Available via license: CC BY-NC 4.0
Content may be subject to copyright.
Targeting the SARS-CoV-2 Main Protease to Repurpose Drugs for
COVID-19
Erol C. Vatansevera, †, Kai Yanga, †, Kaci C. Kratcha, †, Aleksandra Drelichb, Chia-Chuan
Choa, Drake M. Mellotc, Shiqing Xu,a Chien-Te K. Tsengb, and Wenshe Ray Liua,c,d,*
aThe Texas A&M Drug Discovery Laboratory, Department of Chemistry, Texas A&M
University, College Station, TX 77843, USA
bDepartment of Microbiology and Immunology, University of Texas Medical Branch,
Galveston, TX 77555, USA
bDepartment of Biochemistry and Biophysics, Texas A&M University, College Station, TX
77843, USA
cDepartment of Molecular and Cellular Medicine, College of Medicine, Texas A&M
University, College Station, TX 77843, USA
†Contribute equally to the paper.
*Correspondence should be addressed to Wenshe Ray Liu: wliu@chem.tamu.edu.
Keywords: COVID-19, SARS-CoV-2, bepridil, pimozide, ebastine
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
ABSTRACT
Guided by a computational docking analysis, about 30 FDA/EMA-approved small molecule
medicines were characterized on their inhibition of the SARS-CoV-2 main protease (MPro). Of
these tested small molecule medicines, six displayed an IC50 value in inhibiting MPro below 100
M. Three medicines pimozide, ebastine, and bepridil are basic small molecules that are expected
to exert a similar effect as hydroxychloroquine in raising endosomal pH for slowing down the
SARS-CoV-2 entry into human cell hosts. Bepridil has been previously explored in a high dose as
100 mg/kg for treating diseases. Its high dose use will likely achieve dual functions in treating
COVID-19 by both raising the endosomal pH to slow viral entry and inhibiting MPro in infected
cells. Therefore, the current study urges serious considerations of using bepridil in COVID-19
clinical tests.
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
INTRODUCTION
The current worldwide impact of the COVID-19 pandemic has been so profound that it is often
compared to that of 1918 influenza pandemic.1,2 While this manuscript was under preparation, the
total global COVID-19 cases had surpassed 4 million among which close to 300,000 had
succumbed to death.3 A modelling study has predicted that this pandemic will continue to affect
everyday life and the circumstances may require societies to follow social distancing until 2022.4
Finding timely treatment options are of tremendous importance to alleviate catastrophic damages
of COVID-19. However, the time window that is paramount to contain the disease is extremely
challenging to a conventional drug discovery process that requires typically many years to finalize
a drug and therefore might not achieve its goal before the pandemic ceases. In this January, we did
a comparative biochemical analysis between severe acute respiratory syndrome-coronavirus 2
(SARS-CoV-2), the virus that has caused COVID-19, and SARS-CoV that led to an epidemic in
China in 2003 and proposed that remdesivir was a viable choice for the treatment of COVID-19.5,6
We were excited to see that remdesivir was finally approved for emergency use in the United
States and for use in Japan for people with severe symptoms. With only one medicine in stock
right now, the virus may easily evade it, leading us once again with no medicine to use. Given the
rapid spread and the high fatality of COVID-19, finding alternative medicines is imperative. Drug
repurposing stands out as an attractive option in the current situation. If an approved drug can be
identified to treat COVID-19, it can be quickly proceeded to clinical trials and manufactured at a
large scale using its existing GMP lines. Previously, encouraging results were obtained from
repurposing small molecule medicines including teicoplanin, ivermectin, itraconazole, and
nitazoxanide.7-10 These antimicrobial agents were found effective against virus infections.11
However, a common drawback of all these repurposed drugs is their low efficacy level. One way
to circumvent this problem is to combine multiple existing medicines to accrue a synergistic effect.
To be able to discover such combinations, breaking down the druggable targets of the SARS-CoV-
2 to identify drugs that do not cross-act on each other's targets is a promising strategy. For
example, a recent study showed that triple combination of interferon beta-1b, lopinavir-ritonavir,
and ribavirin was safe and superior to lopinavir-ritonavir alone for treating COVID-19 patients.12
In our January paper, we recommended four SARS-CoV-2 essential proteins including Spike,
RNA-dependent RNA polymerase, the main protease (MPro), and papain-like protease as drug
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
targets for the development of novel anti-COVID-19 medicines. Among these four proteins, MPro
that is also called 3C-like protease (3CLpro) provides the most facile opportunity for drug
repurposing owing to the ease of its biochemical assay. MPro is a cysteine protease that processes
itself and then cleaves a number of nonstructural viral proteins from two polypeptide translates
that are made from the virus RNA in the human cell host.13 Its relatively large active site pocket
and a highly nucleophilic, catalytic cysteine residue make it likely to be inhibited by a host of
existing and investigational drugs. Previous work has disclosed some existing drugs that inhibit
MPro.14 However, complete characterization of existing drugs on the inhibition of MPro is not yet
available. Since the release of the first MPro crystal structure, many computational studies have
been carried out to screen existing drugs in their inhibition of MPro and many potent leads have
been proposed.15-18 However, most of these lead drugs have not yet been confirmed
experimentally. To investigate whether some existing drugs can potently inhibit MPro, we have
docked a group of selected FDA/EMA-approved small molecule medicines to the active site of
MPro and selected about 30 hit drugs to characterize their inhibition on MPro experimentally. Our
results revealed that a number of FDA/EMA-approved small molecule medicines do have high
potency in inhibiting MPro and therefore encourage the possible use of these medicines in fighting
COVID-19.
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
RESULTS & DISCUSSION
Deng et al. released the first crystal structure of MPro on Feb 5th, 2020.14 We chose this
structure (the pdb entry 6LU7) as the basis for our initial docking study. MPro has a very large
active site that consists of several smaller pockets for the recognition of amino acid residues in its
protein substrates. Three pockets that bind the P1, P2, and P4 residues in a protein substrate
potentially interact with aromatic and large hydrophobic moieties.19 Although the P1’ residue in a
protein substrate is a small residue such as glycine or serine, the previous studies based on the
same functional enzyme from SARS-CoV showed that an aromatic moiety can occupy the site that
originally bind the P1’ and P2’ residues in a substrate.20 Based on this analysis of the MPro structure,
we selected 55 FDA/EMA-approved small molecule medicines that have several aromatic or large
hydrophobic moieties inter-connected and did a deep docking analysis of their binding to MPro.
Some of the small molecule medicines used in our docking study were previously reported in other
computational studies.15-18 Autodock was the program we adopted for the docking analysis.21 The
covalent ligand and non-bonded small molecules in the structure of 6LU7 was removed for the
preparation of the protein structure for the docking. Four residues His41, Met49, Asnl42, and
Glnl89 that have shown conformational variations in the SARS-CoV enzyme were set as flexible
residues during the docking process. We carried out a genetic algorithm method with 100 runs to
dock each small molecule medicine to the enzyme. We collected the lowest binding energy from
the total 100 runs for each small molecule medicine and summarized them in Table 1. Among all
55 small molecule drugs that we used in the docking study, 29 showed a binding energy lower
than -8.3 kcal/mol. We chose these molecules to do further experimental characterizations.
To express MPro for experimental characterizations of 29 selected small molecule medicines,
we fused the MPro gene to a superfolder green fluorescent protein (sfGFP) gene and a 6xHis tag at
its 5' and 3' ends respectively in a pBAD-sfGFP plasmid that we used previously in the lab. SfGFP
is known to stabilize proteins when it is genetically fused to them. We designed a TEV protease
cleavage site between sfGFP and MPro for the TEV-catalyzed proteolytic release of MPro from
sfGFP after we expressed and purified the fusion protein. We placed the 6xHis tag right after the
MPro C-terminus. The addition of this tag was for straightforward purification with Ni-NTA resins.
We expected that the TEV protease cleavage of sfGFP would activate MPro to cleave the C-terminal
6xHis tag so that a finally intact MPro protein would be obtained. We carried out the expression in
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
TOP10 cells. To our surprise, after expression there was a minimal amount of the fusion protein
that we were able to purify. The analysis of the cell lysate showed clearly the cleavage of a
substantial amount of MPro from sfGFP. Since we were not able to enrich the cleaved MPro using
Ni-NTA resins, the C-terminal 6xHis tag was apparently cleaved as well. TEV protease is a
cysteine protease that cleaves after the Gln residue in the sequence Glu-Asn-Leu-Tyr-Phe-Gln-
(Gly/Ser).22 MPro is known to cleave the sequence Thr-Val-Leu-Gln-(Gly/Ser).23 The two cleavage
sites share a same P1 residue. It was evident in our expression work that MPro is able to efficiently
cleave the TEV protease cutting site to maturate inside E. coli cells. It is likely that MPro has a
substrate promiscuity higher than what we have learnt from the SARS-CoV enzyme. This aspect
needs to be investigated further to help understand the virus replication inside host cells. To purify
the cleaved and maturated MPro, we used ammonium sulfate to precipitate it from the cell lysate
and then used the ion exchange and size exclusion chromatography to isolate it to more than 95%
purity. We designed and synthesized a fluorogenic coumarin-based hexapeptide substrate (Sub1)
and a FRET-based decapeptide substrate (Sub2) and acquired a commercial FRET-based
tetradecapeptide substrate (Sub3) (Figure 1A). The test of enzyme activities on the three substrates
indicated that the enzyme had low activity toward Sub1 under our assay conditions (Figure 1B)
and its activity on Sub3 was higher than that on Sub2 (Figure 1C). Sub3 was subsequently used in
all following inhibition analysis. To identify an optimal enzyme concentration for use in our
inhibition analysis, we tested activities of different concentrations of MPro on 10 µM Sub3, the
detected catalytic rate of the Sub3 cleavage was not proportional to the enzyme concentration
(Figure 1D). When the enzyme concentration decreased from 50 nM to 10 nM, the Sub3 cleavage
rate dropped roughly proportionally to the square of the concentration decrease, characteristics of
second-order kinetics. This observation supports previous claims that the enzyme needs to
dimerize in order to be active. In all the following assays, 50 nM MPro and 10 µM Sub3 were used
throughout.
We purchased all 29 small molecule medicines from commercial providers without further
purification and characterization. Rupintrivir is a previously developed 3C protease inhibitor.24 It
has a key lactone side chain in the P1 residue that has a demonstrated role in tight binding to 3C
and 3CL proteases. Since it has been an investigational antiviral medicine, we purchased it as well
with a hope that it could be a potent inhibitor of MPro. We dissolved most purchased small molecule
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
medicines in DMSO to make 5 mM stock solutions and proceeded to use these stock solutions to
test inhibition on MPro. Except itraconazole that has low solubility in DMSO, all tested small
molecule medicines were diluted to a 1 mM final concentration in the inhibition assay conditions.
We maintained 20% DMSO in the final assay condition to prevent small molecule medicines from
precipitation. The activity of MPro in 20% DMSO was a little lower than that in a regular buffer
but satisfied our assay requirement. An MPro activity assay in the absence of a small molecule
medicine was set up as a comparison. Triplicate repeats were carried out for all tested small
molecules and the control. The results presented in Figure 2 displayed two easily discernable
characteristics. First, about half of the tested compounds showed strong inhibition of MPro at the 1
mM concentration level (itraconazole was at 0.14 mM due to its low solubility in DMSO),
supporting the practical use of a docking method in guiding the drug repurposing research of
COVID-19. Second, several small molecule medicines including fexofenadine, indinavir,
pirenzepine, reboxetine, and doxapram clearly activated MPro (> 15%). This was to the contrary of
what the docking program predicted. This observation strongly suggests that frontline clinicians
need to exhibit caution in repurposing medicines for COVID-19 patients before they are
thoroughly investigated on influencing the SARS-CoV-2 biology. A not-well-understood drug
might deteriorate the already devastating symptoms in COVID-19 patients. Although it is not the
focus of the current study, the observation that MPro can be activated by existing drugs needs to be
further investigated.
We selected 17 small molecule medicines and rupintrivir that displayed strong inhibition of
MPro to conduct further characterizations of their IC50 values in inhibiting MPro by varying the small
molecule concentration from 1 µM to 10 mM. Results collectively presented in Figure 3 identifies
that of the 18 tested compounds, 7 had an IC50 value below 100 µM. These include pimozide,
ebastine, rupintrivir, bepridil, sertaconazole, rimonabant, and oxiconazole. Pimozide, ebastine, and
bepridil were the three most potent FDA/EMA-approved medicines with IC50 values as 42 ± 2, 57
± 12 and 72 ± 12 µM, respectively. Although rupintrivir is a covalent inhibitor that was developed
specifically for 3C and 3CL proteases, its IC50 value (68 ± 7 µM) is higher than that of pimozide
and ebastine. The relatively low activity of rupintrivir in inhibiting MPro might be due to the change
of the amide bond between the P2 and P3 residues to an methyleneketone. This conversion served
to increase the serum stability of rupintrivir, but has likely eliminated a key hydrogen bonding
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
interaction with MPro.14 The repurposing of HIV medicines for the treatment of COVID-19,
particularly those targeting HIV1 protease, has been area of much attention. In fact, the cocktail of
lopinavir and ritonavir was previously tested in China for the treatment of COVID-19.25 The IC50
value of lopinavir in inhibiting MPro is about 500 µM, which possibly explains why this anti-HIV
viral cocktail demonstrated no significant benefit for treating patients. Nelfinavir was previously
shown having high potency in inhibiting the entry of SARS-CoV-2 into mammalian cell hosts. A
cell-based study in inhibiting the SARS-CoV-2 entry indicated a 1 µM EC50 value.26,27 However,
our IC50 determination against MPro resulted in a value of 234 ± 5 µM, highlighting that nelfivavir
likely inhibits another key SARS-CoV-2 enzyme or protein or interferes with key cellular
processes that are required for the SARS-CoV-2 entry into host cells. These possibilities need to
be studied further. Structurally the two most potent medicines pimozide and ebastine share a same
diphenylmethyl moiety. A spatially similar structure moiety N-phenyl-N-benzylamine exists in
bepridil. Our docking results suggested a same binding mode for this similar structure moiety in
all three drugs (Figure 5). The two aromatic rings occupy the enzyme pockets that associate with
the P2 and P4 residues in a substrate. This observation is in line with a crystallographic study that
showed two aromatic rings with a single methylene linker bound to the active site of the SARS-
CoV enzyme.28 We believe that the inclusion of the diphenylmethyl moiety in structure-activity
relationship studies of MPro-targeting ligands will likely contribute to the identification of both
potent and high cell-permeable MPro inhibitors. Figure 4 also revealed large variations in Hill
coefficients of IC50 curves for different small molecule medicines (IC50 values and Hill coefficients
are summarized in Table 2). Duloxetine and zopiclone gave the two highest Hill coefficients with
a gradual MPro activity decrease over an increasing inhibitor concentration. On the contrary,
saquinavir and lopinavir yielded lowest Hill coefficients with highly steep IC50 curves. There are
three possible explanations for the large discrepancy in Hill coefficients. It could be attributed to
different solubility of tested compounds. It is possible that a high DMSO percentage and a
relatively high inhibitor concentration created phase transition for some inhibitors.29 A high Hill
coefficient may also be due to different ligand to enzyme ratios when tested compounds bind to
MPro. An additionally possible reason is the co-existence of the MPro dimer and monomer in the
assay conditions. A previous report showed a Kd value of the MPro dimerization as 2.5 µM.19 In
theory, the catalytically active dimer species was present at a very low concentration in our assay
conditions, leaving the catalytically inactive monomer species as the major form of MPro. In this
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
situation, the inhibitors that preferentially bind to the MPro dimer and the inhibitors that have a
higher affinity to the MPro monomer might yield different Hill coefficients.
From cell biology point of view, our three lead compounds share some similarities with some
proposed COVID-19 treatment options. There are reports on the investigation of using
hydroxychloroquine to treat COVID-19 patients.30,31 A likely mechanism of action for
hydroxychloroquine is its ability to raise endosomal pH that impacts significantly activities of
endosomal proteases that may be required to process the virus membrane proteins.32,33 Our top
three hits pimozide, ebastine, and bepridil are all basic small molecules that can potentiate a similar
effect.34 Among the three drugs, bepridil can be very interesting because it was previously used at
a dose of 12 mg/kg for the treatment of Ebola virus infections.35 In another study, bepridil was
administrated at a dose of 100 mg/kg for managing atrial arrhythmias, indicating that it has a very
low level of cellular toxicity.36 Moreover, a previous study showed that bepridil can increase the
pH of acidic endosomes.37 Administration of a high dose of bepridil is expected to have dual
functions to slow down the virus replication in host cells by both inhibiting MPro and raising the
pH of endosomes. Therefore, we urge clinical tests of bepridil in the treatment of COVID-19. The
same use of pimozide and ebastine needs to be explored as well.
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
CONCLUSION
Guided by a computational docking analysis, we experimentally characterized about 30
FDA/EMA-approved drugs on their inhibition of the essential MPro enzyme of the COVID-19
pathogen SARS-CoV-2. From the study, we identified six FDA/EMA-approved drugs that can
potently inhibit MPro with IC50 value lower than 100 µM. Three lead drugs pimozide, ebastine, and
bepridil are basic molecules. They are expected to exert a similar effect as hydroxychloroquine in
raising endosomal pH as well as provide inhibition to MPro for slowing down the SARS-CoV-2
entry and replication in human cell hosts. Given that bepridil has been previously explored to treat
Ebola infected patients, we urge a serious consideration of its clinical tests in treating COVID-19.
The anti-COVID-19 use of pimozide and ebastine may be explored clinically as well. Our current
study indicates that there is a large amount of FDA/EMA-approved drug space open for
exploration that could hold promise for repurposing existing drugs to target COVID-19.
Performing screening studies on different SARS-CoV-2 protein targets are necessary to unocver
existing medicines that may be combined for cocktail treatments of COVID-19. More explorations
in this direction are imperative.
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
MATERIALS AND METHODS
Chemicals
We purchased econazole nitrate, duloxetine hydrochloride, doxapram hydrochloride
monohydrate, clemastine fumarate salt, sertaconazole nitrate, isavuconazole, rupintrivir, and
zopiclone from Sigma-Aldrich, pimavanserin, trihexyphenidyl hydrochloride, reboxetine
mesylate, sertindole, bepridil hydrochloride, darunavir, nelfinavir mesylate, indinavir sulfate,
lopinavir, tipranavir, saquinavir, pirenzepine hydrochloride, oxiconazole nitrate, pimozide, and
rimonabant from Cayman Chemicals, ebastine and itraconazole from Alfa Aesar, metixene
hydrochloride hydrate and lemborexant from MedChemExpress, fexofenadine hydrochloride from
TCI Chemicals, ketoconazole from Acros Organics, clotiapine from Enzo Life Sciences, and
oxyphencyclimine from Boc Sciences. We acquired Sub3 with the sequence as DABCYL-Lys-
Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu-EDANS from Bachem Inc.
Docking
Autodock 4 was used for all docking analysis. For each small molecule, the genetic algorithm-
based calculation was carried out for 100 runs with each run having a maximal number of
evaluations as 2,500,000.
Mpro Expression and Purification
We constructed the plasmid pBAD-sfGFP-Mpro from pBAD-sfGFP. The MPro gene was
inserted between DNA sequences that coded sfGFP and 6xHis. The overall sfGFP-MPro-6xHis
fusion gene was under control of a pBAD promoter. The antibiotic selection marker was
ampicillin. To express sfGFP-MPro-6xHis, E. coli TOP10 cells were transformed with pBAD-
sfGFP-MPro. A single colony was picked and grew in 5 mL LB medium with 100 µg/mL ampicillin
overnight. In the next day, we inoculated this starting culture into 5 L 2xYT medium with 100
µg/mL ampicillin in 5 separate flasks at 37 °C. When the OD reached to 0.6, we added L-arabinose
(working concentration as 0.2%) to each flask to induce protein expression at 37 °C for 4 h. Then,
the cells were pelleted at 4000 rpm at 4 °C, washed with cold PBS and stored in -80 °C until
purification. To purify the expressed protein, we re-suspended frozen cells in a 125 mL buffer
containing Tris pH 7.5, 2.5 mM DTT, and 1.25 mg lysozyme. We sonicated resuspended cells
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
using a Branson 250W sonicator with 1 second on, 4 second off, and a total 5 min 60% power
output in two rounds. After sonication, we spun down the cellular debris at 16000 rpm for 30 min
at 4 °C. We collected the supernatant and recorded the volume. The whole cell lysate analysis
showed almost all of the fusion protein was hydrolyzed to two separate proteins sfGFP and MPro.
We were able to obtain an insignificant amount of MPro when Ni-NTA resins were used for
purification. Therefore, we did ammonium sulfate precipitation of the whole cell lysate method.
This was done by the addition of a saturated ammonium sulfate solution at 0 °C. We collected the
fraction between 30% and 40% of ammonium sulfate. We dissolved the collected fraction in buffer
A (20 mM Tris, 10 mM NaCl, and 1 mM DTT at pH 8.0) and dialyzed the obtained solution against
the same buffer to remove ammonium sulfate. Then, we subjected this solution to anion exchange
column chromatography using Q sepharose resins. We eluted proteins from the Q sepharose
column by applying a gradient with increasing the concentration of buffer B (20 mM Tris, 1 M
NaCl, and 1 mM DTT at pH 8.0). We concentrated the eluted fractions that contained MPro and
subject the concentered solution to size exclusion chromatography using a HiPrep 16/60 Sephacryl
S-100 HR column with a mobile phase containing 10 mM sodium phosphate, 10 mM NaCl, 0.5
mM EDTA and 1 mM DTT at pH 7.8. The final yield of the purified enzyme was 1 mg/L with
respect to the original expression medium volume. We determined the concentration of the finally
purified Mpro using the Pierce™ 660nm protein assay and aliquoted 10 µM MPro in the size
exclusion chromatography buffer for storage in -80 °C.
The synthesis of Sub1
We loaded the first amino acid (0.5 mmol, 2 equiv.) manually on chlorotrityl chloride resin
(0.52 mmol/g loading) on a 0.25 mmol scale by the addition of DIPEA (3 equiv.). After addition
of the first amino acid, automated Fmoc-based solid phases synthesis was performed using a
Liberty Blue automated peptide synthesizer. Deprotection of the Fmoc group was carried out with
20% piperidine/DMF. Coupling was done with a Fmoc-protected amino acid (0.75 mmol, 3.0
equiv.) and the coupling reagent HATU (0.9 mmol, 3.6 equiv.) and DIEA in NMP (1 mmol, 4.0
equiv.). The final amino acid was capped by the addition of 25% acetic anhydride (v/v) in DMF
and DIEA (0.2mmol, 2.0 equiv.). Coumarin coupling was performed in anhydrous THF using T3P
in EtOAc (50% w/v) (3.0 equiv.), DIEA (3 equiv.) and 7-amino-4methyl-coumarin (0.8 equiv.)
and mixed for 16 h. The solvent was removed and the peptide was dissolved in DCM and washed
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
with H2O (4x) followed by HCl (2x) and brine (1x). The organic layer was dried with Na2SO4,
filtered and concentrated. Global deprotection was then carried out using triisopropylsilane (5%)
and trifluoroacetic acid (30%) v/v in DCM and mixed for 2-3 h to result in the crude substrate.
The peptide was then purified by semi-preparative HPLC and the fractions containing pure product
were pooled, concentrated, and analyzed by LC-MS for purity.
The synthesis of Sub2
We performed automated Fmoc-based solid phase synthesis on a Liberty Blue automated
peptide synthesizer. Synthesis was conducted on a 0.1 mmol scale with Fmoc Rink amide MBHA
resin (0.52 mmol/g loading) and 3 equiv. of protected amino acids. Deprotection of the Fmoc group
was carried out with 20% piperidine/DMF. Coupling was done using the desired Fmoc-protected
amino acid (0.2 mmol, 2.0 equiv.), coupling reagent Oxyma (0.4 mmol, 4.0 equiv.) and DIC (0.4
mmol, 4.0 equiv.). After the final amino acid had been coupled on, the resin was washed trice with
DMF and DCM. Cleavage from the resin was performed using trifluoroacetic acid (95%),
triisopropylsilane (2.5%) and water (2.5%) with agitation for 4 h. The peptide was drained into
cold methyl tert-butyl ether where it precipitated out. We centrifuged the precipitate, decanted
mother liquor, dissolved the pellet in DMF and then purified the peptide by LCMS.
Screening assay
5 mM stock solutions of medicines were prepared in DMSO. The final screening assay
conditions contained 50 nM MPro, 10 µM Sub3, and 1 mM medicine. We diluted enzyme stock
and substrate stock solutions using a buffer containing 10 mM sodium phosphate, 10 mM NaCl,
and 0.5 mM EDTA at pH 7.8 for reaching desired final concentrations. We ran the assay in
triplicates. First, we added 30 µL of a 167 nM MPro solution to each well in a 96-well plate and
then provided 20 µL of 5 mM stock solutions of medicines in DMSO. After a brief shaking, we
incubated the plate at 37°C for 30 min. Then we added 50 µL of a 20 µM Sub3 solution to initiate
the activity analysis. The EDANS fluorescence with excitation at 336 nm and emission at 455 nm
from the cleaved substrate was detected. We determined the fluorescence increasing slopes at the
initial 5 min and normalized them with respect to the control that had no inhibitor provided.
Inhibition analysis
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
The final inhibition assay conditions contained 50 nM MPro, 10 µM Sub3, and a varying
concentration of an inhibitor. Similar to screening assay, we diluted enzyme stock and substrate
stock solutions using a buffer containing 10 mM sodium phosphate, 10 mM NaCl, and 0.5 mM
EDTA at pH 7.8 for reaching desired final concentrations. We ran the assay in triplicates. For the
inhibition analysis, we added 30 µL of a 167 nM MPro solution to each well in a 96-well plate and
then provided 20 µL of inhibitor solutions with varying concentrations in DMSO. After a brief
shaking, we incubated the plate at 37°C for 30 min. Then we added 50 µL of a 20 µM Sub3 solution
to initiate the activity analysis. We monitored the fluorescence signal and processed the initial
slopes in the same way described in screening assay part. We used GraphPad 8.0 to analyze the
data and used the [Inhibitor] vs. response - Variable slope (four parameters) fitting to determine
the values of both IC50 and Hill coefficient.
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
REFERENCES
(1) Gates, B. Responding to Covid-19 - A Once-in-a-Century Pandemic?, N Engl J Med 2020,
382, 1677-1679.
(2) Morens, D. M.; Daszak, P.; Taubenberger, J. K. Escaping Pandora's Box - Another Novel
Coronavirus, N Engl J Med 2020, 382, 1293-1295.
(3) World Health Organization 2020, May 13.
(4) Kissler, S. M.; Tedijanto, C.; Goldstein, E.; Grad, Y. H.; Lipsitch, M. Projecting the
transmission dynamics of SARS-CoV-2 through the postpandemic period, Science 2020,
10.1126/science.abb5793.
(5) Morse, J. S.; Lalonde, T.; Xu, S.; Liu, W. R. Learning from the Past: Possible Urgent
Prevention and Treatment Options for Severe Acute Respiratory Infections Caused by 2019-
nCoV, Chembiochem 2020, 21, 730-738.
(6) Morse, J. S.; Lalonde, T.; Xu, S.; Liu, W. R. Learning from the Past: Possible Urgent
Prevention and Treatment Options for Severe Acute Respiratory Infections Caused by 2019-
nCoV, ChemRxiv 2020, 10.26434/chemrxiv.11728983.v1.
(7) Zhou, N.; Pan, T.; Zhang, J.; Li, Q.; Zhang, X.; Bai, C.; Huang, F.; Peng, T.; Zhang, J.; Liu,
C.; Tao, L.; Zhang, H. Glycopeptide Antibiotics Potently Inhibit Cathepsin L in the Late
Endosome/Lysosome and Block the Entry of Ebola Virus, Middle East Respiratory
Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus
(SARS-CoV), J Biol Chem 2016, 291, 9218-9232.
(8) Strating, J. R.; van der Linden, L.; Albulescu, L.; Bigay, J.; Arita, M.; Delang, L.; Leyssen,
P.; van der Schaar, H. M.; Lanke, K. H.; Thibaut, H. J.; Ulferts, R.; Drin, G.; Schlinck, N.;
Wubbolts, R. W.; Sever, N.; Head, S. A.; Liu, J. O.; Beachy, P. A.; De Matteis, M. A.; Shair,
M. D.; Olkkonen, V. M.; Neyts, J.; van Kuppeveld, F. J. Itraconazole inhibits enterovirus
replication by targeting the oxysterol-binding protein, Cell Rep 2015, 10, 600-615.
(9) Mastrangelo, E.; Pezzullo, M.; De Burghgraeve, T.; Kaptein, S.; Pastorino, B.; Dallmeier,
K.; de Lamballerie, X.; Neyts, J.; Hanson, A. M.; Frick, D. N.; Bolognesi, M.; Milani, M.
Ivermectin is a potent inhibitor of flavivirus replication specifically targeting NS3 helicase
activity: new prospects for an old drug, J Antimicrob Chemother 2012, 67, 1884-1894.
(10) Rossignol, J. F. Nitazoxanide: a first-in-class broad-spectrum antiviral agent, Antiviral Res
2014, 110, 94-103.
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
(11) Mercorelli, B.; Palu, G.; Loregian, A. Drug Repurposing for Viral Infectious Diseases: How
Far Are We?, Trends Microbiol 2018, 26, 865-876.
(12) Hung, I. F.; Lung, K. C.; Tso, E. Y.; Liu, R.; Chung, T. W.; Chu, M. Y.; Ng, Y. Y.; Lo, J.;
Chan, J.; Tam, A. R.; Shum, H. P.; Chan, V.; Wu, A. K.; Sin, K. M.; Leung, W. S.; Law, W.
L.; Lung, D. C.; Sin, S.; Yeung, P.; Yip, C. C.; Zhang, R. R.; Fung, A. Y.; Yan, E. Y.; Leung,
K. H.; Ip, J. D.; Chu, A. W.; Chan, W. M.; Ng, A. C.; Lee, R.; Fung, K.; Yeung, A.; Wu, T.
C.; Chan, J. W.; Yan, W. W.; Chan, W. M.; Chan, J. F.; Lie, A. K.; Tsang, O. T.; Cheng, V.
C.; Que, T. L.; Lau, C. S.; Chan, K. H.; To, K. K.; Yuen, K. Y. Triple combination of
interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to
hospital with COVID-19: an open-label, randomised, phase 2 trial, Lancet 2020.
(13) Baez-Santos, Y. M.; St John, S. E.; Mesecar, A. D. The SARS-coronavirus papain-like
protease: structure, function and inhibition by designed antiviral compounds, Antiviral Res
2015, 115, 21-38.
(14) Jin, Z.; Du, X.; Xu, Y.; Deng, Y.; Liu, M.; Zhao, Y.; Zhang, B.; Li, X.; Zhang, L.; Peng, C.;
Duan, Y.; Yu, J.; Wang, L.; Yang, K.; Liu, F.; Jiang, R.; Yang, X.; You, T.; Liu, X.; Yang,
X.; Bai, F.; Liu, H.; Liu, X.; Guddat, L. W.; Xu, W.; Xiao, G.; Qin, C.; Shi, Z.; Jiang, H.;
Rao, Z.; Yang, H. Structure of M(pro) from COVID-19 virus and discovery of its inhibitors,
Nature 2020, 10.1038/s41586-020-2223-y.
(15) Nguyen, D. D.; Gao, K.; Chen, J.; Wang, R.; Wei, G.-W. Potentially highly potent drugs for
2019-nCoV, bioRxiv 2020, 2020.2002.2005.936013.
(16) Xu, Z.; Peng, C.; Shi, Y.; Zhu, Z.; Mu, K.; Wang, X.; Zhu, W. Nelfinavir was predicted to
be a potential inhibitor of 2019-nCov main protease by an integrative approach combining
homology modelling, molecular docking and binding free energy calculation, bioRxiv 2020,
2020.2001.2027.921627.
(17) Wu, C.; Liu, Y.; Yang, Y.; Zhang, P.; Zhong, W.; Wang, Y.; Wang, Q.; Xu, Y.; Li, M.; Li,
X.; Zheng, M.; Chen, L.; Li, H. Analysis of therapeutic targets for SARS-CoV-2 and
discovery of potential drugs by computational methods, Acta Pharm Sin B 2020.
(18) Ayman, F.; Ping, W.; Mahmoud, A.; Hesham, S. Identification of FDA Approved Drugs
Targeting COVID-19 Virus by Structure-Based Drug Repositioning, ChemRxiv 2020,
10.26434/chemrxiv.12003930.v3.
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
(19) Zhang, L.; Lin, D.; Sun, X.; Curth, U.; Drosten, C.; Sauerhering, L.; Becker, S.; Rox, K.;
Hilgenfeld, R. Crystal structure of SARS-CoV-2 main protease provides a basis for design
of improved alpha-ketoamide inhibitors, Science 2020, 368, 409-412.
(20) Pillaiyar, T.; Manickam, M.; Namasivayam, V.; Hayashi, Y.; Jung, S. H. An Overview of
Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3CL Protease Inhibitors:
Peptidomimetics and Small Molecule Chemotherapy, J Med Chem 2016, 59, 6595-6628.
(21) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson,
A. J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor
flexibility, J Comput Chem 2009, 30, 2785-2791.
(22) Phan, J.; Zdanov, A.; Evdokimov, A. G.; Tropea, J. E.; Peters, H. K., 3rd; Kapust, R. B.; Li,
M.; Wlodawer, A.; Waugh, D. S. Structural basis for the substrate specificity of tobacco etch
virus protease, J Biol Chem 2002, 277, 50564-50572.
(23) Rut, W.; Groborz, K.; Zhang, L.; Sun, X.; Zmudzinski, M.; Hilgenfeld, R.; Drag, M.
Substrate specificity profiling of SARS-CoV-2 M<sup>pro</sup> protease provides basis
for anti-COVID-19 drug design, bioRxiv 2020, 2020.2003.2007.981928.
(24) Dragovich, P. S.; Prins, T. J.; Zhou, R.; Webber, S. E.; Marakovits, J. T.; Fuhrman, S. A.;
Patick, A. K.; Matthews, D. A.; Lee, C. A.; Ford, C. E.; Burke, B. J.; Rejto, P. A.;
Hendrickson, T. F.; Tuntland, T.; Brown, E. L.; Meador, J. W., 3rd; Ferre, R. A.; Harr, J. E.;
Kosa, M. B.; Worland, S. T. Structure-based design, synthesis, and biological evaluation of
irreversible human rhinovirus 3C protease inhibitors. 4. Incorporation of P1 lactam moieties
as L-glutamine replacements, J Med Chem 1999, 42, 1213-1224.
(25) Cao, B.; Wang, Y.; Wen, D.; Liu, W.; Wang, J.; Fan, G.; Ruan, L.; Song, B.; Cai, Y.; Wei,
M.; Li, X.; Xia, J.; Chen, N.; Xiang, J.; Yu, T.; Bai, T.; Xie, X.; Zhang, L.; Li, C.; Yuan, Y.;
Chen, H.; Li, H.; Huang, H.; Tu, S.; Gong, F.; Liu, Y.; Wei, Y.; Dong, C.; Zhou, F.; Gu, X.;
Xu, J.; Liu, Z.; Zhang, Y.; Li, H.; Shang, L.; Wang, K.; Li, K.; Zhou, X.; Dong, X.; Qu, Z.;
Lu, S.; Hu, X.; Ruan, S.; Luo, S.; Wu, J.; Peng, L.; Cheng, F.; Pan, L.; Zou, J.; Jia, C.; Wang,
J.; Liu, X.; Wang, S.; Wu, X.; Ge, Q.; He, J.; Zhan, H.; Qiu, F.; Guo, L.; Huang, C.; Jaki, T.;
Hayden, F. G.; Horby, P. W.; Zhang, D.; Wang, C. A Trial of Lopinavir-Ritonavir in Adults
Hospitalized with Severe Covid-19, N Engl J Med 2020, 382, 1787-1799.
(26) Musarrat, F.; Chouljenko, V.; Dahal, A.; Nabi, R.; Chouljenko, T.; Jois, S. D.; Kousoulas,
K. G. The anti-HIV Drug Nelfinavir Mesylate (Viracept) is a Potent Inhibitor of Cell Fusion
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
Caused by the SARS-CoV-2 Spike (S) Glycoprotein Warranting further Evaluation as an
Antiviral against COVID-19 infections, J Med Virol 2020, 10.1002/jmv.25985.
(27) Yamamoto, N.; Matsuyama, S.; Hoshino, T.; Yamamoto, N. Nelfinavir inhibits replication
of severe acute respiratory syndrome coronavirus 2 in vitro, BioRxiv 2020,
10.1101/2020.04.06.026476.
(28) Williams, A. L.; Yem, D. W.; McGinnis, E.; Williams, L. S. Control of arginine biosynthesis
in Escherichia coli: inhibition of arginyl-transfer ribonucleic acid synthetase activity, J
Bacteriol 1973, 115, 228-234.
(29) Shoichet, B. K. Interpreting steep dose-response curves in early inhibitor discovery, J Med
Chem 2006, 49, 7274-7277.
(30) Chen, Z.; Hu, J.; Zhang, Z.; Jiang, S.; Han, S.; Yan, D.; Zhuang, R.; Hu, B.; Zhang, Z.
Efficacy of hydroxychloroquine in patients with COVID-19: results of a randomized clinical
trial, medRxiv 2020, 2020.2003.2022.20040758.
(31) CHEN Jun, L. D., LIU Li, LIU Ping, XU Qingnian, XIA Lu, LING Yun, HUANG Dan,
SONG Shuli, ZHANG Dandan, QIAN Zhiping, LI Tao, SHEN Yinzhong, LU Hongzhou A
pilot study of hydroxychloroquine in treatment of patients with moderate COVID-19, J
Zhejiang Univ (Med Sci) 2020, 49, 215-219.
(32) Savarino, A.; Boelaert, J. R.; Cassone, A.; Majori, G.; Cauda, R. Effects of chloroquine on
viral infections: an old drug against today's diseases?, Lancet Infectious Diseases 2003, 3,
722-727.
(33) Geleris, J.; Sun, Y.; Platt, J.; Zucker, J.; Baldwin, M.; Hripcsak, G.; Labella, A.; Manson,
D.; Kubin, C.; Barr, R. G.; Sobieszczyk, M. E.; Schluger, N. W. Observational Study of
Hydroxychloroquine in Hospitalized Patients with Covid-19, N Engl J Med 2020,
10.1056/NEJMoa2012410.
(34) Ohkuma, S.; Poole, B. Cytoplasmic vacuolation of mouse peritoneal macrophages and the
uptake into lysosomes of weakly basic substances, J Cell Biol 1981, 90, 656-664.
(35) DeWald, L. E.; Dyall, J.; Sword, J. M.; Torzewski, L.; Zhou, H.; Postnikova, E.; Kollins, E.;
Alexander, I.; Gross, R.; Cong, Y.; Gerhardt, D. M.; Johnson, R. F.; Olinger, G. G., Jr.;
Holbrook, M. R.; Hensley, L. E.; Jahrling, P. B. The Calcium Channel Blocker Bepridil
Demonstrates Efficacy in the Murine Model of Marburg Virus Disease, J Infect Dis 2018,
218, S588-S591.
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
(36) Miura, S.; Sumiyoshi, M.; Tsuchiya, H.; Maruyama, M.; Seigen, I.; Okai, I.; Masaki, Y.;
Okazaki, S.; Inoue, K.; Fujiwara, Y.; Komatsu, K.; Hayashi, H.; Sekita, G.; Tokano, T.;
Nakazato, Y.; Daida, H. The use of serum bepridil concentration as a safe rhythm control
strategy in patients with atrial tachyarrhythmias, Journal of Arrhythmia 2012, 28, 187-191.
(37) Mitterreiter, S.; Page, R. M.; Kamp, F.; Hopson, J.; Winkler, E.; Ha, H. R.; Hamid, R.;
Herms, J.; Mayer, T. U.; Nelson, D. J.; Steiner, H.; Stahl, T.; Zeitschel, U.; Rossner, S.;
Haass, C.; Lichtenthaler, S. F. Bepridil and amiodarone simultaneously target the
Alzheimer's disease beta- and gamma-secretase via distinct mechanisms, J Neurosci 2010,
30, 8974-8983.
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
ACKNOWELDGEMENTS
We thank Profs. Thomas Meek and Kevin Burgess for allowing us to use their instruments.
This work was supported in part by National Institutes of Health (grants R01GM127575 and
R01GM121584) and Welch Foundation (A-1715).
AUTHOR CONTRIBUTIONS
W.R.L. conceived the project. E.C.V., K.Y., K.C.K., C.-C.C., A.D., D.M.M., S.X., C.-T.K.T.,
and W.R.L. designed experiments. E.C.V., K.Y., K.C.K., C.-C.C., A.D., D.M.M. performed the
experiments. E.C.V., K.Y. and W.R.L. wrote the manuscript. All authors approved the final
manuscript before submission.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
FIGURES
Figure 1: Structures of 29 FDA/EMA-approved medicines and rupintrivir whose IC50 values in
inhibiting MPro were determined in the study.
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
Figure 2: Activity of MPro. (A) The structures of three substrates. (B) Activities of 50 nM and 1
M MPro on 10 M Sub1. (C) Activity of 50 nM MPro on 10 M Sub2 and Sub3. The florescence
signals are normalized for easy comparison. (D) Activity of different concentrations of MPro on 10
M Sub3.
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
Figure 3: Initial screening of Mpro inhibition by 29 FDA/EMA-approved medicines and rupintrivir.
1 mM (0.14 mM for Itraconazole due to its low solubility in DMSO) was used for each inhibitor
to perform the inhibition assay. Fluorescence intensity was normalized with respect to the control
that had no small molecule provided. Triplicate experiments were performed for each compound,
and the value was presented as mean ± standard error (SE).
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
Figure 4: IC50 assays for 18 small molecule medicines on their inhibition of Mpro. Triplicate
experiments were performed for each compound, and the IC50 value was presented as mean ±
standard error (SE). GraphPad Prism 8.0 was used to perform data analysis.
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
Figure 5: Pimozide (A), ebastine (B), bepridil (C), and their overlay (D) in the active site of MPro.
The protein surface topography in A, B, and C is presented to show the concaved active site.
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
Table 1: Docking results of small molecule medicines (Compounds whose IC50 values were tested are
asterisked.)
Name
ΔGbinding
(kcal/mol)
Name
ΔGbinding
(kcal/mol)
Rimonabant*
-11.23
Bepridil*
-8.31
Tipranavir*
-10.74
Isoconazole
-8.15
Ebastine*
-10.62
Econazole
-8.14
Saquinavir*
-10.37
Eluxadoline
-8.12
Zopiclone*
-10.10
(R)-Butoconazole
-8.11
Pimozide*
-10.01
(S)-Butoconazole
-8.10
Pirenzepine*
-9.94
Atazanavir
-8.08
Nelfinavir*
-9.67
Cetirizine
-8.01
Doxapram*
-9.55
Efinaconazole
-8.01
Oxiconazole*
-9.18
Amprenavir
-7.99
Indinavir*
-9.13
Hydroxyzine
-7.99
Sertindole*
-9.04
(R)-Tioconazole
-7.98
Metixene*
-9.01
(R)-Carbinoxamine
-7.96
Fexofenadine*
-8.95
Armodafinil
-7.90
Lopinavir*
-8.91
Desipramine
-7.84
Sertaconazole*
-8.87
Ritonavir
-7.74
Reboxetine*
-8.86
Atomoxetine
-7.73
Ketoconazole*
-8.85
Sulconazole
-7.69
Duloxetine*
-8.79
Clotrimazole
-7.67
Isavuconazole*
-8.77
Dipyridamole
-7.67
Lemborexant*
-8.75
Phentolamine
-7.61
Oxyphencyclimine*
-8.74
(S)-Tioconazole
-7.48
Darunavir*
-8.72
Doxylamine
-7.33
Trihexphenidyl*
-8.72
(S)-Carbinoxamine
-7.21
Pimavanserin*
-8.69
Antazoline
-6.86
Clotiapine*
-8.57
Voriconazole
-6.76
Itraconazole*
-8.44
Fluconazole
-6.41
Clemastine*
-8.36
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint
Table 2: IC50 and Hill coefficient values of 18 identified inhibitors
Name
IC50 (µM)
Hill Slope
Pimozide
42 ± 2
3.1 ± 0.4
Ebastine
57 ± 12
1.5 ± 0.2
Rupintrivir
68 ± 7
1.4 ± 0.2
Bepridil
72 ± 3
2.9 ± 1.0
Sertaconazole
76 ± 2
3.5 ± 0.2
Rimonabant
85 ± 3
5.0 ± 0.4
Oxiconazole
99 ± 6
3.8 ± 0.4
Itraconazole
111 ± 35
1.6 ± 0.2
Tipranavir
180 ± 20
1.4 ± 0.2
Nelfinavir
234 ± 15
5.4 ± 1.0
Zopiclone
349 ± 77
1.2 ± 0.2
Trihexyphenidyl
370 ± 53
8.9 ± 6.4
Saquinavir
411 ± 6
26.8 ± 2.6
Isavuconazole
438 ± 11
5.2 ± 0.7
Lopinavir
486 ± 2
29.9 ± 2.4
Clemastine
497 ± 148
11.2 ± 7.3
Metixene
635 ± 43
8.7 ± 5
Duloxetine
3047 ± 634
0.93 ± 0.07
.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (whichthis version posted May 23, 2020. . https://doi.org/10.1101/2020.05.23.112235doi: bioRxiv preprint