Antivirals Against Coronaviruses: Candidate Drugs For Sars-Cov-2 Treatment?

REVIEW
published: 13 August 2020

doi: 10.3389/fmicb.2020.01818
Antivirals Against Coronaviruses:

Candidate Drugs for SARS-CoV-2
Treatment?
Igor de Andrade Santos 1 , Victória Riquena Grosche 1,2 ,
Fernando Rodrigues Goulart Bergamini 3 , Robinson Sabino-Silva 4 and
Ana Carolina Gomes Jardim 1,2*
1
Laboratory of Virology, Institute of Biomedical Science, Federal University of Uberlândia, Uberlândia, Brazil, 2 Institute
of Biosciences, Language and Exact Sciences, São Paulo State University, São José do Rio Preto, Brazil, 3 Laboratory
of Synthesis of Bioinspired Molecules, Institute do Chemistry, Federal University of Uberlândia, Uberlândia, Brazil,
4
Department of Physiology, Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia, Brazil
Coronaviruses (CoVs) are a group of viruses from the family Coronaviridae that can infect
humans and animals, causing mild to severe diseases. The ongoing pandemic of severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a global threat,
urging the development of new therapeutic strategies. Here we present a selection of
Edited by: relevant compounds that have been described from 2005 until now as having in vitro
Anna Kramvis,
and/or in vivo antiviral activities against human and/or animal CoVs. We also present
University of the Witwatersrand,
Johannesburg, South Africa compounds that have reached clinical trials as well as further discussing the potentiality
Reviewed by: of other molecules for application in (re)emergent CoVs outbreaks. Finally, through
Olivier Terrier, rationalization of the data presented herein, we wish to encourage further research
UMR 5308 Centre International
de Recherche en Infectiologie (CIRI), encompassing these compounds as potential SARS-CoV-2 drug candidates.
France
Keywords: antivirals, coronaviruses, COVID-19, SARS-CoV-2, treatment
Maryam Dadar,
Razi Vaccine and Serum Research
Institute, Iran
*Correspondence:
INTRODUCTION
Ana Carolina Gomes Jardim
jardim@ufu.br
Coronaviruses (CoVs) were first identified in 1960 (Kahn and McIntosh, 2005) and were classified
as members of the family Coronaviridae. CoVs are enveloped, single-stranded RNA viruses with
Specialty section: a genome varying from 25 to 32 kb (Payne, 2017). The viral structure is primarily formed by the
This article was submitted to structural spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. The S, M, and
Virology, E proteins are embedded in the viral envelope, which is a lipid bilayer derived from the host cell
a section of the journal membrane. The N protein, on the other hand, interacts with the viral RNA into the core of the
Frontiers in Microbiology virion (Figure 1; Fehr and Perlman, 2015).
Received: 21 April 2020 These viruses can infect vertebrate animals, causing acute to chronic diseases in the respiratory,
Accepted: 10 July 2020 cardiac, enteric, and central nervous systems, both in animals and humans (Weiss and Navas-
Published: 13 August 2020
Martin, 2005). In animals, the most common CoVs are infectious bronchitis virus (IBV), feline
Citation: CoV (FeCoV), and mouse hepatitis virus (MHV), which infect chickens, felines, and rodents,
Santos IA, Grosche VR, respectively (Cui et al., 2019). To date, there are seven known CoVs that cause diseases in humans:
Bergamini FRG, Sabino-Silva R and
HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome
Jardim ACG (2020) Antivirals Against
Coronaviruses: Candidate Drugs
coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and,
for SARS-CoV-2 Treatment? most recently, SARS-CoV-2 (Graham et al., 2013; CDC, 2020a). The CoVs HCoV-229E, HCoV-
Front. Microbiol. 11:1818. NL63, HCoV-OC43, and HCoV-HKU1 cause mild symptoms, similar to a common cold (Payne,
doi: 10.3389/fmicb.2020.01818 2017). However, SARS-CoV, MERS-CoV, and SARS-CoV-2 can cause mild to severe symptoms
Frontiers in Microbiology | www.frontiersin.org 1 August 2020 | Volume 11 | Article 1818

Santos et al. Antivirals Against Human-Animal Coronaviruses
related to upper respiratory infection such as fever, cough, genome is uncoated, and the viral RNA is released. The positive-
dyspnea, pneumonia, and acute respiratory distress syndrome sense RNA viral genome is translated to produce nonstructural
(ARDS), ultimately leading to death (Lai et al., 2020). The proteins (nsps) from two open reading frames (ORFs), ORF1a
severe clinical condition generated especially by SARS-CoV- and ORF1b. The ORF1a encodes the polyprotein pp1a that is
2 has been burdening public health systems worldwide (Hsu cleaved in 11 nsps, while the ORF1b encodes the polyprotein
et al., 2020), evidencing the mandatory need for further research pp1ab, which is cleaved into 15 nsps. The proteolytic cleavage is
encompassing antiviral treatment against CoVs, which has performed by viral proteases nsp3 and nsp5 (Yogo et al., 1977;
somehow, until recently, been relatively ignored by broad phar- Lai and Stohlman, 1981; Kim et al., 2020). The nsps assemble
maceutical and medicinal fields (Lu et al., 2015; Cui et al., 2019). to form a replicase-transcriptase complex (RTC) responsible for
CoVs are linked to a zoonotic transmission due to their RNA synthesis, replication, and transcription of nine subgenomic
ability to infect different species. This can lead to host jumps, RNAs (sgRNAs) (Fehr and Perlman, 2015; Chen W.-H. et al.,
allowing the emergence of new coronaviruses such as SARS- 2020; Kim et al., 2020). The sgRNAs act as mRNAs for structural
CoV, MERS-CoV, and SARS-CoV-2 (Lu et al., 2015; Reusken and accessory genes localized downstream of the replicase
et al., 2016; Andersen et al., 2020). The transmission of CoVs polyproteins. SARS-CoV-2 has six accessory proteins: 3a, 6, 7a,
is based on the fecal-oral route in animals (Kipar et al., 7b, 8, and 10 (Kim et al., 2020). The structural proteins S, E,
2010). In humans, CoV transmission occurs by direct contact and M are translated from the sgRNAs and forwarded to the
with droplets when infected and recipient individuals are in endoplasmic reticulum (ER) and are subsequently inserted into
close contact (about one meter). These infectious oral and an intermediate compartment of ER with Golgi (ERGIC). There,
respiratory droplets produced by talking, coughing, sneezing viral genomes are encapsulated by N proteins and assembled with
need to contact the mucosae (mouth and nose) or conjunctiva the structural proteins to form virions (Siu et al., 2008; Fehr
(eyes) of the recipient person. Additionally, indirect transmission and Perlman, 2015; Li et al., 2020). The M proteins bind to E
can occur by touching a surface with viable CoV and subsequent protein and nucleocapsid, and then, the S protein is incorporated,
contact with mouth, nose, or eyes (van Doremalen et al., forming a complete virion. Finally, the virions are transported to
2020). Viral particles may remain viable on surfaces for several the cell surface in vesicles and released in a pathway mediated by
days, increasing the probability of infection by third parties exocytosis (Figure 2; Fehr and Perlman, 2015; Kim et al., 2020;
(van Doremalen et al., 2020). Li et al., 2020).
Recently, the emergence of SARS-CoV-2 was related to It is important to emphasize that SARS-CoV-2 shows different
zoonotic transmission, but it is still not clear how this virus was epidemiological and clinical features from the epidemics of
first transmitted to humans (Andersen et al., 2020; Gorbalenya SARS-CoV and MERS-CoV (Ceccarelli et al., 2020; Gorbalenya
et al., 2020b). By phylogenetic analysis, the SARS-CoV-2 was et al., 2020a,b). The high transmissibility of SARS-CoV-2 may be
grouped within bat SARS-related coronaviruses, suggesting that related to its entry into host cells (Sun et al., 2020). Although both
a host jump occurred (Cao et al., 2020a; Lai et al., 2020). SARS-CoV and SARS-CoV-2 glycoprotein S attach to ACE2 to
Alarmingly, the high transmissibility of this new CoV allowed the enter the host cells, the binding affinity of SARS-CoV-2 is higher,
rapid and efficient spread of the virus across the world so that it thus enhancing its infectivity (Sun et al., 2020; Yan et al., 2020).
became a pandemic disease in just a few months (CDC, 2020a; Despite the relative homology between S1 and S2 amino acid
Wu et al., 2020). sequences, a 1.2 Å root-mean-square deviation at the 417 position
Due to the novelty of this disease, there is a lack of (Lusvarghi and Bewley, 2016) of S2 protein in SARS-CoV-2 may
understanding of the SARS-CoV-2 replication process in host be related to its higher infectiveness, contributing to a 10- to
cells. The general mechanisms of entry into the host cell, 20-fold higher kinetic affinity of SARS-CoV-2 ectodomain, as
replication, and release follow characteristics that have been evidenced by Wrapp and co-workers, employing surface plasmon
described for other CoVs and have been partially confirmed resonance measurements (Wrapp et al., 2020).
for SARS-CoV-2. To date, it is known that the SARS-CoV-2 Considering the particularities of SARS-CoV-2 and the
virion entries the host cells by the attachment of the S protein emergency caused by its outbreak, several strategies have been
with angiotensin-converting enzyme 2 receptor (ACE2), defining adopted to develop therapeutics and prophylactic measures
SARS-CoV-2 tropism for cells that express this receptor, such against this virus. The strategies employed in these developments
as pulmonary, hepatic, gastrointestinal, and renal human cells include: (i) utilization of bioinformatics for the prediction and
(Chu et al., 2020; Hoffmann et al., 2020; Tai et al., 2020). The investigation of potential ligands toward target molecules in the
interaction of ACE2 with the receptor-binding domain (RBD) viral structure and/or replication (Ahmed et al., 2020, 2; Jeon
of the S protein triggers virion endocytosis and the formation et al., 2020, 2); (ii) employment of cell culture systems, permissive
of an endosome (Rabi et al., 2020). The S protein possesses two to CoVs (Caly et al., 2020; Liu et al., 2020), associated with pseudo
subunits, S1 and S2 (Walls et al., 2020). During endocytosis, particles, subgenomic replicons and/or full-length CoVs, seeking
an acid-dependent proteolytic cleavage of the S1 protein by to assess cellular response or the effects of the compounds on
cellular proteases, like cathepsin, TMPRRS2, and trypsin, exposes the viral replicative cycle (Roberts et al., 2006; Hoffmann et al.,
the S2 subunit, a fusion peptide that allows the fusion of the 2020); (iii) the use of animal models, such as mice, mouse, guinea
viral envelope with the endosome membrane, and consequently, pig, hamster and non-human primates, for evaluating therapeutic
releases the capsid into the cell cytoplasm (Belouzard et al., options or antibody production in immunization (Natoli et al.,
2009; Matsuyama et al., 2020). In the cytoplasm, the CoV viral 2020; Sheahan et al., 2020b), and (iv) clinical trials assessing

FIGURE 1 | Schematic structure of SARS-CoV-2. The viral structure is primarily formed by the structural proteins such as spike (S), membrane (M), envelope (E), and
nucleocapsid (N) proteins. The S, M, and E proteins are all embedded in the viral envelope, a lipid bilayer derived from the host cell membrane. The N protein
interacts with the viral RNA in to the core of the virion.
the administration, distribution, metabolism, and toxicity profiles provide short-term therapies able to reduce the severity of clinical
(ADMeTox) of potential therapeutics as well as immunization outcomes of coronavirus disease 2019 (COVID-19) and to reduce
effects in humans (Clark et al., 2019). the spread of SARS-CoV-2. Here, we summarize compounds
Based in previous results in vaccine development for MERS- described, from 2005 to date, to possess antiviral activity in vitro
CoV and SARS-CoV and the similarity of those viruses with and/or in vivo against CoVs and critically compare molecules
SARS-CoV-2 (Dhama et al., 2020), the current vaccine candidates that could be further investigated by their clinical applicability
are more focused on the S protein, since is a major inducer of (Table 1). We also discuss the compounds that have reached
neutralizing antibodies in infected patients (Walls et al., 2020). clinical trials (Table 2) as well as the potentiality of other
For this reason, efforts are concentrated on using approaches molecules for application in (re)emergent CoVs outbreaks.
such as mRNA, DNA, viral vectors, or virus-like particles vaccines Finally, we aim to encourage further research encompassing these
with a full-length S protein or S1 receptor-binding domain (RBD) compounds as potential SARS-CoV-2 drug candidates.
to stimulate immune response and immunization (Ahmed et al.,
2020; Chen Y. et al., 2020). The most promising vaccines are: (i)
adenovirus-vectored AZD1222 produced by Oxford University INHIBITORS OF THE CoV REPLICATIVE
(Thomas, 2020), a vaccine that is currently in clinical phase 3, CYCLE
being tested in several countries, including the United States,
Brazil, and countries in Asia and Africa; (ii) mRNA-1273 Inhibitors of CoV Entry Into Host Cells
associated with a lipidic nanoparticle (NCT04283461), which The entry of human CoVs into the host cells is mainly related to
is currently in clinical phase 2; and (iii) inactivated virus the binding of viral S protein to the ACE2 receptor (Prabakaran
vaccine, which is currently in clinical phase 1 (Mullard, 2020; et al., 2004; Sun et al., 2020). Therefore, it is reasonable to
Tu et al., 2020). hypothesize that compounds affecting this interaction could be
The high transmissibility and viral variability of the novel potential antivirals (Prabakaran et al., 2004).
SARS-CoV-2, along with the lack of a vaccine or drugs to treat In this context, a survey encompassing in silico studies of more
the infected patients, threaten the global health system. In this than 140 thousand potential S-protein-inhibiting drugs indicated
context, the development of effective antivirals is critical to that the molecule N-(2-aminoethyl)-1 aziridineethanamine

FIGURE 2 | Schematic representation of SARS-CoV-2 replication cycle in host cells. SARS-CoV-2 attaches to the host cells by interaction between the ACE2
receptors and spike proteins. After entry, viral uncoating process results in the release of viral genome and replication stage occurs (translation and transcription).
Structural proteins are produced in intermediate compartment of endoplasmic reticulum with Golgi complex and forwarded to assembly, packaging and virus
release. Compounds with antiviral activity against SARS-CoV-2 are indicated in each step of virus replication cycle.
(NAAE) showed the highest docking grade (-23.7 kcal/mol) (Ramos-Tovar and Muriel, 2019), which was able to inhibit
(Huentelman et al., 2004b). The activity of NAAE was further SARS-CoV entry into Vero cells with an effective concentration
confirmed by employing an in vitro enzymatic inhibitory assay, of 50% (EC50 ) of 300 mg L−1 and a cytotoxicity concentration
using a human recombinant ACE2. In this assay, ACE2 removed of 50% (CC50 ) of >20.000 mg L−1 . GL was less effective
the C-terminal dinitrophenyl moiety that quenched the inherent when the administration occurred during the viral adsorption
fluorescence of the 7-methoxycoumain group, increasing the period than when it was administered after entry into host
fluorescence when ACE2 was active (Huentelman et al., 2004b). cells. Cumulative effects were observed when this compound
The results showed that NAAE inhibited the ACE2 enzymatic was administered both during and after entry into host
activity with the half maximal inhibitory concentration (IC50 ) of cells, which indicates a significantly potent inhibitor against
57 µmol mL−1 (Huentelman et al., 2004b). In addition, 293T the virus under the tested conditions (Cinatl et al., 2003).
cells expressing ACE2 receptor were incubated with NAAE and Additionally, the antiviral activity of 15 GL derivates against
then with S glycoprotein-expressing 293T cells, and measurement SARS-CoV was assessed (Hoever et al., 2005). Conjugation on
of β-galactosidase activity (reported gene in cell-cell fusion) both acidic moieties of the GL disaccharide group with 2-
was performed. NAAE at 0.5 µM inhibited 50% of SARS- acetamido-α-D-glucopyranosylamine, benzylcysteine, and Gly-
CoVs spike protein-mediated cell fusion, suggesting that NAAE Leu peptide generated compounds with an increase of 10-
might be a candidate for treating SARS infection by impairing to 70-fold in anti-SARS-CoV activity when compared to GL
viral attachment via interference with ACE2 (Huentelman et al., itself (Hoever et al., 2005). For the case of 2-acetamido-
2004b). However, a detailed explanation of how NAAE is a α-D-glucopyranosylamine derivative, it was speculated that
more efficient ligand to ACE2 than other compounds was not viral entry was inhibited through N-acetylglycosamine binding
attempted by the authors. onto S-protein carbohydrates. Other derivatives such as the
Ramos-Tovar and Muriel reported the antiviral activity introduction of heterocyclic amides such as 6-amine-thiouracil
of Glycyrrhizin (GL), a major constituent from licorice root induced a higher cytotoxicity profile.

TABLE 1 | Compounds with antiviral activity against human and animal coronaviruses.
Compound Inhibition EC50 or CoVs Advantges and/or limitations References

step inhibition (%)
NAAE Entry 0.5 µM SARS-CoV Synthetic molecule, evaluated in silico, easily produced but Huentelman et al.,
lacks in vivo assays 2004b
Glycyrrhizin Entry 300 mg L−1 SARS-CoV Natural molecule, highly tolerated but lacks in vivo assays Cinatl et al., 2003
2-acetamido-α-D- Entry 40 µM SARS-CoV Semi-synthetic molecule, highly tolerated, and more potent Hoever et al., 2005
Glucopyranosylamine inhibitor but lacks in vivo assays
derivative
Tetrahydroquinoline Entry 273 nM SARS-CoV Synthetic molecule, highly tolerated, easily produced but lacks Shah et al., 2010
oxocarbazate (CID (Cathepsin in vivo assays
23631927) L)
SSAA09E1 Entry 6.7 µM SARS-CoV Synthetic molecule, highly tolerated, easily produced but lacks Adedeji et al., 2013
in vivo assays
in vivo assays
in vivo assays
Emodin Entry and 50 µM SARS-CoV Natural molecule, highly tolerated but lacks in vivo assays Ho et al., 2007;
Post-Entry Schwarz et al.,
2011
Griffithsin (GRFT) Entry 0.16 µg mL−1 HCoV-OC43 Natural molecule, highly tolerated, with a broad-spectrum effect O’Keefe et al.,
(human and animal CoVs); protected against infection and 2010
improved survival in animal assay (Balb/c)
Entry 0.18 µg mL−1 HCoV-229E
Entry 0.61 µg mL−1 SARS-CoV
Entry <0.032 µg mL−1 HCoV-NL63
Entry 0.057 µg mL−1 BCoV
Entry 0.23 µg mL−1 MHV
Eremomycin derivate Entry 5.4 µM FIPV The precursor molecule (Eremomycin) is used to treat bacterial Balzarini et al.,
27 infections; may facilitate clinical assays, but knowledge of the 2006
mechanism of action is lacking
Entry 14 µM SARS-CoV
Eremomycin derivate Entry 12 µM FIPV
39
Entry 22 µM SARS-CoV
Mucroporin-M1 Entry 14.46 µg mL−1 SARS-CoV Synthetic molecule, moderately tolerated, easily produced but Li et al., 2011
lacks in vivo assays
Tyr-Lys-Tyr-Arg-Tyr-Leu Entry 14 mM SARS-CoV Synthetic molecule specifically designed to bind S protein of Struck et al., 2012
SARS-CoV; highly tolerated, does not impair ACE2 activity but
lacks in vivo assays
Entry 14 mM HCoV-NL63
TAPI-2 Entry 65% SARS-CoV Good effects in vitro assays but had no effect on in vivo assays Haga et al., 2010
Monoclonal antibody Entry 0.57 µg mL−1 SARS-CoV-2 Human antibody, specifically to SARS-CoV-2, highly tolerated Wang et al., 2020a
47D11 and easily applicable
AVLQSGFR Replication 2.7 × 10−2 mg mL−1 SARS-CoV Synthetic molecule, highly tolerated, easily produced but lacks Gan et al., 2006
in vivo assays
Phe-Phe dipeptide Replication 0.18 µM SARS-CoV Synthetic molecule, highly tolerated, easily produced but lacks Shie et al., 2005
inhibitor C (JMF1521) in vivo assays
Dipeptidyl EP128533 Replication 3.6 µM or 1.4 µg mL−1 SARS-CoV Synthetic molecule, highly tolerated, easily produced but has Zhang et al., 2006;
contrasting effects in the literature and did not inhibit the virus in Day et al., 2009
in vivo assays
GC373 Replication 0.2 µM HCoV-229E Synthetic molecule, highly tolerated, easily produced, seems to Kim et al., 2012,
interact with SARS-CoV 3CLpro, but there are no in vivo assays 2013
0.3 µM FIPV
2 µM MHV
0.3 µM TGEV
0.7 µM BCV
0.15 µM FCoV-WSU
(Continued)

TABLE 1 | Continued
Compound Inhibition EC50 or CoVs Advantges and/or limitations References

step inhibition (%)
GC376 Replication 0.15 µM HCoV-229E Synthetic molecule, highly tolerated, easily produced, Kim et al., 2012,
seems to interact with SARS-CoV 3CLpro, but there are no 2013
in vivo assays
0.2 µM FIPV
1.1 µM MHV
0.15 µM TGEV
0.6 µM BCV
0.40 µM FCoV-WSU
6-azauridine Replication 32 nM HCoV-NL63 Synthetic molecule, highly tolerated, easily produced, but Pyrc et al., 2006
there are no in vivo assays
2-(benzylthio)-6-oxo-4- Replication NE SARS-CoV Synthetic molecule, highly tolerated, easily produced, but Ramajayam et al.,
phenyl-1,6- there are no in vivo assays 2010
dihydropyrimidine
β-D-N4 -hydroxycytidine Replication 10 µM SARS-CoV Synthetic molecule, highly tolerated, easily produced, and Barnard et al.,
improved pulmonary function and decreased viral load in 2004; Sheahan
lung of infected mice et al., 2020b
Replication 400 nM HCoV-NL63
Replication 0.08–0.3 µM SARS-CoV-2
Replication 0.024 µM MERS-CoV
Ribavirin Replication 20 µg mL−1 SARS-CoV Synthetic molecule, highly tolerated, easily produced, good Saijo et al., 2005;
results in MERS-CoV. However, meta-analyses indicate Barnard et al., 2006
limited efficacy.
Acyclic sugar scaffold of Replication 23 µM MERS-CoV Synthetic molecule, highly tolerated, easily produced, but Peters et al., 2015
acyclovir there are no in vivo assays
8.8 µM HCoV-NL63 Synthetic molecule, highly tolerated, derivate from Acyclovir,
easily produced, but there are no in vivo assays
Niclosamide Replication 0.1 µM SARS-CoV Drug already in use to treat helminthic infections; good Wu et al., 2004;
inhibition in vitro Wen et al., 2007
Mycophenolic acid (MPA) Replication 2.87 µM MERS-CoV Good effects in vitro with MERS-CoV but did not inhibit Cinatl et al., 2003;
SARS-CoV in in vitro and in vivo assays Barnard et al.,
2006; Hart et al.,
2014
TP29 peptide Replication 60 µM MHV Inhibited two species of CoV in mice; also improved survival Wang et al., 2015
and induced INF-I. Inhibited CoV in cell lines. Synthetic
compound designed for nonstructural proteins.
Replication 200 µM SARS-CoV
Bananins Replication <10 µM SARS-CoV Synthetic molecule, highly tolerated, easily produced, but Tanner et al., 2005
there are no in vivo assays
Nitazoxanide Host Enzymes 0.92 µg mL−1 MERS-CoV Drug already in use to treat viral infections; good inhibition Rossignol, 2016
in vitro
Tizoxanide Host Enzymes 0.83 µg mL−1 MERS-CoV Drug derived from Nitazoxanide; good inhibition in vitro Rossignol, 2016
Saracatinib Tyrosine Kinases 2.9 µM MERS-CoV Synthetic molecule, highly tolerated, used to treat Shin et al., 2018
Alzheimer’s disease and easily produced but there are no
in vivo assays
Cyclosporin A (CsA) Hosts Cyclophilin 9–32 µM SARS-CoV, Drug already used to treat several chronic and infectious de Wilde et al.,
Family Enzymes MERS-CoV diseases with broad-spectrum activity among CoVs 2011, 2013;
and MHV Pfefferle et al., 2011
Alisporivir Hosts Cyclophilin 8.3 µM SARS-CoV Analog of CsA and has a strong inhibition in vitro against de Wilde et al.,
Family Enzymes SARS-CoV and other CoVs 2017
Interference RNA (iRNAs) Viral Proteins 70% SARS-CoV Different approach, specific targeting of viral proteins; can Åkerström et al.,
Translation block replication steps and has no cytotoxicity 2007
Viral Proteins 99% SECoV Different approach, specific targeting of viral proteins; can Li et al., 2019
Translation block replication steps and has no cytotoxicity
EC50: effective concentration of 50%; NE: not evaluated.

Frontiers in Microbiology | www.frontiersin.org
Santos et al.
TABLE 2 | Ongoing clinical trials of candidate drugs against SARS-CoV-2 in COVID-19 patients.
Drug Cell culture assays Inhibition step in vitro Animal assays Clinical trials Outcomes in clinical Advantages and/or limitations
trials
Remdesivir Inhibited SARS-CoV, Replication (RdRp) Inhibited EBOV and Clinical case and clinical Did not provide antiviral This is a multicentre, double-blind,
MERS-CoV, and SARS-CoV in both infected trial against SARS-CoV-2 effects or improved clinical placebo-controlled clinical trial, but more
SARS-CoV-2 mice and monkeys outcomes studies might be needed to confirm, since this
includes 255 people, and the drug has some
adverse effects.
Lopinavir and Inhibited SARS-CoV and Replication (protease NE Clinical trial with Did not provide antiviral This drug combination is used for other human
Ritonavir MERS-CoV inhibitor) SARS-CoV-2 effects or improved clinical CoVs, but the study was not multicentre,
outcomes in severe double-blind, and placebo-controlled. More
patients, but, in early studies are necessary to confirm, since it had
infections, clinical only 199 people and the drug showed some
outcomes were improved. adverse effects.
IFN-β Inhibited SARS-CoV, Host Factors (inducing Inhibited SARS-CoV, Clinical trial with Do not have effect alone IFN-β is indicated to be safe, with few adverse
MERS-CoV, MHV, and immune response) MERS-CoV, MHV, and SARS-CoV-2 and is used effects, but in clinical trials, it is only effective
HCoV-229E HCoV-229E for other diseases when associated with other drugs.
Umifenovir Inhibited SARS-CoV NE NE Observational study with 81 Did not provide antiviral This is an observational study and might suffer
patients effects or improved clinical bias from lack and/or loss of information and
outcomes data. It is an applicable study, since it
demonstrates a tendency, and the drug is
already used to treat Influenza viruses.
Corsticosteroids NE Host factors (controlling NE Clinical trial with 454 Reduced death by This is a multicentre, double-blind,
7
(dexamethasone) immune response) treated patients one-third in invasive placebo-controlled clinical trial. More studies
mechanical ventilation are needed to understand better the effect on
patients and one-fifth in different phases of COVID-19. May be a good
oxygen without invasive alternative for treating hyperinflammation and
mechanical ventilation hypersecretion of cytokines.
patients; however, did not
impair mortality in patients
without respiratory support
Ivermectin Inhibited SARS-CoV-2 and Replication (nonstructural NE Clinical trials are beginning NE Ivermectin is safe for use in humans since it is
arboviruses (CHIKV and proteins) used to treat several parasitic infections.
DENV)
Antivirals Against Human-Animal Coronaviruses

Tocilizumab NE Inhibitor of IL-6 NE Ongoing clinical trials with Positive effects: improved Tocilizumab is already used to treat viral
SARS-CoV-2 patients; one inflammatory markers and infections, controlling immune response,
with 100 patients decreased the need for impairing cytokine storms, improving antiviral
August 2020 | Volume 11 | Article 1818
concluded. ventilatory support in response, and providing the best clinical

patients outcomes.
Chloroquine Inhibited HIV, CHIKV, Entry Improved outcomes in Several clinical trials are Impairs virus replication and Chloroquine possesses important side effects
SARS-CoV, and FCoV positive cats being conducted has anti-inflammatory and is indicated only in severe cases. However,
SARS-CoV-2 activities there are some studies with contrasting results
regarding its safety, since it can cause
arrhythmias, hypoglycemia, neuropsychiatric
effects, and depression.
(Continued)
The endossomal cathepsins are essential enzymes in viral entry
compatibility. Antibody titers may interfere with

since it can cause arrhythmias, hypoglycemia,
Administration poses a risk to patients since it
effects in lung and the cardiovascular system.

into host cells (Huang et al., 2006), and cathepsin L has been
is related to donor-dependent variability and

chloroquine treatment, but there are studies
with contrasting results regarding its safety,
outcomes, including when associated with
its activity. In addition, it might cause side

neuropsychiatric effects, and depression.
pointed to as playing a crucial role in membrane fusion with
Hydroxychloroquine improved patients’

the endosomes (Belouzard et al., 2009; Matsuyama et al., 2020).
azithromycin. Less toxic option than

Advantages and/or limitations
In this context, Shah and coworkers demonstrated the effective
activity of tetrahydroquinoline oxocarbazate (CID 23631927),
an oxocarbazate inhibitor of cathepsin L, against SARS-CoV.
Employing a pseudovirus system with a luciferase reporter to
infect 293T cells, the compound inhibited viral entry with an
EC50 of 273 ηM and CC50 > 100 µM (Shah et al., 2010). The
authors also showed that the compound CID 23631927 seems
to bind with a lower inhibition constant (Ki ) to cathepsin L,
improving the compound/cathepsin L interaction. This might
be related to its optimized structure, with stronger hydrophobic
Less toxic option, impairs
Improved outcomes and
interactions and better hydrogen bonds between the compound

Outcomes in clinical
and cathepsin L (Shah et al., 2010).

suppressed viremia.
An extensive study screened a library of compounds

virus replication
following Lipinski’s rule (Lipinski et al., 2001) and identified

three noncytotoxic compounds capable of inhibiting SARS-
trials
CoV pseudoparticle entry into 293T cells (Adedeji et al.,

2013). N-(9,10-dioxo-9,10-dihydroanthracen-2-yl)benzamide
(SSAA09E1) blocked early interactions of SARS-CoV S protein
with ACE2 (EC50 of 6.7 µM and CC50 > 100 µM), whereas
Several clinical trials are
N-[[4-(4-methylpiperazin-1-yl)phenyl]methyl]-1,2-oxazole-5-
being conducted
carboxamide (SSAA09E2) affected cathepsin L activity (EC50 of

Clinical trials
3.1 µM and CC50 > 100 µM). Conversely, [(Z)-1-thiophen-2-

Case report
ylethylideneamino]thiourea (SSAA09E3) prevented the fusion of

the viral envelope with host membrane cells by direct interaction
with spike protein (EC50 of 9.7 µM and CC50 > 20 µM) (Adedeji
et al., 2013). The compound SSAA09E3 presented the highest
cytotoxic, probably due to the interactions with host proteins.
The authors suggested that since these three compounds are
Animal assays
derived from molecules with antiviral activities and presented

good oral bioavailability and rapid systemic distribution in
animal models, they might exhibit interesting pharmacokinetics
(Adedeji et al., 2013).
NE
NE
Other compounds also demonstrated to inhibit CoV entry,

for example, emodin (6-methyl-1,3,8-trihydroxyanthraquinone),
Inhibition step in vitro
a component from Rheum officinale roots, which at 50 µM

inhibited the infectivity of S protein-pseudotype retrovirus from
SARS-CoV in Vero cells by about 80% (Ho et al., 2007).
Besides the entry activity, emodin was described to have an
EC50: effective concentration of 50%; NE: not evaluated.
additional post-entry antiviral action. The authors suggested that

Entry
Entry
emodin might be impairing virus release by affecting 3a viral

protein, which is related to ion channels in infected Vero cells
(Schwarz et al., 2011). This effect may play an important role in
Cell culture assays
immune response.
Inhibited HIV, CHIKV,
The exploitation of other natural compounds such as proteins

SARS-CoV, and
SARS-CoV-2
as potential anti-CoV drugs has also been performed. Griffithsin

(GRFT) is a protein isolated from the red alga Griffithsia sp.
that has shown powerful viral entry inhibition against several
NE
TABLE 2 | Continued
enveloped viruses, such as the human immunodeficiency virus

(HIV). GRFT is capable of binding to terminal mannoses of
oligosaccharides and also to glycans localized on the viral
Convalescent
Hydroxychlo-
envelope glycoproteins (Lusvarghi and Bewley, 2016). GFRT did

roquine
Plasma
not present cytotoxicity in Vero cells, human ileocecal colorectal

Drug
adenocarcinoma cells, human diploid fibroblast cells, and rhesus

monkey kidney cells. Its broad-spectrum antiviral activity in vitro TAPI-2, a compound able to inhibit TACE, has shown potent
was demonstrated against several CoVs such as SARS-CoV antiviral activity, promoting a 65% blockade of SARS-CoV
(EC50 of 0.61 µg mL−1 ), bovine coronavirus (BCoV) (EC50 of entry in HEK-293T cells. However, the compound did not
0.057 µg mL−1 ), MHV (EC50 of 0.23 µg mL−1 ), HCoV-OC43 affect the virus titer in in vivo assays (Haga et al., 2010). The
(EC50 of 0.16 µg mL−1 ), HCoV-229E (EC50 of 0.18 µg mL−1 ), authors suggested that since SARS-CoV attaches to additional
and HCoV-NL63 (EC50 < 0.032 µg mL−1 ) (O’Keefe et al., receptors such as DC-SIGN and L-SIGN (Jeffers et al., 2004;
2010). In another study, GRFT inhibited the early stages of Han et al., 2007), viral entry might be not be impaired
MERS-CoV infection in HEK-293T cells (Millet et al., 2016). by this molecule.
Furthermore, GRFT improved survival in SARS-CoV-infected In addition to amino acid-based inhibitors, monoclonal
mice and protected the Balb/c female mice against infection by antibodies (mAbs) have attracted attention due to their use
binding with S protein (O’Keefe et al., 2010). Altogether, this in infectious and chronic disease treatments (Green et al.,
evidence indicates that GRFT can be considered as a potential 2000; Haynes et al., 2009; Pettitt et al., 2013; D’Amato et al.,
SARS-CoV-2 entry inhibitor with activity against S proteins. 2014), overcoming drawbacks caused in polyclonal Abs therapy,
Antiviral activity by entry inhibition was also evaluated by such as those related to donor compatibility (Marasco and
employing antibacterial chemotherapeutics. Vancomycin, Sui, 2007). Human neutralizing Abs against human CoVs have
eremomycin, and teicoplanin glycopeptide compounds been generated, targeting S glycoproteins to impair viral entry
used to treat infections caused by Gram-positive bacteria (Belouzard et al., 2012; Reguera et al., 2012). Notably, several
(Preobrazhenskaya and Olsufyeva, 2004), as well as hydrophobic mAbs were identified as inhibitors of MERS-CoV and SARS-
derivatives of these drugs, were described to possess antiviral CoV infections both in vitro and in vivo, protecting cells and
activity against HIV (Printsevskaya et al., 2005). A study showed animals when administered 24 h prior to or post-infection
that vancomycin, eremomycin, and teicoplanin were not toxic (Lip et al., 2006; Zhu et al., 2007; Agnihothram et al., 2014;
to Vero and T lymphoblast (CEM) cells. Nonetheless, these Shanmugaraj et al., 2020). The mAbs are developed by merging B
compounds were not able to inhibit feline CoV (FIPV) and lymphocytes and myeloma cells, producing hybridomas capable
SARS-CoV in assays employing such cell lines. Conversely, the of recognizing antigens and producing a single Ab class to
eremomycin derivative molecules labeled 27 and 39 showed bind specific epitopes (Lipman et al., 2005). For that reason,
the best inhibition profiles against FIPV (EC50 of 5.4 and mAb cross-reactivity among different coronaviruses seems to
12 µM, respectively) and SARS-CoV (EC50 of 14 and 22 µM, be ineffective (Totura and Bavari, 2019). In the particular case
respectively) (Balzarini et al., 2006). of SARS-CoV-2, Wang and coworkers produced mAbs using
Cationic antimicrobial peptides (AMPs) are another type 51 lineages of SARS-S hybridoma cells and identified 47D11
of peptides that have been considered as potential broad- H2L2-neutralizing Ab through ELISA assays. This antibody was
spectrum antiviral agents. For instance, mucroporin is an produced using mice cells; therefore, it was further modified to
AMP found in Lychas mucronatus scorpion venom (Dai produce a fully human immunoglobulin IgG1, producing the
et al., 2008). Mucroporin was then optimized synthetically, human monoclonal antibody 47D11. The results showed that
generating mucroporin-M1, which was able to inhibit measles 47D11 bound to the RBD region and inhibited SARS-CoV-
virus (MeV), SARS-CoV, and influenza H5N1. Specifically, 2 entry in Vero cells with an EC50 of 0.57 µg mL−1 (Wang
mucroporin M-1 affected SARS-CoV pseudovirus entry, with et al., 2020a). In this context, this mAb can be used alone or in
EC50 of 14.46 µg mL−1 and CC50 of 61.58 µg mL−1 , by virucidal association with other compounds to treat COVID-19.
activity in HeLa-ACE2 cells (Li et al., 2011). The activity of this
synthetic peptide seems to be related to positive charges of the
hydrophilic site, which can enhance the interaction with the viral Inhibitors of Post-entry Stages of the
surface, inactivating the viral particle. CoV Replicative Cycle
Other potential antiviral peptides were selected by Struck Among the proteins that are pivotal for CoV viral replication
and colleges. Through the exploitation of bioinformatics tools, are the main proteases (Mpro) such as the chymotrypsin-like
the authors were able to predict sixteen peptides with effective protease (3CLpro) and the papain-like proteases (PPL). These
binding onto the receptor-binding domain (RDB) present in S enzymes process viral polyproteins and control replicase complex
proteins of CoVs. These compounds were then synthesized, and activity (Anand et al., 2003), figuring as very attractive targets for
the hexapeptide Tyr-Lys-Tyr-Arg-Tyr-Leu at 14 mM inhibited drug development against CoVs. Several natural products and
SARS-CoV and HCoV-NL63 infection in Vero cells without synthetic peptides have been reported to inhibit Mpro (Cinatl
triggering cytotoxicity (Struck et al., 2012). This peptide was et al., 2005; Vuong et al., 2020).
designed specifically to bind to the site of interaction with S Gan and coworkers used molecular docking methods to select
protein and does not interfere with ACE2 receptor activity, so the octapeptide Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg as Mpro
it might be a good candidate for blocking SARS-CoV-2 entry inhibitor of SARS-CoV and evaluated its antiviral activity in
without impairing host metabolism. Taking into consideration infected Vero cells. The octapeptide presented an EC50 of
that cellular factors such as the Tumor Necrosis Factor-alpha 2.7 × 10−2 mg mL−1 and a CC50 > 100 mg mL−1 , resulting in
(TNF-α) converting enzyme (TACE) facilitate SARS-CoV entry a selectivity index of over 3,704 (Gan et al., 2006). Moreover, five
(Haga et al., 2008), it is reasonable to suggest that TACE Phe-Phe dipeptide inhibitors (A-E) were designed and selected
inhibitors could hinder SARS-CoV infection. In this context, in silico to interact with 3CLpro and showed to be able to protect

Vero cells from the cytopathic effect (CPE) caused by SARS- CC50 > 100 µM) and HCoV-NL63 (EC50 of 400 nM and
CoV. C analog (JMF1521) was obtained by the condensation CC50 > 100 µM) (Barnard et al., 2004). NHC presented a
of Phe-Phe dipeptide unsaturated ester with cinnamic acid and potent antiviral activity against SARS-CoV-2 in infected Vero
exhibited the highest activity, with an EC50 of 0.18 µM and (IC50 of 0.3 µM and CC50 of > 10 µM) and Calu-3 cells (IC50
CC50 > 200 µM (Shie et al., 2005). The authors also performed of 0.08 µM and CC50 > 100 µM) (Sheahan et al., 2020b).
enzymatic assay to evaluate the activity of JMF1521 on 3CLpro The authors assessed the broad-spectrum antiviral activity of
and showed that the peptide inhibited the 3CLpro activity with NHC against MERS-CoV (IC50 0.024 µM) and SARS-CoV
an inhibition constant of 0.52 µM. The results suggested that (IC50 0.14 µM) (Sheahan et al., 2020b) and also evaluated
this analog disposes a rather rigid coplanar structure in the the NHC effect in SARS-CoV- and MERS-CoV-infected mice.
N-terminal motif that results in more effective hydrogen bonds NHC improved pulmonary function and decreased viral load
with the enzyme residues (Shie et al., 2005). in lung, and the authors proposed that NHC might be useful
Another example of a dipeptide-based compound that can for emerging CoVs. Another pyrimidine analog with potential
act as a protease inhibitor is dipeptidyl EP128533 (Zhang et al., antiviral activity is 6-azauridine, which inhibited HCoV-NL63
2006), which showed antiviral activity against SARS-CoV in replication in LLC-MK2 cells with an EC50 of 32 nM and CC50
Vero cells, with EC50 and CC50 values of 3.6 and >100 µM, of 80 µM (Pyrc et al., 2006).
respectively (Zhang et al., 2006). In accordance with that study, it Ribavirin is a synthetic nucleoside analog of guanosine used
was also demonstrated that EP128533 inhibited SARS-CoV with for the treatment of patients chronically infected by the hepatitis
an EC50 of 1.4 µg mL−1 and CC50 > 100 µg mL−1 (Day et al., C virus (HCV) (PubChem, 2005c). The antiviral activities of
2009). However, the compound was not efficient in reducing the ribavirin against several RNA viruses have been described, and
effects of viral replication in BALB/c mice (Day et al., 2009). The it also presents broad-spectrum antiviral activities for CoVs
authors proposed that EP128533 is relatively insoluble and that (Chan et al., 2013; Shen et al., 2016). Its activities were
its lack of activity might be related to a low bioavailability in described for SARS-CoV in vitro (EC50 of 20 µg mL−1 and
the animal models. CC50 > 200 µg mL−1 ) (Saijo et al., 2005). Nevertheless, no viral
The dipeptides GC373 (dipeptidyl aldehyde) and GC376 load reduction was observed in vivo when employing BALB/c
(dipeptidyl bisulfite adduct salt from GC373) were also designed mice (Barnard et al., 2006). The in vitro decrease of ribavirin
and synthesized as protease inhibitors of the 3CLpro enzyme efficacy was demonstrated to be associated with the excision of
(Kim et al., 2012). Their activity was assessed in vitro, and its nucleoside analogs by conserved coronavirus proofreading
the results showed that GC373 inhibited HCoV-229E (EC50 mechanisms (Ferron et al., 2017). Moreover, ribavirin showed
of 0.2 µM), feline infectious peritonitis virus (FIPV, EC50 of good results for the treatment of critical MERS-CoV patients (Al-
0.3 µM), MHV (EC50 of 2 µM), transmissible gastroenteritis Tawfiq et al., 2014), and the combined treatment of ribavirin with
virus (TGEV, EC50 of 0.3 µM), and bovine coronavirus (BCV, type I Interferons (IFN-I) in primate models improved MERS
EC50 of 0.7 µM) (Kim et al., 2012). GC376 also inhibited HCoV- disease symptoms (Falzarano et al., 2013b). Although ribavirin
229E (EC50 of 0.15 µM), FIPV (EC50 of 0.2 µM), MHV (EC50 of has been given as part of treatment regimens for SARS and MERS
1.1 µM), TGEV (EC50 of 0.15 µM), and BCV (EC50 of 0.6 µM). patients, meta-analyses of cases of study have found limited
The 3CLpro activity of these compounds against SARS-CoV was efficacy of its activities in treating patients with highly pathogenic
also analyzed. GC373 and GC376 inhibited enzymatic activity of coronavirus respiratory syndromes (Morra et al., 2018).
SARS-CoV 3CLpro, with inhibition constants of 50% of 3.48 and What is more, a nucleoside analog based on the acyclic
4.35 µM, respectively (Kim et al., 2012). However, the activity sugar scaffold of acyclovir showed antiviral potential against
of these compounds was not evaluated using infected cells or coronaviruses (Tan et al., 2004). Peters and contributors
animal models. Additionally, the effects of GC373 and GC376 demonstrated that this compound has powerful antiviral activity
were assessed against feline coronavirus WSU (FCoV-WSU) against MERS-CoV (EC50 and CC50 of 23 and 71 µM,
(EC50 values for GC373 and GC376 were 0.15 and 0.40 µM, respectively) and HCoV-NL63 (EC50 and CC50 of 8.8 and
respectively) (Kim et al., 2013). Moreover, the authors described 120 µM, respectively) (Peters et al., 2015). However, the authors
that concomitant treatment with these compounds can improve did not suggest mechanisms by which this analog impairs
the antiviral effect against feline coronaviruses and noted that, viral replication, leaving open to question whether it acts like
since the 3CLpro is conserved among CoVs, it might present its precursor acyclovir, impairing viral replication or by an
broad-spectrum activity (Kim et al., 2013). alternative mechanism of action.
RNA-dependent RNA polymerase (RdRp) also figures as a In terms of other drug options for the post-entry stages of
promising target for antivirals. In viral replication, RdRp is the viral replicative cycle, it is possible to report the activities
responsible for catalyzing the replication of the viral RNA using of Niclosamide, a drug used in antihelminthic treatment (Katz,
a complementary RNA as a template. Therefore, compounds 1977). Niclosamide presented antiviral activity on post-entry
that interfere in this process are excellent drug candidates for steps of SARS-CoV infection in Vero cells, with an EC50 of
treating viral infections (Ganeshpurkar et al., 2019). Nucleoside 1–3 µM and CC50 of 250 µM (Wu et al., 2004). Similarly,
analogs of pyrimidine interfere in uridine triphosphate (UTP) this compound suppressed the cytopathic effect of SARS-CoV
metabolism, directly affecting viral replication (Murphy and at a concentration <1 µM and inhibited viral replication with
Middleton, 2012), as demonstrated by β-D-N4 -hydroxycytidine an EC50 value of less than 0.1 µM in Vero E6 cells (Wen
(NHC), which inhibited SARS-CoV (EC50 of 10 µM and et al., 2007). Both authors suggested that Niclosamide impairs

post-entry steps. However, this effect seems to not be related to PI3K/AKT (Thomas and Brugge, 1997), which are strictly related
an interaction with 3CLpro. to CoV infection. Therefore, SFK inhibition might promote
An additional potential compound is mycophenolic acid viral clearance and can be used in association with other drugs
(MPA), an antibiotic derived from penicillium fungal species (Shin et al., 2018).
(PubChem, 2005b), which inhibited MERS-CoV replication in Moreover, Cyclosporin A (CsA), a peptide with activity on
Vero cells with an EC50 of 2.87 µM (Hart et al., 2014). However, the cyclophilin family of host enzymes (isomerases that act as
MPA was not active against SARS-CoV in either in vitro or in vivo chaperones) (PubChem, 2005a; Davis et al., 2010), inhibited
assay (Barnard et al., 2006). The data suggested that MPA inhibits SARS-CoV (100% inhibition at 16 µM), HCoV-229E (75%
the enzyme IMP dehydrogenase, inducing apoptosis on alveolar inhibition at 16 µM), and MHV (100% inhibition at 16 µM) in
macrophages and consequently inhibiting or suppressing cellular human and animal infected cell culture. CsA presented broad-
immune responses that are important for preventing or limiting spectrum antiviral activity against CoVs, and it seems to interfere
viral infection (Barnard et al., 2006). with genome replication/transcription during CoV infections
Bananins, on the other hand, are a class of adamantane-based (de Wilde et al., 2011, 2013; Pfefferle et al., 2011). Alisporivir,
compounds conjugated with a pyridoxal moiety (vitamin B6) a non-immunosuppressive cyclosporin A analog, inhibited the
(Kesel, 2003). These molecules showed effective inhibition of replication of SARS-CoV in Vero E6 infected cells at low-
SARS-CoV in FRhK-4 cells, with EC50 < 10 µM and CC50 of micromolar concentrations (EC50 of 8.3 µM; CC50 > 50 µM).
390 µM. On the basis of both time addition and ATPase assays, This compound also showed broad-spectrum anti-CoV activity,
the authors proposed that the action of bananin is mainly on inhibiting MERS-CoV EMC/2012 (EC50 of 3.6 µM), MERS-CoV
the post-entry step of virus replication and may be related to an N3/Jordan (EC50 of 3 µM), and SARS-CoV MA-15 (EC50 of
effect on the helicase function and/or on components of cellular 1.3 µM) in vitro (de Wilde et al., 2017). However, the authors
pathways (Tanner et al., 2005). demonstrated that Alisporivir did not enhance survival in CoV-
Finally, the nonstructural protein 10 (nsp10) of CoVs was infected mice (de Wilde et al., 2017).
described as being responsible for a stimulatory effect on nsp16, Other biomolecules that are promising as drug antivirals are
a classical S-adenosylmethionine-dependent (nucleoside-2’-O)- interference RNAs (iRNAs). These macromolecules are small
methyltransferase that acts in RNA binding or catalysis. The non-coding RNAs associated with controlling the expression of
peptide TP29 was designed as a ligand to MHV nsp10 and genetic information (Wilson and Doudna, 2013) and have been
presented broad-spectrum activity, inhibiting SARS-CoV (EC50 described as promising candidates for the treatment of hepatitis
of 200 µM) and MHV (EC50 of 60 µM) replication in infected B virus (HBV), HCV, HIV, and human T-cell lymphotropic virus
cell lines (Wang et al., 2015). The authors also assessed TP29 (HTLV) infections (Ma et al., 2007; Shah and Schaffer, 2011;
activity in MHV infected mice and demonstrated that treatment Sanan-Mishra et al., 2017). Short interference RNAs (siRNAs)
improved survival, decreased viral load in liver, and induced were described as being effective for in vitro antiviral treatment
type 1 IFN. Based on these data, it was suggested that TP29 of FIPV, a type of FCoV (McDonagh et al., 2011, 2015). Most
impaired nsp10/nsp16 2’-O-MTase activity, dysregulating the recently, Li and colleagues designed and synthesized siRNAs that
genome replication process. targeted the M and N genes of swine and porcine coronaviruses
(SECoV and PDCoV, respectively). These siRNAs inhibited up
to 99% of the expression of these proteins in both Vero and
Looking Toward Host Machinery: A LLC-PK1 infected cells (Li et al., 2019). Additionally, synthetic
Different Approach to CoV Treatment siRNAs targeting the structural proteins E, M, and N of SARS-
Targeting the host process during viral infection figures as a CoV have also been developed and showed reductions of the
promising alternative for drug development and can play an target gene expressions in Vero cells (Shi et al., 2005). Moreover,
important role in abrogating viral replication (Sayce et al., 2010; siRNAs targeting the structural proteins 7a, 7b, 3a, 3b, and S
Ullah et al., 2019). Nitazoxanide is a broad-spectrum antiviral reduced SARS-CoV progeny in Vero cells by approximately
agent exploited for the treatment of, for instance, influenza A 70% (Åkerström et al., 2007). The different authors propose
and B viruses, as well as Ebola virus (EBOV) (Rossignol, 2014; that treatment with siRNAs can improve treatment-resistance
Jasenosky et al., 2019), with its activity related to the interference among viruses and that these molecules can be designed to target
in host-regulated pathways during viral replication (Rossignol, multiple proteins, aiming at broad-spectrum activity.
2016). In vitro studies demonstrated that Nitazoxanide was
able to inhibit MERS-CoV in LLC-MK2 cells, with an EC50 of
0.92 µg mL−1 . The authors suggested that nitazoxanide affects Ongoing Clinical Evaluations With
pro-inflammatory cytokines and suppresses their overproduction Candidate Drugs Against SARS-CoV-2
(Rossignol, 2016). The current situation of COVID-19 pandemic has accentuated
Another host-target compound is Saracatinib (AZD0530), the urgency of the demand for effective treatments. Based on
a tyrosine kinase (SFK) inhibitor. This compound suppressed previous data concerning activities against other viruses and
the early stages of the MERS-CoV replicative cycle in Huh7 empirical knowledge from treatments used in case reports,
cells (EC50 of 2.9 µM and CC50 > 50 µM), possibly by several drugs have entered clinical trial phases to access their
affecting the SFK pathways (Shin et al., 2018). SFK possesses a therapeutic potential against SARS-CoV-2. In this section, we
central function in signaling pathways such as ERK/MAPK and discuss the current knowledge on the most promising candidates

for the treatment of COVID-19. Data for these drugs are The authors proposed that the low efficacy of Lopinavir with
summarized in Table 2. Ritonavir might be associated with the time of administration,
The nucleoside analog Remdesivir (GS-5734) is a since individuals that were treated at the onset of the disease
monophosphoramidate prodrug that has been described as had improved clinical results (Cao et al., 2020b). Later, it was
having antiviral activity against the EBOV in non-human shown that the association of lopinavir and ritonavir with
primates (Warren et al., 2016, 57). Its activity was assessed in interferon-β1 and ribavirin to treat mild to moderate COVID-19
human airway epithelial (HAE) cells infected with SARS-CoV patients alleviated symptoms and decreased the durations of
(EC50 of 0.069 µM and CC50 > 10 µM) and MERS-CoV (EC50 viral infection and hospital stay (Hung et al., 2020). This might
of 0.074 µM and CC50 > 10 µM) and was demonstrated to be related to their inducing cellular immune response, impairing
inhibit RdRp of these viruses. Also, GS-5734 reduced infectious virus replication.
virus production of bat CoV by 1.5 to 2.0 log10 in HAE cells The type 1 interferons (IFN-I) have also been employed in
and reduced virus titers and virus-induced lung pathologies in a clinical trials. These proteins belong to the cytokine family and
SARS-CoV assay in vivo (Sheahan et al., 2017). This compound are associated with the immune response in viral infections,
also reduced the severity of MERS-CoV disease, virus replication, thus playing major roles in antiviral immunity due to their
and damage in the lungs of rhesus macaques (De Wit et al., 2020). immunomodulatory properties (Samuel, 2001). Therefore, they
The clinical efficacy of GS-5734 has been assessed by several are commonly employed in the treatment of several diseases
clinical trials in different countries like France (NCT04365725), such as Hepatitis C (Kobayashi et al., 1993). There are two
Canada (NCT04330690), and the United States (NCT04292899), subtypes of IFN-I, alpha (IFN-α) and beta (IFN-β) (Samuel,
which have been conducted based on the first reported treatment 2001). IFN-β is associated with more potent activity (Chan
of COVID-19 with Remdesivir in Washington, United States et al., 2015) and is therefore capitalized on in the treatment for
(Holshue et al., 2020). In the first findings from Wang and multiple sclerosis patients (Axtell et al., 2010). Due to its more
coworkers, which were from a randomized, double-blind, potent inhibition profile, it was associated with potent antiviral
multicenter, and placebo-controlled trial with 255 patients, effects against SARS-CoV, MERS-CoV, MHV, and HCoV-229E
Remdesivir did not present significant antiviral effects against in vitro and in vivo (Sperber and Hayden, 1989; Vassão et al.,
SARS-CoV-2, nor did it improve clinical outcomes (Wang et al., 2000; Hensley et al., 2004; Falzarano et al., 2013a; Chan et al.,
2020d). To date, there are several active clinical trials registered 2015). IFN-β, in particular, has a protective effect in endothelial
in the PubMed database involving this compound. However, cells, up-regulating CD73 and consequently stimulating the anti-
most of them presented no conclusive outcomes. inflammatory molecules and maintenance of endothelial barrier
Another two candidates are Lopinavir and Ritonavir, which (Bellingan et al., 2014; Sallard et al., 2020). However, a clinical
are protease inhibitors used in association to treat HIV infections trial with 301 patients showed that this effect was not sufficient
(Cvetkovic and Goa, 2003; Mills et al., 2009). Lopinavir demon- to decrease mortality in SARS patients (Ranieri et al., 2020).
strated antiviral activities, protecting cells from MERS-CoV Therefore, in SARS-CoV-2, IFN-β has been associated with
infection (EC50 of 8 µM) and reducing viral loads in animal other drugs in clinical trials, improving outcomes in COVID-19
assays (de Wilde et al., 2014; Kim et al., 2015). Ritonavir patients as in lopinavir or ribavirin (Hung et al., 2020).
also demonstrated anti-MERS-CoV activities with an EC50 of COVID-19 patients with mild to severe symptoms can develop
24.9 µM (Sheahan et al., 2020a). It is important to point out hyperinflammation and hypercytokinaemia, which can lead to
that these results do not agree with another work that was multiple organ failure and death (Mehta et al., 2020). The
unable to demonstrate in vitro antiviral activity of Lopinavir employment of corticosteroids has shown to be an alternative
against MERS-CoV (Chan et al., 2013). In clinical assays for for overcoming the cytokine storm and hyperinflammation due
MERS-CoV, the association of Lopinavir with Ritonavir reduced to its activities on immune cells (Wilkinson et al., 1991). Such
adverse clinical outcomes and viral load in infected patients a capitalization was previously reported in SARS-CoV patients
(Sheahan et al., 2020a; Yao et al., 2020a). In particular, for during the 2002–2003 epidemic (Chihrin and Loutfy, 2005). For
SARS-CoV, Lopinavir and Ritonavir presented a low to medium SARS-CoV-2, corticosteroids can improve the clinical condition
antiviral activity in vitro, and in vivo assays have not been of patients, reducing hyperinflammation and the development
performed yet (Yao et al., 2020a). In addition, Lopinavir and of ARDS, with faster improvement of symptoms (Wang et al.,
Ritonavir played an important role in the clinical outcome 2020c; Zha et al., 2020). However, contrasting data concerning
of SARS-CoV-infected patients by reducing symptoms and the efficacy of these drugs was described recently, showing
the period of hospitalization, representing a possibility for that corticosteroids did not improve symptoms in COVID-19
the treatment of SARS-CoV-2 (Chu et al., 2004). Cao and patients (Zha et al., 2020). Moreover, dexamethasone emerged
collaborators conducted a randomized clinical trial with 199 as a potential drug for treating COVID-19 patients, as shown
patients with severe COVID-19 (Cao et al., 2020b). Treatment by the results of a randomized, controlled, open-lab, and
of the patients with the association Lopinavir/Ritonavir did not multicenter trial that assessed the effects of dexamethasone in
improve symptoms, nor impaired detectable viral RNA when 454 patients, described to date in pre-print findings (Horby
compared to standard care (supplemental oxygen, noninvasive et al., 2020). Data suggested that dexamethasone reduced death
and invasive ventilation, antibiotic agents, vasopressor support, in one-third of patients in invasive mechanical ventilation
renal-replacement therapy, and extracorporeal membrane and one-fifth of patients in non-invasive oxygen mechanical
oxygenation). Additionally, the treatment generated relevant ventilation. However, it did not impair mortality in patients
adverse effects in some of the patients (Cao et al., 2020b). with no respiratory support (Horby et al., 2020). Other trials

have been conducted, such as NCT043274011, but considering The treatment was observed to cause an effective decrease
the preliminary results, the WHO suggested that treatment in inflammatory markers, radiological improvement, and a
with dexamethasone may be applied during the third phase of reduction in ventilatory support requirements for these patients
COVID-19, when the hyperinflammation is determined, and (Alattar et al., 2020). Additionally, Toniati and collaborators
respiratory support is needed. administered Tocilizumab in 100 patients in Italy (average age
Another antiviral drug assayed toward SARS-CoV-2 is of 62 years old) who had been diagnosed with COVID-19
Umifenovir, a licensed antiviral exploited for the prophylaxis and pneumonia and ARDS and required ventilatory support. Overall,
treatment of influenza viruses (Arbidol), which demonstrated at 10 days of follow-up, the respiratory condition was improved
good pharmacokinetics when absorbed by the organism or stabilized in 77% of the patients, and, based on these data,
(Proskurnina et al., 2020). This drug has an antiviral effect the response to this drug in patients with severe COVID-19
against SARS-CoV in vitro at 50 µg mL−1 (Khamitov et al., was rapid, sustained, and associated with significant clinical
2008). Lian and coworkers coordinated an observational study improvement (Toniati et al., 2020).
with 81 patients with moderate to severe SARS-CoV-2 infection Chloroquine is a 9-aminoquinole that increases the pH in
(Lian et al., 2020) that demonstrated that Umifenovir neither acidic vesicles (Mauthe et al., 2018) and possesses antiviral
shortened the hospitalization period nor improved prognosis in activities against HIV and other viruses (Jacobson et al., 2016;
infected patients (Lian et al., 2020). Al-Bari, 2017). Chloroquine was described as an entry inhibitor
Broad-spectrum drugs used against parasitic infections such of SARS-CoV infection in Vero cells and prevented cell-to-
as Ivermectin (Campbell, 2012; Laing et al., 2017) have also cell spread of the virus (Vincent et al., 2005). Furthermore, it
been investigated due to their antiviral activity against Dengue affected the entry and post-entry stages of the replicative cycle of
virus (DENV), Influenza A viruses, Chikungunya virus (CHIKV), FCoV in Felis catus cells and monocytes. Additionally, an in vivo
and HIV (Tay et al., 2013; Götz et al., 2016; Varghese et al., study in cats demonstrated that treatment with chloroquine
2016; Caly et al., 2020). The activity of Ivermectin is based improved the clinical score of treated groups when compared
on impairing several stages of viral replication, for instance, to the untreated group (Takano et al., 2013). Chloroquine also
interfering with nonstructural proteins (Varghese et al., 2016). had its anti-CoV activities tested in Vero cells (EC50 of 5.47 µM)
Caly and collaborators assessed the effect of Ivermectin on SARS- (Wang et al., 2020b; Yao et al., 2020b). Despite the performance
CoV-2 replication in Vero cells, showing that, at 5 µM, the of chloroquine in vitro, clinical studies conducted in China and
compound presented no toxicity to cells and inhibited up to France showed contradictory clinical data (Chen J. et al., 2020;
99% of viral replication by a possible antiviral effect on viral Chen Z. et al., 2020; Gao et al., 2020; Molina et al., 2020).
release, which is consistent with previous data on its activity Gao and collaborators indicated that chloroquine phosphate was
against other RNA viruses (Tay et al., 2013; Caly et al., 2020). recommended to treat COVID-19-associated pneumonia only
Clinical trials have been conducted in different medical centers during urgent clinical demand because of its antiviral and anti-
in Argentina (NCT04381884), Mexico (NCT04391127), Spain inflammatory activities (Gao et al., 2020). Hydroxychloroquine is
(NCT04390022), and the United States (NCT04374279) to assess an analog of chloroquine that was described as having antiviral
the clinical implications of the use of Ivermectin for COVID-19. activity, inhibiting SARS-CoV-2 in vitro with an EC50 of 0.72 µM
However, to the best of our knowledge, there are no published (Liu et al., 2020; Yao et al., 2020b). In clinical trials, an open-
results on this topic. NCT04343092, a phase 1 clinical trial label non-randomized study by Gautret and colleagues affirmed
in Iraq, was conducted to its completion and evaluated the that hydroxychloroquine reduced symptoms from SARS-CoV-2
efficacy of Ivermectin in COVID-19 patients, so the results might patients and that association with azithromycin could reinforce
be published soon. its effects (Gautret et al., 2020). However, these results have been
According to Guan and colleagues, approximately 15.7% of questioned. The study had a small sample size, and there were
Chinese patients with COVID-19 developed severe pneumonia limitations in the methodologies (Juurlink, 2020).
and cytokine release syndrome (CRS), an important factor Recent studies have been contradicting the safety of
leading to rapid progression of the disease (Chousterman et al., chloroquine and hydroxychloroquine use, as these drugs
2017; Guan et al., 2020). In this context, one of the key cytokines presented severe side effects that interfered with their clinical
involved in infection-induced cytokine storm is interleukin 6 use, even during short-course therapies (Juurlink, 2020; Liu
(IL-6) (Scheller and Rose-John, 2006; Zhang et al., 2020a). et al., 2020). Apart from the mild adverse effects, such as
Tocilizumab is an IL-6 receptor antagonist approved by the pruritus, nausea, and headache, these drugs can predispose
US FDA for the treatment of severe CRS (Grupp et al., 2013) patients to life-threatening arrhythmias, an effect that may be
and figures as an interesting drug to treat the cytokine storm enhanced by concomitant use of azithromycin (Chorin et al.,
caused by SARS-CoV-2 (Zhang et al., 2020b). The treatment of 2020). Both chloroquine and hydroxychloroquine interfere
patients with severe COVID-19 with Tocilizumab presented no with ventricular repolarization, leading to prolongation of
complications in the 21 assisted patients, with an average age of the cardiac QT interval and an increased risk of torsades de
56.8 ± 16.5 and no history of illness deterioration or death. Thus, pointes (TdP), which is a risk especially for patients with cardiac
it immediately improved the clinical outcome and appeared to disease, for children, or for those taking other drugs that delay
be an effective treatment for reducing mortality (Xu et al., 2020). repolarization (Mzayek et al., 2007; Pukrittayakamee et al.,
Another study employing the treatment of COVID-19 patients 2014; Juurlink, 2020; Ursing et al., 2020). Others possible types
with Tocilizumab for 14 days reinforced these observations. of damage are hypoglycemia, even in non-diabetic patients

(Unübol et al., 2011; El-Solia et al., 2018); neuropsychiatric and that could be further exploited for the treatment of human
effects, including agitation, insomnia, confusion, paranoia, and animal CoVs. Furthermore, we described ongoing clinical
depression, psychosis, and suicidal ideation (Mohan et al., 1981); trials for SARS-CoV-2 in order to elucidate the current findings
hypersensitivity reactions, such as severe cutaneous adverse and discussed the relevant features concerning candidate drugs
reactions (Cameron et al., 2014; Girijala et al., 2019); and drug– against SARS-CoV-2.
drug interactions, which are improved by genetic variability As previously mentioned, most human-related CoVs emerged
(genetic polymorphisms of hepatic cytochrome P450 enzyme by zoonotic transmission from animals (Huynh et al., 2012;
2D6 (CYP2D6), responsible for chloroquine metabolization) Coleman and Frieman, 2014; Reusken et al., 2016). Since
(Kirchheiner et al., 2008; Lee et al., 2016). There is a lack of reliable Coronaviridae seem to have a very well conserved genome and
information on target concentrations or doses for COVID-19, structures among their viruses (Huentelman et al., 2004a; Guan
and so doses that proved effective and safe in malaria for both et al., 2012; Yang and Leibowitz, 2015; Madhugiri et al., 2018),
adults and children are considered for the treatment (Smith, it is possible to hypothesize that compounds with antiviral
2020). Recently, the WHO stopped the hydroxychloroquine arm activities against different human and/or animal CoVs (broad-
of the Solidarity trial to treat COVID-19 based on an absence of spectrum activity) could be potential candidates for SARS-CoV-2
effectiveness in reducing the mortality of hospitalized COVID-19 treatment. In a less optimistic scenario, the chemical structures
patients (WHO, 2020c). Besides, the FDA also cautioned against of such compounds and their pharmacological outcomes have
the administration of hydroxychloroquine or chloroquine in the potential to set some light on the drug design of possible
COVID-19 patients, mainly due to the risk of heart rhythm anti-SARS-CoV-2 drugs.
issues (FDA, 2020). From these results, it is evident that the use Among the strategies for drug design, targeting host-immune
of these drugs for COVID-19 requires further investigation. factors or using iRNAs figure as promising alternatives for
An alternative treatment for COVID-19 is the utilization of antiviral drug development. Also, the exploitation of in silico
convalescent plasma (CP) (Chen L. et al., 2020). This treatment studies for drug screening to seek specific targets, as well
refers to plasma therapy based on plasma or plasma derivatives, as for a better comprehension of their interactions with
obtained from donors who were previously infected and have viral biomolecules, has been shown as a promising tool for
developed antibodies. This plasma/derivative is, in its turn, expediting drug development. By narrowing down the number
transfused into individuals with acute SARS-CoV-2 infection of drug candidates, in silico studies have the potential to
(Garraud, 2017; Cao and Shi, 2020). Even though the mechanism avoid the laborious and generally costly synthesis of many of
of action of convalescent plasma therapy is not fully understood, these compounds (Lengauer and Sing, 2006; Villegas-Rosales
it presented great results in the treatment of patients with SARS et al., 2012). Nevertheless, several predicted compounds in the
during the SARS-CoV outbreak in Hong Kong in the early literature have only been screened by in silico and/or interaction
2000s (Cheng et al., 2005). It is possible that the efficacy of CP assays (Chen et al., 2005; Kaeppler et al., 2005; Lee et al., 2005;
therapy is due to the fact that the antibodies from convalescent Kim et al., 2012; Arya et al., 2020; Balasubramaniam and Reis,
plasma might suppress viremia (Chen L. et al., 2020). Duan 2020), which ultimately hinders the proper assessment of the
and colleagues reported CP transfusion to rescue ten severe antiviral activities of the compounds. Therefore, it is imperative
cases of SARS-CoV-2 adult patients. The study showed that that these studies be associated with in vitro and in vivo assays in
one dose (200 mL) of CP significantly increased or maintained order to confirm the predicted activities in biological models and
the neutralizing antibodies at a high level, leading to the also to evaluate pharmacological outcomes (National Research
disappearance of viremia in 7 days. Clinical symptoms rapidly Council (US) Committee on Applications of Toxicogenomic
improved within 3 days, and radiological examination showed Technologies to Predictive Toxicology, 2007). Therefore, this
varying degrees of absorption of lung lesions within 7 days. review encompassed only compounds that have been evaluated
According to these results, CP can also provide a promising by, at least, in vitro models (Table 1).
rescue option for severe COVID-19 (Duan et al., 2020). However, In this context, from the molecules and drugs described as
the author suggested key points to guarantee the effectiveness having in vitro activity, we highlighted the most promising to
of CP therapy: Ab titers and the treatment time point. Firstly, suggest further evaluation using in vivo systems of CoV infection,
taking into consideration previous knowledge from MERS-CoV especially SARS-CoV-2 infection. The compounds are: NAAE,
CP therapy, Abs in plasma donor must have a titer equal or Glycyrrhizin, 2-acetamido-α-D-Glucopyranosylamine derivative,
higher of 1:80 (Ko et al., 2018). This titer is only found in recently Tetrahydroquinoline oxocarbazate (CID 23631927), SSAA09E1,
recovered patients, since antibody levels decrease 4 months after 2 and 3, Emodin, Eremomycin 27 and 29, Mucroporin-M1,
the disease. Secondly, patients receiving CP treatment prior to Monoclonal antibody 47D11, AVLQSGFR, Phe-Phe dipeptide
14 days post-infection responded better than patients treated inhibitor C (JMF1521), GC373 and 376, 6-azauridine, Acyclic
after 14 days (Duan et al., 2020). sugar scaffold of acyclovir, and Bananins. As described above,
these compounds were capable of significantly impairing CoV
infection in cell cultures and might enable important progress
PERSPECTIVES into the treatment of described CoVs as well as viruses that might
be responsible for future viral outbreaks.
This review aimed to summarize and discuss data from the Here, we also described compounds that were evaluated
literature regarding compounds that possess anti-CoVs activities in vivo to elucidate their role in the pathogenesis of CoVs

as well as to assess possible adverse effects. It is important profiles of Chloroquine and Hydroxychloroquine, since they
to emphasize that there is a lack of in vivo model assays, might cause arrhythmia in patients, representing risk for a
representing a delay in anti-CoV drug development, which considerable number of patients (Juurlink, 2020).
directly impacts the SARS-CoV-2 pandemic. Here, we identified Ongoing studies have been evaluating IFN-β and Ivermectin
some studies that employed animal models, such as in Balb/c as treatments against COVID-19. IFN- β can be associated with
mice and C57BL/6, to evaluate the antiviral effect of compounds other drugs, collaborating to control immune response against
in CoV infection (Cinatl et al., 2003; Saijo et al., 2005; Barnard the viral infection (Table 2). On the other hand, corticosteroids,
et al., 2006; Zhang et al., 2006; Day et al., 2009; Hart et al., such as dexamethasone, sound promising, but there are
2014). The in vivo assays allow the gathering of knowledge some issues related to their use. These compounds induce
regarding the ADMeTox profile of these compounds in complex immunosuppression and, when administered during initial
biological systems, the viral titers in different organs, host phases (viral replication), might dysregulate T-cell production
immune responses to the infection, and also potential tissue and activation of B cells for antibody secretion, which are
damage caused by the viruses in the presence or absence of essential for viral clearance (Cohn, 1991; Giles et al., 2018).
candidate drugs, which represents an advance in understanding Furthermore, convalescent plasma therapy is an alternative
pathologies caused by viral infections (Adachi and Miura, approach that presented positive effects in studies on SARS-CoV-
2014). It is also important to emphasize that protocols used 2/COVID-19 patients. However, its safety is not well defined due
in studies of animal-related viruses are not easily translated to donor-dependent variability and compatibility (antibody titers
onto human CoVs, since these viruses are classified to different and other factors vary among donors), which might cause severe
biological safety levels, representing a risk of infection to adverse effects in lung and cardiovascular system and, in some
scientists (Bayot and King, 2020; CDC, 2020b). Additionally, cases, may even transmit diseases (Roback and Guarner, 2020).
the pathologies induced by animal CoVs are mostly related to Despite the finding regarding these drugs, it is important to
gastrointestinal symptoms, differently to what is observed for take some aspects into consideration: i) the trials were generally
human-related CoVs, which mostly affect the upper respiratory conducted with a significant number of patients in each study, but
system (Pedersen et al., 1984; Coleman and Frieman, 2014). The potentially not enough to expand the results to public healthcare;
development of refined and secure protocols to study SARS- ii) some of the studies were observational, which means they were
CoV-2 infection and its treatment options is required. Bearing in based on public data that may not be well documented, leaving
mind the obstacles cited above, assessment of the effect in animal information gaps about particular health issues; additionally, the
models and further translation to humans remains one of the outcomes in patients are defined by their own circumstances,
main challenges. and not by an investigator; iii) some studies were not placebo-
However, some of the studies were able to assess the controlled and double-blind, so the placebo effect cannot be
antiviral effects of some compounds in vivo. The most discarded (Kernan et al., 1999; Hess and Abd-Elsayed, 2019);
relevant compounds we propose that may represent immediate iv) the trials were conducted by selecting a group of COVID-
candidates to clinical trials, considering the urgency of COVID- 19 patients, considering mild, moderate or severe cases, and
19, are Griffithsin (GRFT), β-D-N4 -hydroxycytidine (NHC), different outcomes can be expected in each situation since viral
TP29, Cyclosporin A (CsA), Alisporivir, iRNAs, Saracatinib, load, the progression of the disease, and immune response are
Tizoxanide, Nitazoxanide, Niclosamide, and Ribavirin. These additional factors (Kernan et al., 1999; Hess and Abd-Elsayed,
compounds abrogated CoV infection in vitro and in vivo 2019). Therefore, drugs with no effect in severe cases cannot be
and improved the symptoms and survival of animals. In rejected as a possible treatment in mild to severe cases. When
addition, Saracatinib, Tizoxanide, Nitazoxanide, Niclosamide, these aspects are not considered, the investigators might be open
and Ribavirin are molecules licensed to treat diseases such as to commiting type I or II error in trials (Kernan et al., 1999; Hess
those from viral and helminthic infections or Alzheimer’s disease, and Abd-Elsayed, 2019). For that matter, it is also important to
representing possibilities for clinical trials as repurposed drugs. consider that SARS-CoV-2 is a new virus and that we currently
Regarding clinical trials, most drugs discussed in this review have limited knowledge about its physiopathology. Finally, the
presented adverse effects such as nausea, headache, diarrhea, development of new treatment options is critical, and efforts
urticaria, pathologies related to the gastrointestinal system, have been focused on targeting therapies that aim to improve
and interference with liver enzymes (Ruiz-Irastorza et al., patient outcome by increasing antiviral activity associated with
2010; Takano et al., 2013; Roques et al., 2018; Yao et al., minimal toxicity.
2020a). Remdesivir, Lopinavir and Ritonavir, and Umifenovir Another point to be considered in CoV treatment is that RNA
are drugs employed for the treatment of other viral infections viruses are known to have high levels of mutations (error rate)
such as EBOV and SARS-CoV, but, in the clinical trials with in the replication process (Ganeshpurkar et al., 2019). This can
COVID-19 patients, these treatments did not reduce symptoms result in resistance to antiviral treatment, as observed for HIV,
and/or decrease viral load. Tocilizumab, Chloroquine, and HCV, and Influenza viruses (Laplante and St George, 2014; Li and
Hydroxychloroquine have been demonstrated to inhibit SARS- Chung, 2019; Olearo et al., 2019; Takashita, 2020). A recent study
CoV-2 in vitro and, in some clinical trials, reduced COVID- in pre-print pointed to the genomic variability of SARS-CoV-
19 symptoms, the period of hospitalization, and the viral load 2 and the intra-patient capacity of polymorphic quasispecies,
in patients despite the strong adverse effects of Chloroquine which may offer resistance to antiviral drugs (Karamitros et al.,
(Table 2). Even so, recent studies are contradicting the safety 2020). In addition, previous studies demonstrated that the

use of Chloroquine analogs for decades against malaria has In this context, this review describes drugs that might
established chloroquine-resistant Plasmodium strains (Stocks be overlooked for future analysis and could possibly become
et al., 2002; Al-Bari, 2017; Aguiar et al., 2018). Due to the effective antiviral treatments. As a final remark, we conclude
beneficial immunomodulatory effects of analogs on the severe that, to date, there is no “one hundred percent” effective
inflammatory complications of several viral diseases, such as antiviral therapy against SARS-CoV-2/COVID-19 and that
HIV and SARS-CoV infections, these drugs have been tested further research is needed to achieve the best therapeutic
indiscriminately (Jacobson et al., 2016; Al-Bari, 2017). However, protocol, which may not be based on a unique drug but rather
there is a possibility that prophylactic exposure to pro-apoptotic on a combination of active antivirals.
chloroquine drugs caused natural selection for strains of viruses
and other parasites that have enhanced anti-apoptotic abilities
(Parris, 2004). Despite the side effects, the wide use of some AUTHOR CONTRIBUTIONS
drugs during the SARS-CoV-2 pandemic might raise concerns
regarding the emergence of resistant viral strains in the future, IS: drafting the manuscript and literature review. VG: drafting the
and we emphasize the lack of information on the resistance manuscript and illustration. FB, RS-S, and AJ: critical revision,
associated with these drugs in the treatment of viral infections. editing, and approval of the final version. All of the authors read
and approved the final manuscript.
CONCLUSION FUNDING
The spread of SARS-CoV-2 worldwide is classified as a pandemic The authors received financial support from the FAPEMIG
and represents a threat to global public health. By July 4, 2020, (Minas Gerais Research Foundation APQ-00587-14 – SICONV
SARS-CoV-2 had infected 10,922,324 people and had caused 793988/2013; APQ-02872-16 and APQ-03385-18) and from
523,011 deaths around the world (WHO, 2020b). In this context, CAPES (Coordination for the Improvement of Higher
compounds described to possess antiviral activity against human Education – #1678329P). AJ received a productivity fellowship
and/or animal coronaviruses could provide relevant information from the CNPq (National Counsel of Technological and
for the development of novel SARS-CoV-2 treatments. Herein, Scientific Development – 311219/2019-5). The Brazilian funding
we presented and discussed the most promising compounds that agencies CNPq, CAPES, and FAPEMIG provide financial
can figure as possible candidates for clinical trials. Moreover, support to the National Institute of Science and Technology in
ongoing clinical trials evaluating possible COVID-19 therapies Theranostics and Nanobiotechnology – INCT-Teranano (CNPq-
were also highlighted. 465669/2014-0). RS-S received a fellowship from FAU/UFU and
From what was presented in this review, a plethora of different PrInt CAPES/UFU. AJ, RS-S, and FB are grateful to CAPES –
potential compounds can be capitalized as possible drugs or Prevention and Combat of Outbreaks, Endemics, Epidemics and
even set points for further drug development seeking to mitigate Pandemics - Finance Code #88881.506794/2020-01.
the SARS-CoV-2/COVID-19 outbreak. However, time, resources,
and new experimental protocols are essential for advancing an
efficacious treatment. In addition, and despite the urgency of ACKNOWLEDGMENTS
treatment protocols, it is important to point out the striking
need for the establishment of fail-proof regulatory initiatives that We thank the OSF Preprints database (https://osf.io/preprints/)
could prevent impacts on the healthcare of patients that could, for publishing the pre-print version of this manuscript on May
otherwise, be avoided by a more stringent control. 01, 2020 (https://doi.org/10.31219/osf.io/ycjgq).
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published: 13 August 2020

Antivirals Against Coronaviruses:

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Compound Inhibition EC50 or CoVs Advantges and/or limitations References

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Compound Inhibition EC50 or CoVs Advantges and/or limitations References

EC50: effective concentration of 50%; NE: not evaluated.

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Antivirals Against Human-Animal Coronaviruses

concluded. ventilatory support in response, and providing the best clinical

The endossomal cathepsins are essential enzymes in viral entry

compatibility. Antibody titers may interfere with

Administration poses a risk to patients since it

effects in lung and the cardiovascular system.

is related to donor-dependent variability and

its activity. In addition, it might cause side

Hydroxychloroquine improved patients’

azithromycin. Less toxic option than

Improved outcomes and

interactions and better hydrogen bonds between the compound

and cathepsin L (Shah et al., 2010).

An extensive study screened a library of compounds

following Lipinski’s rule (Lipinski et al., 2001) and identified

CoV pseudoparticle entry into 293T cells (Adedeji et al.,

carboxamide (SSAA09E2) affected cathepsin L activity (EC50 of

3.1 µM and CC50 > 100 µM). Conversely, [(Z)-1-thiophen-2-

ylethylideneamino]thiourea (SSAA09E3) prevented the fusion of

derived from molecules with antiviral activities and presented

Other compounds also demonstrated to inhibit CoV entry,

a component from Rheum officinale roots, which at 50 µM

additional post-entry antiviral action. The authors suggested that

emodin might be impairing virus release by affecting 3a viral

The exploitation of other natural compounds such as proteins

as potential anti-CoV drugs has also been performed. Griffithsin

enveloped viruses, such as the human immunodeficiency virus

envelope glycoproteins (Lusvarghi and Bewley, 2016). GFRT did

not present cytotoxicity in Vero cells, human ileocecal colorectal

adenocarcinoma cells, human diploid fibroblast cells, and rhesus

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