Improving the efficacy of Chloroquine and Hydroxychloroquine against SARS-CoV-2 may require Zinc additives - A better synergy for future COVID-19 clinical trials

Abstract

The recent outbreak of coronavirus disease 2019 (COV-ID-19), has now been officially declared as a pandemic by the World Health Organization. As of now, there is no known effective pharmaceutical agent against the SARS-CoV-2 virus. However, several precautionary measures have been prescribed to prevent further spread of the virus, which include avoidance of social gatherings, proper handwashing, frequently disinfecting of used items and surfaces and so on. More recent studies have highlighted the possibility of treating patients infected with the novel SARS-CoV-2 virus with chloroquine and hydroxychloroquine, of which mechanism of action is not completely understood. We seek to draw the attention of the scientific community to the possibility of drastically reducing the effects of the virus on the affected patients and improving clinical trials outcome through the synergistic action of zinc and chloroquine in patients suffering from the coronavirus disease.

Full text

REVIEWS
192
Improving the efficacy of chloroquine
and hydroxychloroquine against
SARS-CoV-2 may require zinc
additives - A better synergy
for future COVID-19 clinical trials
Mujeeb Olushola Shittu, Olufemi Ifeoluwa Afolami
Biological Science Department, Michigan Technological University, Houghton, Michigan, United States of America
The recent outbreak of coronavirus disease 2019 (COV-
ID-19), has now been ofcially declared as a pandemic
by the World Health Organization. As of now, there
is no known effective pharmaceutical agent against
the SARS-CoV-2 virus. However, several precaution-
ary measures have been prescribed to prevent further
spread of the virus, which include avoidance of social
gatherings, proper handwashing, frequently disinfect-
ing of used items and surfaces and so on. More recent
studies have highlighted the possibility of treating pa-
tients infected with the novel SARS-CoV-2 virus with
SUMMARY
chloroquine and hydroxychloroquine, of which mech-
anism of action is not completely understood. We seek
to draw the attention of the scientic community to the
possibility of drastically reducing the effects of the vi-
rus on the affected patients and improving clinical tri-
als outcome through the synergistic action of zinc and
chloroquine in patients suffering from the coronavirus
disease.
Keywords: coronavirus, COVID-19, chloroquine, hy-
droxychloroquine, zinc, SARS-CoV-2.
Corresponding author
Mujeeb Olushola Shittu
E-mail: mshittu@mtu.edu
n INTRODUCTION
The coronavirus disease named COVID-19 by
the World Health Organization, which orig-
inated from Wuhan, the capital city of Hubei
province in China in December 2019 has sporad-
ically spread throughout the world. As of today,
the 16th of April 2020, over 2 million cases and
134,000 deaths have been reported in 210 coun-
tries and territories around the world [1]. The to-
tal number of cases in the United States, Spain,
Italy, Germany, and France have surpassed the
cases in China where the infection was original-
Le Infezioni in Medicina, n. 2, 192-197, 2020
ly discovered. Currently, comparative genomics
studies have been deployed by some countries
in Europe and North America to trace the origin
of SARS-CoV-2 and to understand its evolution
for proper monitoring of multiple aspects of this
pandemic [2]. The infection is currently consti-
tuting a serious health, economic, social, and
psychological effects on the whole world as the
world is under lock down as a measure to curb
the spread of the virus.
Coronaviruses (CoVs) belong to the family of
Coronaviridae. They have a non-segmented, sin-
gle-stranded, positive-sense RNA genome [3].
The severe acute respiratory syndrome corona-
virus 2 (SARS-CoV-2) that causes COVID-19 is a
zoonotic pathogen, which can infect both human
and animal. This virus is believed to have crossed
the species barrier to infect humans [3]. It has been

193Chloroquine and Hydroxychloroquine against SARS-CoV-2 and Zinc additives
suggested that human contract the SARS-CoV-2
through close contact with the animals, but there
is also the possibility of foodborne transmission
[4]. COVID-19 is thought to spread from person
to person through respiratory droplets produced
when an infected person coughs or sneezes with-
in a proximity to an uninfected person, usually
within 6 feet. Another way of spreading the vi-
rus is by touching your mouth, nose, or eyes after
touching a surface or object that has the virus.
This virus infects the host cell through a non-pH
dependent endocytosis by attaching to the type I
integral membrane receptor angiotensin-convert-
ing enzyme-2 (ACE2) in the alveolar cells in the
lungs with its glycoproteins [5]. Patients affected
with SARS-CoV-2 may progress from the asymp-
tomatic state to Acute Respiratory Distress Syn-
drome (ARDS) and septic shock in severe form
of the disease. The common clinical features of
COVID-19 include cough, sore throat, fatigue,
headache, myalgia, dyspnea, and fever (not in all
cases) [6].
Currently, there is no proven treatment for COV-
ID-19 infection. However, there is a growing evi-
dence that chloroquine and hydroxychloroquine
broadly used as antimalarial and immunomod-
ulatory drugs can be used in the treatment of
patients with COVID-19 infection. Chloroquine
and hydroxychloroquine belong to the same mo-
lecular family. The difference between the two is
the presence of hydroxyl group at the end of the
side chain of hydroxychloroquine. They are both
active against malaria parasite, but hydroxychlo-
roquine is less toxic [7]. Several in vitro studies
and recent clinical trials have shown the efcacy
of chloroquine in patients with COVID-19 at dif-
ferent levels of severity [8-10]. In a recent report,
chloroquine was cited as a potential remedy to al-
leviate exacerbation of pneumonia and mitigate
inammatory response, which improves the dis-
ease outcome [9]. This is not the rst-time chloro-
quine and hydroxychloroquine are being used to
treat a novel emerged virus, there are evidences
for the activities of chloroquine and hydroxychlo-
roquine against Zika virus, Ebola virus, and Chi-
kungunya virus [11-13]. Nevertheless, the mecha-
nism of action of chloroquine on COVID-19 is not
yet fully understood. However, several putative
mechanisms describing the effects of chloroquine
on the replication cycle of SARS-CoV-2 have been
reported [7, 14].
Zinc is another substance that could reduce the
SARS-CoV-2 viral activities when consumed due
to its antiviral effect and perhaps alleviate the res-
piratory tract infection. Zinc is the second most
abundant trace element, which exists in the diva-
lent cation state in the body. Only a little free zinc
exists because it readily binds to protein to form
a metalloprotein. The primary source of zinc is
a diet rich in sh, eggs, dairy products, shellsh
(especially oysters), and red meat. In human, zinc
supplementation is the key to constant supply of
zinc and maintaining homeostasis as the ability of
the body to store zinc is limited [15]. Zinc plays
important roles in immunity and viral infection.
Replication of SARS-coronavirus, hepatitis C vi-
rus, H1N1 inuenza virus has been shown to be
inhibited by zinc oxide and zinc salt. How zinc
exhibits its antiviral activities is not clearly un-
derstood, however, among the possible means
is the inhibition of viral binding to the mucosa,
suppression of inammatory effect, generation of
antiviral interferon and inhibition of important
enzyme in viral replication [16]. Recently, a study
conducted by Kaushik et al. unraveled the ability
of zinc salts in inhibiting Hepatitis E virus rep-
lication through the inhibition of RNA-depend-
ent-RNA-polymerase (RdRp) [17]. Interestingly,
this enzyme also plays a key role in coronavirus
replication. Therefore, in this article, we will be
reviewing the interaction between chloroquine,
hydroxychloroquine, and zinc, and the possibility
of their synergistic administration to mitigate the
exacerbation of COVID-19.
Metal ionophores: their mechanistic interaction
with viral replication and disease progression
Accumulated evidences in past studies have
revealed that metal ionophores are drug com-
pounds that have metal-binding domains which
enable them to act as transporters of cations
such as Ca2+, Zn2+, Na+ and Cu2+ [18-20]. Metal
ions act as ligands that catalyze many down-
stream roles which promotes many key cellular
processes. Deciencies in concentration of metal
ions like zinc, calcium or iron will signicantly
alter cellular signal transduction, DNA synthesis
and mRNA transcription, protein aggregation
and protein function [21, 22]. The ability of met-
al ionophores to reduce metal ion availability in
extracellular matrix (ECM) of living tissues allow
them to move excess ions into the cytosol thereby

194 Mujeeb Olushola Shittu, Olufemi Ifeoluwa Afolami
affecting signal transduction [18, 23]. Drug com-
pounds such as clioquinol, pyrithione (PT), hy-
droxyquinoline, chloroquine (CQ) and hydroxy-
chloroquine (CQ) have been described as metal
ionophores which can transport ion ligands that
drive down stream cell signaling processes from
the ECM into the cell in large amounts [23, 24].
Clioquinol and hydroxyquinoline can bind and
transport Zn2+ and Cu2+ ions into cancer cells that
express excess glucose receptors, causing severe
metal ion toxicities and triggering the apoptot-
ic program [25]. Similarly, metal ionophores act
as weak bases and can bind excess zinc salts in
viral transfected tissues and then directly inter-
fere in synthesis of viral DNA dependent DNA
polymerase or RNA dependent RNA polymerase
[26]. Conversely, certain metal ionophores such
as clioquinol can increase the levels of intracellu-
lar zinc in the lysosomes of cancer cells leading to
lysosome-mediated apoptosis [21].
Metal ionophores may also act as chelators; a
clinical trial investigation showed that drug com-
pounds such as desferrioxamine and tetrathio-
molybdate suppressed tumor clonal expansion,
metastases and angiogenesis [19]. Meanwhile,
clioquinol (5-chloro-7-iodo-8-hydroxyquinoline)
can inactivate superoxide dismutase-1 (SOD1)
and precipitate halt in cancer progression [25].
Similarly, Daniel et al., showed that dithiocarba-
mate requires zinc metal ions to inhibit NF-kappa
B [27]. They study also showed that zinc ions are
need for PT to cause a 10-fold potency for inhibi-
tion of NF-kappa B. The zinc ionophore PT (1-hy-
droxypyridine-2-thionine) has been described to
have antiviral properties and has been proven a
potent industrial biocide [27]. In 2009, Ding and
Lund reported that with adequate zinc additives,
PT when added to cells along with induced ap-
optosis and that a zinc additive-PT treatment of
viral transfected cells can represent a good fron-
tier for clinical trial of antiviral drugs [21]. Fur-
thermore, a later study demonstrated that nov-
el uorinated 8-hydroxyquinoline based metal
ionophore showed potency for amyloid-beta
(Aβ) deposition and stabilization in Alzheimer’s
disease (AD) [20]. Summarily, metal ionophores
have been proven over the years to exert overt
antiviral and anticancer properties especially
when coupled with enough doses of metal ion
additives that will galvanize their functions in
living tissues.
Chloroquine and hydroxychloroquine as zinc
ionophores: indirect interaction with COVID-19
genome replication
Chloroquine (CQ) is a 4-aminoquinoline antima-
laria drug that has also been used over the years
as an anti-inammatory agent and as an antican-
cer drug [28, 29]. CQ and its derivative hydrox-
ychloroquine (HCQ) act as weak bases that can
target key cellular signal transduction organelles
such as lysosomes and Golgi [30, 31]. An accumu-
lated concentration of CQ in these organelles will
catalyze signicant disruption of downstream
signaling processes via increase in the endosomal
and lysosomal pH [28, 31]. Although, continuous
study on the putative mechanism of action of CQ
are still ongoing in molecular medicine, howev-
er, past studies showed that upon administration,
the bioavailability of CQ and HCQ hinges largely
upon their protonation with zinc ions (Zn2+) upon
the cell, which makes them have high afnity
for low-pH organelles [32, 33]. By catalyzing an
increase in the pH, CQ and HCQ impair matu-
ration of cell lysosomes and autophagosomes,
thereby inhibiting antigen presentation tendency
of the host cell [31, 34]. This direct interference
with lysosomal activity upon inhibition triggers
an immunostimulatory response against the host
cell via MHC class II presentation [33, 34]. Xue et
al. showed that CQ and HCQ are zinc ionophores
using human ovarian cancer cell line (A2780)
[33]. They reported that at dose dependent con-
centrations, CQ and HCQ enhanced Zn2+ uptake
by TPEN attenuated A2780 cells in a concentra-
tion-dependent manner. Furthermore, microscop-
ic probe of intracellular zinc distribution demon-
strated that consistent with previous studies, CQ
and HCQ delivered free Zn2+ ions to the lyso-
somes inhibiting lysosomal function. The same
study also suggested that a combination of CQ or
HCQ with zinc enhanced chloroquine’s cytotoxic-
ity and induced apoptosis in A2780 cells [33].
Meanwhile, a study by te Velthuis et al. links an
increase in the intracellular Zn2+ ion concentration
by PT with replication impairments in RNA de-
pendent RNA polymerase viruses such as polio-
virus and inuenza virus [35]. In the same study,
the potency of PT zinc ionophore against these vi-
ruses was attributed to interference with polypro-
tein processing of RNA viruses. Meanwhile the
same study also demonstrated that a combination
of PT zinc ionophore with Zn2+ ions inhibited the

195Chloroquine and Hydroxychloroquine against SARS-CoV-2 and Zinc additives
replication of RNA dependent RNA polymer-
ase viruses; SARS-coronavirus (SARS-CoV) and
equine arteritis virus (EAV) in cell culture. The
RNA-dependent RNA polymerase (RdRp) is a
core enzyme of RNA viruses that enable multipro-
tein replication and transcription complex (RTC)
formation [35]. The same study by te Velthuis et
al. used an activity assay procedure to show that
without Zn2+, PT was unable to effectively hinder
(90%) the RNA-synthesizing activity of the RTCs
of both SARS-CoV or EAV viruses [35]. They also
reported further that enzymatic studies using re-
combinant RdRps of SARS-CoV nsp12 and EAV
nsp9 showed that PT was only a transporter and
that Zn2+ directly inhibited the activity of their
polymerases in vitro. Hence, while PT was an ion-
ophore that carried Zn2+ into the cell, Zn2+ acted
to block the initiation of EAV RNA synthesis and
SARS-CoV RdRp elongation was inhibited so that
RNA template binding was reduced. Conversely,
another study by Kaushik et al. investigated the
effect of zinc salts on RNA replication of hepa-
titis E virus (HEV) using hepatoma cell (Huh7)
cultures [17]. It was reported that zinc salts trans-
ported by PT inhibited the RNA replication of g-3
HEV replicons and g-1 HEV infectious genomic
RNA in a dose-dependent manner [17]. Analy-
sis of a replication-defective mutant of g-1 HEV
genomic RNA showed that zinc salts directly in-
hibit the activity of viral RdRp, leading to inhibi-
tion of viral replication [17]. In summary, zinc ion-
ophores such as CQ, PT and HCQ have demon-
strated promising prospects for successful clinical
trials by in vivo and in vitro studies where their
administration is coupled with zinc supplements.
Combining CQ and HCQ use with zinc
supplements: synergism needed for successful
COVID-19 clinical trials?
A variety of compelling evidences have been
published from early clinical trials in China that
showed the efcacy of CQ and HCQ in the treat-
ment of SARS-CoV-2. The long trail of studies
showed the possibility that CQ and its deriva-
tives may be effective against the novel SARS-
CoV-2 (the pathogen that causes COVID-19 and
shares a close phylogeny with previous species of
coronavirus) [8, 10, 36, 37]. A common consensus
amongst the published clinical trials was that the
SARS-CoV-2 virus requires acidication of endo-
somes and that essential modications to its cap-
sid envelope glycoproteins are needed for viral
replication which occurs within the endoplasmic
and trans-Golgi network vesicles at a low pH in
presence of proteases and glycosyl-transferases
[8, 37-39]. However, this essential prerequisite
for SARS-CoV-2 replication is blocked by CQ and
HCQ since the drugs alter ACE2 glycosylation by
stopping S-protein binding, thereby interfering
with viral replication the cell cytoplasm [9].
Meanwhile, another recent systematic review
on the state of CQ and HCQ clinical trials for
COVID-19 used PubMed and EMBASE data-
bases from inception to 1-March-2020 to nd
information on the efcacy and safety of CQ/
HCQ formulations in patients diagnosed with
SARS-CoV-2 [40]. Their initial search identied
234 sources (156 from PubMed, 73 EMBASE and
5 from other veried sources) amongst which
twenty-three clinical trials were found in the tri-
al registries. However, in all these documented
clinical trials in Europe and China, the pattern
of administration was similar as CQ and HCQ
drugs were used without being combined with
zinc ion supplements. Incidentally, none of
these clinical trials conducted so far has given a
near total positive outcome, which is signicant
enough to trigger an endorsement on a global
scale. Perhaps, consistent with previous studies
that delineated the efcacy of HCQ and CQ as
zinc ionophores, it was rather surprising that
none of these clinical trials so far considered us-
ing a combination of dose depended zinc sup-
plements with HCQ and CQ administration.
n CONCLUSION
Chloroquine can induce the uptake of zinc into the
cytosol of the cell, which is capable of inhibiting
RNA-dependent RNA polymerase and ultimately
halting the replication of coronavirus in the host
cell. Currently, there are several clinical trials that
are currently underway in several countries of
the world to assess the efcacy of chloroquine as
an anti-coronavirus agent. Since chloroquine has
been widely prescribed for use as an anti-malar-
ial, its safety is not in doubt. In view of the fore-
going, clinical trials predicated upon a synergis-
tic administration of Zn supplement with CQ or
HCQ against the novel SARS-CoV-2 virus should
be considered so that better COVID-19 clinical tri-
al outcomes can be obtained going forward.

196 Mujeeb Olushola Shittu, Olufemi Ifeoluwa Afolami
Funding
No funding sources
Conict of interest
None declared
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