Inhibition Mechanism of SARS-CoV-2 Infection by a Cholesterol Derivative, Nat-20(S)-yne

Abstract

The coronavirus disease 2019 (COVID-19) is caused by the etiological agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19, with the recurrent epidemics of new variants of SARS-CoV-2, remains a global public health problem, and new antivirals are still required. Some cholesterol derivatives, such as 25-hydroxycholesterol, are known to have antiviral activity against a wide range of enveloped and non-enveloped viruses, including SARS-CoV-2. At the entry step of SARS-CoV-2 infection, the viral envelope fuses with the host membrane dependent of viral spike (S) glycoproteins. From the screening of cholesterol derivatives, we found a new compound 26,27-dinorcholest-5-en-24-yne-3β,20-diol (Nat-20(S)-yne) that inhibited the SARS-CoV-2 S protein-dependent membrane fusion in a syncytium formation assay. Nat-20(S)-yne exhibited the inhibitory activities of SARS-CoV-2 pseudovirus entry and intact SARS-CoV-2 infection in a dose-dependent manner. Among the variants of SARS-CoV-2, inhibition of infection by Nat-20(S)-yne was stronger in delta and Wuhan strains, which predominantly invade into cells via fusion at the plasma membrane, than in omicron strains. The interaction between receptor-binding domain of S proteins and host receptor ACE2 was not affected by Nat-20(S)-yne. Unlike 25-hydroxycholesterol, which regulates various steps of cholesterol metabolism, Nat-20(S)-yne inhibited only de novo cholesterol biosynthesis. As a result, plasma membrane cholesterol content was substantially decreased in Nat-20(S)-yne-treated cells, leading to inhibition of SARS-CoV-2 infection. Nat-20(S)-yne having a new mechanism of action may be a potential therapeutic candidate for COVID-19.

INTRODUCTION

Coronavirus disease 2019 (COVID-19) is a novel and highly transmissible respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first occurred in Wuhan, China. Since its appearance in December 2019, COVID-19 has rapidly spread worldwide.^1–3) SARS-CoV-2 infection causes inflammation in the respiratory tract and in some cases subsequent severe pneumonia.⁴⁾ In severe cases, systematic infection causes a cytokine storm, followed by multiple organ failure, leading to death.⁵⁾ Although effective vaccines, including a novel mRNA-based vaccines, and antivirals against SARS-CoV-2 have been developed,⁶⁾ COVID-19 remains a global public health problem, with the emergence of new variants of SARS-CoV-2. Thus, the development of new treatments is still required.

Cholesterol derivatives such as oxysterols, e.g. 25-hydroxycholesterol and 27-hydroxycholesterol, are known to have anti-SARS-CoV-2 activities.^7–12) These compounds inhibit various steps of SARS-CoV-2 life cycle, but mainly block the membrane fusion of the entry processes.^9,10) SARS-CoV-2 is an enveloped positive-sense single-stranded RNA virus, belonging to the Betacoronavirus genus, closely related to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV).¹³⁾ In SARS-CoV-2 infection, the receptor-binding domain (RBD) of spike (S) glycoproteins on virions bind to a host cell surface receptor, angiotensin-converting enzyme 2 (ACE2).¹⁴⁾ For entry of viral particles into the host cell, at least two pathways are known to exist: i) S proteins interact with transmembrane protease, serine 2 (TMPRSS2) to cleave S protein, following fusion of the virion with host plasma membrane and ii) virions are endocytosed and then fused with endosomal membranes independent of TMPRSS2.^15,16)

In this study, we focused on cholesterol derivatives to identify the novel anti-SARS-CoV-2 compounds, because the mechanism of action is expected to be different from the existing approved drugs. A unique alkyne-containing cholesterol derivative 26,27-dinorcholest-5-en-24-yne-3β,20-diol (Nat-20(S)-yne) was found as an anti-SARS-CoV-2 agent. Unlike 25-hydroxycholesterol, which modulates cholesterol metabolism in various steps, Nat-20(S)-yne blocked only de novo cholesterol biosynthesis, resulting in strong inhibition of S protein-dependent membrane fusion at the plasma membrane, leading to prevention of SARS-CoV-2 infection. These data suggest that Nat-20(S)-yne may be an interesting prototype for anti-SARS-CoV-2 antivirals with a novel mechanism of action.

MATERIALS AND METHODS

Reagents

All cholesterol derivatives used in this study were purchased from Cayman Chemical Co.; 25-hydroxycholesterol (#11097), 24(S)-hydroxycholesterol (#10009931), 24(R)-hydroxycholesterol (#10217), 22(S)-hydroxycholesterol (#21399), 22(R)-hydroxycholesterol (# 89355), 25(S)-27-hydroxycholesterol (#14791), 25(R)-27-hydroxycholesterol (#14790), 7-dehydrocholesterol (#14612), 8-dehydrocholesterol (#25970), 5α,6β-dihydroxycholesterol (#25538), 7α,25-dihydroxycholesterol (#11032), glycohyodeoxycholic acid (#22643), 3β-hydroxyl-5-cholestenoic acid (#21589), 4β-hydroxycholesterol (#19518), 5α-hydroxyl-6-keto cholesterol (#10007601), 6α-hydroxycholesterol (#10007601), 7β-hydroxycholesterol (#20099), 19-hydroxycholesterol (#15209), 7-keto-25-hydroxycholesterol (#25973), pregnenolone carbonitrile (#16343), and Nat-20(S)-yne (#9001369). pcDNA3.1-Spike plasmids (various strains) were purchased from Genscript Japan Inc. (Tokyo, Japan). Opti-MEM was purchased from Thermo Fisher Scientific (Waltham, MA, U.S.A.). Sandoz 58-035 (#S9318) and U18666A (# 10009085) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.) and Cayman Chemical Co. (Ann Arbor, MI, U.S.A.), respectively.

Cells

VeroE6/TMPRRS2 cells (JCR cell bank, #JCRB1819),¹⁷⁾ VeroE6 cells derived from African green monkey kidney epithelial cells expressing TMPRSS2 protease, were cultured as described previously.¹⁸⁾ HEK293T cells were purchased from ATCC (Manassas, VA, U.S.A.; #CRL-3216). HEK293T cells stably expressing ACE2 (HEK293T/ACE2 cells) were generated by retroviral system using pCX4-pur-hACE2-HA encoding human full length ACE2 (hACE2, GenBank accession number NM_001371415.1, residues 1-805aa) with C-terminal HA tag. HEK293T/ACE2 cells were selected with puromycin (10 µg/mL).

SARS-CoV-2 Strains

SARS-CoV-2 viruses; Wuhan strain (2019-hCoV/Japan/TY/WK-521/2020, GISAID ID: EPI_ISL_408667), delta variant B.1.617.2 (hCoV-19/Japan/TY11-927/2021, GISAID ID: EPI_ISL_2158617), and omicron variant BA.1 (hCoV-19/Japan/TY38-873/2021, GISAID ID: EPI_ISL_7418017) were obtained from National Institute of Infectious Disease (Japan) and handled in biosafety level 3 (BSL-3) facilities.

Syncytium Formation Assay

SARS-CoV-2 S protein-dependent syncytium formation assay was performed as described previously.¹⁸⁾ In brief, VeroE6/TMPRSS2 cells were seeded in 48-well plates (Corning, Corning, NY, U.S.A.) at 5 × 10⁴ cells/well and cultured overnight. Cells were treated with ethanol or 10 µM cholesterol derivatives for 16 h, and then co-transfected with pEGFP-N3 (Clontech) and pcDNA3.1-Spike (delta strain)¹⁸⁾ with PEI MAX transfection reagent (Polysciences, Inc., Warrington, PA, U.S.A.) for 4 h. After washed with phosphate buffered saline (PBS) and further 24 h incubation, cells were observed under a fluorescence microscope.

Cell-to-Cell Fusion Assay

SARS-CoV-2 S protein-dependent cell-to-cell fusion assay was performed as described previously.¹⁸⁾ In brief, HEK293T cells were seeded in 6-well plates (Corning) at 2 × 10⁵ cells/well, and next day cells were co-transfected with pcDNA3.1-Spike (various strains of SARSCoV-2) and pEGFP-N3 plasmids using PEI MAX transfection reagent. After 24 h incubation, cells were washed once with PBS and filled with fresh 10% fetal bovine serum (FBS)-containing medium and further cultured for 16 h. VeroE6/TMPRSS2 cells treated with ethanol or various concentrations of Nat-20(S)-yne for 16 h were layered with 2 × 10⁴ cells/well of above HEK293T cells transiently expressing S protein and green fluorescent protein (GFP), and after 4 h incubation cells were observed under a fluorescence microscope.

SARS-CoV-2 Pseudotype Virus Infection

A vesicular stomatitis virus (VSV)-based pseudotyped virus infection system was used as described previously.¹⁹⁾ In brief, HEK293T cells seeded 4 × 10⁶ cells/dish on 100 mm-dishes (Corning) were transfected with pcDNA3.1-Spike (various strains) plasmid using PEI MAX transfection reagent and cultured for 24 h. After wash with PBS, cells were infected with G-complemented VSV∆G/Luc, washed 3 times with PBS after 2 h, and incubated for 24 h with 10 mL of fresh medium. Cell supernatant containing pseudotyped virus was collected and filtered through a 0.45 µm filter as virus solution. On day 0, VeroE6/TMPRSS2 cells or HEK293T/ACE2 cells were seeded in 48-well plates at 3 × 10⁴ cells/well. On day 1, cells were treated with ethanol or Nat-20(S)-yne for 16 h, and on day 2 pseudotype virus solution were added to the wells. On day 3, culture medium was removed and cells were washed once with PBS. Luciferase assay reagent (PicaGene Meliora Star-LT Luminescence Reagent, TOYO BNET Co., Ltd., Tokyo, Japan) was added to the cells, and luminescence intensity of cell lysates was measured with Luminescencer-PSN (AB-2200) (ATTO, Tokyo, Japan).

SARS-CoV-2 Infection

Infection of VeroE6/TMPRSS2 cells with SARS-CoV-2 virus was performed as described previously.²⁰⁾ In brief, VeroE6/TMPRSS2 cells were seeded in 48-well plates at 5.0 × 10⁴ cells/well. After 24 h of culture, cells were treated with 2.5 and 10 µM of Nat-20(S)-yne for 16 h. SARS-CoV-2 Wuhan, delta or omicron BA.1 strains were then infected at 0.001 TCID50/cell. After 2 h, the mixture of Nat-20(S)-yne and virus was removed and cells were washed three times with culture medium and incubated for 22 h in the presence of Nat-20(S)-yne. Viral RNA was extracted from cells by using the Blood/Cultured cell total RNA kit (Favorgen Biotech Corporation). Real-time quantitative(q)RT-PCR was performed using the THUNDERBIRD probe one-step qRT-PCR kit (Toyobo Co., Ltd., Osaka, Japan) on a Light Cycler 96 (Roche Diagnostics, Basel, Switzerland) with NIID-designed virus specific primers and TaqMan probes (N2 set, Eurofins Genomics, Bayern, Germany).

Quantification of Cellular Cholesterol and Desmosterol Contents

VeroE6/TMPRSS2 cells were seeded in 6-well plates at 2.0 × 10⁵ cells/well. After overnight culture, cells were treated with 10 µM of Nat-20(S)-yne or 25-hydroxycholesterol for 22 h. After washing the cells twice with 1 mL of PBS, cells were collected with 320 µL of PBS and added 800 µL of methanol and 400 µL of chloroform. At this time, 1 nmol of deuterium-labeled cholesterol (cholesterol-d7, Avanti Polar Lipids) and desmosterol (desmosterol-d6, Avanti Polar Lipids) were added as internal standards. Additionally, 400 µL of chloroform and 400 µL of distilled water were added and centrifuged (1000 × g, 15 min, 4 °C), and the lower layer was transferred to a new test tube. The transferred lower layer was evaporated under a stream of nitrogen and dissolved in 100 µL of methanol. Cholesterol and desmosterol in the extract were quantified by a liquid chromatography tandem mass spectrometer system (LC-MS/MS) consisting of a Prominence UFLC system (Shimadzu) and a 3200 QTRAP system (SCIEX, Tokyo, Japan). Ten microliters of the extract was injected onto an InertSustain C18 column (5 μm, 2.1 × 150 mm, GL Science, Tokyo, Japan). Mobile phase A was water containing 5 mM ammonium formate and mobile phase B was methanol containing 5 mM ammonium formate. The flow rate was 0.2 mL/min and gradient elution was performed for 40 min under the following conditions. Mobile phase B, 0–10 min; 75 to 100%, 10–30 min; hold at 100%, 30–33 min; 100 to 75%, 33–40 min; 75% retention. LC-MS/MS analysis was performed in positive ion mode with instrument parameters set to 10 psi curtain gas, 5500 V ion spray voltage, 100 °C temperature, 20 psi nebulizer gas, and 10 psi auxiliary gas, optimized for multiple reaction monitoring (MRM) mode. MRM of each ion pair was monitored for 100 ms with a resolution of 1 unit. MRM transitions corresponding to cholesterol-d7, cholesterol, desmosterol-d6, and desmosterol were m/z = 411.4–376.4, 404.4–369.4, 402.4–367.4, and 408.4–373.4, respectively. Data acquisition and analysis were performed using Analyst Software version 1.6 (SCIEX). Cholesterol and desmosterol content were calculated using standard curves for each internal standard.

Staining of Cell Surface Membrane Cholesterol

VeroE6/TMPRSS2 cells were seeded in 12-well plates at 1 × 10⁵ cells/well and cultured overnight. Cells were treated with 10 µM Nat-20(S)-yne or 25-hydroxycholesterol for 20 h. Cells were washed twice with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. After washing with PBS, cell surface membrane cholesterol was visualized by mCherry-Domain 4 of Perfringolysin O (D4) for 1 h at room temperature and observed by BZ-X710 fluorescence microscopy (KEYENCE, Osaka, Japan).

Metabolic Labeling of Cellular Neutral Lipids

VeroE6/TMPRSS2 cells were seeded in 6-well plates at 2 × 10⁵ cells/well and cultured overnight. Cells were treated with 10 µM Nat-20(S)-yne or 25-hydroxycholesterol. Then, after addition of ³H-acetate to the culture, cells were further cultured at 37 °C for 16 h. The amounts of cellular protein were determined by the BCA assay. Lipids were extracted from the cells and separated by TLC with Silica Gel 60 plates (Merck, Darmstadt, Germany). The plate was developed with hexane/diethyl ether/acetic acid = 70/30/1 (vol/vol). The radioactive lipids on the TLC plates were visualized, and the intensity of each band was quantified using a Typhoon FLA 7000 (GE Healthcare, Chicago, IL, U.S.A.). To compare the relative values of each lipid, the intensity of each band was normalized by the amount of protein. Data were expressed as the relative values of vehicle control (ethanol) and the mean value ± standard deviation (S.D.) was obtained from at least three independent experiments.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 9.5.0 software (GraphPad Inc., San Diego, CA, U.S.A.). Data are presented as mean ± S.D., and differences in means were evaluated using Student’s t-test.

RESULTS

Nat-20(S)-yne Inhibits SARS-CoV-2 S Protein-Dependent Cell Fusion

To explore cholesterol derivatives with anti-SARS-CoV-2 activity, we started to perform SARS-CoV-2 S protein (delta strain)-dependent cell fusion assays using ACE2-positive VeroE6/TMPRSS2 cells treated with each 10 µM of 21 cholesterol derivatives (Figs. 1A, B). Among these, five compounds, 25-hydroxycholesterol, 24(S)-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 25(S)-27-hydroxycholesterol, and Nat-20(S)-yne strongly inhibited syncytium formation (Fig. 1C). Four compounds, excluding Nat-20(S)-yne, have already been reported to have anti-SARS-CoV-2 activity,^7,11,21) whereas Nat-20(S)-yne has not been reported to have antiviral activity against SARS-CoV-2 or other viruses. Thus, we focused on Nat-20(S)-yne and further analyzed its anti-SARS-CoV-2 activity to verify the mechanism of action.

Fig. 1. Effect of 21 Sterol Derivatives on SARS-CoV-2 S Protein-Dependent Syncytium Formation

A, Structural formulae and names of sterol derivatives used in this study. The name is shown at the bottom of the conceptual formula. B, Time course of experiments. The S protein from the delta strain of SARS-CoV-2 was co-expressed with GFP in VeroE6/TMPRSS2 cells treated with ethanol or each sterol derivative (10 µM) for 16 h. At 28 h post-transfection, syncytium formation images visualized by the green signal of GFP were taken. C, The binding of expressed S proteins to ACE2 on the cell surface between neighboring cells leads to cell fusion together, resulting in the appearance of GFP-positive multinucleated cells (syncytium formation). Experiments were repeated three times independently and representative images are shown. Scale bar, 200 µm. Blue letters indicate compounds for which anti-SARS-CoV-2 effects have already been reported. Nat-20(S)-yne (red letter) is a compound with anti-SARS-CoV-2 effect found in this study.

The cytotoxicity of Nat-20(S)-yne was first checked using VeroE6/TMPRSS2 cells. The concentration at which Nat-20(S)-yne inhibited cell survival by 50% (CC₅₀) was 38.69 µM, with no cytotoxicity observed at concentrations up to 30 µM (Supplementary Fig. S1). Next, whether cell-to-cell fusion using S proteins from other strains as well as delta strain is inhibited by Nat-20(S)-yne or not was evaluated by the cell-to-cell fusion assay using ACE2-negative HEK293T cells expressing each S protein from various strains and ACE2-positive VeroE6/TMPRSS2 cells. In the mixed culture of control ethanol-treated VeroE6/TMPRSS2 cells and S protein-expressing HEK293T cells, giant GFP-positive fused cells were observed under each condition using S proteins of Wuhan, delta, omicron BA.1, BA.2.75, and BA.5 strains (Fig. 2A), confirming that cell-to-cell fusion can be occurred depending on S proteins of various SARS-CoV-2 strains. Since omicron strains of SARS-CoV-2 predominantly use the endocytosis pathway to enter into cells^15,16) and this cell-to-cell fusion assay using VeroE6/TMPRSS2 cells largely mimics the viral entry via TMPRSS2-mediated plasma membrane fusion pathway, each subspecies of the omicron strain, BA.1, BA.2.75, and BA.5, have lower cell-to-cell fusion activity in VeroE6/TMPRSS2 cells, compared to Wuhan and delta strains, in ethanol-treated cells (Fig. 2B). Cells treated with Nat-20(S)-yne showed the dose-dependent inhibition of cell-to-cell fusion under all of the conditions using S proteins of Wuhan, delta, omicron BA.1, BA.2.75, and BA.5 strains (Figs. 2B, C).

Fig. 2. Cell-to-Cell Fusion Depending on S Proteins from Various SARS-CoV-2 Strains Is Inhibited by Nat-20(S)-yne

A, Time course of experiments. Detailed procedure was described in Materials and Methods. B, Cell-to-cell fusion assays using S proteins from Wuhan, omicron BA.1, BA.2.75, and BA.5 strains as well as delta strain were performed in the presence of 2.5, 5, and 10 µM of Nat-20(S)-yne. Scale bar, 200 µm. C, GFP-positive area in each image under the conditions in Fig. 2B, showing cell-to-cell fusion activity, was quantitated using Image J software. The values of ethanol-treated cells using S proteins of each strain were set as 100%. Data are shown as mean ± standard deviation (S.D.) (n = 3). Statistical analysis was performed by Student’s t-test (*, p < 0.05, **, p < 0.005).

Nat-20(S)-yne Prevents SARS-CoV-2 Infection at the Entry Step

Since this cell-to-cell fusion assay mimics the viral entry process of SARS-CoV-2 infection, we examined the effect of Nat-20(S)-yne on SARS-CoV-2 pseudotype virus entry into VeroE6/TMPRSS2 cells. Treatment of cells with Nat-20(S)-yne exhibited a dose-dependent inhibitory effect on the entry of pseudotype viruses having S proteins from Wuhan, delta, and omicron subspecies, BA.1, BA.2.75, and BA.5 (Fig. 3A), consistent with the results of cell-to-cell fusion assays (Fig. 2). Inhibition activities of Nat-20(S)-yne against omicron subspecies was slightly weaker than those against Wuhan and delta strains. We then investigated the effect of Nat-20(S)-yne on intact SARS-CoV-2 infection, using VeroE6/TMPRSS2 cells. Cells pretreated with Nat-20(S)-yne for 16 h were infected with Wuhan, delta, and omicron BA.1 strains of SARS-CoV-2 for 2 h and 22 h post-infection cellular SARS-CoV-2 genomic RNA copies were quantitated by real-time qRT-PCR. Nat-20(S)-yne-treated cells exhibited the inhibition of all types of SARS-CoV-2 infection (Fig. 3B). However, higher inhibitory activities against Wuhan and delta strains were observed, compared to that against omicron strain BA.1 (Fig. 3B). In the entry step, the SARS-CoV-2 delta and Wuhan strains predominantly utilize the plasma membrane fusion pathway, and the omicron strain BA.1 predominantly utilizes the endocytosis pathway.^15,16) Hence, we further evaluated the effect of Nat-20(S)-yne on SARS-CoV-2 pseudotype virus entry, using HEK293T/ACE2 cells, into which SARS-CoV-2 enters mainly via the endocytosis pathway.²²⁾ The inhibitory activities by Nat-20(s)-yne against all types of pseudotype viruses tested were obviously weaker in HEK293T/ACE2 cells (Fig. 3C) than in VeroE6/TMPRSS2 cells (Fig. 3A). These results indicate that Nat-20(S)-yne has a limited inhibitory effect on the SARS-CoV-2 entry via the endocytosis pathway and strongly inhibits the SARS-CoV-2 entry via the plasma membrane fusion pathway.

Fig. 3. Nat-20(S)-yne Inhibited SARS-CoV-2 Pseudotype Virus Entry and SARS-CoV-2 Infection

A, SARS-CoV-2 pseudotype viruses having a luciferase reporter gene and S proteins from Wuhan, delta, or omicron subspecies, BA.1, BA.2.75, and BA.5 were used in the entry assay. VeroE6/TMPRSS2 cells treated with control ethanol (EtOH) or 2.5, 5, and 10 µM of Nat-20(S)-yne for 16 h were infected with these pseudotype viruses for one day. Luciferase activity of each cell lysate was measured with a luminometer. Luminescence intensities of ethanol-treated cells in each group of S proteins were set to 100%. Data are shown as mean ± S.D. (n = 3). Statistical analysis was performed by Student’s t-test (*, p < 0.05, **, p < 0.005). B, VeroE6/TMPRSS2 cells treated with control ethanol (EtOH) or 2.5 and 10 µM of Nat-20(S)-yne for 16 h were infected with Wuhan, delta, and omicron BA.1 strains of SARS-CoV-2 for 2 h. SARS-CoV-2 genomic RNA contents in cell lysates were quantitated by qRT-PCR. The viral copy numbers of ethanol-treated cells in each group of S proteins were set to 100%. Data are shown as mean ± S.D. (n = 3). Statistical analysis was performed by Student’s t-test (*, p < 0.05). C, Pseudotype virus infection was carried out in the same method of Fig. 3A, except for the use of HEK293T/ACE2 cells. Data are shown as mean ± S.D. (n = 3). Statistical analysis was performed by Student’s t-test (*, p < 0.05).

The S protein-mediated plasma membrane fusion process and endocytosis of viral particles into host cells are preceded by the binding of host receptor ACE2 protein to the receptor binding domain (RBD) of S protein. Therefore, we next examined whether Na-20(S)-yne directly inhibits the interaction between human ACE2 and the RBDs from various strains of SARS-CoV-2 by ELISA using these recombinant proteins. When we used Wuhan, delta, and omicron BA.1 strain-derived RBDs, no inhibitory effect of Nat-20(S)-yne on RBD-ACE2 binding was observed (Supplementary Fig. S2). These results suggest that Nat-20(S)-yne does not directly inhibit RBD-ACE2 binding, but inhibits rather the subsequent entry steps.

Nat-20(S)-yne Inhibits de Novo Cholesterol Biosynthesis

Cholesterol, which is a major component in cellular membranes, has been reported to promote membrane fusion via the SARS-CoV-2 S protein and viral infection, and cholesterol depletion from host cells leads to a decrease in the membrane fusion and the viral infection.²³⁾ Since a cholesterol derivative Nat-20(S)-yne was shown to strongly inhibit the S protein-dependent membrane fusion activity (Figs. 1, 2) and SARS-CoV-2 infection (Fig. 3), the level of cellular cholesterol in Nat-20(S)-yne-treated cells was determined. As shown in Fig. 4A, cellular cholesterol contents were significantly decreased after the treatment of VeroE6/TMPRSS2 cells with 10 µM of Nat-20(S)-yne as well as 25-hydroxycholesterol, which is well known to have a cellular free cholesterol-lowering activity. Additionally, plasma membrane cholesterol contents were also remarkably reduced in the cells treated with Nat-20(S)-yne as well as 25-hydroxycholesterol, from the cell surface free cholesterol staining with mCherry-D4 (Fig. 4B). Furthermore, the amounts of desmosterol, a cholesterol biosynthesis precursor (Fig. 4C), were markedly decreased in the cells treated with Nat-20(S)-yne and 25-hydroxycholesterol (Fig. 4A). These results indicate that Nat-20(S)-yne may inhibit cholesterol biosynthesis, at least prior to desmosterol production.

Fig. 4. Cellular and Cell Surface Cholesterol and Desmosterol Contents Were Reduced in Nat-20(S)-yne-Treated VeroE6/TMPRSS2 Cells

VeroE6/TMPRSS2 cells were treated with 10 µM of Nat-20(S)-yne or 25-hydroxycholesterol (25-OH) for 22 h. A, Cellular cholesterol and desmosterol contents were then determined by LC-MS/MS. Data are presented as mean ± S.D. (n = 3). Statistical analysis was also performed by Student’s t-test (*:p < 0.05). B, Cell surface cholesterol was stained with a free cholesterol-specific binder mCherry-D4 and observed under a fluorescence microscope. Scale bar, 200 µm. C, Cholesterol biosynthesis pathway in host cells. HMG, 3-hydroxy-3-methylglutaryl; HMG-CoAR, HMG-CoA reductase; DHCR7, 7-dehydrocholesterol reductase; DHCR24, 24-dehydrocholesterol reductase; ACAT, acyl-CoA: cholesterol acyltransferase.

The effects of Nat-20(S)-yne on cholesterol metabolism were further investigated in comparison with that of 25-hydroxycholesterol (Supplementary Fig. S3A). We first determined the expression levels of major enzymes involved in cholesterol biosynthesis. mRNA levels of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGCR), 7-dehydrocholesterol reductase (DHCR7), and 24-dehydrocholesterol reductase (DHCR24) were significantly decreased in the cells treated with Nat-20(S)-yne as well as 25-hydroxycholesterol (Supplementary Fig. S3B). SREBP2, which is a regulatory gene of these cholesterol biosynthesis enzymes, also downregulated in Nat-20(S)-yne-treated cells (Supplementary Fig. S3C). These results revealed that Nat-20(S)-yne inhibits de novo cholesterol biosynthesis. 25-Hydroxycholesterol has other biological activities involved in cellular cholesterol dynamics such as the inhibition of LDL-mediated cellular cholesterol uptake via downregulation of LDL receptor and the enhancement of cholesterol efflux from cells via a cholesterol transporter ABCA1 (Supplementary Fig. S3A). 25-Hydroxycholesterol-treated VeroE6/TMPRSS2 cells showed a decrease in LDL receptor mRNA (LDLR) expression and an increase in ABCA1 mRNA (ABCA1) expression, but Nat-20(S)-yne-treated cells did not (Supplementary Fig. S3C). These results suggested that Nat-20(S)-yne has different effects on cellular cholesterol uptake and efflux than 25-hydroxycholesterol. Cholesterol esterification by acyl-CoA: cholesterol acyltransferase (ACAT) was well known to be strongly enhanced by the treatment of cells with 25-hydroxycholesterol¹⁰⁾ (Supplementary Fig. S3A, Fig. 5A). When VeroE6/TMPRSS2 cells treated with 25-hydroxycholesterol and Nat-20(S)-yne were metabolically labeled with ³H-acetate, cholesteryl ester was highly accumulated in 25-hydroxycholesterol-treated cells, but not in Nat-20(S)-yne-treated cells (Figs. 5B, C). On the other hands, triglyceride was slightly accumulated in Nat-20(S)-yne-treated cells, but not in 25-hydroxycholesterol-treated cells (Figs. 5B, C). These results indicated that Nat-20(S)-yne does not affect cholesterol esterification activity. We next checked the effect of a specific ACAT inhibitor Sandos 58-035 on the SARS-CoV-2 S protein-dependent cell-to-cell fusion activity in 25-hydroxycholesterol-treated cells and Nat-20(S)-yne-treated cells. As anticipated, recovery of cell-to-cell fusion by Sandos 58-035 treatment was observed in 25-hydroxycholesterol-treated cells (Figs. 5D, E), but not in Nat-20(S)-yne-treated cells (Figs. 5F, G) in all cases using S proteins of Wuhan, delta, omicron BA.1, BA.2.75, and BA.5 strains. Taken together, it was demonstrated that the inhibitory effect of Nat-20(S)-yne on SARS-CoV-2 infection was attributed to the inhibition of cholesterol biosynthesis, not to the effects of other cholesterol metabolism.

Fig. 5. Nat-20(S)-yne Has No Effect on ACAT Activity

A, Acyl-CoA: cholesterol acyltransferase (ACAT) is an enzyme catalyzing cholesteryl ester (CE) formation form cholesterol, and its activity is strongly activated by 25-hydroxycholesterol (25-OH). Sandos 58-035, an ACAT inhibitor. B, TLC patterns of metabolically ³H-acetate-labeled cellular neutral lipids in ethanol (EtOH)-, Nat-20(S)-yne-, and 25-OH-treated VeroE6/TMPRSS2 cells. Chol, cholesterol; TG, triglyceride. C, Bands of CE and TG in Fig. 5B were quantitated by Typhoon FLA7000 image analyzer (GE Healthcare). D, E, F, and G, Effect of Sandoz 58-035 on the 25-OH- and Nat-20(S)-yne-mediated inhibition of cell-to-cell fusion activities. VeroE6/TMPRSS2 cells were treated with ethanol (−), or 10 µM of 25-OH (D, E) or 10 µM of Nat-20(S)-yne (F, G) for 16 h in the presence of DMSO (−) or 30 µM of Sandoz 58-035. Cells were then washed and incubated in DMSO or Sandoz 58-035 for 1 h, followed by addition of HEK293T cells transiently co-expressing each S protein and GFP. After 4 h incubation cells were observed by fluorescence microscopy (D, F). Experiments were repeated three times independently and representative images were shown. Scale bar, 200 µm. E and G, GFP-positive area in each image under the conditions in Figs. 5D and F, showing cell-to-cell fusion activity, was quantitated using Image J software, respectively. The values of only ethanol-treated cells in each group using S proteins of each strain were set as 100%. Data are shown as mean ±S.D. (n = 3). Statistical analysis was performed by Student’s t-test (*, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001).

Decrease in Cellular Cholesterol Content by Nat-20(S)-yne Is Critical for Its Inhibition of SARS-CoV-2 Infection

As shown in Fig. 4A, desmosterol and cholesterol contents were significantly decreased in Nat-20(S)-yne-treated cells, and we examined whether cell-to-cell fusion activity could be restored after the addition of exogeneous desmosterol and cholesterol to the Nat-20(S)-yne-treated cells. The addition of cholesterol to Nat-20(S)-yne-treated cells resulted in significant recovery of cell-to-cell fusion activity in all cases using S proteins of Wuhan, delta, omicron BA.1, BA.2.75, and BA.5 strains (Figs. 6A, B). When desmosterol was added to Nat-20(S)-yne-treated cells, recovery trends of cell-to-cell fusion were also observed in all strains except for omicron BA.1. These results suggested that desmosterol, in addition to cholesterol, might be involved in the cell-to-cell fusion activity. To clarify the involvement of desmosterol itself, we evaluated the effects of desmosterol and cholesterol on cell-to-cell fusion activity under the conditions in the presence of an inhibitor of DHCR24, which is an enzyme catalyzing the production of cholesterol from desmosterol (Fig. 7A). Treatment of VeroE6/TMPRSS2 cells with a DHCR24 inhibitor U18666A exhibited the inhibition of cell-to-cell fusion in all cases using S proteins of Wuhan, delta, omicron BA.1, BA.2.75, and BA.5 strains (Figs. 7B, C). The addition of cholesterol to U18666A-treated cells resulted in significant recovery of cell-to-cell fusion activity in all cases, whereas the addition of desmosterol to U18666A-treated cells showed no recovery of the fusion activity (Figs. 7B, C). These results strongly suggested that decrease in cellular cholesterol content, not desmosterol content, is involved in Nat-20(S)-yne-mediated inhibition of SARS-CoV-2 infection.

Fig. 6. Cell-to-Cell Fusion Activity Was Restored by Addition of Desmosterol as Well as Cholesterol to Nat-20(S)-yne-Treated Cells

A, VeroE6/TMPRSS2 cells were treated with ethanol (−), or 10 µM of Nat-20(S)-yne for 16 h. Cells were washed with PBS, and further cultured in ethanol (−), or 10 µM Nat-20(S)-yne-containing medium, followed by addition of HEK293T cells transiently co-expressing each S protein and GFP in the presence of ethanol (−), 100 µM desmosterol (Desm) or 100 µM cholesterol (Chol). After 4 h incubation cells were observed by fluorescence microscopy. Experiments were repeated three times independently and representative images were shown. Scale bar, 200 µm. B, GFP-positive area in each image under the conditions in Fig. 6A, showing cell-to-cell fusion activity, was quantitated using Image J software. The values of only ethanol-treated cells in each group using S proteins of each strain were set as 100%. Data are shown as mean ±S.D. (n = 3). Statistical analysis was performed by Student’s t-test (*, p < 0.05, **, p < 0.01, ***, p < 0.001).

Fig. 7. Decrease in Cellular Cholesterol Content, Not Desmosterol Content, Is Involved in Nat-20(S)-yne-Mediated Inhibition of SARS-CoV-2 S Protein-Dependent Cell-to-Cell Fusion

A, U18666A is an inhibitor of DHCR24, which synthesizes cholesterol from desmosterol. B, VeroE6/TMPRSS2 cells were treated with DMSO (−) or 1 µM U18666A for 24 h. Cells were washed with PBS, and further cultured in ethanol (−), 100 µM desmosterol (Desm)- or 100 µM cholesterol (Cho)-containing medium, followed by addition of HEK293T cells transiently co-expressing each S protein and GFP. After 4 h incubation cells were observed by fluorescence microscopy. Experiments were repeated three times independently and representative images were shown. Scale bar, 200 µm. C, GFP-positive area in each image under the conditions in Fig. 7B, showing cell-to-cell fusion activity, was quantitated using Image J software. The values of ethanol-treated cells in each group using S proteins of each strain were set as 100%. Data are shown as mean ±S.D. (n = 3). Statistical analysis was performed by Student’s t-test (*, p < 0.05, **, p < 0.01).

DISCUSSION

Cholesterol derivatives often modulate cellular cholesterol metabolism and dynamics, and indeed Nat-20(S)-yne-treated cells as well as 25-hydroxycholesterol-treated cells showed a large reduction in cholesterol content at the plasma membrane as well as a reduction in total cellular cholesterol content. Cellular cholesterol-rich membrane regions are known to function as platforms for SARS-CoV-2 entry,^23–25) where entry factors such as ACE2, heparan sulfate proteoglycans, TMPRSS2, CD147 and scavenger receptor class B type I are recruited for their interaction with SARS-CoV-2 S protein, and cholesterol depletion from cellular membranes leads to inhibit SARS-CoV-2 entry and infection.¹⁰⁾ Thus, a decrease in plasma membrane cholesterol content by Nat-20(S)-yne would be critical for its antiviral mechanism of action, similar to that of 25-hydroxycholesterol.^10,12)

Interestingly, Nat-20(S)-yne was found to inhibit only de novo cholesterol biosynthesis (Figs. 4, 5, Supplementary Fig. S3), unlike 25-hydroxycholesterol, which modulates various processes of cholesterol metabolism, including biosynthesis, esterification, cellular uptake and efflux. From the result of Supplementary Fig. S4, Nat-20(S)-yne appears to be mainly distributed to the ER, at which cholesterol is biosynthesized, rather than the plasma membrane, and may therefore be able to efficiently inhibit de novo cholesterol biosynthesis. Further investigations are needed to clarify the detailed mechanism of how Nat-20(S)-yne inhibits cholesterol biosynthesis and the expression of SREBP2, a main regulator of cholesterol synthesis in the future. Nat-20(S)-yne and 25-hydroxycholesterol had comparable anti-SARS-CoV-2 activity. Thus, it is highly expected that Nat-20(S)-yne more mildly affects host cholesterol metabolism, resulting in less adverse effects on the host cells. The potential of cholesterol-lowering approved drugs such as HMG-CoA reductase inhibitors as a therapeutic agent against COVID-19 is actually being investigated.²⁶⁾

Added cholesterol restored the Nat-20(S)-yne-mediated decrease of S protein-dependent membrane fusion activity, while addition of a cholesterol precursor desmosterol failed to restore that, under the conditions that inhibit the enzymatic conversion of desmosterol to cholesterol. These results indicate that cholesterol, not desmosterol, is essential for S protein-dependent membrane fusion activity, and strongly suggested that there is a molecular machinery for strict recognition of the cholesterol structure in the plasma membrane during the S protein-mediated membrane fusion process.

Nat-20(S)-yne has an alkyne group in the side-chain and can be easily modified by click reactions, as we have used in the experiment to observe the subcellular localization of Nat-20(S)-yne, which was visualized by alkyne-azide cycloaddition reaction of it with a fluorescent Alexa Fluor™ 488 Azide. From the structure–activity relationships, it appears that the steroid backbone of Nat-20(S)-yne is important for its antiviral activity and that the side chain can be modified to some extent without loss of its activity. By labeling Nat-20(S)-yne with various probes, we would like to analyze the detailed molecular mechanism involved in its antiviral activity, including the identification of interacting molecules, in the future. Furthermore, Nat-20(S)-yne conjugates with another antiviral probes such like 25-hydroxycholesterol-conjugated EK1 peptide,²¹⁾ which showed a synergistic antiviral activity, may be more potent antiviral candidates. Since oxysterols, such like 25-hydroxycholesterol and 27-hydroxycholesterol, have antiviral activities against different types of viruses,^12,27–30) we would like to further investigate the antiviral activities of Nat-20(S)-yne against various other viruses and its mechanism of action.

In conclusion, a cholesterol derivative Nat-20(S)-yne was found as a novel anti-SARS-CoV-2 agent, mainly inhibiting the S protein-dependent and TMPRESS2-mediated fusion between viral envelope and host plasma membranes at the entry step, by the reduction of cholesterol content in the plasma membrane via down regulation of de novo cholesterol biosynthesis. Nat-20(S)-yne with an alkyne group in the side-chain is useful because its structure can be easily diversified. Nat-20(S)-yne has the potential to be an anti-SARS-CoV-2 drug prototype with a different mechanism from existing approved agents.

Acknowledgments

We thank Ms. Yoko Inamori for technical assistance. The expression plasmid for SARS-CoV-2 spike protein (Wuhan strain) was provided by Drs. Taishi Onodera and Yoshimasa Takahashi (Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases). pCX4-pur vector was kindly provided by Dr. Tsuyoshi Akagi. This work was supported in part by Japan Agency for Medical Research and Development (AMED) under Grant Nos. 21wm0325032j0201 (M.F.) and 21wm0325032s0101 (K.N.), by JSPS KAKENHI (Grants-in-Aid for Scientific Research) Grant No. 22K05551 (S.S.) and by the Grant for Joint Research Program of the Institute for Genetic Medicine, Hokkaido University.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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