molecules

Article
Identification of SARS-CoV-2 Receptor Binding Inhibitors by
In Vitro Screening of Drug Libraries
Alon Ben David 1,† , Eran Diamant 1,† , Eyal Dor 1 , Ada Barnea 1 , Niva Natan 1 , Lilach Levin 1 , Shira Chapman 2 ,
Lilach Cherry Mimran 1 , Eyal Epstein 1 , Ran Zichel 1, * and Amram Torgeman 1, *

1 Department of Biotechnology, Israel Institute for Biological Chemical and Environmental Sciences,
Ness Ziona 7410001, Israel; alonb@iibr.gov.il (A.B.D.); erand@iibr.gov.il (E.D.); eyalo@iibr.gov.il (E.D.);
adab@iibr.gov.il (A.B.); nivan@iibr.gov.il (N.N.); lilachl@iibr.gov.il (L.L.); lilachc@iibr.gov.il (L.C.M.);
eyale@iibr.gov.il (E.E.)
2 Department of Pharmacology, Israel Institute for Biological, Chemical and Environmental Sciences,
Ness Ziona 7410001, Israel; shirac@iibr.gov.il
* Correspondence: ranz@iibr.gov.il (R.Z.); amit@iibr.gov.il (A.T.); Tel.: +972-8-938-1515 (A.T.)
† These authors contributed equally.






Citation: David, A.B.; Diamant, E.;
Dor, E.; Barnea, A.; Natan, N.; Levin,
L.; Chapman, S.; Mimran, L.C.;
Epstein, E.; Zichel, R.; et al.
Identification of SARS-CoV-2
Receptor Binding Inhibitors by In
Vitro Screening of Drug Libraries.
Molecules 2021, 26, 3213. https://
doi.org/10.3390/molecules26113213
Academic Editor: Jan Brezovsky
Received: 2 May 2021
Accepted: 24 May 2021
Published: 27 May 2021
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the
coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell
attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD),
part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug
repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for
COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay
and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the
high-throughput screening of two compound libraries (LOPAC® 1280 and DiscoveryProbeTM). Three
compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an
effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction
assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy
of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these
compounds in the RBD. In light of these findings, the screening system described herein can be
applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.
Keywords: SARS-CoV-2; COVID-19; drug repurposing; spike protein; receptor binding domain
(RBD); angiotensin-converting enzyme 2 (ACE2), high-throughput screening; small molecule in-
hibitors (SMIs)

1. Introduction
Publisher’s Note: MDPI stays neutral
Coronavirus disease 2019 (COVID-19) is a worldwide pandemic caused by severe
with regard to jurisdictional claims in
acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, SARS-CoV-2 has
published maps and institutional affil-
infected over 150 million people, and over 3 million deaths have been reported [1]. The
iations.
emergence of COVID-19 has prompted a global effort to develop vaccines and drugs for
prevention and treatment. These efforts have led to the development of vaccines, several of
which are being administered worldwide under emergency use authorization [2–4]. How-
ever, only a few therapeutics (Remdesivir and monoclonal antibodies) are available under
Copyright: © 2021 by the authors.
emergency-use or conditional marketing authorization by the American and European
Licensee MDPI, Basel, Switzerland.
regulatory authorities [5–7].
This article is an open access article
SARS-CoV-2 infects host cells via the binding of its spike glycoprotein (S) to human
distributed under the terms and
angiotensin-converting enzyme 2 (ACE2) on target cell surfaces [8,9]. The spike protein is
conditions of the Creative Commons
composed of two functional subunits, S1 and S2. S1 includes the ACE2 binding domain
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
(RBD), and the S2 subunit mediates the fusion of the virus to the infected cell membrane [9].
4.0/).
Due to its pivotal role in the virus infection process, the spike and, specifically, the RBD are

Molecules 2021, 26, 3213. https://doi.org/10.3390/molecules26113213 https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3213 2 of 12

major targets for generating vaccines and antibodies aiming to block the ACE2–spike inter-
action [2–4]. However, drug development, optimization, manufacturing, and preclinical
and clinical studies are time consuming [10]. Alternatively, repurposing approved drugs
for treating COVID-19 can potentially promote rapid and safe therapy [11,12].
An efficient strategy for drug repurposing is the high-throughput screening of drug
libraries. Recent library screening studies have reported the discovery of several SARS-
CoV-2 replication inhibitors [13] and compounds that target host proteins and exhibit
antiviral activity by interfering with their interaction with viral proteins [14]. However, no
compounds that can inhibit the RBD-ACE2 interaction have been found in these reports.
Potential RBD-ACE2 inhibitors have been predicted in several in silico library screening
studies only [15–17]. The addition of an in vitro screen of drug libraries can promote the
discovery of potent inhibitors of the RBD-ACE2 interaction by gaining a functional layer
of data.
The urgent need for a treatment prompted us to screen for approved drugs and well-
known pharmaceutically active compounds to discover potential inhibitors of SARS-CoV-2
cell attachment in this study. To this end, an in vitro RBD-ACE2 binding assay was estab-
lished and used to discover inhibitors of the interaction via the high-throughput screening
of two compound libraries. One library, LOPAC® 1280, contains 1280 pharmaceutically
active compounds, and the other, DiscoveryProbeTM , contains 1363 FDA-approved drugs.
We report herein the identification of three compounds (heparin sodium, aurintricarboxylic
acid (ATA), and ellagic acid) that exhibit high inhibition activities. A plaque reduction
assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition
efficacy of heparin sodium and ATA. A possible mechanism via molecular docking to the
RBD is described.

2. Results
2.1. Experimental Design
The identification of compounds that inhibit the binding of RBD to ACE2 was carried
out by high-throughput screening of the LOPAC® 1280 and DiscoveryProbeTM libraries
using an assay designed to specifically measure the RBD-ACE2 interaction. This assay
consisted of the SARS-CoV-2 receptor ACE2 adsorbed to 96-well plates and soluble hFc-
RBD (Figure 1). The first step of the assay included preincubation of hFc-RBD with the
tested compounds. The second step included incubation of the mixtures with the adsorbed
ACE2. Finally, following a washing step, the RBD-ACE2 interaction was quantified by the
addition of a detecting alkaline phosphatase-conjugated goat anti-human-Fc antibody. In
the presence of a compound that could interfere with the RBD-ACE2 interaction, a lower
signal was obtained. The hit compounds were further verified for their inhibitory effect by
a plaque reduction assay conducted in Vero E6 cells with a surrogate 3virus
Molecules 2021, 26, x FOR PEER REVIEW of 12 (rVSV-SARS-
CoV-2-S) expressing the SAR-CoV-2 spike protein.

Figure 1. Schematic illustration of the RBD-ACE2 binding assay. Ninety-six-well plates were
Figure 1. Schematic illustration of the RBD-ACE2 binding assay. Ninety-six-well plates were coated
coated with ACE2 (100 ng/well) and blocked with TSTA buffer. Each compound (10 µ M) from the
with ACE2
libraries (100 ng/well)
was incubated and blocked
with Fc-RBD (1 µ g/mL)with TSTA
for 1 h buffer.
at 25 °C. Each incubation,
Following compoundmixtures
(10 µM) from the libraries
were added to the ACE2-coated plates and incubated for 1 h at 37 °C. After washing, the plates
were incubated for 1 h at 37 °C with the alkaline phosphatase-conjugated goat anti-human Fc frag-
ment. The plates were then washed, and a colorimetric reaction was developed by the addition of
pNPP (measured at 405 nm). Left panel: a maximum signal of an uninterrupted RBD-ACE2 inter-
action was obtained in the presence of noninhibitory compounds. Right panel: a reduced signal of
an inhibited RBD-ACE2 interaction was obtained in the presence of inhibitory compounds.
Molecules 2021, 26, 3213 3 of 12

Figure 1. Schematic illustration of the RBD-ACE2 binding assay. Ninety-six-well plates were
coated with ACE2 (100 ng/well) and blocked with TSTA buffer. Each compound (10 µ M) from the
libraries
was was incubated
incubated with Fc-RBD with Fc-RBDfor(11µhg/mL)
(1 µg/mL) forFollowing
at 25 ◦ C. 1 h at 25 incubation,
°C. Following incubation,
mixtures mixtures
were added
to the ACE2-coated plates and incubated for 1 h at 37 C. After washing, the plates were incubatedplates
were added to the ACE2-coated plates and incubated
◦ for 1 h at 37 °C. After washing, the
were
for 1 hincubated forthe
at 37 ◦ C with 1 halkaline
at 37 °Cphosphatase-conjugated
with the alkaline phosphatase-conjugated goat anti-human
goat anti-human Fc fragment. The plates Fc frag-
ment.then
were Thewashed,
plates were
and a then washed,
colorimetric and awas
reaction colorimetric
developedreaction was developed
by the addition by the addition
of pNPP (measured at of
pNPP
405 (measured
nm). Left panel:ata 405 nm). Left
maximum panel:
signal of anauninterrupted
maximum signal of an interaction
RBD-ACE2 uninterrupted RBD-ACE2
was obtained in inter-
action
the was obtained
presence in the presence
of noninhibitory compounds.of noninhibitory compounds.
Right panel: a reduced Right
signal of panel: aRBD-ACE2
an inhibited reduced signal of
an inhibited RBD-ACE2 interaction was obtained in
interaction was obtained in the presence of inhibitory compounds. the presence of inhibitory compounds.

2.2. Screening the LOPAC®1280 and DiscoveryProbeTM Libraries for RBD-ACE2 Binding
2.2. Screening the LOPAC® 1280 and DiscoveryProbeTM Libraries for RBD-ACE2
Inhibitors
Binding Inhibitors
Thedeveloped
The developed binding
binding assay
assay was to
was used used to screen
screen two compound
two compound libraries
libraries for for inhibi
inhibitors
tors
of theofRBD-ACE2
the RBD-ACE2 interaction.
interaction. Thebinding
The relative relativeinbinding in the
the presence of presence of the tested com
the tested compound
(10
pound
µM) (10wasµcalculated by dividingby
M) was calculated thedividing
absorbance
theofabsorbance
the compound-containing well by
of the compound-containing
that
wellofbythe no-compound
that control wellscontrol
of the no-compound (Figurewells
2A). The z-prime
(Figure parameter
2A). of the parameter
The z-prime screened of the
plates,
screened indicating
plates,the quality and
indicating thepower
qualityof and
the screening
power ofassay, was in the high
the screening assay, acceptable
was in the high
range values (0.65–0.97) (Figure 2B).
acceptable range values (0.65–0.97) (Figure 2B).

Figure2.2.High-throughput
Figure High-throughput screening
screening of theofLOPAC
the LOPAC ® 1280 and DiscoveryProbe
® 1280 and DiscoveryProbeTM compoundTM compound librar
libraries
ies for inhibitors of the RBD-ACE2 interaction. Each compound was tested at 10 µ M. (A) Distribu-
for inhibitors of the RBD-ACE2 interaction. Each compound was tested at 10 µM. (A) Distribution
tion
of theofrelative
the relative RBD-ACE2
RBD-ACE2 bindingbinding
valuesvalues
in the in the presence
presence of the compounds.
of the compounds. (B) Distribution
(B) Distribution
of the
of the Z-prime
Z-primevalues
valuesofofeach
eachplate.
plate.The
Thepurple
purpleandand blue
blue dots
dots correspond
correspond to LOPAC
to LOPAC ® 1280® 1280 and
and
DiscoveryProbe
DiscoveryProbe
TM , respectively.
TM , respectively.

Applying
Applyingananinitial threshold
initial of >40%
threshold inhibition
of >40% (relative
inhibition binding
(relative lower than
binding 60%,
lower than 60%
Figure 2A) resulted in the selection of 30 and 22 compounds from the LOPAC ® 1280 and
Figure 2A) resulted in the selection of 30 and 22 compounds from the LOPAC ® 1280 and
DiscoveryProbeTMTMlibraries, respectively. These hits were reassayed, and three molecules—
DiscoveryProbe libraries, respectively. These hits were reassayed, and three mole
heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid—exhibiting a binding
cules—heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid—exhibiting a
inhibition above 60% were further analyzed at different concentrations to determine
binding
their 50%inhibition
inhibitory above 60% were
concentration further
(IC50 analyzed
) values. The threeat compounds
different concentrations
displayed typ-to deter
mine their 50% inhibitory concentration (IC ) values. The three compounds
ical dose–response curves (Figure 3 and Table 1). ATA yielded the most potent IC50 value
50 displayed
(0.6 µg/mL), followed by ellagic acid (2.5 µg/mL) and heparin sodium (5.5 µg/mL). The
maximal binding inhibition of ATA and ellagic acid was ~80%, whereas heparin sodium
exhibited a 63% inhibition.
 Molecules 2021, 26, x FOR PEER REVIEW 4 of 12
Molecules 2021,
Molecules2021, 26,
26,xxxFOR
2021,26, FOR PEER
FORPEER REVIEW
PEERREVIEW
REVIEW 4 of 12
Molecules 44 of
of 12
12

typical dose–response curves (Figure 3 and Table 1). ATA yielded the most potent IC50
typical dose–response
typicalvalue (0.6 µ g/mL),
dose–response curves (Figure
followed
curves 3 and
by ellagic
(Figure Table
acid 1).
(2.5 ATA
1).µATA
g/mL)yielded the
and heparin most potent
sodium IC
(5.5ICµ50g/mL).
33 and
and Table
Table ATA yielded the most
most potent 50
Molecules 2021, 26, 3213 typical dose–response curves (Figure 1). yielded the potent IC 50
4 of 12
value (0.6
The
value(0.6 µg/mL),
maximal
(0.6µg/mL), followed
binding by
µg/mL),followed
followed by ellagic
inhibition
byellagic acid (2.5
of ATA
ellagicacid
acid µg/mL)
(2.5and and
ellagic
µg/mL) heparin
andacid sodium
was ~80%,
heparin sodium (5.5
whereasµg/mL).
heparin so-
(5.5µg/mL).
µg/mL).
value (2.5 µg/mL) and heparin sodium (5.5
The maximal
The maximal
maximal binding
dium exhibited inhibition
bindingainhibition of ATA
63% inhibition.
inhibition of ATA
ATA andand ellagic
and ellagic acid
ellagic acid was
acid was ~80%,
was ~80%, whereas
~80%, whereas heparin
whereas heparin so-
heparin so-
so-
The binding of
dium
dium exhibited
exhibited aa 63%
63% inhibition.
inhibition.
dium exhibited a 63% inhibition.

Figure 3.
Figure 3. Dose–response
Dose–response relationships
relationships between
between the the hit
hit compound
compoundconcentrations
concentrationsand andRBD-ACE2
RBD-ACE2
Figure
Figure 3.
3. Dose–response
binding. Inhibition
Dose–response relationships
profiles of
relationships between
the RBD-ACE2
between the
the hit compound
interaction
hit compound in concentrations
the presence
concentrations ofand RBD-ACE2
increasing
and concentra-
RBD-ACE2
Figurebinding.
3. Dose–response
Inhibitionrelationships
profiles of thebetween
RBD-ACE2 the hit compound
interaction concentrations
in the and RBD-ACE2
presence of increasing concentrations
binding.
binding. Inhibition
tions of heparin
Inhibition profiles
sodium
profiles of the
ofthe
the RBD-ACE2
(A),RBD-ACE2 interaction
ATA (B), and in
ellagic acid
interaction the
inthe presence
(C),
the determined
presence of increasing concentra-
by the binding
ofincreasing
increasing assay. The
concentra-
binding.
of Inhibition
heparin profiles
sodium of
(A), ATA RBD-ACE2 interaction
(B), and ellagic in
acid (C), presence
determined of
by the binding concentra-
assay. The values
tions of
tionsof heparin
values
ofheparin are
heparinsodiumsodium
the (A),
average
sodium(A), ATA
(A),ATAof
ATA(B),(B), and
triplicates
(B),and ellagic
± SEM.
and ellagicacidacid
The (C),
IC
acid(C), determined
values are
(C), determinedby
50 by
listedthe
bythe in binding
Table
thebinding 1. assay.
bindingassay. The
assay.The
The
tions are the average of triplicates SEM.ellagic
± SEM. The IC 50 valuesdetermined
are listed in Table 1.
values are
valuesare the
arethe average
theaverage
averageof of triplicates
triplicates±±±SEM.
oftriplicates The
SEM.TheTheICIC 50 values are listed in Table 1.
IC50 valuesare arelisted
listedininTable
Table1.1.
values 50values
Table 1. RBD-ACE2 inhibition properties of the hit compounds.
Table Table
1. 1. RBD-ACE2
RBD-ACE2 inhibition
inhibition properties
properties of the of
hitthe hit compounds.
compounds.
Table1.1.RBD-ACE2
Table RBD-ACE2inhibition
inhibitionproperties
propertiesof ofthe
thehithitcompounds.
compounds.
IC50 Binding Inhibition
Compound Structure IC IC Binding Inhibition
Binding Inhibition
Compound
Compound Structure
Structure IC
50 50 BindingInhibition
IC5050[µg/mL]
Binding Inhibition
[%]
Compound
Compound Structure
Structure [µg/mL]
[µg/mL] [%] [%]
[µg/mL]
[µg/mL] [%]
[%]
heparin sodium 5.5 63
heparin
heparin sodium
heparin sodium
sodium 5.5
5.55.5 63
63 63
heparin sodium 5.5 63

aurintricarboxylic acid (ATA) 0.6 80

aurintricarboxylic
aurintricarboxylic acid
acid
aurintricarboxylic (ATA)
(ATA)
acid (ATA) 0.6
0.60.6 80
80 80
aurintricarboxylic acid (ATA) 0.6 80

ellagic acid 2.5 84

ellagic
ellagic acid
acid 2.5
2.52.5 84
84
ellagic acid
ellagic acid 2.5 84 84

2.3. Evaluation of the Selected Compounds by a Plaque Reduction Assay

2.3. Evaluation
2.3.Evaluation
Evaluation of the Selected Compounds by
byaaaPlaque
Plaque Reduction Assay
2.3. Theofof
2.3. Evaluation
theSelected
the
efficacy Selected
of the
of the
Compounds
Compounds
threeCompounds
Selected by
identified Plaque
molecules,Reduction
Reduction
by a Plaque heparin Assay
Assay
Reduction sodium,
Assay ATA, and ellagic acid,
Thewas
The efficacy
furtherof
efficacy of
of the
assessed three
thethree
three identified
byidentified
a plaque reduction
identified molecules,
molecules,assay heparin
heparin sodium,
conducted
sodium, ATA,
inATA,
Veroand
ATA, and ellagic
E6 cells
and using
ellagic acid,a SARS-
acid,
The efficacy
The efficacythe ofplaque
the three molecules,
identified heparin
molecules, sodium,
heparin sodium, ellagic
ATA, acid,
and ellagic
was
was further
CoV-2
further assessed
surrogate
assessed by
by a
virus
a plaque reduction
(rVSV-SARS-CoV-2-S).
reduction assay
assay conducted
The
conducted in
virusin Vero
was
Vero E6 cells using
preincubated
E6 cells using a
a SARS-
with
SARS- various
was further
acid, assessed
was further by aassessed
plaque reduction
by a plaque assay conducted
reduction assay in conducted
Vero E6 cells in using
Vero a SARS-
E6 cells using
CoV-2 surrogate
CoV-2concentrations
surrogate virus virus
virus (rVSV-SARS-CoV-2-S).
of(rVSV-SARS-CoV-2-S).
each molecule to ensure
(rVSV-SARS-CoV-2-S). The
The virus
that
virus was
potential preincubated
interactionswith
was preincubated
preincubated with
between
with various
various the com-
CoV-2 surrogate
a SARS-CoV-2 surrogate virus The
(rVSV-SARS-CoV-2-S). virus was The virus between was preincubatedvarious with
concentrations
pounds and
concentrations of each
of each
each molecule
RBDmolecule
would occur
molecule to ensure
to ensureprior that
ensure that
to
that potential
RBD-ACE2
potential interactions
interaction. between
interactions The
between the
mixtures com-
were then
thebetween
com-
concentrations of
various concentrations of each to molecule topotential
ensure that interactions
potential interactions the com- the
pounds
pounds and
added
and RBDRBD
to
RBD cellswould
would occur
in six-well
occur prior
plates
prior to RBD-ACE2
forRBD-ACE2
to 72 h. The relative
RBD-ACE2 interaction.
interaction. The
reduction mixtures
in plaque
The mixtures
mixtures were then
formation
were then was
pounds and
compounds would
and RBD occur
would prioroccur to prior to RBD-ACE2 interaction. The
interaction. were
The mixtures then
were then
added to cells
calculated, in six-well
and plates for 72 h. The relative reduction in plaque formation was
addedadded
added to cells
to cells
toin in inIC
six-well
six-well
cells was determined
plates
50plates
six-well for 72
for
plates 72for h. The
h. The
72 (FigureThe 4).
relative
h.relative ATA reduction
reduction
reduction
relative and in heparin
in plaque
plaque sodium
formation
formation
in plaque displayed
was was
was
formation po-
calculated,
tent
calculated, and
antiviral
and IC
IC 50 was
was determined
inhibition activities
determined (Figure
in
(Figure 4).
cells4). ATA
(IC
ATA and
values
and heparin
of
heparin5.6 sodium
and
sodium73 µ displayed
g/mL,
displayed po-
respectively).
po-
calculated, and IC
calculated, and50 was
50
IC50determined
was determined (Figure 4). ATA
(Figure 50
4). ATAand and heparinheparin sodiumsodium displayed
displayed po-potent
tent antiviral
For both inhibition
compounds, activities
the ICin in cells (IC values of 5.6 and 73 µg/mL, respectively).
tent antiviral
antiviral inhibition activities ininvalues
cells (IC determined
values ofofusing
5.6 andthe 73 plaque
µg/mL, reduction assay were
respectively).
50
tent antiviral inhibition
inhibition activities
activities 50 cells
cells(IC (IC 50 values
50
50 values of 5.6
5.6 andand 73 73 µg/mL,
µg/mL, respectively).
respectively). For
For both
For both
both compounds,
an compounds,
order
compounds,of magnitude the
the IC
ICIC
50 values
higher
values thandetermined
those determined
determined using
using the plaque
theusingplaque reduction
thereduction
binding
reduction assay
assay,
assay were
maintaining
were
For both compounds, the theIC 50 values
50
50 values determined
determined usingusing the plaque
the plaque reduction assay
assaywere were an
an order
anorder
order of
theirof magnitude
relative
magnitude higher
ranking.
higher A than
than those
dose–response
those determined
determinedcurve using
for
using the
ellagic
the binding
acid
binding couldassay,
not
assay, maintaining
be established,
maintaining as
an of magnitude higher than those determined
order of magnitude higher than those determined using the binding assay, maintaining using the binding assay, maintaining
their relative
theirrelative
relative ranking.
concentrations
ranking. A
inA dose–response
the range
dose–response of 1–120 curve
µ
curve g/mLfor
for ellagic
of this
ellagic acid
compound
acid could
could not
did
not be
not
be established,
inhibit
established, as
rVSV-SARS-
as
their ranking. A dose–response curve for ellagic
their relative ranking. A dose–response curve for ellagic acid could not be established, acid could not be established, as
concentrations
CoV-2-S in the
infection range of
in the
Vero1–120 µg/mL
E6 µg/mL
cells. of
These this compound
results did not inhibit rVSV-SARS-
concentrations
concentrations
as inthe
in
concentrations therangerange
in of1–120
of 1–120
range µg/mL
of 1–120 ofthis
of this
µg/mL compoundofdemonstrate
compound this did didnot
compoundnotthat
inhibit
inhibit ATA
did and
rVSV-SARS-
rVSV-SARS-
not heparin
inhibit so-
rVSV-
CoV-2-S
CoV-2-S diuminfection
are
infection able in
in Vero
to
VeroexertE6
E6 cells.
potent
cells. These
inhibition
These results
results of demonstrate
viral infection,
demonstrate that
that ATA
presumably
ATA and
and heparin
by hindering
heparin so-
so- the
CoV-2-S infection in infection
SARS-CoV-2-S Vero E6 cells. in Vero These results
E6 cells. demonstrate
These that ATA and
results demonstrate that heparin
ATA andso- heparin
dium are
dium sodium able
binding
are able
ableare to
of
totheexert
exert spikepotent
potent inhibition
to cellular
inhibition ACE2. of viral
To
of viral rule
viral infection, presumably
out ainfection,
infection, possible
presumablyreduction by hindering
in plaque
by hindering
hindering the
formation
the the
dium are to exert
able topotent
exert inhibition
potent of
inhibition ofinfection,
viral presumably presumablyby by hindering the
binding
binding dueof the
to
ofthe spike
thecytotoxicity,
spike to cellular
tocellular ACE2.
thecellular
cellular effect
ACE2.ACE2.of To rule
Totherule out
compounds
out a possible
out aat reduction
the highest in plaque
intested formation
in concentrations on
binding binding
of of
spikethe to spike to ACE2. To rule Toout aapossible
rule possible reduction
possible
reduction reduction
in plaque
plaque formation
plaque
formation formation
due to
due totoduecytotoxicity,
cell viability
cytotoxicity,
to cytotoxicity, wasthe effect
theevaluated
effect of the
of the
the effect using
the compounds
alamarBlue
compounds
ofcompounds
the compounds at the
at the highest
reagent, theby
theathighest
highest tested
comparing concentrations
testedtested
highest the viability
concentrations
concentrations on
onof cells
on
due cytotoxicity, the effect of at tested concentrations on
cell viability
incubated
cellviability
viability
cell was
waswith
viability evaluated
wasthe
evaluated evaluatedusing
compound
usingusing alamarBlue
to the
alamarBlue alamarBlue reagent,
viability
reagent, of by
cellscomparing
incubated
bycomparing
reagent, comparing
by comparing the
only viability
with
theviability
viability of
media. cells
ofcells
the viability None
cells of
of cells
cell was evaluated using alamarBlue reagent, by the of
incubated
incubated with
with
incubated the
the
with compound
compound
the compound to
to the
the viability
viability of
of cells
cells incubated
incubated only
only with
with media.
media. None
None of
of
incubated with the compound to the to the viability
viability of cellsofincubated
cells incubated only withonlymedia.
with media.None None of of
the tested compounds were cytotoxic as the cell viability percentages following incubation
with 120 µg/mL of ellagic acid, 169 µg/mL of ATA, and 650 µg/mL of heparin sodium
were 106%, 102%, and 97%, respectively.
Molecules 2021, 26, x FOR PEER REVIEW 5 of 12

Molecules 2021, 26, 3213 the tested compounds were cytotoxic as the cell viability percentages following incubation
5 of 12
with 120 µ g/mL of ellagic acid, 169 µ g/mL of ATA, and 650 µ g/mL of heparin sodium
were 106%, 102%, and 97%, respectively.

Figure 4. Inhibition of viral infection of cells by heparin sodium and ATA. rVSV-SARS-CoV-2-S
Figure 4. Inhibition of viral infection of cells by heparin sodium and ATA. rVSV-SARS-CoV-2-S was
was preincubated for 1 h with the indicated concentrations of heparin sodium (A) and ATA (B).
preincubated for 1 h with the indicated concentrations of heparin sodium (A) and ATA (B). Mixtures
Mixtures were then added to Vero E6 cells and incubated for 72 h. Cells were then stained using
were then
crystal added
violet, andtothe
Vero E6 cells
relative and incubated
plaque reductionforwas72determined.
h. Cells wereThe
then stained
results areusing crystalasviolet,
expressed the
and the relative plaque reduction was determined. The results are expressed as the
percent inhibition relative to that of the virus-only control incubated with the correspondingpercent inhibition
sol-
relative to that of
vent (infection the virus-only
medium withoutcontrol
or withincubated
2% DMSO with
forthe corresponding
heparin sodium orsolvent (infection medium
ATA, respectively). The
values are
without presented
or with 2% DMSOas thefor
mean of 6-well
heparin sodiumreplicates
or ATA,±respectively).
SD. The lower images
The valuesare
arethree representa-
presented as the
tive wells
mean with replicates
of 6-well the indicated compound
± SD. The lower concentrations.
images are three representative wells with the indicated
compound concentrations.
2.4. Molecular Docking Analysis
2.4. Molecular Docking Analysis
Molecular docking was conducted to suggest a mechanism underlying the inhibition
of theMolecular
RBD-ACE2 docking was conducted
interaction by ATA and to suggest
heparinasodium
mechanism underlying
(Figure the inhibition
5). Molecular docking
of the RBD-ACE2 interaction by ATA and heparin sodium (Figure 5).
was based on the published crystal structure of the RBD-ACE2 complex (resolution Molecular docking
of 2.45
was based
Å ) [18]. on compounds
The the published crystal
were structure
allowed of thedock
to freely RBD-ACE2 complex binding
to the receptor (resolution of
motif
2.45 Å) [18]. The compounds were allowed to freely dock to the receptor
(RBM)—the ACE2 binding site within the RBD [19]. The RBM spans amino acids 437 to binding motif
(RBM)—the
507 and forms ACE2 binding
a concave site within
surface the RBDwith
that interacts [19].ACE2.
The RBMThe spans aminowere
compounds acidsdocked
437 to
507 and forms a concave surface that interacts with ACE2. The compounds
to the concave surface of the RBM, implying that their binding to the RBD may disrupt were docked to
the concave surface of the RBM, implying that their binding to the RBD may
the interaction with ACE2. The calculated Gibbs free energy (ΔG) values for the interac- disrupt the
interaction
tion of the with ACE2. The
compounds withcalculated
the RBD Gibbs
were free
−8.9 energy (∆G)
and −7.05 values for
kCal/mL forthe interaction
heparin sodiumof
the compounds with the RBD were −8.9 and −7.05 kCal/mL for heparin sodium and ATA,
and ATA, respectively. The predicted binding site for heparin included Tyr505, which
respectively. The predicted binding site for heparin included Tyr505, which participates in
participates in the interaction of the RBD with ACE2. The predicted binding site for ATA
the interaction of the RBD with ACE2. The predicted binding site for ATA was at a more
was at a more pivotal location as, in addition to Tyr505, it includes Tyr449, Tyr453, Gln493,
pivotal location as, in addition to Tyr505, it includes Tyr449, Tyr453, Gln493, Gln498, and
Gln498, and Asn501, found at the RBD-ACE2 interface.
Asn501, found at the RBD-ACE2 interface.
Molecules 2021, 26, 3213 6 of 12
Molecules 2021, 26, x FOR PEER REVIEW 6 of 12

Figure5.5.AAdocking
Figure dockingmodel
modelof ofthe
thebinding
bindingof ofheparin
heparin sodium
sodium and
and ATA
ATAto tothe
theRBD.
RBD.(A)
(A)RBD-ACE2
RBD-ACE2
interaction(pdb
interaction (pdbcode
code6M0J).
6M0J).The
The RBD
RBD forms
forms a concave
a concave surface
surface with
with a protruding
a protruding loop
loop on one
on one side,
side, to
to which
which ACE2 ACE2 binds
binds (ACE2
(ACE2 is colored
is colored in blue
in light light and
blueisand is shown
shown in a ribbon
in a ribbon presentation;
presentation; RBD is RBD
shown is
shown in surface (gold colored) and ribbon (red colored) representations). Heparin sodium
in surface (gold colored) and ribbon (red colored) representations). Heparin sodium (one disaccharide (one
disaccharide
unit) and ATAunit)
wereand ATAtowere
docked docked
the RBM to the
using the RBM using server
SwissDock the SwissDock
[20]. The server [20]. The
compounds com-
are shown
pounds are shown in stick representation, where oxygen, sulfur, and hydrogen are colored in red,
in stick representation, where oxygen, sulfur, and hydrogen are colored in red, yellow, and white,
yellow, and white, respectively, and carbon atoms are colored in magenta for heparin sodium (B)
respectively, and carbon atoms are colored in magenta for heparin sodium (B) and green for ATA (C).
and green for ATA (C). The figure was prepared using UCSF Chimera [21].
The figure was prepared using UCSF Chimera [21].

3.3.Discussion
Discussion
Theglobal
The globalCOVID-19
COVID-19pandemic pandemicled ledtotoan anurgent
urgentdemand
demandfor fordeveloping
developingvaccinesvaccines
and effective treatments. In view of this need, drug repurposing
and effective treatments. In view of this need, drug repurposing is a rapid approach for is a rapid approach for
discovering beneficial COVID-19 therapeutics by using established
discovering beneficial COVID-19 therapeutics by using established drugs. Inhibiting the drugs. Inhibiting the
RBD-ACE2interaction,
RBD-ACE2 interaction,thereby
therebypreventing
preventingviral viralattachment
attachmentto totarget
targetcells,
cells,isisused
usedas asan an
approachto
approach todevelop
developvaccines
vaccinesand andtherapeutics.
therapeutics.The Thelatter
latterinclude
includeantibodies,
antibodies,aptamers,
aptamers,
peptides,and
peptides, andspecific
specificsmall-molecule
small-moleculeinhibitors inhibitors(SMIs)
(SMIs)[22–28].
[22–28].One Oneeffective
effectiveand andrapid
rapid
strategyfor
strategy fordiscovering
discoveringinhibitors
inhibitors is is
byby screening
screening compound
compound libraries,
libraries, as was
as was recently
recently ap-
applied
plied to find
to find newnew therapeutics
therapeutics for COVID-19.
for COVID-19. For example,
For example, Gordon Gordon
et al. et al. identified
identified 332 new 332
SARS-CoV-2-human
new SARS-CoV-2-human protein–protein
protein–protein interactions and found
interactions two sets
and found two of sets
pharmacological
of pharmaco-
agents
logicalthat displayed
agents antiviral antiviral
that displayed activity [14]. In another
activity [14]. Instudy,
anotherthe study,
ReFRAME (repurposing,
the ReFRAME (re-
focused
purposing,rescue, and accelerated
focused rescue, andmedchem)accelerated drug library was
medchem) screened
drug libraryfor was inhibitors
screenedoffor viral
in-
replication, and 100
hibitors of viral molecules,
replication, andincluding 21 known
100 molecules, drugs,21
including wereknownfound [13].were
drugs, However,
found
no compound
[13]. However,was associated with
no compound blocking the
was associated withRBD-ACE2
blocking the interaction
RBD-ACE2 in either report.
interaction in
Screening for inhibitors, particularly SMIs, that can potentially
either report. Screening for inhibitors, particularly SMIs, that can potentially hamper hamper RBD-ACE2 binding
has been reported
RBD-ACE2 binding in has
several
beeninreported
silico studies. Choudhary
in several et al. identified
in silico studies. Choudhary five etpotential
al. iden-
SARS-CoV-2 cell entry
tified five potential inhibitors that
SARS-CoV-2 cellwere
entryshown to virtually
inhibitors that were bind the spike
shown protein [15].
to virtually bind
Wu
the et al. protein
spike analyzed therapeutic
[15]. targets in
Wu et al. analyzed SARS-CoV-2
therapeutic andinvirtually
targets SARS-CoV-2 screened several
and virtually
compound databases
screened several for the inhibition
compound databases of forthese targets. One
the inhibition compound
of these targets. (hesperidin)
One compound was
suggested to bind the binding interface between the spike
(hesperidin) was suggested to bind the binding interface between the spike and ACE2 and ACE2 [17]. In the study
of Wang
[17]. et al.,
In the studyeight common
of Wang types
et al., eightof common
potentialtypes
inhibitor structures
of potential were outlined
inhibitor structures bywere
ap-
plying a computational screening strategy based on blocking
outlined by applying a computational screening strategy based on blocking S RBD/hACE2 S RBD/hACE2 binding [16].
Despite
bindingthe advantages
[16]. Despite the of the in silico screening
advantages of the inapproach, the in vitro
silico screening screening
approach, the of
indrug
vitro
libraries can add a functional level to the process, further promoting
screening of drug libraries can add a functional level to the process, further promoting the discovery of potent
the
inhibitors of the RBD-ACE2 interaction.
discovery of potent inhibitors of the RBD-ACE2 interaction.
In
Inthe
thecurrent
currentstudy,
study,a newa new binding
bindingassay waswas
assay developed
developedand usedand usedto screen two drug
to screen two
libraries for inhibitors that interfere with the RBD-ACE2 interaction.
drug libraries for inhibitors that interfere with the RBD-ACE2 interaction. Three com- Three compounds
that inhibited over 60% of the interaction were selected. Two compounds, heparin sodium
pounds that inhibited over 60% of the interaction were selected. Two compounds, heparin
and ellagic acid, are FDA-approved drugs. In addition to its anticoagulant activity, hep-
sodium and ellagic acid, are FDA-approved drugs. In addition to its anticoagulant activ-
arin has previously been found to inhibit infection by several viruses, including human
ity, heparin has previously been found to inhibit infection by several viruses, including
immunodeficiency virus (HIV), coronavirus NL63, and Zika virus [29–31]. In line with
human immunodeficiency virus (HIV), coronavirus NL63, and Zika virus [29–31]. In line
our screening results, it has recently been shown that the spike protein of SARS-CoV-2
with our screening results, it has recently been shown that the spike protein of SARS-CoV-
Molecules 2021, 26, 3213 7 of 12

strongly binds immobilized heparin [32]. A docking analysis suggested putative heparin
(and other glycosaminoglycans) binding motifs in the RBD when it is in an open conforma-
tion [32]. Interestingly, Clausen et al. [33] provided evidence that heparan sulfate, a cell
membrane glycosaminoglycan, shifts the RBD to an open conformation to facilitate ACE2
binding. Additionally, it was suggested in their work that heparin could inhibit the role
of heparan sulfate as a coreceptor for SARS-CoV-2 infection [33]. Most recently, heparin
was found to inhibit SARS-CoV-2 infection in Vero cells [34]. The fact that heparin was
specifically identified by the screening assay as a potent inhibitor strongly supports the
reliability of the assay developed and applied herein for discovering effective compounds
capable of directly blocking the RBD-ACE2 interaction. It is important to note that in an
unrelated manner to SARS-CoV-2 inhibition, heparin is administered as an anticoagulant
to COVID-19 patients suffering from coagulopathy and thromboembolism [35]. Thus, it
appears that heparin is able to exert a beneficial synergistic effect in treating COVID-19
patients based on two distinct mechanisms. The third inhibiting compound is ATA, a
polyanionic aromatic compound, which is recognized as a potent DNA topoisomerase II
inhibitor [36]. Additionally, ATA exhibits inhibitory properties against several viruses and
bacteria. Interestingly, among the viruses is the SARS coronavirus [37]. Other pathogens
include HIV-1 [38], influenza virus [39], Vaccinia [40], Zika virus [41], and Yersinia pestis [42].
Ellagic acid, a polyphenol, is an investigational drug used to treat cancer, cardiovascular
disease, and brain injury [43–54]. Similar to heparin and ATA, ellagic acid is also associated
with antiviral activity against various viruses [55–57]. Recently, ellagic acid was identified
by network analysis and molecular docking as a possible modulator of casein kinase II
subunit alpha, a host protein that may be involved in the pathology of SARS-CoV-2 [58].
The three hit compounds were further characterized for their ability to inhibit the
infection of Vero E6 cells by a pseudotyped virus expressing the SARS-CoV-2 spike protein
using a plaque reduction assay. Only ATA and heparin sodium preserved their inhibitory
activity in the cellular system. The IC50 values obtained in the cellular assay were an order
of magnitude higher than those obtained in the binding assay, and in both assays, ATA
demonstrated a higher inhibitory potency. Of note, the IC50 value obtained herein for
heparin sodium is in line with that described in a recent report for the inhibition of the
SARS-CoV-2 virus [34].
The ranking of IC50 values of ATA and heparin coincides with the results of the
docking analysis. The higher potency of ATA can be explained in light of the prediction
that ATA binds multiple residues of RBD at the interface of the RBD-ACE2 interaction,
whereas heparin binds only one residue. The docking analysis may also serve to predict the
binding of the identified compounds to SARS-CoV-2 variants. Of note, it has been reported
that Asn501 is a critical residue for ACE2 binding by the RBM of SARS-like viruses [19].
This residue, according to the docking model, is part of the binding site of ATA. In this
regard, several SARS-CoV-2 variants of concern listed by the CDC [59] (B.1.1.7, B.1.351, and
P.1) contain an Asn501Tyr mutation. Although it can be hypothesized that a mutation in
Asn501 might alter the binding of ATA to the RBD, the impact of the Asn501Tyr mutation on
the potency of ATA is yet unknown. In the case of heparin, the docking analysis predicted
that the binding site does not include residues mutated in the SARS-CoV-2 variants of
concern. This suggests that the heparin binding to RBD of SARS-CoV-2 variants may not
be altered.
No effective inhibition of the rVSV-SARS-CoV-2-S virus could be demonstrated in
Vero E6 cells by ellagic acid. It should be noted that the elimination of hit compounds
between the primary and secondary screens is a characteristic of the screening funnel.
However, we cannot rule out the possibility that, although ineffective in cells, ellagic acid
could still be potent in vivo. Eubanks et al., for example, reported that two botulinum toxin
inhibitors were effective in vivo even though they were ineffective in a cellular assay [60].
Further investigation is required to determine the inhibitory activity of ellagic acid against
SARS-CoV-2.
Molecules 2021, 26, 3213 8 of 12

In the absence of a specific anti-SARS-CoV-2 therapy, repurposing approved drugs

may be useful for treating COVID-19 [11,61]. The current study can expand the limited
drug toolkit by identifying approved drugs and pharmaceutically active compounds that
can potentially be repurposed to inhibit the viral cell attachment step. The results provide
a basis for further research with SARS-CoV-2 to evaluate the efficacy of hit compounds.
Future structure–activity research (SAR) may allow one to design modifications that en-
hance the affinity of the compounds, required to achieve improved therapeutic efficacy
and selectivity. Interestingly, as these drugs have been reported to counteract additional
viruses as well, they may serve as broad-spectrum antiviral therapeutics. These drugs can
also be considered for use in a combined administration to increase the treatment efficacy,
which may be further advantageous as their doses in a combined regimen can be reduced,
thus minimizing the risk of side effects. Moreover, the binding assay developed herein can
be applied for the high-throughput screening of additional compound libraries to facilitate
the discovery of additional potent compounds.

4. Materials and Methods

4.1. Expression and Purification of ACE2 and Fc-RBD
A synthetic gene encoding the peptidase domain of Homo sapiens ACE2 (amino acids
19-615, GenBank: BAB40370.1), with optimized codon usage for expression in Escherichia
coli and a C-terminal His-tag, was prepared by GenScript (Piscataway, NJ, USA) and cloned
into pET-9a between the NdeI and BamHI sites. The plasmid pET-9a-ace2 was transformed
into E. coli BL21(DE3). To express the protein, E. coli BL21(DE3)+pET-9a-ace2 was grown at
18 ◦ C in TB media and was harvested after 40 h of incubation. To purify the protein, the cell
pellet was resuspended in binding buffer (20 mM of sodium phosphate, 0.5 M of NaCl, and
20 mM of imidazole; pH 7.4; 100 mL per 25 g of wet cells) and disrupted by three passages
in a cell homogenizer at 25 kPa (C series cell disruptor 1.1 KW, Constant Systems). The
cell extract was centrifuged (14,000 g, 30 min) and the supernatant was loaded onto a 5 mL
HisTrap FF column (GE Healthcare, Chicago, IL, USA) mounted on an AKTA Explorer
FPLC system (GE Healthcare). The column was washed with 10 CV of binding buffer and
10 CV of binding buffer containing 40 mM of imidazole. The protein was eluted from
the column with elution buffer (20 mM of sodium phosphate, 0.5 M of NaCl, 500 mM of
Imidazole, pH 7.4). The purified protein was dialyzed against PBS and stored at −70 ◦ C.
The recombinant hFc-RBD fusion protein, expressed as recently described [22], was kindly
provided by Dr. Ohad Mazor Lab (IIBR, Ness Ziona, Israel).

4.2. RBD-ACE2 Binding Assay

Ninety-six-well plates (Maxisorp, Nunclon) were coated with ACE2 (100 ng/well) for
1 h at 37 ◦ C. The plates were blocked for 1 h at 37 ◦ C with TSTA buffer (2% (w/v) bovine
serum albumin (BSA), 0.9% NaCl, 0.05% Tween 20, 50 mM of Tris, pH 7.6). For initial
screening, a concentration of 10 µM of each compound from the LOPAC® 1280 (Sigma,
St. Louis, MO, USA) and DiscoveryProbeTM FDA-approved drug (APExBIO) libraries
was incubated with 1 µg/mL of Fc-RBD in a total reaction mixture volume of 50 µL. For
the IC50 experiments, 3-fold serial dilutions of the indicated compound (1 mM to 5.6 nM)
were incubated with Fc-RBD (1 µg/mL). The mixtures (50 µL) were incubated for 1 h
at 25 ◦ C. Following incubation, the mixtures were added to the ACE2-coated plates and
incubated for 1 h at 37 ◦ C. After washing, the plates were incubated for 1 h at 37 ◦ C with
that alkaline phosphatase-conjugated goat anti-human Fc fragment (Sigma, St. Louis, MO,
USA), diluted 1:1000 in blocking solution. The plates were washed, and the colorimetric
reaction was developed using p-nitrophenyl phosphate (pNPP, Sigma, St. Louis, MO,
USA). The absorbance was measured at 405 nm with a Synergy HTX ELISA reader (Biotek,
USA). The assay included a rabbit anti-SARS-CoV-2 RBD polyclonal antibody as a positive
control. The antibody was kindly provided by Dr. Ohad Mazor Lab (IIBR, Ness Ziona,
Israel). Naïve rabbit serum served as a negative control. The IC50 values were calculated
Molecules 2021, 26, 3213 9 of 12

using a 4-parameter logistic fit by Gen5 software (Biotek, Winooski, VT, USA). The z-prime
parameter for each plate was calculated by Gen5 software.

4.3. Plaque Reduction Assay

The assay was conducted in Vero E6 cell monolayers (ATCC® CRL-1586™). The cells
were grown in growth medium (Dulbecco’s modified Eagle’s medium (DMEM) containing
10% fetal bovine serum (FBS), 1% MEM nonessential amino acids (NEAA), 2 mM of
L-glutamine, and 0.5% Pen/Strep, all from Biological Industries, Kibbutz Beit-Haemek,
Israel) and were cultured at 37 ◦ C and 5% CO2 . The cells were infected with 60 PFU/well
of rVSV-SARS-CoV-2-S, which served as a SARS-CoV-2 surrogate virus. This virus is a
replication-competent recombinant VSV-∆G-spike virus in which the glycoprotein of VSV
(vesicular stomatitis virus) is replaced by the spike protein of SARS-CoV-2 [62]. Cells were
seeded in 6-well plates (7 × 105 cells/well) and grown overnight in growth medium. A
fixed dose (600 PFU/mL) of rVSV-SARS-CoV-2-S was incubated with different inhibitor
concentrations of ATA, ellagic acid, and heparin (1:1, v/v) in infection medium (MEM
containing 2% FBS with 1% NEAA, 2 mM of glutamine, and 0.5% P/S) and was used to
infect Vero E6 monolayers in six replicates (200 µL/well). After incubation for 1 h at 37 ◦ C,
3 mL/well of overlay (MEM containing 2% FBS and 0.4% tragacanth (Merck, Herzliya
Pituach, Israel)) was added to each well, and the plates were incubated at 37 ◦ C and 5%
CO2 for 72 h. The media were then aspirated, and the cells were fixed and stained with
3 mL/well of crystal violet solution (Biological Industries, Kibbutz Beit-Haemek, Israel).
The number of plaques in each well was determined, and the reduction in infection was
calculated relative to the control wells containing no compound. The IC50 values were
calculated using GraphPad Prism.

4.4. Cytotoxicity assaY

A cytotoxicity assay was performed in Vero E6 cell monolayers using the alamarBlue
cell viability reagent (Thermo Fisher Scientific). Cells (2 × 104 cells/well in 100 µL) were
seeded in 96-well plates. After an overnight incubation at 37 ◦ C, 80 µL of medium was
aspirated, and 20 µL of inhibitors was added in triplicate to the wells. After 1 h of
incubation, 20 µL was aspirated, 150 µL of overlay was added, and the cells were incubated
at 37 ◦ C for three days. Then, 19 µL of alamarBlue reagent was added per well and
incubated for 2.5 h at 37 ◦ C. The fluorescence results were determined by excitation at
530 nm and the collection of emission at 580 nm was determined using a CLARIOstar®
reader (BMG labtech, Ortenberg, Germany). The cell viability was calculated by dividing
the fluorescence results of the compound-containing wells by those of the control wells
(containing no compounds).

4.5. Molecular Docking Analysis

Molecular docking of the selected compounds was performed using the SwissDock
server (www.swissdock.ch accessed on 7 October 2020 [20]). The RBD of pdb file 6M0J [18]
was set as a target, and the 3D structures of the hits were from the ZINC database [63].

Author Contributions: Conceptualization, A.B.D., E.D. (Eran Diamant), R.Z. and A.T.; methodology,
A.B.D., E.D. (Eran Diamant), R.Z. and A.T.; investigation, A.B.D., E.D. (Eran Diamant), E.D. (Eyal
Dor), A.B., N.N., L.L., S.C., L.C.M., E.E. and A.T.; writing—original draft preparation, A.B.D., E.D.
(Eran Diamant) and A.T.; writing—review and editing, R.Z. and A.T. All authors have read and
agreed to the published version of the manuscript.
Funding: This work was supported by the Israel Institute for Biological Chemical and Environmental
Sciences (SB182).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Molecules 2021, 26, 3213 10 of 12

Data Availability Statement: The data presented in this study are available on request from the
corresponding authors.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
Sample Availability: Samples of the compounds are available from the authors.

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