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viruses
Article
The Combination of Bromelain and Acetylcysteine (BromAc)
Synergistically Inactivates SARS-CoV-2
Javed Akhter 1, 2, †, Grégory Quéromès3 ,†, Krishna Pillai 2,† , Vahan Kepenekian 1 ,4 ,† , Samina Badar 1,5,
Ahmed H. Mekkawy 1,2,5, Emilie Frobert 3, 6, ‡, Sarah J. Valle 1,2,5,‡ and David L. Morris 1,2,5,*,‡
Citation: Akhter, J.; Quéromès, G.;
Pillai, K.; Kepenekian, V.; Badar, S.;
Mekkawy, A.H.; Frobert, E.; Valle, S.J.;
Morris, D.L. The Combination of
Bromelain and Acetylcysteine
(BromAc) Synergistically Inactivates
SARS-CoV-2. Viruses 2021,13, 425.
https://doi.org/10.3390/v13030425
Academic Editors:
Kenneth Lundstrom and Alaa A.
A. Aljabali
Received: 31 January 2021
Accepted: 1 March 2021
Published: 6 March 2021
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Copyright: © 2021 by the authors.
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Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Department of Surgery, St. George Hospital, Sydney, NSW 2217, Australia;
Javed.Akhter@health.nsw.gov.au (J.A.); vahan.kepenekian@chu-lyon.fr (V.K.);
samina.badar@unsw.edu.au (S.B.); z3170073@ad.unsw.edu.au (A.H.M.); sarah.valle@mucpharm.com (S.J.V.)
2Mucpharm Pty Ltd., Sydney, NSW 2217, Australia; panthera6444@yahoo.com.au
3CIRI, Centre International de Recherche en Infectiologie, Team VirPatH, Univ Lyon, Inserm, U1111,
UniversitéClaude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007 Lyon, France;
gregory.queromes@univ-lyon1.fr (G.Q.); emilie.frobert@chu-lyon.fr (E.F.)
4Hospices Civils de Lyon, EMR 3738 (CICLY), Lyon 1 Université, F-69921 Lyon, France
5St. George & Sutherland Clinical School, University of New South Wales, Sydney, NSW 2217, Australia
6Laboratoire de Virologie, Institut des Agents Infectieux (IAI), Hospices Civils de Lyon,
Groupement Hospitalier Nord, F-69004 Lyon, France
*Correspondence: david.morris@unsw.edu.au; Tel.: +61-(02)-91132590
† These authors contributed equally to this work.
‡ These authors contributed equally to this work.
Abstract:
Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is the cause of
a worldwide pandemic, currently with limited therapeutic options. The spike glycoprotein and
envelope protein of SARS-CoV-2, containing disulfide bridges for stabilization, represent an attractive
target as they are essential for binding to the ACE2 receptor in host cells present in the nasal mucosa.
Bromelain and Acetylcysteine (BromAc) has synergistic action against glycoproteins by breakage of
glycosidic linkages and disulfide bonds. We sought to determine the effect of BromAc on the spike
and envelope proteins and its potential to reduce infectivity in host cells. Recombinant spike and
envelope SARS-CoV-2 proteins were disrupted by BromAc. Spike and envelope protein disulfide
bonds were reduced by Acetylcysteine. In
in vitro
whole virus culture of both wild-type and spike
mutants, SARS-CoV-2 demonstrated a concentration-dependent inactivation from BromAc treatment
but not from single agents. Clinical testing through nasal administration in patients with early
SARS-CoV-2 infection is imminent.
Keywords: SARS-CoV-2; Bromelain; Acetylcysteine; BromAc; drug repurposing
1. Introduction
The recently emergent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
is the causative agent of coronavirus disease 2019 (COVID-19), which can range from
asymptomatic to severe and lethal forms with a systemic inflammatory response syndrome.
As of 21 February 2021, over 111 million confirmed cases have been reported, with an
estimated overall mortality of 2.2% [
1
]. There are currently few therapeutic agents proven
to be beneficial in reducing early- and late-stage disease progression [
2
]. While there are
fortunately many vaccine candidates, their widespread availability for vaccination may
not be immediate, the length of immune protection may be limited [
3
,
4
], and the efficacy of
the vaccines may be reduced by novel SARS-CoV-2 variants. The continued exploration of
effective treatments is therefore still needed.
Structurally, SARS-CoV-2 contains surface spike proteins, membrane proteins, and
envelope proteins, as well as internal nucleoproteins that package the RNA. The spike
protein is a homotrimer glycoprotein complex with different roles accomplished through
Viruses 2021,13, 425. https://doi.org/10.3390/v13030425 https://www.mdpi.com/journal/viruses
Viruses 2021,13, 425 2 of 11
dynamic conformational modifications, based in part on disulfide bonds [5]. It allows the
infection of target cells by binding to the human angiotensin-converting enzyme (ACE2)
receptors, among others, which triggers proteolysis by transmembrane protease serine 2
(TMPRSS2), furin, and perhaps other proteases, leading to virion and host cell membrane
fusion [6,7].
The entry of viruses into mammalian cells, or “virus internalization”, is a key mecha-
nism of enveloped virus infection and is based on dynamic conformational changes of their
surface glycoproteins, namely, as mediated by disulfide bond reduction and regulated by
cell surface oxydoreductases and proteases [
5
,
8
–
11
]. SARS-CoV-2 entry into host cells has
been shown to start with destabilization of the spike protein through allosteric mechan-
ical transition, which induces a conformational change from the closed “down” state to
open “up” state of the receptor binding domain (RBD) of the spike protein [
12
,
13
]. The
conformational changes of RBD and virus binding are induced by TMPRSS2 or Cathepsin
L, which trigger the transition from the pre-fusion to post-fusion state [
5
,
12
,
13
]. The energy
liberated by disulfide bond reduction increases protein flexibility, which is maximal when
the reduced state is complete [
8
], thus allowing the fusion of host–virus membranes, which
is otherwise impossible due to the repulsive hydration forces present before reduction [
5
].
Bromelain is extracted mainly from the stem of the pineapple plant (Ananas comosus)
and contains a number of enzymes that give it the ability to hydrolyze glycosidic bonds
in complex carbohydrates [
14
]. Previous studies have indicated that Bromelain removes
the spike and hemagglutinin proteins of Semliki Forest virus, Sindbis virus, mouse gas-
trointestinal coronavirus, hemagglutinating encephalomyelitis virus, and H1N1 influenza
viruses [
15
,
16
]. As a therapeutic molecule, it is used for debriding burns. Acetylcysteine is a
powerful antioxidant that is commonly nebulized into the airways for mucus accumulation
and is also used as a hepatoprotective agent in paracetamol overdose. Most importantly in
the present context, Acetylcysteine reduces disulfide bonds [
17
]. Moreover, the association
of the spike and envelope proteins by their respective triple cysteine motifs warrants the
hypothesis of impacting virion stability following disulfide bridge disruption by the action
of Acetylcysteine [
18
]. The combination of Bromelain and Acetylcysteine (BromAc) exhibits
a synergistic mucolytic effect that is used in the treatment of mucinous tumors [
19
,
20
] and
as a chemosensitizer of several anticancer drugs [
21
]. These different actions are due to
the ability of BromAc to unfold the molecular structures of complex glycoproteins, thus
allowing binding to occur because of the high affinity between RBD and ACE2.
Therefore, in the current study we set out to determine whether BromAc can disrupt
the integrity of SARS-CoV-2 spike and envelope proteins and subsequently examine its
inactivation potential against
in vitro
replication of two viral strains, including one with a
spike mutant alteration of the novel S1/S2 cleavage site.
2. Materials and Methods
2.1. Materials
Bromelain API was manufactured by Mucpharm Pty Ltd (Kogarah, Australia) as a
sterile powder. Acetylcysteine was purchased from Link Pharma (Cat# AUST R 170803;
Warriewood, Australia). The recombinant SARS-COV-2 spike protein was obtained from
SinoBiological (Cat# 40589-V08B1; Beijing, China). The recombinant envelope protein was
obtained from MyBioSource (Cat# MBS8309649; San Diego, CA, USA). All other reagents
were from Sigma Aldrich (St. Louis, MO, USA).
2.2. Recombinant Spike and Envelope Gel Electrophoresis
The spike or envelope proteins were reconstituted in sterile distilled water according
to the manufacturer’s instructions, and aliquots were frozen at
−
20
◦
C. Two and a half
micrograms of spike or envelope protein were incubated with 50 or 100
µ
g/mL Bromelain,
20 mg/mL Acetylcysteine, or a combination of both in Milli-Q water. The control contained
no drugs. The total reaction volume was 15
µ
L each. After 30 min incubation at 37
◦
C,
5µL
of sample buffer was added into each reaction. A total of 20
µ
L of each reaction was
Viruses 2021,13, 425 3 of 11
electrophoresed on an SDS-PAGE (Cat# 456-1095; Bio-Rad Hercules, CA, USA). The gels
were stained using Coomassie blue.
2.3. UV Spectral Detection of Disulfide Bonds in Spike and Envelope Proteins
The method of Iyer and Klee for the measurement of the rate of reduction of disulfide
bonds has been used to detect disulfide bonds in spike and envelope proteins [
22
]. The
recombinant SARS-CoV-2 spike protein at a concentration of 3.0
µ
g/mL in phosphate-
buffered saline (PBS) (pH 7.0) containing 1 mM ethylenediaminetetraacetic acid (EDTA)
was incubated with 0, 10, 20, 40, and 50 µL of Acetylcysteine (0.5 M), agitated at 37 ◦C for
30 min followed by equivalent addition of Dithiothreitol (DTT) (0.5 M), and agitated for a
further 30 min at 37
◦
C. The spike protein was incubated in parallel only with DTT (0.5 M)
as before without any Acetylcysteine and agitated at 37
◦
C for 30 min. The absorbance was
then read at 310 nm. UV spectral detection of disulfide bonds in the envelope protein was
performed in a similar manner.
2.4. SARS-CoV-2 Whole Virus Inactivation with BromAc
Fully respecting the World Health Organization (WHO) interim biosafety guidance
related to the coronavirus disease, the SARS-CoV-2 whole virus inactivation tests were
carried out with a wild-type (WT) strain representative of early circulating European viruses
(GISAID accession number EPI_ISL_578176). A second SARS-CoV-2 strain (denoted as
∆
S), reported through routine genomic surveillance in the Auvergne-Rhône-Alpes region
of France, was added to the inactivation tests due to a rare mutation in the spike S1/S2
cleavage site and its culture availability in the laboratory (GISAID accession number
EPI_ISL_578177).
These tests were conducted with incremental concentrations of Bromelain alone (0, 25,
50, 100, and 250
µ
g/mL), Acetylcysteine alone (20 mg/mL), and the cross-reaction of the
different Bromelain concentrations combined with a constant 20 mg/mL Acetylcysteine
formulation, against two virus culture dilutions at 105.5 and 104.5 TCID50/mL. Following
1 h
of drug exposure at 37
◦
C, all conditions, including the control, were diluted 100-fold to
avoid cytotoxicity, inoculated in quadruplicate on confluent Vero cells (CCL-81; ATCC
©
,
Manassas, VA, USA), and incubated for 5 days at 36
◦
C with 5% CO
2
. Cells were main-
tained in Eagle’s minimal essential medium (EMEM) with 2% Penicillin-Streptomycin, 1%
L-glutamine, and 2% inactivated fetal bovine serum. Results were obtained by daily optical
microscopy observations, an end-point cell lysis staining assay, and reverse-transcriptase
polymerase chain reaction (RT-PCR) of supernatant RNA extracts. Briefly, the end-point
cell lysis staining assay consisted of adding Neutral Red dye (Merck KGaA, Darmstadt,
Germany) to cell monolayers, incubating at 37
◦
C for 45 min, washing with PBS, and
adding citrate ethanol before optical density (OD) was measured at 540 nm (Labsystems
Multiskan Ascent Reader, Thermo Fisher Scientific, Waltham, MA, USA). OD was directly
proportional to viable cells, so a low OD would signify important cell lysis due to virus
replication. In addition, RNA from well supernatants was extracted by the semi-automated
eMAG
®
workstation (bioMérieux, Lyon, FR), and SARS-CoV-2 RdRp IP2-targeted RdRp
Institute Pasteur RT-PCR was performed on a QuantStudio
™
5 System (Applied Biosys-
tems, Thermo Fisher Scientific, Foster City, CA, USA). Log
10
reduction values (LRV) of
viral replication were calculated by the difference between treatment and control wells per
condition divided by 3.3 (as 1 log10 ≈3.3 PCR Cycle thresholds (Ct)).
2.5. Replication Kinetics by Real-Time Cell Analysis
To compare the
in vitro
replication capacity of both WT and
∆
S SARS-CoV-2 strains,
replication kinetics were determined by measuring the electrode impedance of microelec-
tronic cell sensors on the xCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument
(ACEA Biosciences, Inc., San Diego, CA, USA). Vero cells were seeded at 20,000 cells per
well on an E-Plate 16 (ACEA Biosciences, Inc., San Diego, CA, USA) and incubated with
the same media conditions as described previously at 36
◦
C with 5% CO2. After 24 h,
Viruses 2021,13, 425 4 of 11
SARS-CoV-2 culture isolates were inoculated in triplicate at a multiplicity of infection of
10
−
2. Mock infections were performed in quadruplicate. Electronic impedance data (cell
index) were continuously collected at 15-min intervals for 6 days. Area under the curve
analysis of normalized cell index, established at time of inoculation, was then calculated at
12-h intervals. At each interval, cell viability was determined by normalizing against the
corresponding cell control. Tukey multiple comparison tests were used to compare each
condition on GraphPad Prism (software version 9.0; San Diego, CA, USA).
3. Results
3.1. Alteration of SARS-CoV-2 Spike and Envelope Proteins
Treatment of the spike protein with Acetylcysteine alone did not show any alteration
of the protein, whereas concentrations of Bromelain at 50 and 100
µ
g/mL and BromAc
at 50 and 100
µ
g/20 mg/mL resulted in protein alteration (Figure 1A). Treatment with
Acetylcysteine on the envelope protein did not alter the protein, whereas treatment with
Bromelain at 50 and 100
µ
g/mL and BromAc at 50 and 100
µ
g/20 mg/mL also resulted in
near complete and complete fragmentation, respectively (Figure 1A).
Viruses 2021, 13, x FOR PEER REVIEW 4 of 12
To compare the in vitro replication capacity of both WT and ∆S SARS-CoV-2 strains,
replication kinetics were determined by measuring the electrode impedance of microelec-
tronic cell sensors on the xCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument
(ACEA Biosciences, Inc., San Diego, CA, USA). Vero cells were seeded at 20,000 cells per
well on an E-Plate 16 (ACEA Biosciences, Inc., San Diego, CA, USA) and incubated with
the same media conditions as described previously at 36°C with 5% CO2. After 24 hours,
SARS-CoV-2 culture isolates were inoculated in triplicate at a multiplicity of infection of
10−2. Mock infections were performed in quadruplicate. Electronic impedance data (cell
index) were continuously collected at 15-minute intervals for 6 days. Area under the curve
analysis of normalized cell index, established at time of inoculation, was then calculated
at 12-hour intervals. At each interval, cell viability was determined by normalizing against
the corresponding cell control. Tukey multiple comparison tests were used to compare
each condition on GraphPad Prism (software version 9.0; San Diego, CA, USA).
3. Results
3.1. Alteration of SARS-CoV-2 Spike and Envelope Proteins
Treatment of the spike protein with Acetylcysteine alone did not show any alteration
of the protein, whereas concentrations of Bromelain at 50 and 100 µg/mL and BromAc at
50 and 100 µg/20mg/mL resulted in protein alteration (Figure 1A). Treatment with Ace-
tylcysteine on the envelope protein did not alter the protein, whereas treatment with Bro-
melain at 50 and 100 µg/mL and BromAc at 50 and 100 µg/20mg/mL also resulted in near
complete and complete fragmentation, respectively (Figure 1A).
Figure 1. (A) Bromelain and Acetylcysteine present a synergistic effect on severe acute
respiratory syndrome coronavirus (SARS-CoV-2) spike and envelope protein destabilization.
0 1020304050
0.0
0.2
0.4
0.6
0.8
1.0
μl (DTT , Ac + DTT)
OD 310 nm
DTT
Ac + DTT
Best-fit values
Slope
Y-intercept
X-intercept
1/slope
DTT
0.006171
0.2082
-33.74
162.0
Ac + DTT
0.002599
0.2261
-87.02
384.8
0 1020304050
0.0
0.2
0.4
0.6
0.8
1.0
μl (DTT, Ac + DTT)
OD 310 nm
DTT
Ac + DTT
Best-fit values
Slope
Y-intercept
X-intercept
1/slope
DTT
0.01293
0.2885
-22.31
77.34
Ac + DTT
0.007866
0.2679
-34.05
127.1
BC
A
Spike protein (150 KDa)
Envelope protein (25 KDa)
- + 50 50 100 100
-+-+- +
Acetylcysteine (20 mg/mL)
Bromelain (μg/mL)
123456
Figure 1.
(
A
) Bromelain and Acetylcysteine present a synergistic effect on severe acute respiratory syndrome coronavirus
(SARS-CoV-2) spike and envelope protein destabilization. SDS-PAGE of the recombinant SARS-CoV-2 spike protein S1
+ S2 subunits (150 kDa) and envelope protein (25 kDa). Proteins were treated with 20 mg/mL Acetylcysteine alone,
100 and
50 µg/mL
Bromelain alone, and a combination of 100 and
50 µg/20 mg/mL
BromAc. (
B
) Disulfide reduction
of recombinant SARS-CoV-2 spike protein by Acetylcysteine. The differential assay between Acetylcysteine (Ac) and
Dithiothreitol (DTT) for the reduction of disulfide bonds found on the spike protein indicates that Acetylcysteine reduces
42% of the disulfide bonds before the addition of DTT. The remaining bonds are reduced by DTT to produce the chromogen
detected at 310 nm. (
C
) Disulfide reduction of recombinant SARS-CoV-2 envelope protein by Acetylcysteine. The differential
assay between Acetylcysteine (Ac) and Dithiothreitol (DTT) for the reduction of disulfide bonds found on the envelope
protein indicates that Acetylcysteine reduces 40% of the bonds before the addition of DTT.
Viruses 2021,13, 425 5 of 11
3.2. UV Spectral Detection Demonstrates the Alteration of Disulfide Bonds in Spike and
Envelope Proteins
The comparative reduction of disulfide bonds on the spike protein between DTT alone
and DTT with Acetylcysteine demonstrated a 42% difference (Figure 1B), based on the slope
of the graphs [0.002599/0.006171 (100) = 42 %]. Acetylcysteine was thus able to reduce 58%
of the disulfide linkages in the sample, after which the remaining disulfide bonds were
reduced by DTT to produce the chromogen that was monitored in the spectra. Similarly,
the differential assay between Acetylcysteine and DTT for the reduction of disulfide bonds
found in the envelope protein [0.007866/0.01293 (100) = 60%] indicates that Acetylcysteine
reduces 40% of the disulfide bonds before the addition of DTT (Figure 1C).
3.3. In Vitro SARS-CoV-2 Inactivating Potential of Bromelain, Acetylcysteine, and BromAc
For both SARS-CoV-2 strains tested, the untreated virus controls at 10
5.5
and
104.5 TCID50/mL
yielded typical cytopathic effects (CPE), and no cytotoxicity was ob-
served for any of the drug combinations on Vero cells. Optical CPE results were invariably
confirmed by end-point Neutral Red cell staining. Overall, Bromelain and Acetylcysteine
treatment alone showed no viral inhibition, all with CPE comparable to virus control
wells, whereas BromAc combinations displayed virus inactivation in a concentration-
dependent manner (Figure 2). Treatment on 10
4.5
TCID
50
/mL virus titers (Figure 2B,D)
yielded more consistent inhibition of CPE for quadruplicates than on 10
5.5
TCID
50
/mL
virus titers (Figure 2A,C).
Based on the virus inactivation guidelines established by the WHO, a robust and
reliable process of inactivation will be able to reduce replication by at least 4 logs [Log
10
re-
duction value (LRV) = (RT-PCR Ct treatment – RT-PCR Ct control)/3.3; as 1
log10 ≈3.3 Ct]
.
As such, RT-PCR was performed on the RNA extracts to directly measure virus replication.
For the wild-type (WT) strain at 10
4.5
TCID
50
/mL, successful LRV > 4 were observed
with 1 out of 4 wells, 2 out of 4 wells, 3 out of 4 wells, and 4 out of 4 wells for 25, 50,
100 and
250 µg/20 mg/mL
BromAc, respectively (Figure 3). It is worth noting that at
105.5 TCID50/mL, LRV were slightly below the threshold at, on average, 3.3, with 3 out of
4 wells and 2 out of 4 wells for 100 and 250
µ
g/20 mg/mL BromAc, respectively
(Table 1
).
For the spike protein mutant (
∆
S) at 10
4.5
TCID
50
/mL, no successful LRV > 4 was observed
for 25
µ
g/20 mg/mL BromAc, but it was observed in 4 out of 4 wells for 50, 100, and
250 µg/20 mg/mL
BromAc (Figure 3). Of note, at 10
5.5
TCID
50
/mL, LRV were slightly
below the threshold at, on average, 3.2, with 1 out of 4 wells, 2 out of 4 wells, and 4 out
of 4 wells for 50, 100, and 250
µ
g/20 mg/mL BromAc, respectively (Table 1). Overall,
in vitro
inactivation of both SARS-CoV-2 strains’ replication capacity was observed in
a dose-dependent manner, most strongly demonstrated at 100 and 250
µ
g/20 mg/mL
BromAc against 104.5 TCID50/mL of virus.
Table 1.
Log
10
reduction values (LRV) of
in vitro
virus replication 96 h after BromAc treatment on
WT and
∆
S SARS-CoV-2 strains at 5.5 and 4.5 log
10
TCID
50
/mL titers. LRV were calculated with the
following formula: LRV = (RT-PCR Ct of treatment – RT-PCR Ct virus control)/3.3; as 1 log
10 ≈
3.3 Ct.
Each replicate is described. TCID
50
/mL = Median Tissue Culture Infectious Dose;
WT = wild-type;
∆S = S1/S2 spike mutant.
BromAc (µg/20 mg/mL) Virus Titer
5.5 log10TCID50 /mL 4.5 log10TCID50 /mL
WT
25 0.033 0.104 0.250 0.213 0.463 0.356 4.390 0.173
50 0.050 0.304 0.446 0.698 0.471 4.378 0.404 4.651
100 3.415 3.323 0.360 3.313 4.418 4.463 0.423 4.508
250 0.033 3.423 0.200 3.389 4.496 4.370 4.419 4.506
∆S
25 0.010 0.153 NA 0.414 0.330 0.313 0.172 0.075
50 3.252 0.297 0.278 0.275 4.762 4.612 4.618 4.571
100 3.191 3.260 0.210 0.301 6.054 4.518 5.155 4.747
250 3.287 3.298 3.308 3.308 4.333 4.302 4.410 4.361
Viruses 2021,13, 425 6 of 11
Viruses 2021, 13, x FOR PEER REVIEW 6 of 12
Figure 2. Cell lysis assays demonstrated in vitro inactivation potential of Acetylcysteine and
Bromelain combined (BromAc) against SARS-CoV-2. Cell viability was measured by cell staining
with Neutral Red, where optical density (OD) is directly proportional to viable cells. Low OD
would signify important cell lysis due to virus replication. The wild-type (WT) SARS-CoV-2 strain
at 5.5 and 4.5 log10TCID50/mL titers (A and B, respectively) showed no inhibition of cytopathic
effect (CPE) for single agent treatment, compared to the mock treatment virus control condition.
BromAc combinations were able to inhibit CPE, compared to the mock infection cell controls.
Treatment of a SARS-CoV-2 spike protein variant (∆S) with a mutation at the S1/S2 junction at 5.5
and 4.5 log10TCID50/mL titers (C and D, respectively) showed similar results. Bars represent the
average of each quadruplicate per condition, illustrated by white circles. Ordinary one-way
ANOVA was performed, using the mock treatment virus control as the control condition (****p <
0.0001, ***p < 0.0005, **p < 0.003, and *p < 0.05).
Based on the virus inactivation guidelines established by the WHO, a robust and re-
liable process of inactivation will be able to reduce replication by at least 4 logs [Log10
0.0
0.5
1.0
1.5
2.0
2.5
OD 540 nm
****
***
cell
CT virus
CT 25
-
25
+
50
-
50
+
100
-
100
+
250
-
250
+Acetylcysteine
Bromelain (
μg/mL)
+
-
0.0
0.5
1.0
1.5
2.0
2.5
OD 540 nm
** ** ***
****
cell
CT virus
CT 25
-
25
+
50
-
50
+
100
-
100
+
250
-
250
+Acetylcysteine
Bromelain (
μg/mL)
+
-
0.0
0.5
1.0
1.5
2.0
2.5
OD 540 nm
****
***
*
cell
CT virus
CT 25
-
25
+
50
-
50
+
100
-
100
+
250
-
250
+Acetylcysteine
Bromelain (
μg/mL)
+
-
0.0
0.5
1.0
1.5
2.0
2.5
OD 540 nm
**** ****
**** ****
cell
CT virus
CT 25
-
25
+
50
-
50
+
100
-
100
+
250
-
250
+Acetylcysteine
Bromelain (
μg/mL)
+
-
A
B
C
D
Figure 2.
Cell lysis assays demonstrated
in vitro
inactivation potential of Acetylcysteine and Brome-
lain combined (BromAc) against SARS-CoV-2. Cell viability was measured by cell staining with
Neutral Red, where optical density (OD) is directly proportional to viable cells. Low OD would
signify important cell lysis due to virus replication. The wild-type (WT) SARS-CoV-2 strain at
5.5 and
4.5 log10TCID50 /mL
titers (
A
and
B
, respectively) showed no inhibition of cytopathic ef-
fect (CPE) for single agent treatment, compared to the mock treatment virus control condition.
BromAc combinations were able to inhibit CPE, compared to the mock infection cell controls. Treat-
ment of a SARS-CoV-2 spike protein variant (
∆
S) with a mutation at the S1/S2 junction at 5.5 and
4.5 log10TCID50 /mL
titers (
C
and
D
, respectively) showed similar results. Bars represent the average
of each quadruplicate per condition, illustrated by white circles. Ordinary one-way ANOVA was per-
formed, using the mock treatment virus control as the control condition (**** p< 0.0001,
*** p< 0.0005,
** p< 0.003, and * p< 0.05).
Viruses 2021,13, 425 7 of 11
Viruses 2021, 13, x FOR PEER REVIEW 7 of 12
reduction value (LRV) = (RT-PCR Ct treatment – RT-PCR Ct control)/3.3; as 1 log10 ≈ 3.3
Ct]. As such, RT-PCR was performed on the RNA extracts to directly measure virus rep-
lication. For the wild-type (WT) strain at 104.5 TCID50/mL, successful LRV > 4 were ob-
served with 1 out of 4 wells, 2 out of 4 wells, 3 out of 4 wells, and 4 out of 4 wells for 25,
50, 100 and 250 µg/20mg/mL BromAc, respectively (Figure 3). It is worth noting that at
105.5 TCID50/mL, LRV were slightly below the threshold at, on average, 3.3, with 3 out of 4
wells and 2 out of 4 wells for 100 and 250 µg/20mg/mL BromAc, respectively (Table 1).
For the spike protein mutant (∆S) at 104.5 TCID50/mL, no successful LRV > 4 was observed
for 25 µg/20mg/mL BromAc, but it was observed in 4 out of 4 wells for 50, 100, and 250
µg/20mg/mL BromAc (Figure 3). Of note, at 105.5 TCID50/mL, LRV were slightly below the
threshold at, on average, 3.2, with 1 out of 4 wells, 2 out of 4 wells, and 4 out of 4 wells for
50, 100, and 250 µg/20mg/mL BromAc, respectively (Table 1). Overall, in vitro inactivation
of both SARS-CoV-2 strains’ replication capacity was observed in a dose-dependent man-
ner, most strongly demonstrated at 100 and 250 µg/20mg/mL BromAc against 104.5
TCID50/mL of virus.
Figure 3. Threshold matrix of log10 reduction values (LRV) of in vitro virus replication 96 h after
BromAc treatment on WT and ∆S SARS-CoV-2 strains at 5.5 and 4.5 log10TCID50/mL titers. LRV
were calculated with the following formula: LRV = (RT-PCR Ct of treatment—RT-PCR Ct virus
control)/3.3; as 1 log10 ≈ 3.3 Ct. The color gradient matrix displays the number of quadruplicates
per condition yielding an LRV>4, corresponding to a robust inactivation according to the WHO.
WT = wild-type; ∆S = S1/S2 spike mutant.
WT ΔSWTΔS
25 µg/mL
50 µg/mL
100 µg/mL
250 µg/mL
5.5 log
10
TCID
50
/mL 4.5 log
10
TCID
50
/mL
21304/ 4 with LRV > 4
Figure 3.
Threshold matrix of log
10
reduction values (LRV) of
in vitro
virus replication 96 h after
BromAc treatment on WT and
∆
S SARS-CoV-2 strains at 5.5 and 4.5 log
10
TCID
50
/mL titers. LRV
were calculated with the following formula: LRV = (RT-PCR Ct of treatment—RT-PCR Ct virus
control)/3.3; as 1 log10
≈
3.3 Ct. The color gradient matrix displays the number of quadruplicates
per condition yielding an LRV > 4, corresponding to a robust inactivation according to the WHO.
WT = wild-type; ∆S = S1/S2 spike mutant.
Real-time cell analysis demonstrated comparable replication kinetics for both WT and
∆
S SARS-CoV-2 strains (Figure 4). No significant difference in cell viability was observed
between WT and
∆
S at any time point. From 48 h post-infection, WT and
∆
S cell viability
were significantly different compared to the mock infection (p< 0.05).
Viruses 2021, 13, x FOR PEER REVIEW 8 of 12
Table 1. Log10 reduction values (LRV) of in vitro virus replication 96 hours after BromAc treat-
ment on WT and ∆S SARS-CoV-2 strains at 5.5 and 4.5 log10TCID50/mL titers. LRV were calculated
with the following formula: LRV = (RT-PCR Ct of treatment – RT-PCR Ct virus control)/3.3; as 1
log10 ≈ 3.3 Ct. Each replicate is described. TCID50/mL = Median Tissue Culture Infectious Dose; WT
= wild-type; ∆S = S1/S2 spike mutant.
BromAc (µg/20mg/mL) Virus Titer
5.5 lo
g
10TCID50/mL 4.5 lo
g
10TCID50/mL
WT
25 0.033 0.104 0.250 0.213 0.463 0.356 4.390 0.173
50 0.050 0.304 0.446 0.698 0.471 4.378 0.404 4.651
100 3.415 3.323 0.360 3.313 4.418 4.463 0.423 4.508
250 0.033 3.423 0.200 3.389 4.496 4.370 4.419 4.506
∆S
25 0.010 0.153 NA 0.414 0.330 0.313 0.172 0.075
50 3.252 0.297 0.278 0.275 4.762 4.612 4.618 4.571
100 3.191 3.260 0.210 0.301 6.054 4.518 5.155 4.747
250 3.287 3.298 3.308 3.308 4.333 4.302 4.410 4.361
Real-time cell analysis demonstrated comparable replication kinetics for both WT
and ∆S SARS-CoV-2 strains (Figure 4). No significant difference in cell viability was ob-
served between WT and ∆S at any time point. From 48 hours post-infection, WT and ∆S
cell viability were significantly different compared to the mock infection (p < 0.05).
Figure 4. SARS-CoV-2 replication capacity of WT and ∆S SARS-CoV-2 measured by Real-Time
Cell Analysis. Data points correspond to area under the curve analysis of normalized cell index
(electronic impedance of RTCA established at time of inoculation) at 12-hour intervals. Cell viabil-
ity was then determined by normalizing against the corresponding cell control. WT = wild-type;
∆S = S1/S2 spike mutant.
4. Discussion
The combination of Bromelain and Acetylcysteine, BromAc, synergistically inhibited
the infectivity of two SARS-CoV-2 strains cultured on Vero cells. Protein confirmation and
its molecular properties are dependent on its structural and geometric integrity, which are
dependent on both the peptide linkages and disulfide bridges. Acetylcysteine, as a good
reducing agent, tends to reduce the disulfide bridges and hence alter the molecular prop-
erties of most proteins. This property has been widely exploited in the development of
several therapies (chronic obstructive pulmonary disease, allergic airways diseases, cystic
fibrosis, pseudomyxoma peritonei, etc.) [20,23–27]. More recently, Acetylcysteine has
been used in the development of therapies for respiratory infections such as influenza and
COVID-19 [28–30], where the integrity of the spike protein is vital for infection [12,13]. A
hypothesized mechanism of action could be the unfolding of the spike glycoprotein and
the reduction of its disulfide bonds.
0 1224364860728496108120
0
10
20
30
40
50
60
70
80
90
100
Time post-infection (hours)
Cell viability (%)
from normalized cell index
WT
ΔS
Figure 4.
SARS-CoV-2 replication capacity of WT and
∆
S SARS-CoV-2 measured by Real-Time Cell
Analysis. Data points correspond to area under the curve analysis of normalized cell index (electronic
impedance of RTCA established at time of inoculation) at 12-h intervals. Cell viability was then
determined by normalizing against the corresponding cell control. WT = wild-type;
∆
S = S1/S2
spike mutant.
4. Discussion
The combination of Bromelain and Acetylcysteine, BromAc, synergistically inhibited
the infectivity of two SARS-CoV-2 strains cultured on Vero cells. Protein confirmation and
its molecular properties are dependent on its structural and geometric integrity, which
are dependent on both the peptide linkages and disulfide bridges. Acetylcysteine, as a
good reducing agent, tends to reduce the disulfide bridges and hence alter the molecular
properties of most proteins. This property has been widely exploited in the development
of several therapies (chronic obstructive pulmonary disease, allergic airways diseases,
cystic fibrosis, pseudomyxoma peritonei, etc.) [
20
,
23
–
27
]. More recently, Acetylcysteine has
been used in the development of therapies for respiratory infections such as influenza and
Viruses 2021,13, 425 8 of 11
COVID-19 [
28
–
30
], where the integrity of the spike protein is vital for infection [
12
,
13
]. A
hypothesized mechanism of action could be the unfolding of the spike glycoprotein and
the reduction of its disulfide bonds.
The SARS-CoV-2 spike protein is the cornerstone of virion binding to host cells and
hence represents an ideal therapeutic target. A direct mechanical action against this spike
protein is a different treatment strategy in comparison to most of the existing antiviral
drugs, which prevents viral entry in host cells rather than targeting the replication machin-
ery. BromAc acts as a biochemical agent to destroy complex glycoproteins. Bromelain’s
multipotent enzymatic competencies, dominated by the ability to disrupt glycosidic link-
ages, usefully complement Acetylcysteine’s strong power to reduce disulfide bonds [
17
].
Amino acid sequence analysis of the SARS-CoV-2 spike glycoprotein identified several
predetermined sites where BromAc could preferentially act, such as the S2’ site rich in
disulfide bonds [
31
], together with three other disulfide bonds in RBD [
32
]. In parallel, the
role of the glycosidic shield in covering the spike, which is prone to being removed by
BromAc, has been highlighted as a stabilization element of RBD conformation transitions
as well as a resistance mechanism to specific immune response [5,33,34].
Mammalian cells exhibit reductive functions at their surface that are capable of cleav-
ing disulfide bonds, and the regulation of this thiol-disulfide balance has been proven to
impact the internalization of different types of viruses, including SARS-CoV-2 [
8
,
35
–
38
].
Both ACE2 and spike proteins possess disulfide bonds. When all the spike protein RBD
disulfide bonds were reduced to thiols, ACE2 receptor binding to spike protein became
less favorable [
8
]. Interestingly, the reduction of ACE2 disulfide bonds also induced a
decrease in binding [
8
]. Moreover, other reports suggested that Bromelain alone could
inhibit SARS-CoV-2 infection in VeroE6 cells through an action on disulfide links [
39
,
40
].
As such, the loss of SARS-CoV-2 infectivity observed after pre-treatment with BromAc
could be correlated to the cumulative unfolding of the spike and envelope proteins, with a
significant reduction of their disulfide bonds by Acetylcysteine, demonstrated in vitro.
Interestingly, a similar effect of BromAc was observed against both WT and
∆
S SARS-
CoV-2. The main difference in amino acid sequences between SARS-CoV-2 and previous
SARS-CoV is the inclusion of a furin cleavage site between S1 and S2 domains [
41
]. This
distinct site of the spike protein and its role in host spill-over and virus fitness is a topic of
much debate [
41
–
44
]. Of note,
∆
S, which harbors a mutation in this novel S1/S2 cleavage
site and alters the cleavage motif, exhibits no apparent difference in replication capacity
compared to the WT strain. The slightly increased sensitivity of
∆
S to BromAc treatment is
therefore not due to a basal replication bias, but the mutation could perhaps be involved in
enhancing the mechanism of action of BromAc. These results would nevertheless suggest
that, from a threshold dose, BromAc could potentially be effective on spike mutant strains.
This may be a clear advantage for BromAc over specific immunologic mechanisms of a
spike-specific vaccination [3,4].
To date, different treatment strategies have been tested, but no molecules have demon-
strated a clear antiviral effect. In addition, given the heterogeneous disease outcome of
COVID-19 patients, the treatment strategy should combine several mechanisms of ac-
tion and be adapted to the stage of the disease. Thus, treatment repurposing remains
an ideal strategy against COVID-19, whilst waiting for sufficient vaccination coverage
worldwide [
45
,
46
]. In particular, the development of early nasal-directed treatment prone
to decreasing a patient’s infectivity and preventing the progression towards severe pul-
monary forms is supported by a strong rationale. Hou et al. demonstrated that the first
site of infection is the nasopharyngeal mucosa, with secondary movement to the lungs
by aspiration [
47
]. Indeed, the pattern of infectivity of respiratory tract cells followed
ACE2 receptor expression, decreasing from the upper respiratory tract to the alveolar
tissue. The ratio for ACE2 was five-fold greater in the nose than in the distal respiratory
tract [
40
]. Other repurposing treatments as a nasal antiseptic have been tested
in vitro
, such
as Povidone-Iodine, which has shown activity against SARS-CoV-2 [
48
]. In the present
study, we showed the
in vitro
therapeutic potential of BromAc against SARS-CoV-2 with
Viruses 2021,13, 425 9 of 11
a threshold efficient dose at 100
µ
g/20 mg/mL. As animal airway safety models in two
species to date have exhibited no toxicity (unpublished data), the aim is to test nasal ad-
ministration of the drug in a phase I clinical trial (ACTRN12620000788976). Such treatment
could help mitigate mild infections and prevent infection of persons regularly in contact
with the virus, such as health-care workers.
Although our results are encouraging, there are a number of points to consider
regarding this demonstration. Namely, the
in vitro
conditions are fixed and could be
different from
in vivo
. Any enzymatic reaction is influenced by the pH of the environment,
and even more so when it concerns redox reactions such as disulfide bond reduction [
9
]. The
nasal mucosal pH is, in physiological terms, between 5.5 and 6.5 and increases in rhinitis
to 7.2–8.3 [
49
]. Advanced age, often encountered in SARS-CoV-2 symptomatic infections,
also induces a nasal mucosa pH increase [
49
]. Such a range of variation, depending
on modifications typically induced by a viral infection, may challenge the efficacy of
our treatment strategy. Further
in vitro
experiments to test various conditions of pH
are ongoing, but ultimately, only clinical studies will be able to assess this point. Our
experiments were led on a monkey kidney cell line known to be highly permissive to
SARS-CoV-2 infectivity. With the above hypothesis of S protein lysis thiol-disulfide balance
disruption, BromAc efficacy on SARS-CoV-2 should not be influenced by the membrane
protease pattern. Reproducing this experimental protocol with the human pulmonary
epithelial Calu-3 cell line (ATCC
®
HTB-55
™
) would allow these points to be addressed, as
virus entry is TMPRSS2-dependent and pH-independent, as in airway epithelium, while
virus entry in Vero cells is Cathepsin L-dependent, and thus pH-dependent [50].
Overall, results obtained from the present study in conjunction with complementary
studies on BromAc properties and SARS-CoV-2 characterization reveal a strong indication
that BromAc can be developed into an effective therapeutic agent against SARS-CoV-2.
5. Conclusions
There is currently no suitable therapeutic treatment for early SARS-CoV-2 aimed at
preventing disease progression. BromAc is under clinical development by the authors for
mucinous cancers due to its ability to alter complex glycoprotein structures. The potential
of BromAc on SARS-CoV-2 spike and envelope proteins stabilized by disulfide bonds
was examined and found to induce the unfolding of recombinant spike and envelope
proteins by reducing disulfide stabilizer bridges. BromAc also showed an inhibitory effect
on wild-type and spike mutant SARS-CoV-2 by inactivation of its replication capacity
in vitro
. Hence, BromAc may be an effective therapeutic agent for early SARS-CoV-2
infection, despite mutations, and even have potential as a prophylactic in people at high
risk of infection.
Author Contributions:
Conceptualization, J.A., K.P., S.J.V., and D.L.M.; methodology, J.A., G.Q., K.P.,
S.B., and A.H.M.; validation, J.A., G.Q., K.P., V.K., S.B., and A.H.M.; investigation, J.A., G.Q., K.P.,
V.K., S.B., and A.H.M.; writing—original draft preparation, G.Q., K.P., V.K, A.H.M., E.F., and S.J.V.;
supervision, D.L.M. and E.F.; project administration, S.J.V.; funding acquisition, S.J.V. and D.L.M. All
authors have read and agreed to the published version of the manuscript.
Funding: This research is partly funded by Mucpharm Pty Ltd., Australia.
Data Availability Statement:
A preprint of this manuscript was archived on www.biorxiv.org
(accessed on 31 January 2021) due to the emergency of COVID-19.
Conflicts of Interest:
David L. Morris is the co-inventor and assignee of the Licence for this study
and director of the spin-off sponsor company, Mucpharm Pty Ltd. Javed Akhter, Krishna Pillai,
and Ahmed Mekkawy are employees of Mucpharm Pty Ltd. Sarah Valle is partly employed by
Mucpharm for its cancer development and is supported by an Australian Government Research
Training Program Scholarship. Vahan Kepenekian thanks the Foundation Nuovo Soldati for its
fellowship and was partly sponsored for stipend by Mucpharm Pty Ltd.
Viruses 2021,13, 425 10 of 11
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