Anti-SARS-CoV-2 Activity of Extracellular Vesicle Inhibitors: Screening, Validation, and Combination with Remdesivir

The coronavirus disease 2019 (COVID-19) pandemic severely impacts health, economy, and society worldwide. Antiviral drugs against SARS-CoV-2 are urgently needed to cope with this global crisis. It has been found that the biogenesis and release mechanisms of viruses share a common pathway with extracellular vesicles (EVs). We hypothesized that small molecule inhibitors of EV biogenesis/release could exert an anti-SARS-CoV-2 effect. Here, we screened 17 existing EV inhibitors and found that calpeptin, a cysteine proteinase inhibitor, exhibited the most potent anti-SARS-CoV-2 activity with no apparent cytotoxicity. Calpeptin demonstrated the dose-dependent inhibition against SARS-CoV-2 viral nucleoprotein expression in the infected cells with a half-maximal inhibitory concentration (IC50) of 1.44 µM in Vero-E6 and 26.92 µM in Calu-3 cells, respectively. Moreover, calpeptin inhibited the production of infectious virions with the lower IC50 of 0.6 µM in Vero E6 cells and 10.12 µM in Calu-3 cells. Interestingly, a combination of calpeptin and remdesivir, the FDA-approved antiviral drug against SARS-CoV-2 viral replication, significantly enhanced the anti-SARS-CoV-2 effects compared to monotherapy. This study discovered calpeptin as a promising candidate for anti-SARS-CoV-2 drug development. Further preclinical and clinical studies are warranted to elucidate the therapeutic efficacy of calpeptin and remdesivir combination in COVID-19.

SARS-CoV-2 (SARS-CoV-2/01/human/Jan2020/Thailand) was isolated in Vero cells from the nasopharyngeal swab of a patient with COVID-19 in Thailand (GenBank: QYZ85362.1) [30,31]. Viral stocks were propagated in Vero E6 cells, as previously described [30,31]. Briefly, the virus was adsorbed onto a monolayer of Vero E6 cells at 37 • C for 1 h before replacing the infection media with 2% FBS (Gibco) in DMEM (Gibco). Infected cells were then incubated at 37 • C, at 5% CO 2 , until a cytopathic effect (CPE) was observed. Virus-containing supernatant was collected as the virus stocks and was titrated in quadruplicate in 96-well microtiter plates on Vero E6 cells in 4-fold serial dilution to obtain 50% tissue culture infectious dose (TCID50). The viral titer was calculated with the Reed and Münch endpoint method. All the experiments with live SARS-CoV-2 virus were conducted at a certified biosafety level 3 facility at the Department of Microbiology, Faculty of Science, Mahidol University, Thailand.

In Vitro Anti-SARS-CoV-2 Assays
In vitro anti-SARS-CoV-2 assays were performed as previously described [30,31]. Vero E6 cells were seeded at 1 × 10 4 cells per well, and Calu-3 cells were seeded at 5 × 10 4 cells per well in a 96-well black plate (Corning, Corning, NY, USA) and left to adhere overnight at 37 • C, 5% CO 2 . For primary screening, compounds were diluted in a culture medium to achieve a final concentration of 10 µM with DMSO < 0.5%. For dose responses and drug combinations, compounds were diluted in a culture medium to get the desired concentration with DMSO < 0.5%. All the compounds for in vitro drug screenings were prepared before the start of the infection. Cells were washed with phosphate-buffered saline (PBS) and adsorbed with SARS-CoV-2 at 25TCID50 for 2 h at 37 • C. The viral inoculum was then removed, and cells were washed twice with PBS. After cell infection, a fresh culture medium containing the drug at the indicated concentration was added. Positive convalescent serum (heat-inactivated at 56 • C for 30 min) of a COVID-19 patient was used as a positive control for viral inhibition. Cells were then maintained at 37 • C, at 5% CO 2 , for 48 h. After 48 h post-infection, the infected cells were fixed and permeabilized with ice-cold acetone:methanol (1:1) (Sigma-Aldrich) for 20 min and subjected to detect SARS-CoV-2 nucleoprotein (NP) expression, using a high-content imaging system. At the same time, the culture supernatants were collected to quantify the viral output.

High-Content Imaging System for SARS-CoV-2 Nucleoprotein Detection
Following acetone:methanol fixation, the cells were washed with phosphate-buffered saline with 0.5% Tween (PBST) three times. The cells were then blocked by 2% (w/v) BSA in PBST for 1 h, at room temperature. Cells were incubated with 1:500 rabbit anti-SARS-CoV-2 NP monoclonal antibody (Sino Biological Inc., Beijing, China) at 37 • C for 1 h. Thereafter, cells were washed with PBST three times, followed by incubation with 1:500 of the goat anti-rabbit IgG Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA, USA). After washing, cells were stained with Hoechst dye (Thermo Fisher Scientific). The fluorescent signals were detected and analyzed by the high-content imaging system Biomedicines 2021, 9, 1230 4 of 14 (Operetta, PerkinElmer, Waltham, MA, USA) at 40× magnification. The percentage of the infected cells in each well was automatically obtained from 16 images per well, using Harmony software (PerkinElmer). Data were normalized to the infected control, and the IC50 value was calculated by GraphPad Prism 7 (GraphPad Company, San Diego, CA, USA).

Virus Output Assay
Viral output (or the number of infectious virions released from infected cells) was measured by the plaque assay. The Vero cell monolayer was prepared in a 6-well plate 24 h before infection. The cells were infected with a serial dilution of the virus and incubated for 1 h at 37 • C. Then, the cells were overlaid with 3 mL/well of overlay medium containing MEM supplemented with 5% FBS and 1% agarose. The culture was incubated at 37 • C, in 5% CO 2 , for three days. Plaque phenotypes were visualized by 0.33% Neutral Red staining (Sigma-Aldrich, St. Luis, MO, USA) for 5 h. Plaque numbers were counted as plaque-forming units per milliliter (PFUs/mL) and presented as the percentage of plaque reduction. This experiment was performed in two biological replicates, and the data were presented as the mean of two independent experiments.

Cell Viability Assay
Vero E6 cells were seeded at 1 × 10 4 cells per well, and Calu-3 cells were seeded at 5 × 10 4 cells per well in a 96-well plate and allowed to adhere overnight at 37 • C. Cells were treated with serial dilutions of compounds in a medium for 48 h. Cell viability was examined by the MTT colorimetric assay (Sigma-Aldrich). In brief, the medium was replaced with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] at a final concentration of 0.5 mg/mL and incubated for 4 h, at 37 • C, with 5% CO 2 . The MTT solution was removed, and DMSO (Sigma-Aldrich) was added to the cell to dissolve the formazan crystals. Absorbance was measured at a wavelength of 570 nm by an EnVision Multilabel reader (PerkinElmer). Data were normalized to the solvent control, and then 50% cytotoxic concentration (CC50) values were calculated by using GraphPad Prism 7 (GraphPad Company).

Statistical and Data Analysis
The curve was fitted by using non-linear regression, and the IC50 and CC50 values were calculated by GraphPad Prism 7 (GraphPad Company). All statistical tests were performed by using GraphPad Prism version 5. Multiple comparisons were performed by one-way analysis of variance (ANOVA) with the Tukey post hoc test to compare differences among groups.

Screening of 17 EV Inhibitors for Anti-SARS-CoV-2 Activity
Since EVs and viruses have been found to cross paths in biogenesis [20], it has been postulated that EV inhibitors could serve as the antiviral agents of host-targeting strategy [27,28]. Therefore, this study focused on elucidating whether any of the 17 known EV inhibitors [29] exert an anti-SARS-CoV-2 effect. Chemical structures of EV inhibitors are shown in Figure 1.
The infected Vero E6 cells were used for a primary screen of the 17 EV inhibitors at a fixed concentration of 10 µM. The positive convalescent serum of a COVID-19 patient was included as a positive control for viral inhibitory effects. The criteria were set to identify the hit compound that led to a decrease in viral infection by >50% and had low to modest toxicity (>50% cell viability). As a result, calpeptin was the only EV inhibitor that met the hit compound criteria, achieving up to 95% reduction of the infected cells (Figure 2a Table S2). Thus, its anti-SARS-CoV-2 activity should be excluded ( Figure 2b). Accordingly, only calpeptin was subjected to further validations of SARS-CoV-2 inhibitory activity.  Table S1.
The infected Vero E6 cells were used for a primary screen of the 17 EV inhibitor fixed concentration of 10 µ M. The positive convalescent serum of a COVID-19 patien included as a positive control for viral inhibitory effects. The criteria were set to id the hit compound that led to a decrease in viral infection by >50% and had low to m toxicity (>50% cell viability). As a result, calpeptin was the only EV inhibitor that m hit compound criteria, achieving up to 95% reduction of the infected cells (Figure 2a Table S2). Thus, its anti-SARS-CoV-2 ac should be excluded ( Figure 2b). Accordingly, only calpeptin was subjected to furthe idations of SARS-CoV-2 inhibitory activity.  Table S1.

Calpeptin Exerts the Dose-Dependent Antiviral Activity against SARS-CoV-2 Infection
Next, we validated the hit compound from the primary screening by the dose-response analyses. Vero E6 cells were infected with SARS-CoV-2 at 25TCID50 for 2 h and subsequently treated with calpeptin at the varied concentrations of 0.008 to 100 µM. The anti-SARS-CoV2 activity was determined by high-content imaging of fluorescent NP-positiveinfected cells. As a result, calpeptin exerted an anti-SARS-CoV-2 effect in a dose-dependent manner ( Figure 3a) with IC50 of 1.44 µM (Figure 3b), while the toxicity of calpeptin, as determined by the MTT assay, showed no apparent cytotoxic with CC50 > 100 µM (Figure 3b). In addition, the viral output study was performed to determine the inhibitory activity of calpeptin against infectious virions released. Calpeptin also exhibited the dose-dependent inhibition of viral output in the culture supernatant of Vero E6 with IC50 of 0.60 µM ( Figure 3c). Moreover, the antiviral activity of calpeptin against SARS-CoV-2 was also determined in a human cell line representing the human-lung epithelial cell (Calu-3). Biomedicines 2021, 9, x FOR PEER REVIEW 6 of 14  tin, as determined by the MTT assay, showed no apparent cytotoxic with CC50 > 100 µ M (Figure 3b). In addition, the viral output study was performed to determine the inhibitory activity of calpeptin against infectious virions released. Calpeptin also exhibited the dosedependent inhibition of viral output in the culture supernatant of Vero E6 with IC50 of 0.60 µ M (Figure 3c). Moreover, the antiviral activity of calpeptin against SARS-CoV-2 was also determined in a human cell line representing the human-lung epithelial cell (Calu-3).

Combination of Calpeptin and Remdesivir Increases Antiviral Activity against SARS-CoV-2
The combination of drugs targeting multiple steps in viral life cycle has potential benefits against SARS-CoV-2 infection. In this direction, we further evaluated whether or not calpeptin (possibly targeting viral release) combined with remdesivir (targeting viral replication) exhibited a synergistic anti-SARS-CoV-2 effect. Vero E6 and Calu-3 human lung epithelial cells were infected with SARS-CoV-2 at 25TCID50 for 2 h and subsequently treated with a combination of remdesivir and calpeptin. We decided to evaluate calpeptin (1 and 25 µM for Vero E6 and Calu-3, respectively) and remdesivir (0.5 and 0.1 µM for Vero E6 and Calu-3, respectively) at the concentrations below the IC50 values based on the high-content imaging studies in the corresponding cell lines (Figures 3b and 4b). The dose-dependent effects of remdesivir and IC50 in the infected Vero E6 and Calu-3 cells are provided in Supplementary Materials Figure S1.
In Vero E6 cells, we found that the combination of calpeptin and remdesivir had a higher anti-SARS-CoV-2 effect than monotherapy, either calpeptin or remdesivir treatment alone, as quantified by %infected cells using the high-content imaging system (Figure 5a,c) and the viral output assay (Figure 5d). Consistently, this synergistic antiviral effect was observed in the infected Calu-3 cells (Figure 5b,f,g). In addition, the combination of calpeptin and remdesivir no apparent cytotoxicity in both Vero E6 and Calu-3 cells (Figure 5e,h). Our results indicated that the combination of calpeptin and remdesivir, even at the suboptimal dosages, substantially enhanced their inhibitory effects against SARS-CoV-2 infection. treated with a combination of remdesivir and calpeptin. We decided to evaluate calpeptin (1 and 25 µ M for Vero E6 and Calu-3, respectively) and remdesivir (0.5 and 0.1 µ M for Vero E6 and Calu-3, respectively) at the concentrations below the IC50 values based on the high-content imaging studies in the corresponding cell lines (Figures 3b and 4b). The dose-dependent effects of remdesivir and IC50 in the infected Vero E6 and Calu-3 cells are provided in Supplementary Materials Figure S1.
In Vero E6 cells, we found that the combination of calpeptin and remdesivir had a higher anti-SARS-CoV-2 effect than monotherapy, either calpeptin or remdesivir treatment alone, as quantified by %infected cells using the high-content imaging system (Figure 5a,c) and the viral output assay (Figure 5d). Consistently, this synergistic antiviral effect was observed in the infected Calu-3 cells (Figure 5b,f,g). In addition, the combination of calpeptin and remdesivir no apparent cytotoxicity in both Vero E6 and Calu-3 cells (Figure 5e,h). Our results indicated that the combination of calpeptin and remdesivir, even at the suboptimal dosages, substantially enhanced their inhibitory effects against SARS-CoV-2 infection.  Calu-3 (g) cells were quantified by plaque assay, and data were presented as the percentage of the control. The percentage of cell viability of Vero E6 (e) and Calu-3 (h) are shown. The data are presented as the mean ± SEM of three biological replicates. Statistical analysis was performed by using one-way ANOVA with Tukey post hoc test: * p < 0.05, ** p < 0.005, and *** p < 0.001, ns, not significant. Scale bar: 20 µm.

Discussion
EVs have been shown to cross paths in biogenesis with viruses [20]. Therefore, inhibiting EV biogenesis and release may interfere SARS-CoV-2 infection, mainly via the viral budding and production. Our study was performed to explore the anti-SARS-CoV-2 activity of the existing EV inhibitors [29]. Of 17 EV inhibitors screened, calpeptin stands out with the most potent anti-SARS-CoV2 activity, with no apparent cytotoxicity observed ( Figure 2). Then, validations of antiviral activity of calpeptin were rigorously performed using two assays (high-content imaging detection of SARS-CoV-2 NP expression and the viral output studies) in two cell lines (Vero E6 kidney cells and Calu-3 lung epithelial cells) in order to confirm SARS-CoV-2 inhibitory activity of calpeptin with high confidence (Figures 3 and 4).
Our findings are in line with previous studies [33][34][35][36][37]. Barnard et al. [33] reported that calpain inhibitors, including calpeptin, could inhibit SARS-CoV replication in vitro. Ma et al. [34] utilized an enzyme kinetic assay to demonstrate that calpeptin could target SARS-CoV-2 M pro viral protease, and thus calpeptin may inhibit viral replication. The IC50 values of calpeptin against SARS-CoV in Vero cells (20 µM) [33] and SARS-CoV-2 M pro (10.69 µM) [34] were shown in a similar trend as the IC50 value reported in this study (1.44 µM), using Vero E6 cells. However, the IC50 value of calpeptin was higher in the infected Calu-3 cells (26.92 µM). This finding might be explained by phenotypic differences between Vero E6 and Calu-3 cells that led to differ antiviral efficacies. Both Vero E6 and Calu-3 cells are susceptible to SARS-CoV-2 infection [30,31], but Vero E6 (monkey kidney) cells lack genes encoding type-I interferons in response to viral infections [38]. In contrast, Calu-3 cells are human-lung epithelial cells with intact immune responses [39]. A recent work by Hoffmann et al. [32] also demonstrated that anti-SARS-CoV-2 activity of small molecules can be cell-type dependent. Chloroquine, which efficiently blocked SARS-CoV-2 viral entry of Vero (kidney) cells, did not appreciably inhibit the SARS-CoV-2 infection of Calu-3 human-lung epithelial cells [32]. Therefore, these results indicated that calpeptin exerted antiviral activity against SARS-CoV-2 in a cell-type independent manner, suggesting that it might be a candidate for developing an effective and safe antiviral agent.
We propose that calpeptin is supposed to mainly inhibit viral release rather than viral replication, as evidenced by the IC50 values of calpeptin based on the viral output studies in both Vero E6 and Calu-3 cells that were >2-fold lower than the IC50 values based on the percentage of infected cells (Figures 3 and 4). Nonetheless, it should be emphasized that this cysteine protease inhibitor may also inhibit coronaviral entry via inhibition of host cysteine protease [35,36]. Recently, Mediouni et al. [37] found that calpeptin may exert dual effects of SARS-CoV-2 inhibition at the viral entry and post-entry processes. Thus, our findings contribute to the current evidence that the post-entry action of calpeptin is responsible by the inhibition of SARS-CoV-2 infectious virion release (Figures 3c and 4c).
To date, little is known about the SARS-CoV-2 egress. SARS-CoV-2 may use the multivesicular body (MVB)-like structure for packaging virions that will be released to the extracellular space via exocytosis [28,[40][41][42][43][44]. Alternatively, SARS-CoV-2 may release from infected cells via unconventional egress by lysosomal exocytosis instead of the conventional biosynthetic secretory pathway [42,45], by which it will likely be released as microvesicles [28,43]. SARS-CoV-2 may also be released through a shedding microvesicles bud from infected cells [43,44]. The release of microvesicles can be triggered by Ca 2+ activating calpain, calcium-dependent cysteine proteases [29]. Microvesicle generation is then inhibited by calpeptin, a protease inhibitor of calpains, as it has been found to reduce bleb formation in hepatocytes [46]. Calpeptin has also been demonstrated to inhibit the shedding of microvesicles in several cell lines [47][48][49]. Moreover, calpeptin has been illustrated to inhibit multivesicular body release [50]. Thus, we propose that calpeptin might inhibit SARS-CoV-2 infection by suppressing viral production through a shared pathway of EV release. Further mechanistic studies are needed to confirm this hypothesis. Nonetheless, the potential viral release inhibition by targeting host-cell machinery of calpeptin is explicitly usable as a good adjunct to the mainstay treatment using the viral replication inhibitors.
In this direction, we then evaluated the combination of calpeptin, the host-targeting inhibitor, and remdesivir, the direct virus-targeting drug authorized for emergency use to treat COVID-19 [13,15,16]. Our results showed that combination treatment of calpeptin with remdesivir boosted the antiviral activity against SARS-CoV-2 compared to monotherapy ( Figure 5). This finding supports future investigations of calpeptin and remdesivir combination in preclinical animal models and early phase clinical studies. Our proposed model of this synergistic effect of calpeptin and remdesivir is illustrated in Figure 6.
Since remdesivir has been extensively used for COVID-19 treatment, the virus might develop the resistant strains under the drug pressure, due to the mutations in the RdRp [51]. To address this issue, the combination therapy using drugs targeting both virus and host factors could be an attractive strategy to prevent and overcome resistance. Additionally, targeting host proteins that are shared in a common pathway with viruses can potentially offer a broad-spectrum antiviral effect [52]. However, one should be aware that drugs targeting host cellular proteins have a higher risk of developing toxicities and side effects, as the drugs could interfere with the normal cellular functions [52]. In this regard, we found that the combination of remdesivir and calpeptin, even at the suboptimal dosages, exhibited substantially high inhibitory effects against SARS-CoV-2 infection. This finding allows us to propose the low-dose drug combination strategy, which may have great benefits on the cost-effectiveness and the safety profile, for further preclinical and clinical studies.
Biomedicines 2021, 9, x FOR PEER REVIEW 1 the potential viral release inhibition by targeting host-cell machinery of calpeptin plicitly usable as a good adjunct to the mainstay treatment using the viral replicatio hibitors.
In this direction, we then evaluated the combination of calpeptin, the host-targ inhibitor, and remdesivir, the direct virus-targeting drug authorized for emergency u treat COVID-19 [13,15,16]. Our results showed that combination treatment of calp with remdesivir boosted the antiviral activity against SARS-CoV-2 compared to m therapy ( Figure 5). This finding supports future investigations of calpeptin remdesivir combination in preclinical animal models and early phase clinical studies proposed model of this synergistic effect of calpeptin and remdesivir is illustrated in ure 6. Since remdesivir has been extensively used for COVID-19 treatment, the virus m develop the resistant strains under the drug pressure, due to the mutations in the R [51]. To address this issue, the combination therapy using drugs targeting both viru host factors could be an attractive strategy to prevent and overcome resistance. Add ally, targeting host proteins that are shared in a common pathway with viruses ca tentially offer a broad-spectrum antiviral effect [52]. However, one should be aware drugs targeting host cellular proteins have a higher risk of developing toxicities and effects, as the drugs could interfere with the normal cellular functions [52]. In this re we found that the combination of remdesivir and calpeptin, even at the suboptimal ages, exhibited substantially high inhibitory effects against SARS-CoV-2 infection. finding allows us to propose the low-dose drug combination strategy, which may This study has several limitations. Calpeptin is an investigational drug and has not been approved for clinical use at this moment. In addition, the study was conducted only in vitro. To address these issues, in vivo animal experiments should be performed to evaluate the efficacy and safety of calpeptin, including the combination with remdesivir. This data will be critical for future research, especially the clinical studies. Lastly, we tested the antiviral activity of calpeptin on SARS-CoV-2 infection and demonstrated the inhibitory effect in Vero E6 and Calu-3 cells; however, in-depth mechanistic studies of calpeptin may be more persuasive and may lead to new therapeutic targets focusing on host proteins.
In conclusion, this study screened the existing EV inhibitors to find calpeptin as a promising candidate for further anti-SARS-CoV-2 development. Combining calpeptin with remdesivir is an attractive treatment strategy that should be pursued in future preclinical and clinical studies.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/biomedicines9091230/s1. Figure S1: IC50 and CC50 of remdesivir in Vero E6 (a) and Calu-3 (b) cells. Vero E6 and Calu-3 cells were infected with SARS-CoV-2 at 25TCID50 for 2 h and then post-infection-treated with calpeptin at concentrations ranging from 0.0008 to 2.5 µM for 48 h. The infected cells were fixed and stained for viral nucleoproteins with anti-SARS-CoV NP mAb. The SARS-CoV-2 infected cells were detected by high-content imaging. The percentage of inhibition of infected cell was calculated as percentage of the control conditions. The data are presented as the mean ± SEM of three biological replicates. Table S1: EV inhibitors for screening as anti-SARS-CoV-2 agents in this study. Table S2: