Introduction
To date, more than 17 million cases of coronavirus disease 2019 (COVID-19) and 900,000
deaths have been reported globally (World Health Organization, 2020). Major collaborative
efforts focusing on the development of anti-SARS-CoV-2 vaccines and antiviral therapeutics
are accelerated at an unprecedented rate. Various therapeutic agents quickly taken
into clinical trials are largely based on existing drugs with non-specific antiviral
activities or compounds pharmacologically speculated to be effective in enhancing
the overall clinical outcome of COVID-19 patients. To name a few, these include the
nucleoside inhibitor prodrug remdesivir (GS-5734), the antiparasitic drug ivermectin,
the HIV protease inhibitor nelfinavir, the anti-inflammatory drug cepharanthine, the
antimalarial drug hydroxychloroquine, the general steroid dexamethasone, and others
(Caly et al., 2020; Ledford, 2020; Ohashi et al., 2020; Vanden Eynde, 2020; Wang Y.
et al., 2020). However, only dexamethasone and remdesivir appear to confer promising
treatment outcomes by reducing deaths or time to clinical improvement in the severe
COVID-19 patients (Grein et al., 2020; Ledford, 2020; Wang Y. et al., 2020). The world
is extremely desperate for a cure or prophylaxis against SARS-CoV-2 with the hope
of saving more lives. Nevertheless, little has been described for antiviral peptides
(AVPs) or alternatively known as antimicrobial peptides (AMPs) that possess antiviral
activities. AVPs are a class of short (8–40 amino acids in length) polycationic antivirals
with potent broad spectrum antiviral activities (Chang and Yang, 2013; Skalickova
et al., 2015; Ahmed et al., 2019; Nyanguile, 2019; Sala et al., 2019; Vilas Boas et al.,
2019). Interestingly, there are AVPs demonstrated to exert prophylactic and therapeutic
effects against coronaviruses (CoVs). In this opinion paper, we describe the potential
use of AVPs against COVID-19 based on the documented evidence against SARS-CoV2, SARS-CoV,
MERS-CoV, SARS-related CoVs, and other respiratory viruses that shall warrant further
development of this class of compounds in the face of the current pandemic threat.
Mechanism of Viral Infection
SARS-CoV-2 is an enveloped virus with the characteristic spike (S) glycoprotein to
mediate viral entry via the cell surface receptor angiotensin-converting enzyme-2
(ACE2) (Zhou et al., 2020). The S1 subunit of S protein is the receptor-binding domain
responsible for ACE2 binding (Xia et al., 2020b). Subsequently, a cellular serine
protease TMPRSS2 is required for S protein priming, which induces proteolytic cleavage
of S protein at S1/S2 and S2’ sites (Hoffmann et al., 2020). Following cleavage, both
heptapeptide repeat 1 (HR1) and heptapeptide repeat 2 (HR2) regions of the S2 subunit
of S protein interact to form the 6-helix bundle (6HB) fusion core (Liu et al., 2004;
Kang et al., 2020). The formation of 6HB is critical in facilitating the viral membrane
fusion process for viral entry into the host cell via endocytosis. During the late
endosomal stage, endosomal acidification would induce virus-endosome membrane fusion,
thereby leading to viral uncoating (viral RNA release) for the initiation of viral
replication and infection (Du et al., 2009; Das et al., 2010; Letko et al., 2020).
Notably, the S1 subunit of SARS-CoV-2 showed a 10- to 20-fold higher ACE2 binding
affinity comparing to SARS-CoV, which may explain the higher transmissibility and
infectivity of COVID-19 (Wrapp et al., 2020).
Targeting the Viral Envelope
Mucroporin-M1 (LFRLIKSLIKRLVSAFK) is a peptide analog designed with four residual
mutations at positions G3R, P6K, G10K, and G11R from the parent peptide mucroporin
(LFGLIPSLIGGLVSAFK) isolated from the venom of the scorpion Lychas mucronatus (Dai
et al., 2008; Li et al., 2011). Such increase in cationicity enhanced the dipolar
characteristic of the peptide helix, which contributed to the drastic increment in
antiviral activity against SARS-CoV (50% virus infection, EC50 of 14.46 µg/ml), influenza
H5N1 (EC50 of 2.10 µg/ml), and measles (EC50 of 7.15 µg/ml) pseudoviruses (Li et al.,
2011). Based on the observations that mucroporin-M1 directly interacted with measles
viral particles and a greater antiviral effect with pre-treatment, mucroporin-M1 was
proposed to serve the role of a molecular blocker, which must find its target before
viral attachment to the host cells (
Figure 1
). Following peptide-virus binding, the strong electrostatic affinity of mucroporin-M1
could allow interaction and disruption of the viral envelope, thereby exerting a direct
virucidal effect against SARS-CoV, MERS-CoV, and influenza H5N1 viruses (Li et al.,
2011).
Figure 1
Mechanism of actions of AVPs with potential anti-SARS-CoV-2 activities. Several AVPs
target the key structural components of the virus to exert antiviral effects: mucroporin-M1
acts by disrupting viral envelope, HR2P-M2 targets the viral S protein-mediated fusion,
EK1 and EK1C4 block the HR1 domain of viral S2 subunit, and P9 peptide inhibits late
endosomal acidification and thus preventing viral RNA release. There are AVPs which
confer antiviral protection to the host: RTD-1 is a potent antiviral immunomodulator
to trigger protective immunity, and HD5 binds to and shields ACE2 from viral recognition
and binding.
Targeting the Spike (S) Glycoprotein
EK1 (SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL) is a pan-CoV fusion inhibitor designed
by Xia and colleagues. This 36-residue peptide showed high cross-reactivity against
all SARS-CoV, MERS-CoV, and three SARS-related CoVs from bats tested (Xia et al.,
2019). As shown in
Figure 1
, EK1 acts by blocking the HR1 domain to disrupt the formation of the 6HB core, which
causes inhibition of viral fusion entry into the host cell (Xia et al., 2020b). Intranasal
application of EK1 further protected mice from pre- and post-challenges by the HCoV-OC43
alphacoronavirus and MERS-CoV challenges. Such dual therapeutic and prophylactic effects
displayed by a single compound against various closely related CoVs are highly desirable.
The broad spectrum anti-CoV activity strongly suggests that EK1 could be useful against
SARS-CoV-2 infection. Recently, a highly potent lipopeptide variant EK1C4 was generated
by C-terminally conjugating EK1 with a cholesterol moiety using a glycine/serine linker
and a polyethylene glycol (PEG) spacer (-EK1-GSGSG-PEG4-chol) (Xia et al., 2019).
The results were exciting, where SARS-CoV-2 cell-cell fusion was severely impaired
at 50% inhibitory concentration (IC50) of 1.3nM, while anti-SARS-CoV-2 pseudoviruses
infection activity was recorded at 15.8 nM. Notably, the SARS-CoV-2 HR1 protein binding
affinity of EK1C4 was drastically enhanced by 226-fold, which also explained the observed
149-fold higher in antiviral activity of EK1C4 as compared to EK1. It was hypothesized
that the conjugated cholesterol might have a profound role in facilitating EK1C4 binding
toward the HR1 protein. Indeed, cholesterol conjugation enhancement of antiviral activity
had previously been reported with the HIV-1 fusion inhibitor C34 (Hollmann et al.,
2013). Besides EK1C4, HR2P-M2 was a peptide designed based on the 6HB core of MERS-CoV
HR1 and HR2 domains (Channappanavar et al., 2015). This peptide acted by blocking
the S protein-mediated membrane fusion mechanism of MERS-CoV. Of note, intranasal
administration of HR2P-M2 was found to significantly reduce lung viral titers by >1,000-fold
and protected the mice from MERS-CoV infection (Channappanavar et al., 2015). However,
the narrow specificity of HR2P-M2 against MERS-CoV but not SARS-CoV may indirectly
suggest an insufficient cross-reactivity with HR2P-M2 against other betacoronaviruses.
Inhibition of Endosomal Acidification
The mouse β-defensins-4 derived P9 (NGAICWGPCPTAFRQIGNCGHFKVRCCKIR) was shown to bind
to the MERS-CoV S2 subunit and remained co-localized with the viruses without inhibiting
viral entry into the host cell via endocytosis (Zhao et al., 2016). While in the endosomes,
the polycationic property of P9 induced a basic microenvironment to prevent endosomal
acidification of the late endosomes. Without endosomal acidification, the pH-dependent
activation of viral fusion proteins to initiate viral-host endosomal membrane fusion
failed to occur and thus the critical step in viral uncoating prior to viral RNA release
(
Figure 1
). This mechanism of action was indeed highly effective, broadly inhibiting SARS-CoV,
MERS-CoV, and a diverse panel of influenza viruses H1N1, H3N2, H5N1, H7N7, and H7N9.
Such target specificity nature of P9 also partly explained the low cytotoxic property
of P9 (IC50 of 380 µg/ml) against the mammalian Madin-Darby canine kidney cells tested.
The broad therapeutic windows of P9 would be a significant advantage in the future
development of P9 as an antiviral agent. Notably, P9 displayed both prophylactic and
therapeutic effects as observed in an in vivo SARS-CoV mouse model of infection (Zhao
et al., 2016).
For the Host: Shielding the Cell Receptors
ACE2 is well-known as the primary receptor for CoV at the first step of viral infection.
It was found that HD5 (ATCYCRTGRCATRESLSGVCEISGRLYRLCCR), a natural lectin-like human
defensins-5 (HD5) peptide secreted by the Paneth cells in the crypts of Lieberkuhn,
could interact with glycosylated proteins and lipid components (Wang C. et al., 2020).
Based on the structural and biochemical properties, HD5 was initially hypothesized
to recognize and inhibit SARS-CoV-2 S protein, host cell ACE2 or both of these target
components (Wang C. et al., 2020). However, it was later confirmed that HD5 competitively
blocked ACE2 receptors on the host cells instead of targeting the viral S1 subunit
(
Figure 1
). The high-affinity binding between HD5 and the ligand-binding domain of ACE2 through
the formation of multiple hydrogen bonds effectively shielded (protected) the host
cells from viral recognition and infection.
Antiviral Immunomodulatory Effect
The cyclic peptide RTD-1 (GFCRCLCRRGVCRCICTR) from rhesus macaque leukocytes was reported
to decrease disease pathogenesis of SARS-CoV infection in mice, as observed with a
substantial reduction in perivascular infiltrate and necrotizing bronchiolitis (Wohlford-Lenane
et al., 2009). Interestingly, neither did RTD-1 inhibit the virus or interact with
the host cell receptors to exert an antiviral effect. In contrast, it was noticed
that the virus titers and lung tissue nucleocapsid (N) gene antigen expression were
similar to the untreated control mice. Together with an increase in cytokine levels
of interleukin-6, keratinocyte chemoattractant, and granulocyte colony-stimulating
factor in lung cell homogenates, RTD-1 was suggested to act as an immunomodulatory
effector molecule via a blunted proinflammatory cytokine response in eliminating SARS-CoV
(Wohlford-Lenane et al., 2009).
Discussion
Prioritization and emergency preparedness in responding to the emerging infectious
diseases associated with outbreaks and pandemics are of utmost urgency. In search
of an effective antiviral against COVID-19, we believe AVPs could represent one of
the potential classes of new antiviral agents against SARS-CoV-2. It is fascinating
to discover how AVPs, composed principally of short and simple amino acid sequences,
could interact with and specifically target the different viral components to achieve
potent antiviral effects. Here, we would like to bring the attention to a number of
AVPs with highly promising anti-CoV activities: mucroporin-M1 disrupts viral envelope,
HR2P-M2 targets the viral S protein-mediated fusion mechanism, EK1 and EK1C4 block
the HR1 domain of viral S2 subunit, and P9 peptide inhibits late endosomal acidification
and thus preventing viral RNA release. Besides, there are AVPs that confer protection
to the host: RTD-1 is an antiviral immunomodulator that triggers protective immunity;
HD5 binds to and shields host ACE2 receptor to prevent viral recognition and attachment.
Recent findings have gathered evidence on the potential role of a host serine protease
TMPRSS2 in facilitating the S protein priming process of SARS-CoV-2 that is crucial
for the subsequent viral membrane fusion events (Hoffmann et al., 2020). Furthermore,
the newly discovered S protein furin-like cleavage site and the novel CD147-mediated
viral entry pathway could have important implications in SARS-CoV-2 viral pathogenicity
(Coutard et al., 2020; Wang K. et al., 2020; Xia et al., 2020a). These key viral components
could represent valuable targets in the development of novel antiviral agents. Cocktail
therapy using a selected combination of AVPs or as supplemental therapeutics in combination
with other classes of antiviral agents could be a promising treatment strategy that
would worth further clinical investigations (Vilas Boas et al., 2019). Such optimism
is not unfounded, as enfuvirtide, a 36-amino acid fusion inhibitor AVP, was approved
by the US Food and Drug Administration in 2003 for treatment against human immunodeficiency
virus in combination with other antiretroviral drugs (Lalezari et al., 2003; Fung
and Guo, 2004; Poveda et al., 2005). It acts by blocking the HR1 domain of the viral
envelope glycoprotein 41. Sifuvertide, an AVP with enhanced potency and a lower threshold
for resistance than enfuvirtide, is currently under phase III trials in China (He
et al., 2008; Wang et al., 2009; Su et al., 2015; Yu et al., 2018). Although clinical
trials of AVPs against CoVs have not been reported thus far, the dire need for an
effective anti-SARS-CoV-2 agent could potentially reshape the drug discovery landscape
as with the initiation of Operation Warp Speed by the US government in April 2020
for COVID-19 vaccine, therapeutic, and diagnostic development (Cohen, 2020). In conclusion,
AVPs are structurally and functionally versatile owing to its simple primary structure
and could serve as the molecular templates for the generation of fast-track therapeutic
candidates in the face of COVID-19 or emerging outbreaks posing severe public health
threats in the unpredictable future. Given its unique and promising antiviral activity,
the potential use of AVPs in clinical treatment and as prophylaxis against COVID-19
should be further explored.
Author Contributions
AM, YL, C-MF, H-SL, and CL conceived the idea. AM and CL wrote the article. YL, C-MF,
and H-SL provided critical comments on the article. AM, YL, C-MF, H-SL, and CL revised
the article. All authors contributed to the article and approved the submitted version.
Funding
The current work was supported by the Fundamental Research Grant Scheme (FRGS), Ministry
of Education Malaysia (MOE), grant number FRGS/1/2018/STG05/UNIM/03/1.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.