TYPE Original Research

PUBLISHED 06 December 2022

DOI 10.3389/fchem.2022.1062352

Therapeutic potential of
OPEN ACCESS compounds targeting
EDITED BY
Mohamed M. Radwan,
University of Mississippi, United States
SARS-CoV-2 helicase
REVIEWED BY
Zhonglei Wang, Matthew T. J. Halma 1,2†, Mark J. A. Wever 3,4†, Sanne Abeln 5,
Qufu Normal University, China
Shizhong Dai,
Didier Roche 4 and Gijs J. L. Wuite 1*
Stanford University, United States 1
Department of Physics and Astronomy, Vrije Universiteit Amsterdam, Amsterdam, Netherlands,
2
*CORRESPONDENCE LUMICKS B. V., Amsterdam, Netherlands, 3DCM, University of Grenoble Alpes, Grenoble, France,
4
Gijs J. L. Wuite, Edelris, Lyon, France, 5Department of Computer Science, Vrije Universiteit Amsterdam, Amsterdam,
[email protected] Netherlands

†
These authors have contributed equally
to this work and share first authorship

SPECIALTY SECTION
This article was submitted to Medicinal The economical and societal impact of COVID-19 has made the development
and Pharmaceutical Chemistry, of vaccines and drugs to combat SARS-CoV-2 infection a priority. While the
a section of the journal
SARS-CoV-2 spike protein has been widely explored as a drug target, the SARS-
Frontiers in Chemistry
CoV-2 helicase (nsp13) does not have any approved medication. The helicase
RECEIVED 05 October 2022
ACCEPTED 25 November 2022 shares 99.8% similarity with its SARS-CoV-1 homolog and was shown to be
PUBLISHED 06 December 2022 essential for viral replication. This review summarizes and builds on existing
CITATION research on inhibitors of SARS-CoV-1 and SARS-CoV-2 helicases. Our analysis
Halma MTJ, Wever MJA, Abeln S, on the toxicity and specificity of these compounds, set the road going forward
Roche D and Wuite GJL (2022),
Therapeutic potential of compounds
for the repurposing of existing drugs and the development of new SARS-CoV-
targeting SARS-CoV-2 helicase. 2 helicase inhibitors.
Front. Chem. 10:1062352.
doi: 10.3389/fchem.2022.1062352
KEYWORDS
COPYRIGHT
© 2022 Halma, Wever, Abeln, Roche SARS-CoV-2, helicase, nsp13, drug repurposing, small-molecule inhibitors, natural
and Wuite. This is an open-access article products, COVID-19
distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or 1 Introduction
reproduction in other forums is
permitted, provided the original
author(s) and the copyright owner(s) are The global coronavirus disease (COVID-19) pandemic is caused by severe acute
credited and that the original respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronaviruses, named after the
publication in this journal is cited, in
accordance with accepted academic similarity of the viral capsid on microscopy to the solar corona (Author anonymous,
practice. No use, distribution or 1968), are widespread and can cause mild infection similar to the common cold. In fact, all
reproduction is permitted which does
four human coronaviruses: HCoV-OC43, HCoV-HKU-1, HCoV-299E, and HCoV-
not comply with these terms.
NL63, are endemic and continuously circulate the human population (Corman et al.,
2018). Three previous coronavirus outbreaks, albeit much smaller than the COVID-19
outbreak, have been reported: SARS-CoV-1, MERS-CoV, and coronavirus HuPn-2018.
Similar to COVID-19, all of these are zoonotic diseases, initially transmitted to humans
via animal hosts (Ye et al., 2020). In contrast to previous outbreaks, COVID-19 has caused
massive disruptions to the lives of virtually every person since the emergence in late 2019.
As of 4 November 2022, COVID-19 has caused 6.60 million deaths globally (Ritchie et al.,
2020). The significant death toll and the impact on society have resulted in large-scale
campaigns to develop vaccines and antivirals to prevent and combat COVID-19.
There should be no doubt about the positive outcomes of this research effort; multiple
vaccines, e.g., AstraZeneca, Moderna, Pfizer/BioNTech, have been developed and

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Halma et al. 10.3389/fchem.2022.1062352

FIGURE 1
Binding sites of SARS-CoV-Nsp13 helicase. Panel (A) Structure of SARS-CoV-Nsp13 helicase (PDB ID: 7NN0) (Newman et al., 2021). V570, the
single different residue from SARS Helicase (I570) is highlighted in red. The residues constituting the ATP binding site are shown in the enlarged
window bound with AMP-PNP, an AMP analog. Panel (B) Possible binding pockets from Nsp13 fragment screening. Reproduced from Newman et al.,
2021 under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

deployed in many countries. The three vaccines mentioned (Andrews et al., 2022). Furthermore, vaccines may be less
all target the SARS-CoV-2 spike protein, either as an mRNA effective or even dangerous for immunocompromised
or inactivated adenovirus vaccine (Dai and Gao, 2021). Concerns individuals (Marra et al., 2022). Moreover, certain individuals
have been raised about the emergence of vaccine-resistant SARS- are allergic to components of vaccines (Cabanillas and Novak,
CoV-2 variants, most notably the BA.4 and BA.5 omicron 2021), and adverse events are being reported (Karlstad et al.,
subvariants (Jian et al., 2022). These strains have mutations 2022). Lastly, with the likelihood of the virus to become, and
in the spike protein, and various sources report higher attack remain, endemic (Lavine et al., 2021), and given the range of
rates and infectivity for these mutants. Vaccine-produced confirmed animal reservoirs of SARS-CoV-2 infection (Prince
antibodies were shown to have less neutralizing potential et al., 2021), a variety of strategies to combat SARS-CoV-
against omicron as compared to alpha- and delta variants 2 infection are required.

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Halma et al. 10.3389/fchem.2022.1062352

1.1 Current antivirals unwinding the RNA secondary structure so that it can be either
replicated by RNA-dependent RNA polymerase or translated by
In the early days of the pandemic, there were no approved the host ribosome.
antiviral compounds against SARS-CoV-2 (World Health
Organization, 2020). This changed in October 2020, when
remdesivir (brand name: Veklury; Gilead Sciences) was 1.2 Drug repurposing
granted emergency use authorization (EUA) by the US Food
and Drug Administration (FDA) for treatment of hospitalized Responding to emerging and pandemic viral illnesses
patients (World Health Organization 2020). Remdesivir was the requires a multifaceted approach, one strategy is drug
only approved medicine until the EUA of molnupiravir (Merck repurposing. Drug repurposing is the use of approved drugs
and Ridgeback) and paxlovid (Pfizer) in December 2021 (U.S. for novel targets and diseases. First, finding a useful medication
Food and Drug Administration, 2021). amongst already existing drugs obviates the need to create novel
The approved drugs have different mechanisms of action; drugs, thus saving time in disease response. Moreover, the side-
remdesivir, a nucleotide analogue, acts by stalling SARS-CoV- effects of marketed drugs, having undergone clinical trials and
2 RNA-dependent RNA polymerase (RdRp) (Kokic et al., 2021). prescribed use, are extensively researched and documented.
Remdesivir exhibited conflicting impact in studies, showing Lastly, the manufacturing process is already known, and needs
improvement in time to recovery in the initial study cited only to be scaled. Drug repurposing has previously found success,
during authorization (Beigel et al., 2020), but later studies for example in sildenafil, an angina medication, that was
showed either no statistically significant effect (Wang et al., successfully repurposed for erectile dysfunction as Viagra ®
2020), or a statistically significant but clinically minor effect (Pushpakom et al., 2019).
(Spinner et al., 2020). Concerns over renal toxicity (Gérard et al., One example of a successfully repurposed and widely
2021; Wu et al., 2022), as well as a cardiac safety signal (Rafaniello available medication for treatment of COVID-19 is
et al., 2021) challenge the safety of the drug. The second drug fluvoxamine, a well-tolerated and selective serotonin reuptake
under EUA, molnupiravir, was approved based on a study inhibitor. Fluvoxamine is commonly used as an antidepressant
showing a reduction in hospitalization and death (Jayk Bernal (Sukhatme et al., 2021). It has been shown to reduce
et al., 2022). Molnupiravir, in addition to remdesivir, targets hospitalization in a large-scale randomized control trial (Reis
RNA-dependent RNA polymerase and increases the frequency of et al., 2022). Being a repurposed drug, fluvoxamine, which was
mutations during SARS-CoV-2 replication (Kabinger et al., first approved by the FDA in 1994 (trade name: Luvox), has the
2021). Concerningly, it has also been shown to induce advantage of decades of safety data surrounding its use. Unlike
mutations in mammalian cells (Zhou et al., 2021). The molnupiravir and paxlovid where a treatment course costs
mechanism of action of molnupiravir is concerning as it has a approximately 700 and 500 USD, respectively (Goswami et al.,
possibility of driving new variants (Kabinger et al., 2021; 2022; Morrison Ponce et al., 2022), fluvoxamine is accessible at
Hashemian et al., 2022), as a result, its use is cautioned by the 4 USD per course (Wang et al., 2021). Remdesivir is also
World Health Organization (World Health Organization, 2022). expensive at over 2000 USD per 5-day treatment course
The third approved antiviral, paxlovid acts as a 3CL protease (Carta and Conversano, 2021). The price and availability of
inhibitor. 3CL protease is necessary for viral replication (Marzi drugs is an important consideration, especially considering
et al., 2022). Paxlovid displays a reasonable safety profile, that developing nations have far lower vaccination rates than
although patients often report a “paxlovid rebound” where developed nations (Bollyky et al., 2020). As of 25 July 2022, 73.2%
there is a resurgence of symptoms, often worse than the initial of EU citizens have completed a full course with an EU-approved
bout (Charness et al., 2022). Moreover, drug-drug interactions vaccine1 and 55.0% have received at least one booster shot
have been shown to cause adverse events (Burki, 2022). Drug (Ritchie et al., 2020). For comparison, in Africa 42.7% of
resistance is also a concern, as mutations have been characterized individuals have been vaccinated and only 2.5% have received
which drastically reduce the effectiveness of paxlovid (Zhou et al., at least one booster shot (Ritchie et al., 2020).
2022). Finally, other concerns shape the adoption of a particular
Depending on the drug target, medication is tailored for pharmacological compound in response to a global pandemic;
different stages in infection. Different proteins can be targeted for these include intellectual property concerns, current and future
therapy depending on the stage of infection. Compounds availability, distribution, and (un)known side-effects. Ultimately,
targeting the spike protein will inhibit entry of SARS-CoV-
2 into cells, whereas compounds targeting RNA-dependent
RNA polymerase will inhibit the replication process, but will
not prevent entry into the cell. Therefore, depending on the
1 i.e., Two doses of Moderna MRNA-1273, two doses of Pfizer-BioNTech
clinical course, certain compounds can be used at different stages BNT162B2, two doses of Oxford-AstraZeneca ChAdOx1 or a single
of infection. The helicase, being a replication protein, is active in dose of Johnson & Johnson Ad26.COV2.S.

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Halma et al. 10.3389/fchem.2022.1062352

an effective treatment of COVID-19 is preferred, that is widely specific tumor backgrounds for hypersensitization of tumors to
available, inexpensive and without significant toxicity. pharmacological inhibition, a concept which is known as
synthetic lethality (Datta and Brosh, 2018).

1.3 SARS-CoV-2 helicase (nsp13)

2 Main considerations
Drug repurposing is mostly a phenotypic approach, meaning
that protein target and mechanism of action are often unknown. In 2.1 Target stability
contrast, target-based approaches seek to first identify protein targets
(chemical biology) and to subsequently develop small-molecule As previously mentioned, SARS-CoV-2 helicase is among the
inhibitors (medicinal chemistry) for the target. In principle, every most conserved proteins in the SARS-CoV-2 genome (Martin et al.,
SARS-CoV-2 protein can be considered a target, but it is preferable 2021; Newman et al., 2021). Throughout the pandemic, it has
to target essential and/or conserved proteins. A previous review has remained largely stable. Phylogenetic evidence demonstrates
already reviewed and postulated the main drug targets for COVID- increasing negative, i.e., purifying, selection over time, making it
19 (Gil et al., 2020), while this report focuses on the helicase of a stable target (Figure 2A). The development of drug resistance is an
SARS-CoV-2. The SARS-CoV-2 nsp13 gene encodes a molecular issue that undermines many treatments, most notably anti-biotics.
motor, which is a 5′ to 3′-translocating helicase, belonging to Under the selection pressure of a drug treatment, the target protein
superfamily 1B. Helicases act on (deoxy)-ribonucleic acid can mutate such that the compound no longer binds (Richman,
substrates and are fueled by (deoxy)-nucleotide triphosphates 1994; Menéndez-Arias and Richman, 2014). It was evaluated
(Figure 1A). The primary functions of helicases are in DNA whether the mutations observed through genomic surveillance of
repair, replication, recombination, and transcription. COVID-19 cases (Kumari et al., 2022) altered the initial protein
Nsp13 is one of the most conserved genes in the SARS-CoV- sequence (Newman et al., 2021). For a drug to retain effectiveness
2 genome, having one of the lowest mutation rates of any of the over time, the major mutations would not alter binding affinity of
essential SARS-CoV-2 proteins (Martin et al., 2021; Newman the drug compounds, thus maintaining drug effectiveness against
et al., 2021). The SARS-CoV-2 helicase differs from the SARS- mutations. Possibly, conservation of structure may enable
CoV-1 helicase by only one amino acid residue, i.e., V570 in production of pan-beta coronaviral inhibitors to guard against
SARS-CoV-2 helicase (Figure 1A, highlighted in red) compared future zoonotic coronaviral outbreaks (Li et al., 2021; Munshi
to I570 in SARS-CoV-1 helicase, allowing drugs discovered for et al., 2022). This possibility is supported by the low level of
SARS-CoV-1 to potentially be re-used. Potential binding pockets nsp13 genetic variation within beta-coronaviruses, as
of Nsp13 were explored via crystallographic fragment screening demonstrated by the phylogenetic tree shown in Figure 2B.
(Figure 1B), presenting a starting point for structure-based drug The technique used here to identify mutations is exploratory,
discovery (Newman et al., 2021). Moreover, the helicase plays a in that the predicted energetic shift was used as a proxy for
critical role in replication of the viral genome (Jia et al., 2019). conformational change. It has been assessed whether there are
The combination of these two argues for the functional any changes likely to significantly impact the structural
importance of SARS-CoV-2 helicase and makes it an conformation of SARS-CoV-2 helicase. If a mutation was near
attractive target for the development of antivirals. This is also a binding site and significantly shifted the energetic stability of
evidenced by an upcoming CACHE challenge2 that aims to the protein, it is likely that the mutation alters compound
discover new molecules that target SARS-CoV-2 helicase. binding. Selection was determined using the toolkit made
The viral helicase is not a new target in drug discovery, for from the GISAID database3, for all SARS-CoV-2 genomes up
example the helicases of herpes simplex virus and hepatitis C to 2 January 2022. In Figure 2C, site selection in terms of fixed-
virus have been targeted, as reviewed by Shadrick et al. (Shadrick effects likelihood (FEL) (Kosakovsky Pond and Frost, 2005) is
et al., 2013). More recent reports feature the helicases of displayed (blue and red stem plots), FEL is a measure of selection
polyomaviruses, Zika virus, and MERS-CoV (Bonafoux et al., pressure in phylogenetic trees and is calculated by comparing the
2016; Kumar et al., 2020; Zaher et al., 2020; Mehyar et al., 2021b). expected number of non-synonymous mutations with the actual
Additionally, human helicases have also attracted research observed rate. In short, observing a higher than expected
interest, and inhibitors for DDX and BLM, among others, frequency of non-synonymous mutations suggests positive
have been reported (Datta and Brosh, 2018). This approach selection, i.e., evolutionary pressure for the protein to change.
aims to use small molecule inhibitors to sensitize cancer cells Observing fewer than expected non-synonymous mutations is
to chemotherapy and DNA-damaging agents and/or to utilize

3 https://observablehq,com/@spond/revised-sars-cov-2-analytics-
2 https://cache-challenge.org/competitions/competition-2. page.

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Halma et al. 10.3389/fchem.2022.1062352

FIGURE 2
Suitability of the nsp13 protein as a drug target. Panel (A) Time course of selection pressures on SARS-CoV-2 helicase from August 2020 to
January 2022. Blue lines show the extent of negative selection, defined as the number of sites under negative selection normalized by kilobase of
gene length and the internal tree length. Red lines how the positive selection force, defined as the number of positively selected sites with the same
normalization. Over the time history, more sites show negative (purifying) selection, suggesting evolutionary stability. Panel (B) Phylogenetic
tree of the coronavirus family based on nsp13 protein sequences. Legend: alpha-CoV (blue), beta-CoV (black), delta-CoV (red), and gamma-CoV
(green). Within the beta-CoVs, there is high nsp13 conservation shown by the short tree lengths. Given the low variance amongst this clade, it may be
possible that a SARS-CoV-2 nsp13 inhibitor also inhibits the other clade members. Panel (C) Energetics and selection on residues in SARS-CoV-
2 nsp13 helicase. Stem plots show positive (red) or negative (blue) selection, expressed as FEL rate. Color plot shows the average energetic change in
kcal/mol of all mutations at the site.

evidence of negative purifying selection, whereby mutants are not selection and an energetically destabilizing impact (upward stems in
likely to survive and reproduce. Figure 2C). These sites should be monitored for development of drug
Additionally, a color plot depicts the average change in energetic resistance and ideally a drug will either act on a different location, or
stability of the protein resulting from the set of possible mutations at the destabilization is significant enough to render the protein non-
that site (Kwasigroch et al., 2002). Most mutations result in a slight functional.
destabilization of the helicase protein, suggesting a high level of
structural optimization. While this makes it less likely that the protein
will develop a drug resistant mutation, it is not certain. Residues 2.2 Current inhibitors: In vitro, in vivo and
where mutations have a destabilizing effect are more likely to alter the in silico assessment
helicase structure, which affects the binding of compounds. The
limitation of this approach is the lack of experimental data to support Having established the validity of the helicase as a drug target,
the generated model. Our assessment shows potentially worrisome multiple methods can be applied for the discovery of inhibitors. In
loci for future drug resistance, where there is a confluence of positive silico screening is experimentally less intense, requiring mostly

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Halma et al. 10.3389/fchem.2022.1062352

computational power. This methodology requires the availability requirement for further experimentation to investigate the
of an X-ray or cryo-EM structures. The crystal structure for SARS- inhibition and selectivity of flavonoids and synthetic analogues
CoV-1 helicase was solved in 2019 (Jia et al., 2019), whereas for thereof on SARS-CoV-1 helicase. Aryl di-keto acids are derived
SARS-CoV-2 helicase structural information was first published in from flavonoids, and were also shown to inhibit SARS-CoV-
2021 (Newman et al., 2021). Earlier in silico research made use of 1 helicase and various other targets, e.g., hepatitis C virus RNA
homology models based on either SARS-CoV-1 or MERS-CoV polymerase (Lee et al., 2009a). Lastly, four compounds (17–20)
helicase to perform molecular modelling studies. Orthogonal to in have been published but there was no information on related
silico, is in vitro, the screening of compounds directly on the compounds. SSYA-10–001 (18) has additionally been reported as
protein of interest. This methodology can be low- (1–100), an inhibitor of hepatitis C virus RNA polymerase and MERS-CoV
medium- (100–10.000) or high- (>10.000) throughput, helicase (Adedeji et al., 2012, 2014).
depending on the equipment used and assay deployed. The
most common in vitro assay performed for helicases is an 2.2.2 SARS-CoV-2 helicase
ATP-turnover assay, there is, however, a high risk for false The first reports on inhibitors of SARS-CoV-2 helicase were
positives, e.g., aggregators or DNA-binders, when running these compounds that have previously been investigated for SARS-CoV-
experiments (McGovern et al., 2003; Acker and Auld, 2014). 1 helicase, namely bismuth complexes (5, 21–24) (Supplementary
Another common in vitro assay for helicase activity is to Table S2, Supplementary Figure S2). Ranitidine Bismuth Citrate (5)
measure the unwound fraction by using a DNA construct with was validated with sub-micromolar helicase and ATPase IC50’s
a double stranded region formed by an annealed oligonucleotide. If (Yuan et al., 2020) and exhibited greater activity compared to
the helicase is active, it will separate the oligonucleotide from the Bismuth (III) tetraphenylpoprhyrinate (23) and Bismuth (III)
construct, and a lighter band will show up on the gel. Form the tetra-4-pyridiylporphyrinate (24). Moreover, 5 relieved virus-
intensity of this band, the unwound fraction and subsequent associated pneumonia in a golden Syrian hamster model.
helicase activity can be calculated (Kim and Seo, 2009). Disulfiram (25) and Ebselen (26) (Supplementary Table S2) are
other Zinc-ejector drugs that have been validated on SARS-CoV-
2.2.1 SARS-CoV-1 helicase 2 helicase (Chen et al., 2021).
The first reports of compounds with SARS-CoV-1 helicase White et al. have identified a hit list of 368 FDA-approved drugs,
activity date back to 2005, when Tanner et al., described a group of from which cepharanthine (27), IC50 = 400 µM and lumacaftor (28),
adamantane-derived bananins (1-4, Supplementary Table S1, IC50 = 300 µM) were confirmed in an ATPase assay (White et al.,
Supplementary Figure S1) with low micromolar ATPase and 2020). Cepharanthine (27) has previously been reported as a SARS-
helicase inhibitor activities (Tanner et al., 2005). These CoV-1 inhibitor, however at the time the target enzyme was not
pyridoxal-conjugated trioxa-adamantanes were shown to be known (Zhang et al., 2005). Vapreotide (29), grazoprevir (30) and
non-competitive inhibitors by DNA- and ATP-competition simeprevir (31) are other FDA-approved drugs discovered by
assays and did not exhibit inhibitory activity on E. coli DnaB phenotypic screening that inhibit SARS-CoV-2 helicase in vitro.
helicase. To the best of our knowledge, compounds 1-4 have not Their activities were confirmed by a DNA-unwinding activity assay
been further investigated. Structurally different Ranitidine with IC50 values of ≈10, ≈2.5, and ≈1.25 µM, respectively (Muturi
Bismuth Citrate (5, Supplementary Table S1) inhibits ATPase et al., 2022). All three compounds have also been reported as virtual
and DNA-duplex unwinding activity, IC50 = 0.3 and 0.6 µM, hits (Borgio et al., 2020; Gurung, 2020). Furthermore, a high-
respectively (Yang et al., 2007b). Compound 5 is the most throughput screening of five thousand known pharmaceuticals by
potent from a series of bismuth complexes (Yang et al., 2007a), Zeng et al., mentions the inhibitory activity of FPA124 (32), IC50 =
whose mechanism of action involves the displacement of Zinc ions 8.5 µM) and suramin (33, IC50 = 0.94 µM). These hits were
from the ATP-binding site (Yuan et al., 2020). Furthermore, confirmed by a fluorescence resonance energy transfer (FRET)
flavonoids have been shown to inhibit SARS-CoV-1 helicase. based helicase assay in the presence of Tween-20. Tween-20 is a
Myricetin (6), baicalein (7), quercetin (8), and scutellarein (9) non-ionic detergent that stops potential colloid formation. Both
all are natural products that inhibit helicase and/or ATPase activity compounds still inhibited helicase activity in this assay at IC50 =
in the low micromolar range (Lee et al., 2009b; Yu et al., 2012; 8.4 µM and 1.1 µM, respectively, and viral inhibition was confirmed
Keum et al., 2013). Flavonoids have been ascribed many potential in vivo on Vero E6 cells (Zeng et al., 2021). SARS-CoV-1 inhibitors
health benefits, including antineoplastic and antiviral. However, myricetin (6) and SSYA-100–01 (18) were used as a comparison in
there have also been multiple reports characterizing flavonoids as these experiments and were confirmed to be active on SARS-CoV-
false positives and protein aggregators in biological assays. 2 helicase. Research from the EXSCALATE4COV (E4C)4 project on
Myricetin (6) has been reported to inhibit many other targets a natural product library once more confirmed the activity of SSYA-
including E. coli DnaB helicase and DNA polymerase (Griep et al.,
2007). The activity of flavonoids on SARS-CoV-1 helicase has
further been validated by the design and synthesis of compounds
10–15 (Lee et al., 2009b; Kim et al., 2011). There is still a 4 www.exscalate4cov.eu.

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TABLE 1 the nine most promising SARS-CoV-2 helicase inhibitors for Abidi et al., 2021; Sundar et al., 2021; Alanazi et al., 2022;
further development and drug repurposing.
Azmoodeh et al., 2022) or natural products (Kousar et al., 2020;
Name (#) Classification Reference Naik et al., 2020; Ahmad et al., 2021; James et al., 2021; Vivek-
Ananth et al., 2021; Bhargavi et al., 2022; Hossain et al., 2022;
Bananin (4) synthetic product Tanner et al. (2005) Samdani et al., 2022). Other published works make use of fragments
Ranitidine Bismuth Citrate (5) pharmaceutical drug Yang et al. (2007b) (Freidel and Armen, 2021) or publicly available compound libraries
Yuan et al. (2020) (Mirza and Froeyen, 2020; García et al., 2021; El Hassab et al., 2022;
Myricetin (6) natural product Yu et al. (2012) Pitsillou et al., 2022). It is recognized that multi-targeted approaches
Zeng et al. (2021) are often carried out, most notably including RNA-dependent RNA
Corona et al. (2022) polymerase and 3CL protease, to have dual-target SARS-CoV-
SSYA10-001 (18) synthetic product Adedeji et al. (2012) 2 inhibitors. The best scoring helicase inhibitors resulting from
Zeng et al. (2021) in silico approach, and without in vitro data, are shown in
Corona et al. (2022) Supplementary Table S3. One particularly large study performed
Disulfiram (25) pharmaceutical drug Chen et al. (2021) ultra-large virtual screening of one billion molecules on fifteen
Vapreotide (29) pharmaceutical drug Borgio et al. (2020) SARS-CoV-2 proteins, for each target the top 1,000 and top one
Muturi et al. (2022) million (0.1%) are publicly available online5 (Gorgulla et al., 2021).
Grazoprevir (30) pharmaceutical drug Gurung (2020) All publications mentioned in this paragraph, however, lack the
Muturi et al. (2022) biological validation that is required to confirm activity. The
FPA124 (32) synthetic product Zeng et al. (2021) occurrence of false positives in virtual screening is still high and
Epirubicin HCl (38) natural product Mehyar et al. (2021b) results do often not translate to in vitro assays, as was recently shown
by Cerón-Carrasco (Cerón-Carrasco, 2022). Thus, it remains critical
to validate ‘virtual’ hits and to refrain from the use of thereof in
100–01 (18) and identified five flavonoids with low micromolar determining structure-activity relationships.
activity: myricetin (6), quercetin (8), kaempferol (34), flavanone (35),
and licoflavone C (36) (Corona et al., 2022). Moreover, Mehyar et al.
(2021a) report on the repurposing of sulphoxide- and sulphone- 2.3 Toxicity analysis
containing FDA-approved compounds. Zafirlukast (37) was the only
compound with inhibitory activity, interestingly 37 was also reported The potential side-effects of any treatment are a concern for
by Zeng et al., 2021), but was not selected for further analysis (Zeng medical practitioners when making a choice of which therapy to
et al., 2021). Mehyar et al. (2021a) also report SARS-CoV-2 helicase implement. Certainly, drugs with minimal off-target toxicity are
inhibitory activity for five previously identified MERS-CoV helicase preferred. While toxicity information exists for some compounds
inhibitors (37–42). Lastly, Newman et al. identified 65 fragments by in the included tables, many have limited application as
crystallographic fragment screening. Although there were no treatments and therefore little associated data on side-effects.
inhibitory values published for these fragments, the crystal Toxicity prediction applies machine learning to chemical
structures show binding in the ATP binding site as well as the structures with known toxicity tests on model organisms.
RNA/DNA-entry tunnel. These crystal structures have been made Based on chemical similarities, the toxicity of untested
publicly available and can be seen as a starting point for fragment compounds can be predicted. Toxicity prediction is a useful
growing (Newman et al., 2021). More recently, Romeo et al. tool for evaluating potential harmful side-effects before taking
identified multiple inhibitors with predicted binding to the RNA/ the drug through costly pre-clinical and clinical trials.
DNA-entry tunnel in vitro. (Romeo et al., 2022). It was not possible to use the same assay or toxicity prediction
Although in vitro and in vivo assays are the gold standard for hit for all compounds. Individual studies often use different assays and
validation, virtual screening allows for rapid identification of ‘virtual’ thus report different values. Additionally, the toxicity prediction
hits. The screening of ultra-large chemical spaces in silico has greatly software was not always successful, and therefore several different
increased the possibilities of modern drug discovery (Warr et al., tools were used: the Quantitative Structure-Activity Relationship
2022), but biological assays are still required to validate these hits. (QSAR) toolbox, developed by the Organization for Economic
Not all laboratories, however, have the means to perform in vitro Cooperation and Development (OECD) (Dimitrov et al., 2016);
assays, thus making molecular modeling a more accessible method the Toxicity Estimation Software Tool (TEST) software developed
for initial target investigation. The SARS-CoV-2 helicase has been by the US Environmental Protection Agency (US-EPA) (Martin
screened, virtually, in many instances (Supplementary Table S3). et al., 2008); and the lazar toxicity prediction web server (Maunz
From our analysis it was observed that most publications have
performed virtual screening on commercially available drugs
(Balasubramaniam and Schmookler Reis 2020; Borgio et al.,
2020; Gurung 2020; Iftikhar et al., 2020; Ugurel et al., 2020; 5 See: https://vf4covid19.hms.harvard.edu/.

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Halma et al. 10.3389/fchem.2022.1062352

FIGURE 3
Structures of the nine most promising SARS-CoV-2 helicase inhibitors for further development and drug repurposing.

et al., 2013). For some compounds, particularly pharmaceutical doses of compounds differ, it is more beneficial to take a selective
drugs, toxicity data was accessible from public documents for their ratio against a toxicity endpoint.
approval by either the FDA or the European Medicines Agency.
Many of the natural products included have long histories of use in
food as well as herbal medicines (Wang and Yang, 2020, 2021; 3 Discussion
Yang and Wang, 2021; Wang et al., 2022). Many are found in
common foods and show strong association with positive health In Supplementary Tables S1,S2,S3, the source information of
outcomes (Kumar and Pandey, 2013), including possible antiviral SARS-CoV-2 inhibitors is found, referring to where the compound
and antineoplastic (Rodriguez-García et al., 2019) properties. Since can be extracted, synthesized or otherwise procured. Three
these products have been consumed for millennia, it is unlikely categories are presented: Natural Products (NP), Synthetic
that they exhibit toxicity, although this may of course be different Products (SP) and Pharmaceutical Drugs (PD). Natural
when the active compound becomes highly concentrated. The products need only be extracted from their source organism,
retrieved experimental toxicities and/or the predicted toxicity typically a plant; pharmaceutical drugs are approved molecules
values for every compound are provided in Supplementary for the treatment of diseases, though some may be off-market.
Tables S1,S2,S3 for the reader’s consideration. For most assays, Synthetic products are typically only produced in very specific
acute toxicity values were reported, this certainly has its drawbacks, contexts, typically a research study. For natural products, the
as compounds may exhibit toxicity at much lower doses. These source organism(s) are indicated, whereas for pharmaceutical
toxicities should not be overly interpreted, since the effective IC50 drugs the tradename and manufacturers are mentioned.

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Halma et al. 10.3389/fchem.2022.1062352

Contrary to natural products and pharmaceutical drugs, synthetic Data availability statement
products often do not yet have a known toxicity profile.
From the compounds in Supplementary Tables S1,S2, the nine The original contributions presented in the study are
most promising compounds for further development are shown in included in the article/Supplementary Material, further
Table 1 and Figure 3. They have been determined based on inquiries can be directed to the corresponding author.
inhibitory activity, number of orthogonal assays and structural
diversity. The first compound, bananin, was discovered, along
with several other related compounds, to inhibit the helicase of Author contributions
SARS-CoV-1 (Tanner et al., 2005). As such, it presents a scaffold on
which lead optimization can be performed. Two other synthetic MH conceptualized the idea and proof outline. MH wrote the
products, SSYA10-001 and FPA124, offer promising scaffolds to target stability and toxicity sections. MW wrote the current
develop into pharmaceutical drugs, should they have a reasonable inhibitors section. MH and MW wrote the manuscript with
biodistribution and safety profile. Ranitidine Bismuth Citrate (RBC) input from all authors. All authors contributed to the article
is a promising compound showing inhibition in helicase unwinding and approved the submitted version.
assays, as well as in vivo activity in a Syrian hamster model (Yuan
et al., 2020). RBC has a higher level of validation than the other
compounds, and its previous use as a pharmaceutical (TRITEC, Funding
GlaxoSmithKline) make it a promising drug for repurposing. Other
pharmaceutical drugs for potential repurposing are disulfiram, This project has received funding from the European Union’s
vapreotide and grazoprevir. These are distinct enough that they Horizon 2020 research and innovation program under the Marie
can be developed as independent scaffolds. Among the natural Skłodowska-Curie grant agreement No 859853.
products, myricetin, has the lowest IC50 (0.41 µM) of flavonoid
compounds against SARS-CoV-2 (Supplementary Table S2). Its
safety, wide use, and availability make it a promising compound for Acknowledgments
development. Another natural product, Epirubicin HCl, is included
for its low IC50 (0.31 µM), while still being distinct enough from We would like to thank Christoph Waldhart and Restuan
myricetin to develop it as a distinct scaffold. Lubis for their help in compiling a chemical list. We would like to
This review summarizes and builds on the work on discovery thank Mohamed Ayaou for discussion and early toxicity
of therapeutics targeting SARS-CoV-2 helicase, a vital replication predictions for this project and Mattijs de Groot for discussion.
protein. We demonstrate that this protein is highly conserved
and resistant to drug-inactivating mutations. Additionally, the
high degree of conservation within the coronavirus family, and Conflict of interest
particularly the beta-coronavirus clade, make coronaviral helicases
attractive targets for future coronaviral outbreaks. The authors declare that the research was conducted in the
We have aimed to provide a complete overview of drugs, absence of any commercial or financial relationships that could
natural products, and synthetic products targeting the SARS- be construed as a potential conflict of interest.
CoV-2 helicase, at several levels of discovery. A broad range of
compounds either computationally predicted to bind to the target
or with higher levels of validation, such as in vitro or even in vivo Publisher’s note
assays, have been covered. Furthermore, a summary of clinical trials
for COVID-19 that involve these compounds can be found as All claims expressed in this article are solely those of the
Supplementary Table S4. Toxicity information on compounds was authors and do not necessarily represent those of their affiliated
provided and predicted for those with absent literature values. organizations, or those of the publisher, the editors and the
Overall, SARS-CoV-2 helicase is an attractive drug target for reviewers. Any product that may be evaluated in this article, or
COVID-19. The potential of immune escape of future SARS-CoV- claim that may be made by its manufacturer, is not guaranteed or
2 strains from the immunity imparted by the current vaccination endorsed by the publisher.
program motivates the development of backup treatment options
(Harvey et al., 2021; Lazarevic et al., 2021). Finally, while vaccines
are a preventive measure, there is still a need for acute therapeutic Supplementary material
interventions, for which there is currently a paucity of options.
Both targeting the SARS-CoV-2 helicase by drug repurposing or The Supplementary Material for this article can be found
new drug discovery may provide acute interventions for COVID- online at: https://www.frontiersin.org/articles/10.3389/fchem.
19 in the future. 2022.1062352/full#supplementary-material

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Halma et al. 10.3389/fchem.2022.1062352

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