Many
people have been exposed to the spike protein by either Covid-19 and or the
shot and many have been damaged and or damage is ongoing.
You
may have probably already heard, but in case you have not, a report just came
out that indicates that a combination of Bromelain
and NAC (N-Acetyl L-Cysteine) creates a synergistic effect that destroys
the spike protein.
I know many were coerced into introducing spike
proteins into their body. God is a very loving and kind and merciful God and can provide a way of escape for those who ask forgiveness. And probably many of us have gotten COVID from exposure
to others. Maybe this synergy, through God’s grace, can be a way of
restoration for those who have been damaged.
The
Combination of Bromelain and Acetylcysteine (BromAc) Synergistically
Inactivates SARS-CoV-2
by
1Department of Surgery, St. George Hospital, Sydney, NSW 2217,
Australia
2Mucpharm Pty Ltd., Sydney, NSW 2217, Australia
3CIRI, Centre International de Recherche en Infectiologie, Team
VirPatH, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS,
UMR5308, ENS de Lyon, F-69007 Lyon, France
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
*Author to whom correspondence should be addressed.
†These authors contributed equally to this work.
‡These authors contributed equally to this work.
Viruses 2021, 13(3),
425; https://doi.org/10.3390/v13030425
Received: 31 January 2021 / Revised: 25 February
2021 / Accepted: 1 March 2021 / Published: 6 March 2021
(This article belongs to the Special Issue Vaccines and
Therapeutics against Coronaviruses)
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 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 mechanism 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 [5,8,9,10,11].
SARS-CoV-2 entry into host cells has been shown to start with destabilization
of the spike protein through allosteric mechanical 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
gastrointestinal 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 µg/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 µL each. After 30 min incubation
at 37 °C, 5 µL of sample buffer was added into each reaction. A total of 20 µL
of each reaction was 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 µg/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 µg/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% CO2. Cells were maintained 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 Biosystems, Thermo Fisher Scientific, Foster
City, CA, USA). Log10 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 microelectronic 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, 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 µg/mL and BromAc at 50 and
100 µg/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 µg/mL and BromAc at 50
and 100 µg/20 mg/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. 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.
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 105.5 and 104.5 TCID50/mL yielded typical cytopathic effects (CPE),
and no cytotoxicity was observed 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 104.5 TCID50/mL virus titers (Figure 2B,D)
yielded more consistent inhibition of CPE for quadruplicates than on 105.5 TCID50/mL virus titers (Figure 2A,C).
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 reliable process of inactivation will be
able to reduce replication by at least 4 logs [Log10 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 replication. For the wild-type
(WT) strain at 104.5 TCID50/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 µg/20 mg/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/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 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/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 µg/20 mg/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.
Table 1. 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. Each
replicate is described. TCID50/mL = Median Tissue
Culture Infectious Dose; 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).
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.) [20,23,24,25,26,27].
More recently, Acetylcysteine has been used in the development of therapies for
respiratory infections such as influenza and COVID-19 [28,29,30],
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 machinery. BromAc acts as a biochemical agent to destroy complex
glycoproteins. Bromelain’s multipotent enzymatic competencies, dominated by the
ability to disrupt glycosidic linkages, 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 cleaving 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 [8,35,36,37,38].
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 [41,42,43,44].
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 demonstrated a clear antiviral effect. In
addition, given the heterogeneous disease outcome of COVID-19 patients, the
treatment strategy should combine several mechanisms of action 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 pulmonary 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 a threshold efficient dose at 100 µg/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 administration 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.
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MDPI and ACS Style
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
AMA Style
Akhter J, Quéromès G, Pillai K, Kepenekian V,
Badar S, Mekkawy AH, Frobert E, Valle SJ, Morris DL. The Combination of
Bromelain and Acetylcysteine (BromAc) Synergistically Inactivates
SARS-CoV-2. Viruses. 2021; 13(3):425.
https://doi.org/10.3390/v13030425
Chicago/Turabian Style
Akhter, Javed, Grégory Quéromès, Krishna
Pillai, Vahan Kepenekian, Samina Badar, Ahmed H. Mekkawy, Emilie Frobert, Sarah
J. Valle, and David L. Morris. 2021. "The Combination of Bromelain and
Acetylcysteine (BromAc) Synergistically Inactivates SARS-CoV-2" Viruses 13,
no. 3: 425. https://doi.org/10.3390/v13030425
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