Discussion
Since the outbreak of the COVID-19 pandemic, numerous single herbs and
herbal formulations as well as single compounds isolated from the herbal
extracts have been reported to exhibit significant anti-SARS-CoV-2
effects through the inhibition of SARS-CoV-2 life cycle by targeting
viral proteins such as 3CLpro, PLpro, RNA-dependent RNA polymerase
(RdRp), and S protein, and cellular proteins such as ACE2, cathepsin L,
and TMPRSS2 (Adeleye et al., 2022;
Benarba & Pandiella, 2020;
Lee, Park & Cho, 2022). Saponins
including triterpenoid saponins derived from various medicinal plants
have also been investigated for their therapeutic potential against
SARS-CoV-2 (Ebob, Babiaka & Ntie-Kang,
2021). But, most are based on in silico studies
(Diniz, Perez-Castillo, Elshabrawy, Filho
& de Sousa, 2021; Falade, Adelusi,
Adedotun, Abdul-Hammed, Lawal & Agboluaje, 2021;
Sinha et al., 2021;
Yang et al., 2020) and it is only
recently that their inhibitory activities have been demonstrated using
pseudotyped or authentic SARS-CoV-2. Licorice-saponin A3, a triterpenoid
saponin isolated from licorice, was reported to inhibit SARS-CoV-2 by
targeting the NSP7, a core component of RdRp
(Yi et al., 2022). Hui and co-workers
very recently showed that 3-O-β-chacotriosyl ursolic acid saponins they
synthesized displays anti-SARS-CoV-2 activity by binding to S protein
(Li et al., 2022). However, to our
knowledge, there have been few studies verifying the role of natural
triterpenoid saponins in preventing SARS-CoV-2 infection at the stage of
membrane fusion. We previously reported that PD, a triterpenoid saponin
derived from PG, inhibits SARS-CoV-2 infection and proposed the blockade
of membrane fusion as a possible mechanism of action by demonstrating
its ability to inhibit a well-known membrane fusion event of spontaneous
inhibitory postsynaptic currents in acute brain slices
(Kim et al., 2021). Here, we discovered
that LA, a triterpenoid saponin isolated from CL and has a very similar
chemical composition to PD, exhibits inhibitory activity against
SARS-CoV-2. To provide direct evidence that LA blocks the membrane
fusion mediated by S protein of SARS-CoV-2, we established cell fusion
assay systems based on S protein-ACE2 interaction. The results from
these assays revealed that LA indeed hinders the S-mediated membrane
fusion event, thereby preventing SARS-CoV-2 infection.
Cholesterol is an essential component of cell membranes involved in
virus-host cell fusion and cell-cell fusion for SARS-CoV-2 entry and
pathological syncytia formation (Barrantes,
2022; Sanders et al., 2021). Thus,
cholesterol-targeting therapeutics have been proposed for the treatment
of COVID-19 (Palacios-Rápalo et al.,
2021). The role of membrane cholesterol in SARS-CoV-2 infection was
highlighted by recent studies showing that 25-hydroxycholesterol
inhibits SARS-CoV-2 entry by blocking membrane fusion through depleting
accessible cholesterol from plasma membrane
(Wang et al., 2020;
Zang et al., 2020;
Zu et al., 2020). It was also reported
that exogenously added 27-hydroxycholesterol accumulates in the plasma
membrane lipid rafts, leading to inhibition of SARS-CoV-2 entry
(Marcello et al., 2020;
Palacios-Rápalo et al., 2021). Together,
these studies suggest that transient changes in cholesterol distribution
in plasma membranes can exert inhibitory effects on membrane fusion and
viral entry. In this regard, we investigated the effects of LA on
membrane cholesterol and observed that upon treatment with LA,
cholesterol levels at the plasma membrane were significantly increased,
implying that LA blocks S-mediated membrane fusion by altering
cholesterol distribution in plasma membranes.
In this study, we also explained the structure-activity relationship by
comparing the antiviral activity of three different triterpenoid
saponins isolated from CL such as LA, LB, and LD. These compounds share
structure similarities consisting of 30-carbon triterpene backbone with
a single sugar at position C3 and an oligosaccharide chain attached at
position C28, but the number of sugar units and the presence or absence
of a branched sugar in the oligosaccharide chain are different each
other (Figure 1c). Both pSARS-CoV-2 entry assay and cell-to-cell fusion
assay identically demonstrated that LA consisting of linearly linked
four sugar residues exhibited the most potent antiviral activity
compared to LB and LD that are composed of a branched and/or a shorter
chain of sugar residues, indicating that both length and linearity of
the conjugated sugar residues of triterpenoid saponins are critical for
such inhibitory actions. Previously, our molecular modeling studies
predicted that triterpene backbone of PD are embedded in the lipid
bilayer membrane with an oligosaccharide chain sticking out of the
membrane, highlighting the importance of glycosylated group attached at
C28 position in the antiviral activity. Thus, the present study
corroborates our previous results, further supporting the idea that the
linear sugar residues of triterpenoid saponins create a protrusion from
the cell membrane and play critical roles in hindering membrane fusion
events. Intriguingly, the potency of LA, LB, and LD against SARS-CoV-2
is well correlated with their ability to increase membrane cholesterol
levels (Figure 1d, 3d, and S3), raising an interesting possibility that
the attached oligosaccharide chain of LA might also be responsible for
the alteration of membrane cholesterol. The more detailed studies of the
relationships of structure and mechanism of action of triterpenoid
saponins including LA are needed to address this hypothesis.
There are many advantages of membrane fusion blockers over other viral
entry inhibitors. Because most reported mutations of SARS-CoV-2 variants
are clustered near the RBD of S protein, compounds that bind to
ancestral S protein and block its interaction with ACE2 might exhibit
reduced inhibitory activity against SARS-CoV-2 variants as monoclonal
antibodies targeting the RBD of the S protein of ancestral virus showed
reduced neutralizing activity against new variants of SARS-CoV-2
(Bekliz et al., 2022;
Takashita et al., 2022). Moreover,
SARS-CoV-2 entry inhibitors that specifically act on the interaction
between S protein and ACE2 do not provide cross-reactivity to other
coronaviruses that recognize different cellular receptors, such as
MERS-CoV that uses the receptor dipeptidyl peptidase 4 (DPP4)
(Mou, Raj, van Kuppeveld, Rottier,
Haagmans & Bosch, 2013) and novel enveloped viruses that might utilize
a different cellular receptor to enter host cells. In contrast, viral
membrane fusion blockers have a broader application since the membrane
fusion process is shared in all enveloped viruses including SARS-CoV-2
and its variants. Supporting this notion, we here demonstrated that LA
effectively inhibits the infection of the ancestral SARS-CoV-2 and its
variants including Omicron with similar IC50 values
ranging from 2.23 to 3.37 μM. Another advantage is that, since the
membrane fusion is a common event in the virial entry pathways, fusion
blockers can prevent two main SARS-CoV-2 entry routes with a similar
efficiency. In agreement with this hypothesis, we found that LA inhibits
the cell surface and endosomal pathways of SARS-CoV-2 entry with similar
IC50 values in experiments using pseudotyped and
authentic viruses (Figure 2f).
The S-mediated membrane fusion is critical, not only for SARS-CoV-2
entry into host cells but also for syncytia formation. The important
roles of syncytia formation in SARS-CoV-2 Infection include evasion from
neutralizing antibodies and viral spread by cell-to-cell transmission
(Rajah, Bernier, Buchrieser & Schwartz,
2021; Zeng et al., 2022). Moreover,
syncytia can lead to cell death via apoptosis or pyroptosis, releasing
virus particles to re-infect neighboring cells and triggering an
inflammatory response (Sanders et al.,
2021; Santana et al., 2021). Here, we
observed that LA effectively inhibits S protein-mediated syncytia
formation and cellular apoptosis, indicating that LA is able to block
SARS-CoV-2 pathological effects. Overall, LA is a natural viral fusion
blocker that effectively prevents SARS-CoV-2 and all newly emerging
variants from infecting host cells and syncytia formation by hindering
the S-mediated membrane fusion. We propose here that LA can be a
broad-spectrum antiviral drug not only against SARS-CoV-2 but also
against other novel enveloped viral pathogens that might arise in the
future.
Author contributions: Conceptualization, T.Y.K., D.S.J., S.K.,
and C.J.L.; Experimental execution, T.Y.K., S.J., M.K., Y.E.D, and
S.-R.S. Methodology and formal analysis, T.Y.K., S.J.; writing-original
draft preparation, T.Y.K and S.J.; writing-review and editing, T.Y.K.,
D.S.J., S.K., and C.J.L. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was supported by the Institute for Basic
Science (IBS), Center for Cognition and Sociality (IBS-R001-D2) to
C.J.L., National Research Foundation of Korea (NRF) with grants funded
by the Korean government (MSIT) (NRF-2017M3A9G6068245 to S.K. and
NRF-2019R1A2C1083945 to D.S.J.).
Data Availability Statement: Materials are available upon a
reasonable request from the corresponding author
Acknowledgement: We appreciate Mr. Chan-yong Kwak for providing
the root of Codonopsis lanceolata , which motivated the present
study. We also thank Haejin Jung and Ph.D. Taek Seung Kim, senior
engineers at the Research Solution Center (RSC) in IBS, for performing
the flow cytometry analysis and imaging cell-to-cell fusion using Leica
DMi8 microscope, respectively.
Conflict of interest s: The authors declare no conflict of
interests.