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N-(4-Hydroxyphenyl) Retinamide Suppresses SARS-CoV-2 Spike Protein-Mediated Cell-Cell Fusion by a Dihydroceramide Δ4-Desaturase 1-Independent Mechanism

ABSTRACT The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N-(4-Hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeative ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells than in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. IMPORTANCE Sphingolipids could play an important role in SARS-CoV-2 S-mediated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S-expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1, and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.

Sphingolipids are critical to all stages of the viral life cycle, such as the binding in human rhinovirus (5), entry in influenza virus (6), replication in hepatitis C virus (7), and cell lysis and release in adenovirus (8). We found that sphingomyelin synthase 2, involved in the synthesis of sphingomyelin (SM), can promote HIV-1 envelope-mediated membrane fusion (9). Recently, Vitner et al. (10) reported that the glucosylceramide synthase (GCS) inhibitors Genz-123346 and Genz-667161 block the infection of RNA viruses, including the neuroinvasive Sindbis virus, West Nile virus, influenza A virus, and SARS-CoV-2. This study suggested that sphingolipids are involved in SARS-CoV-2 infection; however, the lipid profiles of inhibitor-treated cells were not examined. Therefore, the precise function of sphingolipids in SARS-CoV-2 infection remains unclear.
SARS-CoV-2 must penetrate the cell membrane to infect cells. Therefore, in the present study, we tested the ability of inhibitors against the enzymes of the sphingolipid metabolism, for altering the membrane environment, making the virus penetration difficult, and subsequently preventing infection. To understand the function of sphingolipids in SARS-CoV-2 infection, we analyzed the quantitative sphingolipid metabolome of the inhibitor-treated cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
We assessed the effects of the 13 compounds against the SARS-CoV-2 S proteinmediated cell-cell fusion at nontoxic concentrations of each compound ( Fig. 2C and Table 1). The analysis of cell-cell fusion revealed that 4-HPR, threo-PPMP, and erythro-PPMP inhibited DSP activity by more than 50%, compared to that in the solvent-only control containing dimethyl sulfoxide (DMSO). Luciferase activity in cells harboring the preformed DSP1-7/DSP8-11 reporter complex was not affected by 4-HPR, threo-PPMP, and erythro-PPMP (Fig. 2D), indicating that the suppression of luciferase activity reflects the inhibition of cell-cell fusion by these compounds. However, little or no antimembrane fusion activity was found in the myriocin, fumonisin B1, GT11, CBE, HPA12, GW4869, amitriptyline, ceranib-2, SKI-II, and FTY720 treatments.
The levels of sphingolipid in inhibitor-treated cells were investigated by quantifying the sphingolipids using LC-MS/MS. Treatment with myriocin and fumonisin B1-inhibitors of de novo sphingolipid biosynthesis-significantly decreased the levels of most species with a distinct acyl chain in Cer, GlcCer, and SM (panels A to F in Fig. 3 to 5) and total levels of Cer, GlcCer, and SM ( Fig. 6A to C, upper panels) compared with that in the DMSO treatment. Cells treated with myriocin and fumonisin B1 showed a reduction of approximately 65% and 79% in total sphingolipid levels, respectively (Fig. 6D). However, these changes had little effect on SARS-CoV-2 S protein-mediated cell-cell fusion, indicating that the total quantity of sphingolipid was not involved.
Contrary to our expectations, cells treated with 10 mM erythro-PPMP, an inactive stereoisomer of the GCS inhibitor threo-PPMP, exhibited decreased levels of GlcCer compared to the DMSO-treated cells, although to a lesser extent than the cells treated with threo-PPMP (Fig. 6B, upper panel). Myriocin and fumonisin B1 reduced the levels of all GlcCer species at levels comparable to or more than that by threo-PPMP ( Fig. 4A to F); however, no correlation was observed between the GlcCer levels and the inhibition efficiency against cell-cell fusion. The inhibition of cell-cell fusion caused by 10 mM threo-PPMP and 10 mM erythro-PPMP could be potential off-target effects.
The levels of all species with a distinct acyl chain in DHCer, DHGlcCer, and DHSM (panels G to L in Fig. 3 to 5) and the total levels of DHCer, DHGlcCer, and DHSM ( Fig.  6A to C, lower panels) increased in the 5 mM 4-HPR-treated cells, compared to that in the 10 mM GT11-treated cells, despite the fact that the two compounds inhibit the same enzyme, DES1. The ratio of saturated sphinganine-based lipids to total sphingolipids was close to 20% in 5 mM 4-HPR-treated cells and 10% in cells treated with 2.5 mM 4-HPR or 10 mM GT11 (Fig. 6E). The increased ratio was consistent with the inhibition efficiencies of cell-cell fusion (Fig. 2C and Fig. 6E).

4-HPR inhibits SARS-CoV-2 infection in vitro.
We examined the antiviral activity of three compounds, 4-HPR, threo-PPMP, and erythro-PPMP, which inhibited SARS-CoV-2 S protein-mediated cell-cell fusion by more than 50%. In addition to these compounds,  myriocin and GT11 were also used to investigate the function of sphingolipids in viral infection. When VeroE6 TMPRSS2 cells were exposed to various concentrations of the compounds for 3 days, myriocin, at a concentration above 20 mM, exhibited no effect on cell viability (Fig. 7A). In contrast, threo-PPMP and erythro-PPMP exhibited cytotoxicity at concentrations above 20 mM, while 4-HPR and GT11 showed cytotoxicity above 10mM.
The analysis of sphingolipid levels with LC-MS/MS revealed that the ratio of saturated sphinganine-based lipids to total sphingolipids in 4-HPR-treated cells increased in a concentration-dependent manner, exhibiting 25% for 5 mM 4-HPR, 17% for 2.5 mM 4-HPR, and 12% for 1.3 mM 4-HPR treatment (Fig. 7C). No significant difference in the ratio of saturated sphinganine-based lipids to total sphingolipids was observed between 1.3 mM 4-HPR and 5 mM GT11 treatments (Fig. 7C). These results were consistent with the inhibition efficiencies of viral infection (Fig. 7B).
Myriocin, threo-PPMP, and erythro-PPMP did not exhibit antiviral activity (Fig. 7B). The cells treated with myriocin exhibited reduction in the total levels of Cer (Fig. 7D), GlcCer (Fig. 7E), and SM (Fig. 7F) and approximately 50% reduction in the total sphingolipid levels (Fig. 7G). The cells treated with threo-PPMP showed approximately 60% reduction in total GlcCer levels (Fig. 7E), compared to the cells treated with DMSO, indicating that myriocin and threo-PPMP inhibited their respective target enzymes. These results indicate that the total quantities of sphingolipids and GlcCer are not involved in SARS-CoV-2 infection.
4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion by a DES1independent mechanism. To elucidate the role of DES1 in SARS-CoV-2 S-mediated membrane fusion, we examined the effects of complementation with Cer, a reaction product of DES1, in 4-HPR treatment. The exogenously added cell-permeative C 8 -Cer, the short-chain Cer analog containing short fatty acids (C 8 ), increased the cellular levels of C 8 -Cer, C 8 -GlcCer, and C 8 -SM (data not shown), in a concentration-dependent manner. C 8 -Cer treatment increased the levels of the native-chain-length C 16 -, C 18 -, and C 20 -Cers ( Fig. 8A to F), which are reacylated via the salvage pathway (26). In addition, C 8 -Cer treatment decreased the levels of the native DHCer ( Fig. 8G to L), because C 8 -Cer suppresses the de novo biosynthesis of sphingolipids, through SPT inhibition (27). The ratio of saturated sphinganine-based lipids to total sphingolipids in the 4-HPR-and 10 mM C 8 -Cer-treated cells decreased by approximately 74%, compared to that in the 4-HPR treated cells; this indicated that the changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous C 8 -Cer (Fig. 8M). However, there was no significant differences in the cell-cell fusion efficiencies in the cells treated with 4-HPR and C 8 -Cer, compared to that in the cells treated with 4-HPR (Fig. 8N).
4-HPR-treated cells display decreases in membrane fluidity. Finally, we examined the cellular effect of 4-HPR to obtain mechanistic insights into the inhibitory effect of 4-HPR on SARS-CoV-2 S protein-mediated cell-cell fusion and viral infection. Cell-cell fusion and viral infection were strictly dependent on the cell surface levels of the SARS-CoV-2 receptor ACE2, and therefore, we examined the effect of 4-HPR on the expression of ACE2 in 293FT/ACE2/TMPRSS2/DSP1-7 cells and VeroE6 TMPRSS2 cells. No significant differences were observed in ACE2 expression levels in 293FT/ACE2/TMPRSS2/ DSP1-7 cells treated with 5 mM 4-HPR compared with cells treated with DMSO or 10 mM GT11 (Fig. 10A). Similar results were obtained with VeroE6 TMPRSS2 cells (Fig. 11A). These results indicate that the inhibition of cell-cell fusion and viral infection by 4-HPR is not attributable to ACE2 expression levels on the cell surface.
Lipid rafts are the detergent-resistant sphingolipid-rich microdomains of cellular membranes (28). Several viral receptors are localized in the lipid rafts and are involved in efficient viral infection (29,30). We examined whether 4-HPR treatment affected the localization of ACE2 in the membrane microdomain. When we treated 293FT/ACE2/ TMPRSS2/DSP1-7 cells with the nonionic detergent Triton X-100, ACE2 was found in the detergent-solubilized membrane fraction in which the nonraft marker transferrin receptor protein was localized, but not flotillin, the raft marker protein (Fig. 10B). Furthermore, ACE2 was not redistributed to the lipid raft domains even in the presence of SARS-CoV-2 S protein stimulation (Fig. 10B). Similar results were obtained with VeroE6 TMPRSS2 cells (Fig. 11B). We observed no dramatic difference in ACE2 localization in the membrane microdomain between the DMSO and 4-HPR treatments (Fig. 10B), indicating that the inhibition of cell fusion by 4-HPR was not attributable to ACE2 localization on the membrane microdomain.
Membrane fluidity is associated with HIV-1 entry (31). We therefore examined the effect of 4-HPR on cellular membrane fluidity using lipophilic pyrene probes, which undergo excimer formation with increasing membrane fluidity (32). Cells treated with 5 mM and 2.5 mM 4-HPR exhibited decreased membrane fluidity by about 50% and 20%, respectively, compared with that of the DMSO-treated cells (Fig. 10C). In addition, membrane fluidity was not significantly different between cells treated with 2.5 mM 4-HPR and 10 mM GT11 (Fig. 10C), as well as the wild-type (WT) HEK293T and DES1-KO (Fig. 10D) cells. These results were consistent with the inhibition efficiencies of SARS-CoV-2 S protein-mediated cell-cell fusion (Fig. 2C and Fig. 9C). Similar results were obtained with VeroE6 TMPRSS2 cells (Fig. 11C). These findings suggest that a decrease in cellular membrane fluidity by 4-HPR might be a major cause of the inhibition of SARS-CoV-2 infection.

DISCUSSION
In this study, we assessed several inhibitors of the enzymes functioning in sphingo- of sphingolipids did not appear to influence the SARS-CoV-2 infection. ACE2 is localized within the nonraft domains in Chinese hamster ovary cells (33) and VeroE6 cells (34), while there are some reports on the ACE2 localization within raft domains in VeroE6 cells (35,36). The cause of the discrepancies between these findings is unknown. In this study, ACE2 did not redistribute to the lipid raft domains even in the presence of SARS-CoV-2 S protein stimulation. Therefore, we propose that ACE2 localization on the membrane microdomain is not essential for SARS-CoV-2 entry into host cells, at least in our infection study. 4-HPR suppressed SARS-CoV-2 S-mediated membrane fusion by a DES1-independent mechanism. The antiviral activity of 4-HPR is proved in different viruses, such as vesicular stomatitis virus (data not shown), HIV (37), dengue virus (DENV) (38), and Zika virus (39). 4-HPR inhibited DENV replication through blocking the association of the viral RNA-dependent RNA polymerase (nonstructural protein 5) and the host's nuclear transport factors, importin a/b1 (38); however, the mechanism underlying the inhibition of viral entry by 4-HPR remains unclear. 4-HPR decreased the cell membrane fluidity; however, the direct cause of the reduced membrane fluidity and the relationship between fluidity and reduced viral infection are currently unclear. It was known that 4-HPR increased the levels of reactive oxygen species (ROS) intracellularly (40), and lipid peroxides generated by ROS caused membrane fluidity (41). We hypothesize that the inhibitory effect of 4-HPR on viral infections may be related to ROS production. Further investigation is needed to elucidate the precise mechanism underlying the inhibition in SARS-CoV-2 S-mediated membrane fusion, by 4-HPR.
4-HPR potently inhibited SARS-CoV-2 S protein-mediated membrane fusion in a cell-cell fusion assay system (EC 50 = 4.1mM) and the viral infection in vitro (EC 50 = 4.4mM). 4-HPR is a synthetic derivative of all-trans-retinoic acid, which is widely investigated as a cancer treatment (42)(43)(44)(45). In a phase I study, where 4-HPR was administered as an oral powder to patients with relapsed/refractory neuroblastoma, the day 6 mean peak plasma concentration was 21mM 4-HPR at a dose of 1,500 mg/m 2 per day (42). The EC 50 value for 4-HPR against SARS-CoV-2 infection was below its plasma concentration (4.4mM versus 21mM) in this study, and therefore, 4-HPR could be a potential therapeutic agent for COVID-19, as predicted previously (46). 4-HPR exhibited a low-toxicity profile in many clinical trials and in long-term treatments (42)(43)(44)(45). Therefore, the results from this study, together with the accumulated clinical data regarding the safety of 4-HPR, make it a potential candidate drug to treat COVID-19. In addition, 4-HPR could be used for prophylactic therapy against SARS-CoV-2 infection. It could be administered via an inhaler or a spray to the airways, perhaps by selfadministration, unlike the vaccines. This could relieve the pressure on the health care system.

FIG 10 Legend (Continued)
(1) and S (2), respectively. Sucrose gradients were harvested in 1-ml fractions (fraction 1, top of the gradient; fraction 11, bottom of the gradient), and each fraction was analyzed using SDS-PAGE and immunoblotted with anti-ACE2, anti-transferrin receptor (nonraft marker), or antiflotillin (raft marker) antibodies. One representative experiment is shown, and similar results were obtained in three independent experiments. (C and D) Membrane fluidity was examined using a fluorescent lipophilic pyrene probe. Results are normalized to the membrane fluidity in the vehicle/ DMSO-treated cells. Values represent the mean 6 SD from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett test versus vehicle/DMSO-treated cells; NS, not significant. Individual data points are shown as a scatterplot.