogram representative of cell staining with Annexin V/PI. Viable cells are in the bottom left quadrant, nonviable necrotic cells are in the top left quadrant, nonviable advanced apoptotic cells are in the top right quadrant, and early apoptotic cells are in the bottom right quadrant. Viability (B), early apoptosis (C), advanced apoptosis (D), and necrosis (E), respectively. The results represent the mean ± SEM of three to four independent experiments and are expressed as % of the control. 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.
Figure 7. Viability, early apoptosis, advanced apoptosis, and necrosis in lymphocytes exposed to Eb and (PhSe)2. Human PBMCs (0.5 × 106 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 AccuriTM C6 flow cytometer. (A) Histogram representative of cell staining with Annexin V/PI. Viable cells are in the bottom left quadrant, nonviable necrotic cells are in the top left quadrant, nonviable advanced apoptotic cells are in the top right quadrant, and early apoptotic cells are in the bottom right quadrant. Viability (B), early apoptosis (C), advanced apoptosis (D), and necrosis (E), respectively. The results represent the mean ± SEM of three to four independent experiments and are expressed as % of the control. 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.
Vaccines 11 01222 g007
Vaccines 11 01222 g008 550
Figure 8. Cell cycle phases of human lymphocytes exposed to Eb and (PhSe)2. Cell cycle distribution of PBMCs exposed to (PhSe)2 or Eb at 2.5 µM. (A) Representative histogram showing cell numbers present in the different phases (G0–G1, S, or G2/M). (B) Percentage of resting PBMCs in G0–G1 phases, or S or G2/M phases (proliferative phases). Results were analyzed by one-way ANOVA followed by Tukey’s multiple test and represent the mean ± SEM of six independent experiments.
Figure 8. Cell cycle phases of human lymphocytes exposed to Eb and (PhSe)2. Cell cycle distribution of PBMCs exposed to (PhSe)2 or Eb at 2.5 µM. (A) Representative histogram showing cell numbers present in the different phases (G0–G1, S, or G2/M). (B) Percentage of resting PBMCs in G0–G1 phases, or S or G2/M phases (proliferative phases). Results were analyzed by one-way ANOVA followed by Tukey’s multiple test and represent the mean ± SEM of six independent experiments.
Vaccines 11 01222 g008
Table
Table 1. EC50 (µM), CC50 (µM), and SI of Eb and (PhSe)2 in Calu-3 cells.
Table 1. EC50 (µM), CC50 (µM), and SI of Eb and (PhSe)2 in Calu-3 cells.
MOI 0.01MOI 0.1
24 hpi48 hpi24 hpi48 hpi
MoleculesCC50EC50SIEC50SIEC50SIEC50SI
(PhSe)2>2003.9 ± 0.451.283.4 ± 0.358.824.2 ± 0.747.623.5 ± 1.057.14
Eb>2003.8 ± 0.552.632.6 ± 0.576.923.1 ± 0.664.523.9 ± 0.651.28
CC50: Drug concentration that promotes the death of 50% of treated cells. EC50: Drug concentration capable of reducing the viral effect in 50% of cells. SI: Selectivity index (calculated by the ratio of CC50 and EC50 values).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Wildner, G.; Tucci, A.R.; Prestes, A.d.S.; Muller, T.; Rosa, A.d.S.; Borba, N.R.R.; Ferreira, V.N.; Rocha, J.B.T.; Miranda, M.D.; Barbosa, N.V. Ebselen and Diphenyl Diselenide Inhibit SARS-CoV-2 Replication at Non-Toxic Concentrations to Human Cell Lines. Vaccines 2023, 11, 1222. https://0-doi-org.brum.beds.ac.uk/10.3390/vaccines11071222

AMA Style

Wildner G, Tucci AR, Prestes AdS, Muller T, Rosa AdS, Borba NRR, Ferreira VN, Rocha JBT, Miranda MD, Barbosa NV. Ebselen and Diphenyl Diselenide Inhibit SARS-CoV-2 Replication at Non-Toxic Concentrations to Human Cell Lines. Vaccines. 2023; 11(7):1222. https://0-doi-org.brum.beds.ac.uk/10.3390/vaccines11071222

Chicago/Turabian Style

Wildner, Guilherme, Amanda Resende Tucci, Alessandro de Souza Prestes, Talise Muller, Alice dos Santos Rosa, Nathalia Roberto R. Borba, Vivian Neuza Ferreira, João Batista Teixeira Rocha, Milene Dias Miranda, and Nilda Vargas Barbosa. 2023. "Ebselen and Diphenyl Diselenide Inhibit SARS-CoV-2 Replication at Non-Toxic Concentrations to Human Cell Lines" Vaccines 11, no. 7: 1222. https://0-doi-org.brum.beds.ac.uk/10.3390/vaccines11071222

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop