Identification of bis-benzylisoquinoline alkaloids as SARS-CoV-2 entry inhibitors from a library of natural products
Chang-Long He , Lu-Yi Huang , Kai Wang , Chen-Jian Gu , Jie Hu , Gui-Ji Zhang , Wei Xu , You-Hua Xie , Ni Tang , Ai-Long Huang
PMID: 33758167 PMCID: PMC7985570
DOI: 10.1038/s41392-021-00531-5
Coronavirus disease 2019 (COVID-19) caused by severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major
public health issue. The spike (S) protein mutation D614G
became dominant in SARS-CoV-2 during a global pandemic,
which displayed increased infectivity.1 Entry of a virus into host
cells is one of the most critical steps in the viral life cycle. Since
blockade of the entry process is a promising therapeutic option
for COVID-19, research attention has been focused on the
discovery of viral entry inhibitors. Although SARS-CoV-2 entry
inhibitor development is very attractive, no candidates have
progressed into clinical trials yet.
Using a luciferase-expressing pseudovirus encoding SARSCoV-2 S (G614) protein, a library of 188 natural compounds
(Supplementary Tab. S1) was screened in 293T-ACE2 cells (HEK
293T cells overexpressing human angiotensin-converting
enzyme 2) to find novel anti-SARS-CoV-2 entry inhibitors.
Vesicular stomatitis virus G (VSV-G) pseudovirus was used as a
control to exclude compounds targeting the lentiviral backbone. A workflow chart of screening is shown in Fig. 1a. After a
preliminary screening, 41 compounds associated with a relative
infection rate <30% (Fig. 1b) were identified. We selected 19
compounds with low cytotoxicity for further testing (Supplementary Tab. S2, Fig. S1). Among the 19 hits, nine compounds
(SC9, SC161, SC171, SC182–187) with relatively high activity
(EC50 < 10 μM), low cytotoxicity (CC50 > 20 μM), and high
specificity (SI > 10, VSV-G EC50 > 20 μM) were selected for
subsequent analyses. Notably, all these compounds were bisbenzylisoquinoline alkaloids except SC171.
Next, we analyzed the relationship between the antiviral
efficacy of the nine selected compounds against S-G614
pseudovirus and the timing of treatment (Supplementary Fig.
S2). We divided the pseudovirus-based entry assay into three
stages: pretreatment (pre-entry), viral entry, and post-entry stage.
In total, eight experimental groups were set up for each
compound, including seven treatment groups (A–G) and a control
group. Importantly, pretreatment with each compound (group B)
significantly inhibited S-G614 pseudovirus infection. In the viral
entry stage (group C), the compounds exerted similar suppressive
effects. However, in the post-entry stage (group D), none of the
compounds showed any inhibitory effect. These data demonstrated that the nine selected compounds showed high blockade
efficacy presenting in both pre-entry and entry steps, indicating
that they target host factors during viral infection.
Cell lines mimicking important aspects of respiratory epithelial
cells should be used when analyzing the anti-SARS-CoV-2
activity. Hence, we determined their EC50 values against SG614 pseudovirus in Calu-3 and A549 cells (Supplementary Fig.
S3a–i). Five compounds (SC9, SC161, SC171, SC182, and SC185)
with EC50 < 10 μM in all three cell lines were selected for
subsequent experiments To determine whether these compounds have broad-spectrum
antiviral effects against other betacoronaviruses as well as recently
emerged SARS-CoV-2 variants, we constructed S-D614, N501Y.V1
(B.1.1.7), N501Y.V2 (B.1.351), S-SARS, and S-MERS pseudoviruses
using the same lentiviral system as S-G614, and then determined
the EC50 values of SC9 (cepharanthine, Fig. 1c), SC161 (hernandezine, Fig. 1d), SC171 (Fig. 1e), SC182 (tetrandrine, Fig. 1f), and
SC185 (neferine, Fig. 1g) against these pseudoviruses in 293T cells
expressing ACE2 or dipeptidyl peptidase 4 (DPP4) (Fig. 1h).
Interestingly, SC9, SC161, SC171, and SC185 exhibited highly
potent pan-inhibitory activity against S-pseudotyped coronaviruses including two emerging SARS-CoV-2 variants N501Y.V1
and N501Y.V2, reported in the United Kingdom and South Africa
(Supplementary Fig. S3j). As SARS-CoV and SARS-CoV-2 have been
reported to enter host cells via binding to ACE2, and while DPP4 is
critical for MERS-CoV entry, it could be ruled out that these five
compounds interfere with ACE2 to block pseudovirus entry.
Then, we used competitive ELISAs and thermal shift assays to
determine whether these five compounds interact with the
receptor-binding domain (RBD) in the S protein of SARS-CoV-2.
SBP1, a peptide derived from the ACE2 α1 helix, bound RBD of
SARS-CoV-2 and exhibited a weak ability to inhibit the entry of SG614 pseudovirus (Supplementary Fig. S4a), whereas the interaction between SC9, SC161, SC171, or SC185 and RBD was negligible
(Supplementary Fig. S4b–d). Thus, the blockade of virus entry by
these candidate compounds is not related to the interaction with
RBD of SARS-CoV-2.
Following attachment to the host receptor, the membrane
fusion process mediated by the S protein of SARS-CoV-2 plays an
important role in viral entry. Our data indicated that the above
five compounds may target host cells to inhibit coronavirus
entry. Therefore, we examined whether these compounds
perturb SARS-CoV-2 induced cell fusion. Cell-cell fusion assay
exhibited that SC9, SC161, SC182, and SC185 at 5 μM potently
inhibited SARS-CoV-2 S-mediated membrane fusion of 293TACE2 cells with approximately 90% decrease of fusion rates
(Fig. 1i, Supplementary Fig. S4e). Since calcium ion (Ca2+) plays a
critical role in SARS-CoV or MERS-CoV S-mediated membrane
fusion,2 calcium channel blockers (CCBs), originally used to treat
cardiovascular diseases, are supposed to have a high potential to
treat SARS-CoV-2 infections.3 Consistently, calcium-free medium
or intracellular Ca2+ chelation with BAPTA-AM significantly
diminished SARS-CoV-2 pseudovirus infection (Fig. 1j, Supplementary Fig. S4f–i), suggesting that Ca2+ is also required for
SARS-CoV-2 entry. The identified bis-benzylisoquinoline alkaloids
had been reported as CCBs.4 Herein, bis-benzylisoquinoline
alkaloids may abolish S–ACE2-mediated membrane fusion by
targeting the host calcium channel. Upon pretreatment with
BAPTA-AM, the bis-benzylisoquinoline CCBs had approximately
10-fold higher EC50 values than those without BAPTA-AM
pretreatment (Fig. 1k–l, Supplementary Fig. S4j–k). Besides,perturbation of the cholesterol biosynthesis pathway with the
CCB amlodipine reduced viral infection.5 Consistent herewith,
the bis-benzylisoquinoline CCBs upregulated intracellular cholesterol level (Supplementary Fig. S4l), which also likely
contributed to the inhibition of viral infection. These data
indicated that blockade of S-G614 pseudovirus entry by
bis-benzylisoquinoline CCBs mainly depends on calcium
homeostasis.
Finally, the antiviral activities of SC9 (cepharanthine), SC161
(hernandezine), SC171, and SC185 (neferine) were confirmed in Vero
E6 cells infected with native SARS-CoV-2. Virus-induced cytopathogenic effect and the viral RNA levels were partially inhibited by these
compounds, with SC9 (cepharanthine) at the highest efficacy (Fig.
1m–n). The results showed that these compounds inhibited SARSCoV-2 to varying degrees and may be useful as leads for SARS-CoV-2
therapeutic drug development.
In summary, we reported a set of bis-benzylisoquinoline
alkaloids as pan-coronavirus entry inhibitors. These hosttargeted inhibitors effectively protected different cell lines
(293T-ACE2, Calu-3, and A549) from infection by different
coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2 [S-D614,
S-G614, and N501Y variants]) in vitro. The compounds blocked
host calcium channels, thus inhibiting Ca2+-mediated fusion and
suppressing virus entry. Considering the effectiveness of CCBs in
the control of hypertension, our study provided clues to support
that CCBs may be helpful for treating coronavirus infection in
patients with hypertension.
