2.1 Materials

Cysteamine (MEA), dithiothreitol (DTT), diamide, and methoxy polyethylene glycol 5000 maleimide (mPEG-mal5000) were purchased from Merck KGaA (Darmstadt, Germany). C4-GSH was synthesized by Gluos Srl (Urbino, Italy).²⁰

I-152 and I-152SdAc were synthesized as described previously resulting in comparable yields and identical NMR spectra.^{13, 21}

Anti S1 antibody, recombinant SARS-CoV-2 Spike protein RBD domain (aa 304-526) as well as HEK293 cells stably expressing the Spike S1 subunit (with D614G mutation) or the RBD domain were a generous gift of Diatheva SRL (Cartoceto, Italy). The proteins were engineered to carry an N-terminal signal peptide for secretion and a C-terminal His tag for purification.

SARS-CoV-2 Receptor Binding Domain (RBD, GenBank: QHD43416, Sigma-Aldrich, St. Louis, USA) was used in SPR and spectrometry studies. Recombinant Human Angiotensin-converting Enzyme 2 (ACE2, Acc.N: Q9BYF1, RayBiotech, Peachtree Corners, USA); Sensor chip CM5, HBS-EP+ buffer (10 mM HEPES pH 7.4; 150 mM NaCl; 3 mM EDTA; 0.05% P20) and Acetate Buffer pH 5.5 were purchased from Cytiva (Cytiva life sciences, Marlborough, USA).

2.2 In vitro incubation of recombinant SARS-CoV-2 Spike protein RBD domain with thiol molecules

The recombinant SARS-CoV-2 Spike protein RBD (1–5 μg) domain was incubated at 37°C with the LMW thiol molecules in phosphate-buffered saline (PBS) for 1 h. At the end of the incubation, a 5-fold molar excess of Iodoacetamide was added to the mixture to inactivate the molecules and alkylate protein -SH groups, and the samples were further incubated for 1 h at room temperature in the dark. The samples were then used for western immunoblotting or mass spectrometry-based analyses.

For SPR measurements, RBD and thiols (final concentration 2 mM) were incubated in HBS-EP+ buffer at 37°C for 1 h. To limit the effects of the thiols on the measurements, after incubation, thiol concentration was reduced by repeated steps of dilution and ultrafiltration with Amicon Ultra 0.5 (3 kDa MWCO, Millipore, Merck Group, Darmstadt, Germany) and RBD final concentration adjusted to 1 × 10⁻⁶M (stock solution).

2.3 Western immunoblotting analysis of recombinant SARS-CoV-2 Spike protein RBD domain after incubation with the thiol molecules

The samples incubated as described above were diluted in non-reducing Sodium Dodecyl Sulphate (SDS) sample buffer, heated at 100°C, and resolved by SDS-PAGE. Fully reduced [R] and non-reduced [NR] RBD used as a reference were obtained by diluting the protein in SDS sample buffer containing or not 4% (v/v) β-mercaptoethanol, respectively. For detection, the protein was blotted onto PVDF membranes and immune-stained with anti 6X His tag rabbit polyclonal antibody (OriGene Technologies Inc, Rockville, MD, USA). Detection was performed using a goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Bio-Rad, Hercules, CA, USA) and the enhanced chemiluminescence detection kit WesternBright ECL (Advasta, San Jose, CA, USA). Immunoreactive bands were visualized in a ChemiDoc MP Imaging System (Bio-Rad).

2.4 Mass spectrometry-based analysis

LC–MS/MS was performed by the Orbitrap Exploris™ 240 Mass Spectrometer (Thermo Fisher Scientific) coupled with the Ultimate 3000 LC system. After RBD treatment, samples were digested by Trypsin/LysC and the obtained peptides were BCA assayed. Ten ng of each sample was loaded onto the trap column AcclaimTM PepMapTM 100 C18 75 μm × 2 cm LC Columns (Thermo Scientific™; Waltham, MA, USA) at a flow rate of 10 μl/min and were then separated with a Thermo RSLC Ultimate 3000 (Thermo Scientific™; Waltham, MA, USA) on a Thermo Easy-SprayTM PepMap RSLC C18 2 μm, 100 Å, 75 μm × 15 cm column (Thermo Scientific™; Waltham, MA, USA) with a step gradient of 4%–60% solvent B (0.1% FA in 80% ACN) from 3–45 min and of 60%–90% solvent B for 45–50 min at 300 nL/min and 35°C, resulting in a 60 min total run time.

MS data were acquired using a data-dependent Top20 method dynamically choosing the most abundant precursor ions from the survey scan (300–1500 Th) for HCD fragmentation. For MS1orbitrap resolution was set to 120.000 (at m/z 200), target automatic gain control (AGC) values of 3 × 10⁶, and auto maximum injection times. For MS2 orbitrap resolution was set to 60.000 (at m/z 200), target automatic gain control (AGC) values of 7.5 × 10⁴, and 40 ms of maximum injection times. Fragmentation was performed with a normalized collision energy of 30. Charge survey (2–6) and dynamic exclusion options (mass tolerance 10 ppm) were employed.

2.5 Spike/ACE2 interaction inhibition assay

Inhibition of SARS-CoV-2 spike and ACE2 interaction by thiol molecules was tested using the SARS-CoV-2 Spike-ACE2 interaction Inhibitor Screening Assay kit from Cayman Chemical (MI, USA). To evaluate whether the LMW thiols could disrupt the native form of S1 RBD, the protocol, was slightly modified: the recombinant SARS-CoV-2 spike S1 RBD bound to the pre-coated plate, was incubated with LMW thiols at 37°C for 1 h, and after several washes, the His-tagged ACE2 protein was added. All the remaining procedures were conducted as indicated by the manufacturer.

2.6 Surface plasmon resonance assay

Kinetics studies were performed on a Biacore X100 (Cytiva). S protein (acc. #YP_009724390.1, R&D Systems, Biotechne, MSP, USA) was immobilized on a CM5 sensor chip by the amine coupling method by using Acetate pH 5.0 and a final protein concentration of 50 μg/ml as described in manufacturer instructions. HBS-EP+ (10 mM HEPES buffer pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) was used as running and dilution buffer. Serial dilutions of the analyte (ACE2) were injected at 25°C with a flow rate of 30 μl/min while the RBD solution concentration (treated with the thiol molecules or untreated) was kept at 25 nM. The surface was regenerated between samples with a 70% ethylene glycol solution. All data were zero adjusted and the reference (blank) was subtracted. The quality of the fitted results was evaluated by the χ² parameter. The inhibition of ACE2 binding to RBD was calculated according to the equation:

$$$$ \mathrm{ACE}2\ \mathrm{i}\mathrm{nhibition}\ \left(\%\right)=\frac{K_{\mathrm{RBD}\mathrm{i}}-{K}_0}{K_{\mathrm{RBD}}-{K}_0}\bullet 100 $$$$

where K_RBDi, K_RBD, and K₀ represent the dissociation constants of the interaction ACE2-S protein measured in the presence of RBD pre-treated with the molecules, the functional RBD or the ACE2 alone, respectively, and the overall reaction is depicted below:

The interaction between the S protein and ACE2 was assessed in the conditions described above by injections of ACE2 without RBD. The reported values (Figure S1 and Table S1) are consistent with the manufacturer and the kinetics and thermodynamics constants for the ACE2-S protein interaction present in the literature.²²

2.7 Cell culture

HEK293 and Vero E6 cells were cultured in DMEM and MEM medium, respectively, supplemented with 10% (v/v) FBS, 2 mM glutamine, 100 μg/ml streptomycin, and 100 μg/ml penicillin.

2.8 Analysis of RBD protein production/secretion in HEK293 cells after incubation with the thiol molecules

HEK293 cells expressing SARS-CoV-2 RBD recombinant protein were seeded at 120.000 cells/well in 24 well plates and after 2 days treated with different molecule concentrations for 24 h. Control cells received only fresh medium. After incubation, the medium was collected and centrifuged to remove any detached cells. The supernatant was transferred into a new tube while the pellet was washed with PBS and pooled with the cell lysate. Cells were lysed directly on the dish by adding 100 μl of SDS-lysis buffer (50 mM Tris–HCl, pH 7.8, 0.25 M sucrose, 2% (w/v) SDS, 10 mM N-ethylmaleimide (NEM)), supplemented with protease inhibitors (Complete, Roche, Basel, Switzerland). An aliquot of the culture medium (supernatant) and the cell lysate was analyzed by western immunoblotting using an anti-His tag antibody, as described above. Quantification of the immunoreactive bands was performed with the Image Lab software (Bio-Rad, Hercules, CA, USA). β-actin was stained as a loading control (Cell Signaling Technologies, Danvers, MA, USA).

2.9 Polyethylene glycol maleimide (PEGmal) alkylation of sulfhydryl groups

HEK293 cells expressing SARS-CoV-2 S1 recombinant protein were treated with the molecules for 4 h. In control dishes, cells were incubated for 30 min with either 5 mM diamide to fully oxidize or 5 mM DTT to fully reduce S1. As control of the pegylation assay, untreated cells were incubated with 20 mM NEM in PBS for 20 min on ice. Cells were then washed in cold PBS and lysed in 10% (v/v) trichloroacetic acid (TCA) and 50% (v/v) acetone. After centrifugation, the pellet was washed three times in 80% (v/v) acetone, dried, and solubilized in a buffer consisting of 0.1 M Tris–HCl, pH 6.8, 8 M urea, 5 mM mPEGmal5000 and 1 mM ethylenediaminetetraacetic acid, adjusted to pH 7.0. The lysate was incubated for 2 h at room temperature in the dark and the reaction was stopped by adding 0.1 M DTT. Protein concentration was determined by the Bradford assay using BSA as standard. Ten μg of proteins were resolved on 6.5% (v/v) polyacrylamide gel and immunoblotted as described above using an anti-S1 monoclonal antibody.

2.10 Glutathione and thiol species determination in HEK293 cells

HEK293 cells expressing SARS-CoV-2 RBD recombinant protein were seeded at 400.000 cells/well in 6 well plates and after 3 days treated with different molecule concentrations for 2 h. Thiol content was assayed as previously described.²³ Briefly, after treatment, cells were lysed with a buffer consisting of 0.1% (v/v) Triton X-100, 0.1 M Na₂HPO₄, 5 mM EDTA, pH 7.5. Then, 0.1 N HCl and precipitating solution (100 ml containing 1.67 g of glacial metaphosphoric acid, 0.2 g of disodium EDTA, and 30 g of NaCl) were added. After centrifugation, the pellet was dissolved in 0.1 M NaOH and protein concentration was determined by the Bradford assay; the thiol species were determined in the supernatant by high-performance liquid chromatography (HPLC) method, validated according to USA and European standards. The method was based on separation coupled with ultraviolet detection and pre-column derivatization with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) as previously described.²⁴

2.11 Cytotoxicity assay

The cytotoxicity of LMW thiols was evaluated in Vero E6 cells by trypan blue exclusion. The data were used to calculate the CC50 (Cytotoxic concentration of the molecule that causes death to 50% of viable cells) value.

2.12 Anti-viral activity

Virus. SARS-CoV-2 (hCoV-19/Italy/CDG1/2020/EPI_ISL_412973), isolated from a nasopharyngeal swab by the Department of Infectious Diseases, National Institute of Health Rome, Italy was propagated in Vero E6 cells to produce viral working stocks. The concentration of infectious virus was determined by plaque-forming titer assay. All the experiments with SARS-CoV-2 were performed in the Biological Safety Level 3 (BSL-3) laboratory of the Scientific Department, Army Medical Center, Rome, Italy.

Viral-entry inhibition assay. Two different protocols were performed. In the first one, SARS-CoV-2 (0.01 m.o.i.) was incubated with MEM containing the molecules for 1 h at 37°C in a 5% CO₂ incubator. Then, the mixtures were used to infect cells monolayer for 1 h at 37°C. After viral adsorption, the inoculum was removed, and a plaque assay was performed.

In the second protocol, it was evaluated the amount of infective viral particles released in the supernatants of the cells infected as above. Briefly, after 1 h incubation with the mixture (thiol molecule+virus), the medium was removed and replaced with fresh medium supplemented with 2% FBS and cells were maintained in a 5% CO₂ incubator for the following 24 h. Then, supernatants were collected and used to infect cells monolayer for 1 h at 37°C. After viral adsorption, the inoculum was removed, and a plaque assay was performed. Untreated SARS-CoV-2 was used as a positive control of viral infection in both protocols.

Post-infection treatment assay. Vero E6 cells seeded in a 24-well plate were infected for 1 h at 37°C with SARS-CoV-2 (0.01 m.o.i.); after 1 h incubation, the medium was removed and replaced with fresh medium containing the thiol molecules, supplemented with 2% FBS. After 24 h, the supernatants were collected and used to infect cells monolayer for 1 h at 37°C. After the viral adsorption period, the inoculum was removed, and a plaque assay was performed. Infected-untreated cells were used as a positive control of viral infection.

2.13 Plaque assay

Vero E6 cells were overlaid with a mixture of MEM (no glutamine, no phenol-red-GIBCO), 1.5% Tragacanth (SIGMA), NaHCO₃ 7% (Gibco), L-glutamine 1X (Gibco), MEM NEAA 1x (Gibco), 0.02 M Hepes (Euroclone), DMSO (Sigma-Aldrich) and 2% FBS (final concentration). To calculate PFU, 5 days post-infection the mixture was removed carefully, plates were washed with saline solution, and stained with 1% crystal violet for 20 min. The plaque reduction ratio was calculated as (100 − N/N₀ × 100) where N is the PFU count of the treated sample, and N₀ is the PFU count of the control sample.

The plaque assay was also used to determine the concentration of molecule that inhibits 50% of plaques in each well (IC50).

2.14 Protein extraction and western immunoblotting analysis of viral proteins

Vero E6 cells were lysed with RIPA buffer [20 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% (v/v) Triton X-100, 0.5% (w/v) sodium dodecyl sulfate (SDS) and 1% (w/v) sodium deoxycholate] supplemented with phenylmethylsulfonyl fluoride, protease and phosphatase inhibitor cocktails (Sigma-Aldrich, Milan, Italy) in BSL-3 facilities. Cell lysates were centrifuged (13 000 rpm, 30 min, 4°C) and the supernatants were diluted in SDS sample buffer containing DL-Dithiothreitol (DTT 0.1 M). The total extract was analyzed by SDS-PAGE followed by western immunoblotting. Membranes were stained with anti-Spike (GeneTex Cat No. GTX632604) or anti Nucleocapsid (Rockland Cat. No. 200-401-A50) antibodies and then incubated with HRP-linked anti-mouse or anti-rabbit (Jackson ImmunoResearch, Newmarket, UK) antibodies as secondary antibodies. The membranes were developed using Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA).

2.15 Statistical analysis

Statistical analysis was performed by GraphPad Prism™ 6.0 software (GraphPad Software Inc., San Diego, California, USA) using a one-way Anova test. A p-value <.05 was considered statistically significant. For MS data, statistical evaluation was performed by Friedman test followed by Dunn's multiple comparisons test (Dunn correction, Alpha .05).

3.1 Thiol molecules cause disulfide bond reduction in recombinant SARS-CoV-2 Spike protein RBD domain and inhibit RBD/ACE2 interaction

The thiol molecules investigated for their ability to disrupt disulfide bonds of the Spike protein were the monothiols C4-GSH and I-152; moreover, the S-deacetylated dithiol derivative of I-152 (I-152SdAc)²¹ was added to our analyses based on the virucidal activity results of mono and dithiol molecules recently reported⁵ (Figure 1A). To evaluate the capacity of our thiols to interfere with the receptor-binding processing of the Spike protein, we chose to perform our investigations by using the RBD domain instead of the entire trimeric Spike considering the critical role of the four S-S bridges of the RBD in the Spike RBD–ACE2 interaction before fusion process.⁶ Most studies previously conducted on the activity and mechanism of action of thiol-based reducing agents against SARS-CoV-2 recognition and entry were based on RBD/ACE2 recognition-based assays.^{5, 6, 25}

After incubation of the recombinant RBD protein domain with different molecule concentrations, samples were subjected to non-reducing denaturing polyacrylamide gel electrophoresis (PAGE) where proteins with S-S bonds maintain a more compact conformation than the reduced protein and thus run faster.²⁶ RBD detection was performed on blotted gels with an anti-His tag antibody. Under these conditions, an evident mobility shift of the RBD protein band, from the non-reduced [NR] toward the reduced [R] form was observed as the molecule concentration increased (Figure 1B). In addition, since the NR form was less immunoreactive compared to the reduced one, probably due to epitope masking, reduction of S-S bonds was also observed as changes in the intensity of the chemiluminescent signal which becomes more intense as disulfides are progressively broken and the His tag becomes more accessible to the antibody. Results of Figure 1B show that all the tested molecules, although to a different extent, were able to change the electrophoretic mobility of the protein. The most evident mobility shifts and increase in signal intensity were observed with I-152 and I-152SdAc, while C4-GSH, had modest effects that could be appreciated only after prolonged exposure of the blot (Figure 1B, dark exposure).

To identify the disulfides targeted by thiol molecules, peptide mapping analysis by mass spectrometry was performed. For this purpose, the 2-Iodacetamide (IAM) modified cysteines, in controls and after treatments, were studied. In Figure 1C the amounts of IAM alkylated discovered cysteines, are reported. Compared to the control condition, the I-152 and I-152SdAc-treated samples showed more alkylated residues compared to the C4-GSH ones (Dunn's test, remarkably the C4-GSH condition was examined 10-fold more concentrated). This evidence supports the concept that the proposed thiol molecules caused disulfide bond reduction in the RBD protein.

To test if the perturbation in the RBD redox assessment induced by the LMW thiols could reduce the affinity of the protein for ACE2, the RBD/ACE2 interaction was quantified by a SARS-CoV-2 Spike-ACE2 interaction inhibitor screening assay. We found that all three tested molecules inhibited RBD binding to ACE2 in a dose-dependent manner, but their potency varied according to the number of the thiol groups present in the molecule (Figure 2A). In fact, the most marked binding inhibition was found with I-152SdAc and I-152 followed by C4-GSH, which was tested at higher doses in agreement with the results shown in Figure 1. Half maximal inhibitory concentration (IC50) values calculated for the thiol molecules were 2.6, 3.9, and 9.7 mM for I-152SdAc, I-152, and C4-GSH, respectively. Interaction between ACE2 and RBD pre-incubated with the thiol molecules, which had shown the strongest activity in the previous tests (i.e., I-152 and I-152SdAc), was further characterized by SPR measurements. The binding activity of the RBD (in solution, pre-incubated or not with the thiol molecules, at 2 mM concentration) was assessed by measuring the interaction of ACE2, the analyte, with the functional S protein immobilized on the chip. The SPR sensorgrams show a decrease in the curvature, which is indicative of the interaction phase, moving from the fully functional RBD (Untreated) toward the I-152SdAc-reduced RBD (I-152SdAc) (Figure 2B). The apparent KD measurements follow the same trend, with values of 3.53 × 10⁻⁷ M, 1.33 × 10⁻⁷ M, and 5.45 × 10⁻⁸ M for RBD, I-152-treated RBD, and I-152SdAc-treated RBD, respectively. The calculated decrease in the binding activity of ACE2 to I-152-treated RBD was about 63% compared to that measured in the presence of untreated RBD, but the highest inhibition was measured with I-152SdAc (85%).

3.2 Thiol molecules inhibit SARS-CoV-2 entry and replication in Vero E6 cells

Based on the results obtained by cell-free experimental models (Figures 1 and 2), SARS-CoV-2 suspensions, pre-incubated with I-152, I-152SdAc and MEA (2 mM) or C4-GSH (20 mM), (thiol molecule+virus), were used to evaluate the virucidal activity of the molecules. In these tests, MEA effectiveness was also investigated since it is a metabolite of I-152 and I-152SdAc within cells and it has been previously described as an effective SARS-CoV-2 entry inhibitor.²⁷ Moreover, MEA is an approved drug, being the specific targeted therapy for cystinosis treatment.^{28, 29}

All the reducing agents were able to significantly impair the infectivity of SARS-CoV-2. Pre-incubation of the virus with I-152 and I-152SdAc caused a decrease in the number of plaques by about 86% and 95% respectively, while about 80% reduction was obtained with MEA and C4-GSH, demonstrating that these reducing molecules strongly impair virus entry (Figure 3A). The antiviral effect was confirmed by the western blot analysis of the viral proteins, Spike (S) and Nucleocapsid (N), in lysates obtained from the cells infected with the virus pre-incubated with the thiol molecules (Figure 3B). We can observe that the thiol compounds reduced S protein to undetectable levels, and N protein almost disappeared with I-152SdAc treatment while it was strongly reduced by I-152 and, to a less extent, by MEA and C4-GSH. Calculation of the amount of infective viral particles released in the supernatant of the infected cells nearly confirmed the results reported in the previous test (Figure 3C). In agreement with the results reported in Figures 1 and 2, the most effective compound was I-152SdAc indicating that the number of thiol groups of the molecules may affect the potency of the thiols to impair the viral entry into the cell.

In our previous works we have demonstrated that one mechanism by which the thiol molecules could inhibit viral infection was by interfering with protein maturation/secretion.^{14, 19} Hence, we explored whether the thiol molecules also inhibited intracellular SARS-CoV-2 life cycle's step. To this aim, Vero E6 cells were infected with SARS-CoV-2 and the thiol molecules were added for the following 24 h (I-152, MEA, I-152SdAC at the dose of 0.25 mM and C4-GSH at the dose of 10 mM). The plaque assay showed that all the LMW thiols impaired viral replication, although with different efficiencies (Figure 3D). Treatment with I-152 and I-152SdAc led to over 90% reduction in virus production while in the case of MEA and C4-GSH the inhibition was by about 50% (Figure 3D).

Similarly, viral protein expression analysis revealed that thiols were able to interfere with viral replication and confirmed the highest inhibition mediated by I-152SdAc and I-152 (Figure 3E). Thus, the inhibitory effect of I-152 and I-152SdAc was further explored in Vero E6 cells infected and treated for 24 h with the two molecules by evaluating the IC50 values, which were found to be 0.128 mM and 0.077 mM for I-152 and I-152SdAc, respectively, markedly lower than CC50 values that were 1.89 mM and 0.7 mM for I-152 and I-152SdAc respectively (not shown), suggesting that the antiviral effect observed is not due to toxicity of the molecules.

3.3 Thiol molecules increase intracellular thiol content and affect Spike protein domains folding/production in HEK293 cells

The mechanisms by which the thiol molecules could inhibit SARS-CoV-2 replication were explored in HEK293 cells stably expressing the RBD/S1(D614G) domains since both the biogenesis of CoV-2 viral proteins and the production of recombinant proteins in mammalian cells require an efficient host's secretory protein factory which is known to be sensible to redox shifts.^{30, 31}

Cells were treated for 24 h with different molecule concentrations and the amount of intracellular and secreted RBD was evaluated. Results shown in Figure 4A demonstrate that I-152 and I-152SdAc at 1 mM concentration were able to significantly reduce the amount of the secreted protein. A 40% reduction compared to control was also observed with 0.25 mM I-152, albeit not statistically significant. The decreased amount of secreted protein was paralleled by an intracellular accumulation of the protein only in the case of I-152SdAc (Figure 4B). As already demonstrated in other cell models,³² analysis of the free thiol pools showed that within cells I-152 and I-152SdAc release high amounts of thiol species in the form of NAC, MEA, and cysteine and at the same time increase GSH concentration, thus creating a thiol-rich environment made of multiple thiol species which may interfere with oxidative protein folding (Figure 4C). On the other hand, C4-GSH-based redox change is mainly due to an increase in GSH (in the form of C4-GSH) and cysteine (Figure 4C). To shed light on the molecular mechanism affecting protein production/secretion, HEK293 cells expressing the S1 protein domain were treated with either I-152 or I-152SdAc, and the redox state of sulfhydryl S1 protein groups was assessed by thiol PEGylation and subsequent western immunoblotting analysis with an anti S1 antibody (Figure 4D). As a reference, cells were incubated with reducing (DTT) and oxidant (diamide) agents to generate fully reduced and fully oxidized forms of the S1 subunit. In this assay, thiol groups undergo alkylation that shifts electrophoretic mobility to a higher molecular weight, whereas disulfide bonds remain without alkylation. Mal-PEG alkylation of a single sulfhydryl results in an apparent molecular mass shift of ∼15 kDa as observed by SDS-PAGE.^{33, 34} These experiments demonstrated a dose-dependent switch from the oxidized to the reduced state of the S1 protein in cells treated with I-152 and I-152SdAc, thus highlighting the potential of these molecules and/or of their metabolites to directly/indirectly interfere with spike protein folding.