Fluoxetine Inhibits Enterovirus Replication by Targeting the Viral 2C Protein in a Stereospecific Manner

Article abstract of DOI:10.1021/acsinfecdis.9b00179

Enteroviruses (family Picornaviridae) comprise a large group of human pathogens against which no licensed antiviral therapy exists. Drug-repurposing screens uncovered the FDA-approved drug fluoxetine as a replication inhibitor of enterovirus B and D speci

Full text of DOI:10.1021/acsinfecdis.9b00179

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Article
Fluoxetine inhibits enterovirus replication by targeting
the viral 2C protein in a stereospecific manner
Lisa Bauer, Roberto Manganaro, Birgit Zonsics, Jeroen R.P.M Strating, Priscila
El Kazzi, Moira Lorenzo Lopez, Rachel Ulferts, Clara van Hoey, Maria J. Maté,
Thierry Langer, Bruno Coutard, Andrea Brancale, and Frank J.M. van Kuppeveld
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00179 • Publication Date (Web): 15 Jul 2019
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Fluoxetine inhibits enterovirus replication by targeting the viral 2C protein in a
stereospecific manner
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Lisa Bauer1,6, Roberto Manganaro2,6, Birgit Zonsics2,6, Jeroen R.P.M. Strating1,† Priscila El Kazzi3, Moira
,
,‡
Lorenzo Lopez2, Rachel Ulferts1 , Clara van Hoey4, Maria J. Maté3, Thierry Langer4, Bruno Coutard3,5,
Andrea Brancale2, Frank J.M. van Kuppeveld1,*
1
Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine,
Utrecht University, 3584CL Utrecht, The Netherlands
2
School of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff
CF10 3NB, United Kingdom
3
Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques, UMR 6098 Centre
National de la Recherche Scientifique, Université de la Méditerranée and Université de Provence, Case 925,
163 Avenue de Luminy, 3288 Marseille CEDEX 9, France
4 Department of Pharmaceutical Chemistry, Faculty of Life Sciences, University of Vienna, Althanstraße 14,
A-1090 Vienna, Austria
5 Unité des Virus Emergents, (UVE: Aix-Marseille Univ-IRD 190-Inserm 1207-IHU Méditerranée Infection),
Marseille, France
6 These authors contributed equally
† Present address: Viroclinics, Rotterdam, The Netherlands
‡ present address: The Francis Crick Institute, London, United Kingdom
* Corresponding author: f.j.m.vankuppeveld@uu.nl
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Enteroviruses (family Picornaviridae) comprise a large group of human pathogens against which no licensed
antiviral therapy exists. Drug-repurposing screens uncovered the FDA-approved drug fluoxetine as replication
inhibitor of enterovirus B and D species. Fluoxetine likely targets the non-structural viral protein 2C, but
detailed mode-of-actions studies are missing because structural information of 2C of fluoxetine-sensitive
enteroviruses is lacking. We here show that broad-spectrum anti-enteroviral activity of fluoxetine is
stereospecific concomitant with binding to recombinant 2C. (S)-fluoxetine inhibits with 5-fold lower EC50 than
racemic fluoxetine. Using a homology model of 2C of the fluoxetine-sensitive enterovirus coxsackievirus B3
(CVB3) based upon a recently elucidated structure of a fluoxetine-insensitive enterovirus, we predicted stable
binding of (S)-fluoxetine. Structure-guided mutations disrupted binding and rendered CVB3 resistant to
fluoxetine. The study provides new insights into the anti-enteroviral mode-of-action of fluoxetine. Importantly,
using only (S)-fluoxetine would allow for lower dosing in patients, thereby likely reducing side effects.
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Keywords:
antiviral, enteroviruses, drug-repurposing, virus replication, molecular modelling,
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The genus Enterovirus within the Picornaviridae family includes many medically and socioeconomically
important pathogens, which are among the most common infections in mankind. Four enterovirus (EV) species
(EV-A, -B, -C and -D) and three rhinovirus (RV) species (RV-A, -B and -C) include serotypes that are known
to cause human infections, like poliovirus, coxsackie A and B viruses, echoviruses, numbered enteroviruses
(e.g. EV-A71 and EV-D68) and rhinovirus. Infections with enteroviruses can cause a broad spectrum of
diseases ranging from hand-foot-and-mouth disease, conjunctivitis, aseptic meningitis, severe neonatal sepsis-
like disease, to acute flaccid paralysis, whereas infection with rhinoviruses cause the common cold, as well as
exacerbations of asthma and chronic obstructive pulmonary disease (COPD) 1. These viral infections are often
self-limiting but can also result in severe complications especially in young children. To date, no antiviral
therapy to treat enterovirus infections has been approved and treatment remains limited to supportive care.
Worldwide vaccination campaigns have almost eradicated poliomyelitis. However, the vaccines against
poliovirus and a recently in China approved EV-A71 vaccine, are currently the only ones developed against
enteroviruses. Vaccination is likely not a feasible general strategy to prevent enterovirus infections given the
enormous amount (>250) of enterovirus (sero)types. Hence, the development of broad-spectrum anti-
enteroviral drugs could be a promising alternative.
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Enteroviruses are small, non-enveloped, positive-sense, single-stranded RNA viruses with an icosahedral
capsid. The genome of ~ 7.5 kb encodes a single polyprotein that is auto-processed into structural proteins
(VP1, VP2, VP3, VP4), non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C, 3D) and several functional processing
intermediates. The viral non-structural proteins, particularly the protease 3Cpro and the RNA-dependent RNA
polymerase 3Dpol, are attractive targets for antiviral drug development 2.
The viral protein 2C is the most conserved non-structural protein among picornaviruses, which makes it
particularly interesting for broad-spectrum anti-enteroviral drug design. The viral 2C protein functions as
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ATPase 3-5, ATPase-dependent RNA helicase and as an ATPase-independent RNA chaperone , all of these
enzymatic functions are indispensable for the viral life cycle. The ATPase domain of the protein belongs to
the superfamily of SF3 helicases of the AAA+ ATPases and contains Walker A and Walker B motifs and motif
C 7. Besides the ATPase domain, 2C harbors an N-terminal membrane-associated helical domain, a cysteine-
rich motif and putative RNA binding motifs. 2C has been implicated in pleiotropic functions such as uncoating
8, cellular membrane rearrangement 9-12, RNA binding 13-15, RNA replication 16-21, immune evasion and
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encapsidation 23-26. Although 2C has a central role in the viral life cycle, the exact details of its involvement
remain poorly understood.
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Over the past decades, structurally disparate 2C inhibitors such as guanidine hydrochloride (GuaHCl), 2-(-
hydroxybenzyl)-benzimidazole HBB, MRL-1237 and TBZE-029 have been identified 2, 27-30. An emerging
concept to discover new antivirals is drug repurposing. This strategy offers an attractive alternative to de novo
drug development, as profound pharmacological and toxicological profiles of the compounds are already
available. Furthermore, when the repurposed drug can be used at a similar dosage as for the original indication
it may directly enter phase 2 clinical trials 31-32, thereby reducing development cost and time. Several drug-
repurposing screens have uncovered FDA-approved drugs as inhibitors of enterovirus replication 2. Some of
these compounds are thought to inhibit the non-structural protein 2C because non-synonymous resistance
mutations occur in 2C. Fluoxetine (Prozac®), a selective serotonin reuptake inhibitor (SSRI) that is FDA-
approved for the treatment of major depression and anxiety disorders, was identified as a potent inhibitor of
EV-B and -D species but EV-A, EV-C or rhinovirus species remained unaffected 33-35. Besides its anti-
enteroviral activity, fluoxetine was also shown to inhibit dengue virus and hepatitis C virus, two members of
the Flaviviridae family, where it likely acts as a host-targeting rather than a direct-acting antiviral as it is the
case for enteroviruses 36-37. Fluoxetine has already been successfully used to treat an immunocompromised
child with life-threatening chronic enterovirus encephalitis 38, underscoring the potential of fluoxetine for the
application as an anti-enteroviral compound. Although various 2C inhibitors have been discovered over the
years, their mode-of-action is still poorly understood.
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Here, we set out to investigate how fluoxetine targets 2C of coxsackievirus B3 (CVB3), a virus model
commonly used as a prototype for enterovirus B species. Fluoxetine has one chiral center, resulting into 2
enantiomers and we experimentally showed that only the S-enantiomer inhibits enterovirus replication by
directly binding to the viral protein 2C. Based on the recently published crystal structure of the catalytic domain
of EV-A71 2C protein 39, a homology model for the corresponding part of CVB3 2C was generated. Two
pockets flanking a stretch of amino acids that often mutate to convey resistance against 2C inhibitors
(224AGSINA229) were identified. Molecular dynamics simulations predicted a stable interaction for the (S)-
fluoxetine in only one of these pockets. Mutations of residues deep in the predicted binding pocket confer
resistance to fluoxetine and contribute to the understanding of the antiviral mode-of-action. Thus, we identified
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for the first time a putative binding pocket for antiviral compounds in the non-structural enterovirus protein
2C.
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1.1
(S)-fluoxetine inhibits CVB3 replication by binding to the non-structural protein 2C
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Fluoxetine is clinically used as racemic mixture (1:1 enantiomeric ratio) and both enantiomers are of equal
pharmacological activity towards the serotonin transporter SERT 40. The racemic compound was identified in
drug-repurposing screens as an inhibitor of replication of EV-B and EV-D species 33-34. Since fluoxetine has
one chiral center, we investigated the antiviral properties of both enantiomers (Figure 1A). Coxsackievirus B3
(CVB3), a member of the EV-B genus, causes a readily observable cytopathic effect (CPE), apparent as
rounding, detachment and eventually dying of the cell. The racemic mixture and both enantiomers purchased
from two different vendors (Sigma Aldrich and Carbosynth), were tested in a multicycle CPE-reduction assay
to elucidate whether the compounds inhibit virus replication and thereby prevent the development of CPE. In
parallel, cytotoxicity of the compounds was determined using an MTS assay.
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Figure 1. Fluoxetine inhibits CVB3 replication in a stereospecific manner
(A) The two enantiomers of fluoxetine. (B) Multicycle CPE reduction assay to determine the antiviral activity
of fluoxetine enantiomers. HeLa R19 cells were treated with serial dilutions of racemic, (S)-, or (R)-fluoxetine
and infected with coxsackievirus B3 (CVB3) at MOI 0.001. In parallel, cells were treated with compound only
to assess cytotoxicity. After three days, cell viability was determined using an MTS assay. Data are from one
experiment representative of at least three independent experiments. (C) In a single cycle assay, HeLa R19
cells were infected with Renilla luciferase (RLuc)-CVB3 reporter virus, treated with serial dilutions of racemic,
(S)-, or (R)-fluoxetine, and luciferase activity was determined at seven hours post infection as a quantitative
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measure of replication. (D) In parallel, uninfected cells were treated with compound and cell viability was
determined using an MTS assay. Data are from one experiment representative of two independent experiments.
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Hela R19 cells were infected with CVB3 at MOI 0.001, which yields complete cell death within three days of
incubation. The racemic mixture inhibited CVB3 with 50% effective concentration (EC50) of 3.2 ± 0.95 µM,
while the S-enantiomer inhibited with an EC50 of 0.4 ± 0.15 µM (Figure 1B). In contrast, the R-enantiomer did
not show any protection against CVB3 (Figure 1B). To validate these findings in a single cycle assay, HeLa
R19 cells were infected with RLuc-CVB3 and the cells were treated with serial dilutions of the corresponding
compounds. Cells were lysed 7 hours post infection and luciferase activity was measured as a quantitative and
sensitive readout for viral replication. At the same time, cytotoxicity of the compounds was determined with
an MTS assay and the CC50 of the compounds in HeLa R19 cells ranges from 23 µM to 28 µM (Table 1). The
racemic mixture and the S-enantiomer exerted antiviral activity with a ∼5-fold higher potency for the S-
enantiomer (EC50 0.42 ± 0.17 µM) compared to the racemic mixture (EC50 2.02 ± 0.94 µM) (Figure 1C). Again,
the R-enantiomer did not show an antiviral effect (Figure 1C, D).
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Table 1. Antiviral activity of stereoisomers of fluoxetine
Shown are EC50 and CC50 values in µM. Data represents mean values ± SD calculated from at least three
different experiments. NA = not active. SI= Selectivity index (CC50/EC50)
Virus
Species
Strain
Racemic
(S)-fluoxetine
(R)-fluoxetine SIRacemic SI(S)-fluoxetine
EV-A71
EV-A
BrCr
NA
NA
NA
NA
NA
14.51
NA
NA
71.56
NA
CVB3
PV-1
EV-B
EV-C
EV-D
RV-A
RV-B
Nancy
Sabin1
Fermon
2.02 ± 0.52
NA
0.42 ± 0.17
NA
NA
EV-D68
HRV-A2
HRV-B14
CC50
1.85 ± 0.10
NA
0.67 ± 0.22
7.95 ± 0.39
6.34 ± 1.02
28.63 ± 1.02
NA
21.72
NA
42.73
3.60
4.52
NA
NA
NA
NA
29.32 ± 0.35
23.63 ± 1.40
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1.2
Antiviral effect of (S)-fluoxetine against other enteroviruses
Previously, it was shown that the racemic mixture of fluoxetine inhibits enterovirus B and D replication
prototyped by CVB3 (strain Nancy) and EV-D68 (strain Fermon) 33-34. As the S-enantiomer is more active
towards CVB3 than the racemic mixture, we reassessed the antiviral activity of (S)-fluoxetine against a panel
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of enteroviruses. As expected, the racemic mixture is only active against enterovirus B and D, eg CVB3 and
EV-D68 (strain Fermon) species (Table 1) 34, 41. (S)-fluoxetine is not only more potent than the racemic mixture
towards CVB3 but also towards EV-D68. Strikingly, while the racemic mixture does not confer any antiviral
effect against rhinoviruses, the S-enantiomer inhibits rhinovirus 2 (HRV-2) or HRV-14 replication, with EC50
of 7.95  0.39 μM and 6.34  1.02 μM respectively (Table 1). The S-enantiomer was not active against EV-
A71 (strain BrCr) and poliovirus (strain Sabin) at concentration up to 30 µM. Higher concentrations could not
be reached due to cytotoxicity of (S)-fluoxetine. We cannot exclude that (S)-fluoxetine would also inhibit other
enteroviruses at higher concentrations in other systems in which the CC50 is much higher.
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1.3
Fluoxetine directly binds to recombinant 2C protein in vitro.
To gain further insights into the fluoxetine mode-of-action we next investigated whether it directly binds to
2C protein. Production and purification of full-length 2C protein usually leads to a polydisperse preparation,
which is problematic for binding assays. By removing the first 36 amino acids of the N-terminus, a
homogenous preparation of monomeric protein can be obtained and used for binding assays, namely thermal
shift assay (TSA) and isothermal titration calorimetry (ITC) 35. First, TSA was used to detect a direct binding
of the racemic mixture and the enantiomers of fluoxetine to CVB3 2C. In this assay, the increase of the melting
temperature (Tm) of the protein reflects the binding of a ligand. The racemic mixture was able to induce a dose-
dependent increase of the Tm of 2C protein in a range of 10 µM to 250 µM (Figure 2A) while at higher
concentrations the Tm decreased. By contrast, the S-enantiomer of fluoxetine was able to thermally stabilize
the 2C protein in a concentration-dependent manner (Figure 2A) with no destabilization at high concentrations
of compound, suggestive of a direct binding of (S)-fluoxetine to the protein. As positive control we used
dibucaine, an established 2C inhibitor 35.
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ne (µM)
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(S)-fluoxeti
A
C
3
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Racemate
(S)-fluoxetine
(R)-fluoxetine
Dibucaine
1
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0.4
0.1
0.01
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-1
-2
-3
DMSO
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0.01 0.1
1
10 100
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0
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ine (µM)
(R)-fluoxet
fluoxetine (mM)
B
(S)-fluoxetine
(R)-fluoxetine
Concentration fluoxetine mM
Concentration fluoxetine mM
Figure 2. (S)-fluoxetine binds to the non-structural protein 2C in vitro.
(A) The binding of racemic, (S)-, and (R)-fluoxetine to recombinant CVB3 2C was determined by thermal shift
assay. The thermal stabilization of 2C by racemic and (S)-fluoxetine, represented by an increase in melting
temperature, indicates binding of the compounds to 2C. (B) The binding of (S)- and (R)-fluoxetine to 2C were
determined by isothermal calorimetry. As positive control the know 2C inhibitor dibucaine was used 35. Raw
data are depicted at the top, and the integrated data are depicted at the bottom. Data are shown fitted to a one-
site binding model. (C) Hela R19 cells were infected with RLuc-CVB3 and the cells were treated with a fixed
concentration of (S)-fluoxetine combined with serial dilutions of (R)-fluoxetine. Error bars depict standard
error of the mean calculated from biological triplicates. Data representative of two independent experiments.
Interestingly, the R-enantiomer appeared to have a destabilizing effect on the protein at high concentrations
(Figure 2A). The unexpected pattern of the racemic mixture may be explained by the collective effects of (S)-
fluoxetine (stabilization) and (R)-fluoxetine (destabilization). To confirm these results, we next quantified the
binding of (S)- and (R)-fluoxetine to 2C protein by ITC. The S-enantiomer bound to 2C with a dissociation
equilibrium constant (Kd) of ~9.5 µM (Figure 2B). 2C partly aggregated during the titration of (R)-fluoxetine,
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in agreement with the results obtained by TSA. It was therefore not possible to retrieve a precise Kd, but when
fitting the experimental data to a one-site binding model the observed Kd was higher than 200 µM (Figure 2B).
Thus, together with the data obtained in the antiviral assay, binding of (S)-fluoxetine is likely responsible for
the 2C-mediated antiviral effect. However, given the difference of binding of (S)-fluoxetine to the recombinant
protein (10 µM) and the EC50 in cell-based assays (<1 µM), off-target effects cannot be formally ruled out.
Still, (R)-fluoxetine did affect the 2C protein in the TSA and ITC at high concentrations. Therefore, we
investigated whether (R)-fluoxetine can exert any additional effect on virus replication in combination with
(S)-fluoxetine. To this end, the effect of different concentrations of (S)-fluoxetine combined with increasing
concentrations of the R-enantiomer on CVB3 replication was determined. At high concentrations of (S)-
fluoxetine (4 µM and 10 µM), replication of CVB3 was completely inhibited and as expected no additional
effect of (R)-fluoxetine could be observed (Figure 2C). At low concentrations of (S)-fluoxetine CVB3
replication was not impaired, nor could an additional effect of the R-enantiomer be observed (Figure 2C), in
line with the absence of antiviral activity of (R)-fluoxetine alone (Figure 1B, 1C). Strikingly, when cells were
treated with the approximate EC50 concentration of (S)-fluoxetine (0.4 µM), (R)-fluoxetine exerted a clear
dose-dependent inhibition of virus replication (Figure 2C). Hence, (R)-fluoxetine can apparently exert weak
antiviral activity that could come from the destabilization of the 2C protein or from the broad-spectrum
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antiviral effect induced by the targeting of a cellular partner 36-37
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1.4
Fragment screening identifies key parts of fluoxetine involved in targeting 2C
To gain more information about which chemical moieties of fluoxetine are involved in exerting the antiviral
activity, we tested six different fragments of fluoxetine (Figure 3A) in a CPE reduction assay using CVB3.
Fragments 1, 2 and 4 were synthesized, whereas fragments 3, 5 and 6 were purchased and tested as received
without further purification. Only fragment 1 showed a weak antiviral activity in the multicycle assay at
concentrations close to cytotoxic concentrations (Figure 3B). To exclude that inhibition of the virus is caused
by an unspecific cytotoxic effect, the fragments were also tested in a single cycle assay using a RLuc-CVB3.
HeLa R19 cells were infected with RLuc-CVB3, treated with 100 µM or 10 µM of each fragment respectively,
and luciferase activity at 7 hours post infection was determined as a quantitative and sensitive readout for virus
replication. In this single cycle assay, fragment 1 showed an antiviral effect at 100 µM (Supplementary Figure
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1A). In parallel, acute cytotoxicity of the fragments was excluded using an MTS assay (Supplementary Figure
1B). To further affirm that the observed weak antiviral activity of fragment 1 is specific and not due to
cytotoxicity, we tested whether two other fluoxetine-insensitive viruses were affected by fragment 1. Cells
were infected with EV-A71 or Renilla luciferase-expressing encephalomyocarditis virus (RLuc-EMCV) 42-43
and as positive controls guanidium chloride (Gua) a replication inhibitor for EV-A71 and CVB3 and
dipyridamole (DIP) a replication inhibitor for EMCV were used. The virus titers and replication were
determined by endpoint titration and a luciferase assay, respectively. Both viruses were not inhibited by
fragment 1 at 100 µM (Figure 3C), indicating that the inhibitory effect of fragment 1 on CVB3 replication is
specific.
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Figure 3. Fragment screening identifies key parts of fluoxetine involved in targeting 2C
(A) Molecular structures of fluoxetine and the different fragments that were used. (B) CPE-reduction assay as
performed in Figure 1B. (C) Hela R19 cells were infected with either CVB3, EV-A71 and RLuc-EMCV in
order to exclude an unspecific antiviral effect of fragment 1. The replication inhibitor guanidium chloride
(GuaHCl, 2 mM) was used in the case of CVB3 and EV-A71 as positive control. Dipyridamole (DIP, 100 µM)
was used as positive control replication inhibitor during RLuc-EMCV infection.
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Finally, we investigated binding of the fluoxetine fragments to recombinant 2C using a thermal shift assay
(TSA). The binding of low molecular weight molecules usually has a moderate effect on protein stability (<1°C
in protein stabilization) and concentrations should be >100 µM to observe protein stabilization 44. Therefore,
we tested the fragments in the TSA at a concentration range from 100 µM to 400 µM. Neither fragment 1 nor
any other fragment was able to stabilize the 2C protein at the indicated concentrations (Supplementary Figure
1C). At present we can only speculate why fragment 1 exerts weak antiviral activity but does not stabilize 2C
in the TSA. Possibly, the amount of recombinant 2C protein in the in vitro assay is higher than in an infection
setting, which would require a higher concentration of the compound to induce a shift in the melting
temperature of 2C. It can however be noticed that the effect of fragments on Tm is usually very modest because
the energy of binding is weak 44. Taken together, fragment 1 was able to inhibit CVB3 replication, but a direct
binding could not be observed in the TSA.
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1.5 Identification of a potential binding pocket for fluoxetine on 2C
After having investigated chemical properties of fluoxetine that are important for its antiviral activity, we next
wanted to further characterize the possible intermolecular interactions between (S)-fluoxetine and 2C. Because
structures of 2C of fluoxetine-sensitive viruses were not available we resorted to an in silico modelling
approach. We first generated a homology model of CVB3 2C (Figure 4A) based on the crystal structure of a
part of 2C of the fluoxetine-insensitive virus EV-A71 39. The crystalized part of EV-A71 2C covers amino
acids 116-329. In this region, the sequence identity and similarity between EV-A71 and CVB3 2C is 62% and
80%, respectively.
Enterovirus 2C belongs to a family of AAA+ ATPases, which oligomerize in hexameric ring structures in
which the ATP is coordinated by two monomers 39. Such ring structures were observed in low resolution
electron microscopy structures of 2C proteins from poliovirus and food-and-mouth disease virus, a
picornavirus from the aphthovirus genus 45-46. The published EV-A71 2C structure (PDB: 5GRB) shows a C-
terminal interaction between two adjacent 2C molecules to form a bipartite binding site for the ATP. In total
there are six protein chains in the crystal structure of which only chain A and B are fully resolved, whereas all
the others have at least one gap 39. The co-crystallized ATP molecules adopt different conformations for each
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chain and only chains A and F crystallize in a conformation resembling a bipartite binding site with both
monomers forming hydrogen bonds with the ATP. Therefore, chain A was used as a template to generate the
homology model of CVB3 2C (Figure 4A). Superimposition onto the EV-A71 2C structure resulted in a CVB3
2C model with a reasonably good fit and an overall RMSD of 0.30 Å from the template with the highest
deviation for residues N257 (1.65 Å) and D274 (2.08 Å).
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The CVB3 2C homology model was used to search for pockets in which fluoxetine may bind. Because
mutations in the flexible 224AGSINA229 loop confer resistance to fluoxetine 34, we focused on pockets near
this loop. Two potential binding pockets flanking the 224AGSINA229 loop were identified, termed site A and
site B (Figure 4B). Site A faces away from the ATP binding site, is confined by the 224AGSINA229loop on
one side and hydrophilic residues (D245, R295 and R296) on the other side. Site A is a deep, lipophilic pocket,
whereas site B is a rather shallow pocket and lies between the 224AGSINA229 loop and the 175-183 loop,
which is downstream of the Walker B motif. In the crystal structure of EV-A71 and in the CVB3 homology
model both sites might considerably change their shapes due to loop movements. However, site A is not
affected to the point of blocking the binding of fluoxetine, while site B is. Both enantiomers of fluoxetine were
docked into each of the two pockets for further computational investigations (Figure 4B). In site A both
enantiomers docked in proximity of the residues A229 and I227 of the 224AGSINA229 loop. The 4-
(trifluoromethyl)benzene moiety occupied the hydrophobic bottom of the pocket consisting of the residues
L178, C179, V187 and F190. Instead, in site B both enantiomers were mostly exposed to the solvent.
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(S)-fluoxetine
(R)-fluoxetine
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Figure 4. Predicted fluoxetine binding sites on CVB3 2C protein
(A) Homology model of the 2C protein of CVB3 built on the crystal structure of EV-A71. Ribbon and carbon
atoms of 224AGSINA229 loop are in blue, the 175-183 loop in violet and the 158-164 loop in cyan (B) S- and
R-enantiomers of fluoxetine docked into sites A and B of the homology model. (C) View of (S)-fluoxetine in
site A as identified in the molecular dynamics simulations, comprising the residues L126, L178, C179, V187,
F190, I227, A229, L238, F242, D245. The trifluoromethyl moiety of fluoxetine is buried deep inside the
hydrophobic pocket. (D) Three possible entrances of (S)-fluoxetine to reach C179 (green line surface).
1.5.1 Molecular dynamics simulations reveal stable positioning of (S)-fluoxetine in pocket A.
After the selection of the possible pockets near the AGSINA motif, the binding sites containing both
enantiomers were subjected to molecular dynamics simulations. The 224AGSINA229 loop is thought to be
flexible thereby alternating the shape of the two identified pockets, resulting in different predicted binding
modes compared to the docking. To evaluate how stable the fluoxetine enantiomers docked into the pockets
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and to address why mutations in this loop can cause resistance towards fluoxetine, the docking models of (S)-
fluoxetine and (R)-fluoxetine in site A and site B were subjected to molecular dynamics simulations. For each
enantiomer docked into either site, three independent molecular dynamic simulations of 100 ns were
performed. The calculated binding energies of (S)- or (R)-fluoxetine into site A and site B from the molecular
dynamics simulations are listed in Table 2. The simulations showed that (S)-fluoxetine bound stronger to 2C
than (R)-fluoxetine in both site A and site B. We observed that (R)-fluoxetine dissociated from pocket A as
well as pocket B in one of the three independent simulations, suggesting that (R)-fluoxetine cannot engage in
stable interaction with 2C. In site B, movement of the 224AGSINA229 loop was observed, making the
hydrophobic pocket accessible for the 4-(trifluoromethyl)benzene moiety of fluoxetine. However, visual
inspection and the binding energies of the molecular dynamics suggested that site A provides a more stable
binding pocket for (S)-fluoxetine, defined by the residues L126, L178, V187, F190, L238, I227, A229, F242
(Figure 4C).
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Table 2. Binding energies of the protein ligand complexes during MD simulations
Values indicated are calculated ΔGbinding average values over the 100 ns of each of the three independent
molecular dynamics simulations (MD) for the indicated ligands in site A or B (kJ/mol). The most favorable
complex according to the simulations is (S)-fluoxetine in site A. For all the other complexes in at least one
simulation the ligand dissociated from the protein (indicated by *).
Compound
(R)-fluoxetine
(S)-fluoxetine
(R)-fluoxetine
(S)-fluoxetine
MD1
-29.71*
-4163
MD2
-42.70
-42.28
-42.95
-29.88
MD3
-19.85*
-41.54
-27.38
-30.83*
Site A
Site B
-29.59*
-34.79
In each independent repetition of the molecular dynamics simulation on (S)-fluoxetine in site A, the compound
was oriented such that the 4-(trifluoromethyl)benzene moiety occupied the hydrophobic pocket (Figure 4C).
During the molecular dynamics simulations, a hydrogen bond repeatedly formed between the amino acid D245
and the positively charged amino group of (S)-fluoxetine (Figure 4C). We conclude, in line with the antiviral
data and binding assays from the in silico approach that (S)-fluoxetine binds stronger to 2C than the R-
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enantiomer. Furthermore, on the basis of our in silico analyses we conclude that (S)-fluoxetine most likely
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1.5.2 Mutations in the identified binding pocket confer resistance to (S)-fluoxetine
The triple amino acid substitution A224V-I227V-A229V (AVIVAV) in CVB3 2C, clustered in the
224AGSINA229 region, was previously shown to confer resistance to the racemic fluoxetine mixture 34 and
to several other 2C inhibitors 29. Likewise, the triple mutant provided resistance to (S)-fluoxetine (Figure 5A).
This resistance is specific, as AVIVAV mutations did not confer resistance to BF738735, a compound that
inhibits enterovirus replication via a different mechanism, namely by targeting the cellular protein PI4KIIIβ,
which is essential for enterovirus replication 47.
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BF738735 +GuaHCl
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Figure 5. Mutations in the identified binding pocket confer resistance towards (S)-fluoxetine.
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(A) HeLa R19 cells were infected with WT CVB3 or the AVIVAV mutant (A224V-I227V-A229V triple
mutant) at a MOI 0.1 and treated with 1 µM (S)-fluoxetine or 1 µM BF738735 as a control replication inhibitor
that acts via the host protein PI4KBIIIβ 48. Eight hours post infection, cells were freeze-thawed and virus titers
were determined by endpoint titration. Means and standard deviations were calculated from biological
triplicate. (B) The individual A224V and I227V mutations were tested for resistance towards (S)-fluoxetine as
in (A). (C) The guanidine chloride-dependent virus A229V was tested for dependency on (S)-fluoxetine.
Experiments were performed similar as in (A). Because the A229V virus is dependent on GuaHCl, sensitivity
to the inhibitors was also tested in the presence of 1 mM GuaHCl (blocked bars). (D) Residues in the
hydrophobic binding pocket were mutated (C179F and F190L) and tested for resistance towards (S)-fluoxetine
as in (A). (E) Residues at the surface of the hydrophobic binding pocket (V187M and D245N) were substituted
in the Renilla luciferase virus and sensitivity to (S)-fluoxetine was determined as in Figure 1C. (F) The binding
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of (S)-fluoxetine to recombinant WT CVB3 2C or 2C harboring the resistance mutations C179F, I227V and
A229V was tested using thermal shift assay as in Figure 2A. In all panels data are shown from one experiment
representative of at least two independent experiments. Error bars depict standard error of the mean calculated
from biological triplicates
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To dissect the contribution of the individual mutations to the resistance, several CVB3 mutant viruses
containing either the single mutations A224V, I227V and A229V were made. The A224V mutation alone did
not confer resistance to the compounds (Figure 5B). The single mutation I227V provided a high level of
resistance towards (S)-fluoxetine, but not the control compound BF738735 (Figure 5B). This is in concordance
with a recently published report, which raised resistant mutants towards racemic fluoxetine and identified the
mutations I227V and N228S 41. The A229V single mutant virus was previously described to be dependent on
all structurally divergent 2C inhibitors tested (GuaHCl, HBB, TBZE-029 and MRL-1237) 29, meaning that
these mutant viruses are not inhibited by the compounds but instead strictly require them for efficient
replication. Strikingly, the A229V virus was not dependent on (S)-fluoxetine at the concentration tested (Figure
5C). To test whether the A229V virus is resistant to (S)-fluoxetine we assessed replication in the presence of
both GuaHCl (to allow replication) and (S)-fluoxetine. However, the A229V mutation does not confer
resistance to (S)-fluoxetine.
To find experimental support for the predicted binding pocket, we next investigated the importance of key
residues in site A pocket by mutational analysis. We first mutated two amino acids that are located deep within
site A and tested whether they could confer resistance to (S)-fluoxetine. These mutations are C179F, which –
like C179Y - provides resistance towards several structurally different 2C inhibitors 41, and F190L, which
raises resistance to a novel 2C inhibitor 49. In line with our model, viruses containing the C179F or the F190L
mutation in 2C were highly resistant to (S)-fluoxetine (Figure 5D). We next investigated two amino acids that
are located near the edge of site A. V187 was substituted by a M, because the corresponding amino acid 187
in the fluoxetine-insensitive virus EV-A71 is a methionine and we hypothesized that this methionine may
contribute to the insensitivity of EV-A71 to fluoxetine. The amino acid D245 displayed repeated interactions
with the positively charged amino group of (S)-fluoxetine in the molecular dynamics simulations (Figure 4C).
We therefore decided to remove the charge of this residue by replacing it with asparagine. However, neither
the V187M nor the D245N mutations provided resistance to (S)-fluoxetine (Figure 5E), which seems to
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contradict our computation-based hypothesis that (S)-fluoxetine binds to site A interacting with C187 and
D245. Fluoxetine may bind in a slightly different conformation within the pocket that does not involve
interactions with these two residues, thus mutations would not confer resistance. That would still explain why
the C179F and F190L mutations confer resistance. Alternatively, fluoxetine may access residues C179 and
F190 from a third entrance site (Site C), schematically depicted in Figure 4D (cyan arrow), and therefore being
unaffected by the mutations in site A and the 224AGSINA229 loop. However, neither in the homology model
nor in the EV-A71 crystal structure the supposed site C presents an obvious cavity for potential ligand entrance
towards the residues C179 and F190. Because of flexibilities in the loops ranging from amino acid residue 158
to 163, 175 to 183 and 224 to 229 other conformations likely exist so that site C may become accessible. In
that case the occurrence of mutations in the 224AGSINA229 loop and how they can confer resistance is more
difficult to explain. We propose the hypothesis that conformational changes over a longer range could affect
the shape of the pocket and therefore slight changes in the distant 224AGSINA229 loop might be sufficient to
convey resistance. Clearly, actual crystallographic data of (S)-fluoxetine bound to 2C is needed to definitively
resolve this point.
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Finally, we wanted to address whether resistance mutations in 2C affect binding of (S)-fluoxetine to 2C.
Recombinant 2C proteins harboring the resistance mutations I227V or C179F or the A229V mutation were
produced and binding was tested by TSA (Figure 5F). (S)-fluoxetine was not able to stabilize any of the
mutated 2C proteins from thermal denaturation, implying that the substitutions abrogated fluoxetine binding
to 2C. Unexpectedly, the C179F substitution made 2C more sensitive to thermal denaturation in the presence
of (S)-fluoxetine when compared to the C179F mutant without compound (ΔTm -2.5°C). The A229V
substitution nullified thermal stabilization of 2C by (S)-fluoxetine, suggesting that this mutation abrogated
binding of (S)-fluoxetine. However, the GuaHCl-dependency of the A229V virus required combining GuaHCl
and (S)-fluoxetine in the TSA experiment, which may add confounding effects (Figure 5C).
Summarized, mutations at position I227 in the 224AGSINA229 loop, as well as the C179F and F190L
mutations at the bottom of the binding site A confer resistance to (S)-fluoxetine, while the V187M and D245N
mutations, which are more at the edge of the pocket, do not.
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DISCUSSION
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Enteroviruses are a major global health burden, but currently no antiviral therapy is available. The high degree
of conservation makes the enterovirus 2C protein an attractive target for the development of broad-spectrum
enterovirus inhibitors (Supplementary Figure 3). Several compounds, including a number of repurposed drugs,
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have been identified as inhibitors of enterovirus replication by targeting 2C (reviewed in ), but to date the
molecular mechanisms underlying the antiviral effects are lacking. Fluoxetine, one of the identified
compounds, is an FDA-approved drug that is used as a highly selective inhibitor of SERT for the treatment of
major depression and anxiety disorders. In this study, we provide new insights into how the repurposed drug
fluoxetine acts as antiviral compound against CVB3.
Fluoxetine has one chiral center and only the S-enantiomer of fluoxetine has anti-enteroviral activity and binds
to 2C in vitro. In previous studies the racemic mixture of fluoxetine inhibited EV-B and EV-D species, but not
EV-A, EV-C and rhinoviruses 33-34. Here we show that the S-enantiomer, but not the R-enantiomer, has a clearly
increased antiviral potency compared to the racemic mixture against CVB3 and also EV-D68, while the
cytotoxicity in cell culture is comparable (Table 1). Unexpectedly, we observed that the S-enantiomer also
exerts antiviral activity against rhinoviruses. Presumably, the antiviral activity of the racemic mixture against
rhinoviruses is so weak that it cannot be separated from cytotoxicity effects. We cannot exclude that (S)-
fluoxetine would also inhibit EV-A or EV-C species at even higher concentrations, but this cannot be tested in
the current model systems due to cytotoxicity. Another explanation for why EV-A and EV-C species are not
sensitive to fluoxetine is that there could be strain-specific differences in the sensitivity towards inhibitors. For
example, we show that also (S)-fluoxetine does not inhibit EV-A71 (strain BrCr). It is unknown why EV-A71
is resistant to (S)-fluoxetine, further in-depth investigations are needed to gain a better understanding of the
underlying mechanism of resistance of EV-A71. However, a different EV-A71 strain was shown to be sensitive
to racemic fluoxetine 41. Thus, it is possible that different EV-A and EV-C strains are sensitive towards (S)-
fluoxetine.
The in vivo efficacy of fluoxetine towards enterovirus infections has until now been relatively poorly studied.
Recent outbreaks of acute flaccid myelitis (AFM) in the US are – at least in part – associated with EV-D68 50.
In a mouse model for EV-D68 associated paralysis, fluoxetine did not have an effect on motor impairment of
mice or viral load in muscle and spinal cord, but instead seemed to slightly aggravate disease 51. Because in
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human AFM cases treatment options other than supportive care are lacking, several clinicians have tried off-
label use of fluoxetine to treat pediatric patients. A retrospective study of safety and efficacy of fluoxetine to
treat AFM revealed no beneficial effect of fluoxetine 52. Instead, fluoxetine-treated patients had somewhat
more severe symptoms, suggesting a negative effect of fluoxetine on AFM, in line with the mouse model 51.
However, this retrospective study had some limitations that make it difficult to draw definitive conclusions.
For example, patients had been treated with different dosing regimens. Furthermore, fluoxetine treatment was
started only after onset of AFM symptoms when irreversible neuronal damage may already have been inflicted.
Moreover, viral loads were not tested, leaving the question unanswered whether the virus was still actively
replicating at the time of treatment and – if so – whether fluoxetine had any effect on viral loads. In contrast,
in a pancreatitis mouse model the racemic fluoxetine mixture reduced the levels of CVB4 infectious particles
in heart and pancreas and reduced pancreatitis severity when it was given one day prior to CVB4 infection and
subsequently every day post infection 53. Finally, in a case study of an immunocompromised child with chronic
EV-B induced encephalitis, off-label use of fluoxetine eliminated the virus and led to recovery of the patient
38. These seemingly opposing results stress the importance of further in vivo studies of fluoxetine to combat
different enterovirus and treat different types of enterovirus-associated diseases. The plasma concentration of
fluoxetine ranges from 91 to 300 ng/ml after 30 days of dosing 40 mg/day54. The metabolite norfluoxetine,
which also shows antiviral activity 33, reaches a plasma concentration from 72 to 258 ng/ml. The slow
elimination of fluoxetine together with the metabolite norfluoxetine should reach a sufficient plasma
concentration that correspond with the EC50 in cell culture (~1 μM, which corresponds to ~150 ng/ml).
In contrast to the SSRI activity, leading to the antidepressant effect of fluoxetine, the 2C-mediated antiviral
activity of fluoxetine is stereoselective, lending further support to the idea that the antiviral activity is unrelated
to the known SSRI activity of fluoxetine. This implies that, when fluoxetine is used as an antiviral treatment,
any potential SSRI-related side effects can be reduced by providing only (S)-fluoxetine, which is then at lower
overall concentration. However, when only (S)-fluoxetine is used, the advantages of drug repurposing of
fluoxetine, which is licensed as a racemic mixture, are nullified and new safety studies are needed. Fragment
1, which contains the 4-(trifluoromethyl)benzene moiety and the amino group, gave the first indication for the
importance of these chemical features for the antiviral effect. From there on, structure activity relationship
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studies could help to design new molecules with even a stronger antiviral effect, but reduced or suppressed
SERT inhibition.
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To investigate the mode-of-action of how fluoxetine binds to enterovirus 2C, we built a homology model of
CVB3 based on the crystal structure of the fluoxetine-insensitive EV-A71 2C. In the predicted binding model,
the 4-(trifluoromethyl)benzene group of fluoxetine occupies the deep hydrophobic pocket close to residues
L126, L178, V187, F190, L238, I227, A229, F242. Mutational studies to test the binding hypothesis were
designed and mutations at the bottom of the predicted pocket reduced (S)-fluoxetine binding by providing
resistance. The mutations on the borders of the pocket did not confer resistance to (S)-fluoxetine therefore we
cannot exclude alternative entrance sites to the hydrophobic cavity. Crystallographic data is needed to yield
ultimate clarity about the binding mode.
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2C is a member of the SF3 helicases of the AAA+ ATPase superfamily and contains Walker A and B motifs,
which are important for recognizing the triphosphate of ATP, and a short motif C that is located on top of
Walker B 7. The residues L178 and C179 in the predicted binding pocket are located within the Walker B motif
or immediately thereafter. Furthermore, the 224AGSINA229 loop, which harbors resistance mutations and
lines the edge of the predicted pocket, is directly C-terminal to the motif C. Residues L238, F242 and D245
are in proximity to the arginine fingers (R240 and R241), which play an important role in the ATPase domain
and are required for ATP hydrolysis. Hence, it is conceivable that fluoxetine inhibits ATPase activity
allosterically through relatively short-range effects although alternative modes, e.g. through long-range effects,
cannot be excluded. Still, the exact molecular details of how fluoxetine inhibits the ATPase activity of 2C
remain to be determined.
The 224AGSINA229 loop forms a hot-spot for resistance mutations against fluoxetine and many other 2C
inhibitors, while fewer mutations have been found in the predicted fluoxetine binding pocket. The residues in
the pocket are evolutionarily highly conserved, implying that there is little room for variation that could induce
resistance in the pocket residues without affecting virus fitness. In contrast, the 224-229 loop diverges more
between enterovirus species, suggesting that the loop allows for more sequence diversity that could yield
resistance (Supplementary Figure 3). The 224AGSINA229 loop is conserved between the fluoxetine sensitive
viruses CVB3 and EV-D68, but the motif differs in the fluoxetine resistant viruses EV-A71 and poliovirus
(Supplementary Figure 3). The corresponding 224AGSINA229 loops might be more rigid in these latter
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viruses, thereby interfering with fluoxetine binding. Mutations in the 224AGSINA229 loop confer resistance
not only against fluoxetine, but also against several other compounds, including TBZE-029, HBB and MRL-
1237 and GuaHCl 29. Resistance mutations may favor a conformation of the 224AGSINA229 loop in which
the binding pocket is not accessible for fluoxetine anymore. Furthermore, the mutations may change the
flexibility of the loop in such a way that the inhibitory effect of fluoxetine is circumvented. Further biophysical
studies are needed to decipher how the resistance mutations affect the overall stability of the protein or the
accessibility of the binding pocket. However, addressing the possible role of S-fluoxetine in the hexamerisation
of 2C is presently not possible because the production of homogenous 2C protein in its biologically relevant
oligomerization state has not yet been achieved.
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In conclusion, this study sheds new light onto how the 2C inhibitor fluoxetine may target the enterovirus 2C
protein. In particular, the discovery of the stereoselective activity will fuel further mode-of-action studies and
support the rational design of novel, fluoxetine-derived broad-spectrum enterovirus inhibitors.
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3.1
Cells and Reagents
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Buffalo Green Monkey cells (BGM) and HeLa R19 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM, Lonza) supplemented with 10 % fetal bovine serum (FBS, Lonza). Huh7-Lunet 7/T7, a
stable cell pool expressing T7 RNA polymerase and blasticidin S-deaminase 55, were cultured in DMEM
supplemented with 10% FBS and 10 µg/ml blasticidin (Sigma-Aldrich). All cell lines were grown at 37°C in
5 % CO2. Guanidine hydrochloride (GuaHCl) was purchased from Sigma-Aldrich. Racemic mixture of
fluoxetine was purchased from Sigma-Aldrich. The S- and R-enantiomers were purchased either from Sigma-
Aldrich or Carbosynth. BF738735 was provided by Galapagos NV 48. Dibucaine was purchased from Sigma-
Aldrich. GuaHCl was dissolved in water at 2M stock concentration and all other compounds were dissolved
in DMSO at 10 mM stock concentration.
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3.2
Viruses
EV-A71 (strain BrCr), PV1 (strain Sabin, ATCC) and EV-D68 (strain Fermon) were obtained from the
National Institute for Public Health and Environment (RIVM) in the Netherlands. HRV-2 and HRV-14 were
obtained from Joachim Seipelt from the Medical University of Vienna in Austria. RLuc-CVB3, which contains
a Renilla luciferase gene upstream of the capsid coding region, was obtained by transfecting Huh7-Lunet/T7
cells with MluI-linearized pRLuc-53CB3/T7 plasmid as described 56. RLuc-encephalomyocarditis virus
(EMCV, strain mengovirus), encoding a Renilla luciferase gene upstream of the capsid-coding region, was
described before 42-43 CVB3 (strain Nancy) and CVB3 2C mutant viruses were obtained by transfecting BGM
cells with RNA transcripts derived from the full-length infectious clones p53CB3/T7 as described in 57. The
mutations 2C[A224V], 2C[I227V], 2C[A229V], 2C[A224V/I227V], 2C[A224V/A229V], 2C[I227V/A229V],
2C[A224V/I227V/A229V], 2C[C179F] and 2C[F190L] were introduced into the p53CB3/T7 infectious clone
and 2C[V187M] and 2C[D245N] were introduced into the pRLuc-53CB3/T7 using side directed mutagenesis.
In vitro transcribed RNA transcripts were transfected into Hela R19 cells to obtain virus. To ensure that the
introduced mutations are retained in the generated virus, viral RNA was isolated with NucleoSpin® RNA Virus
kit (Macherey-Nagel) according to the manufacturer’s protocol and the presence of the desired mutations was
confirmed by Sanger sequencing. Virus titers were determined by endpoint dilution titration and calculated
according to the method of Reed and Muench 58 and expressed as 50% cell culture infective dose (CCID50).
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Single-cycle virus infection
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Virus infections were performed by incubating subconfluent HeLa R19 cells with virus at a multiplicity of
infection (MOI 0.1) at 37°C for 30 min. Next, the medium was removed and fresh (compound-containing)
medium was added to the cells. At the indicated time points, the medium was discarded and cells were lysed.
For measurements of infectious particles, virus was released from the cells by three freeze-thawing cycles.
Virus titers were determined by end-point dilution assay and calculated by the method of Reed and Muench
58. In the case of infection with RLuc-CVB3 and RLuc-EMCV, cells were lysed 6-7 hrs post infection and the
Renilla luciferase Assay System (Promega) was used to determine the luciferase activity. Where indicated,
cell viability was determined in parallel using the AQueous One Solution Cell Proliferation Assay (Promega)
according to the manufacturer’s protocol. Optical density at 490 nm was determined using a microplate reader.
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3.4
Multicycle virus infection
Subconfluent layers of HeLa R19 cells were seeded in 96-wells and treated with serial dilutions of the
corresponding compounds. Cells were infected with CVB3 at the lowest possible MOI (MOI 0.001) resulting
in full CPE within 3 days. Subsequently the cells were incubated at 37°C for 3 days until full CPE was observed
in the virus infected untreated cell controls. Cell viability was determined in parallel using the AQueous One
Solution Cell Proliferation Assay (Promega) according to the manufacturer’s protocol. The optical density at
490 nm was determined using a microplate reader. Raw OD values were converted to percentage of untreated
and uninfected cell control after subtraction of the background.
3.5
Binding of fluoxetine to recombinant WT and mutant 2C proteins
The DNA fragment coding for CVB3 2C (amino acids 37 to 329) was cloned downstream of a cleavable
thioredoxin-hexahistidine tag. Mutations were introduced into the 2C coding sequence by PCR-based site-
directed mutagenesis. The recombinant WT and mutant proteins were produced in Escherichia coli T7 Express
(New England BioLabs) at 17°C. Protein purification and tag removal were performed under nondenaturing
conditions as previously described 35. The final size-exclusion chromatography step was performed with a
buffer containing 10 mM HEPES and 300 mM NaCl (pH 7.5). The binding of fluoxetine or corresponding
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fragments on WT and mutant 2C proteins was monitored by fluorescence-based thermal shift assay (TSA)
using a Bio-Rad CFX Connect. TSA plates were prepared by dispensing into each well the 2C protein (final
concentration of 15 μM in 50 mM Tris, 300 mM NaCl, pH 8) which was mixed with 1 μL of fragment or
fluoxetine (from 20 mM stock in 100% DMSO, 1 mM final concentration in 4% DMSO) and a SYPRO orange
solution in concentrations recommended by the manufacturer in a final volume of 25 μL. The experiments
were performed under a temperature gradient ranging from 20 to 95°C (incremental steps of 0.2°C/12 seconds).
The denaturation of the proteins was monitored by following the increase of the fluorescence emitted by
SYPRO orange that binds exposed hydrophobic regions of the denatured protein. The melting temperature
(Tm) was calculated as the mid-log of the transition phase from the native to the denatured protein using a
Boltzmann model (Origin software). The reference unfolding temperature of proteins in 4% DMSO (T0) was
subtracted from the values in the presence of fragment (Tm) to obtain thermal shifts, ΔTm = Tm – T0.
The binding of (S)- and (R)-fluoxetine to WT CVB3 2C was further characterized by isothermal titration
calorimetry (ITC) using a MicroCal iTC200 instrument (Malvern). Experiments were carried out at 20°C in a
solution containing 10 mM HEPES, 300 mM NaCl, and 0.8% DMSO (pH 7.5). The 2C protein concentration
in the cell was 80 μM whereas the fluoxetine concentration in the syringe was 400 μM. For (R)-fluoxetine, two
injections were necessary. Heats of dilution were measured by injecting the ligand into the protein solution.
Titration curves were fitted by using MicroCal Origin software, assuming one set of sites, and enthalpy changes
(ΔH), dissociation equilibrium constants (Kd), and stoichiometry were extracted.
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3.6
Molecular modelling
The computational studies were carried out on 1.80 GHz Intel Xeon (8 cores) processor-based system, running
Ubuntu 14.04 LTS, using Molecular Operating Environment (MOE) 2015.10 (Chemical Computing Group
Inc. 2016) and Maestro v11.4 (Schrödinger LLC, New York, NY, 2017). The homology model was generated
with MOE using integrated sequence alignment and structure preparation tools for the template. Preparation
of the structure for docking and molecular dynamic simulations and subsequent data analysis was carried out
with Maestro. Docking experiments were performed using the GlideSP module in Maestro, running the default
settings. The molecular dynamics simulations were performed using Desmond package (Desmond Molecular
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Dynamics System, D. E. Shaw Research, New York, NY, 2018. Maestro-Desmond Interoperability Tools,
Schrödinger, New York, NY, 2018). Pictures of molecular modelling were prepared using MOE.
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Homology modelling
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The protein sequence of CVB3 (strain Nancy) 2C protein was downloaded from Uniprot (ID: P03313 amino
acids 1101-1429). The structures reported in 39, especially 5GRB, was used as a starting point in this study and
was retrieved from the Protein Data Bank. 5GRB contains ATPγS and was the structure used for the
computational studies. The sequence of CVB3 was aligned to the sequence of the crystalized EV-A71 using
MOE. 5GRB chain A was used as a structural template for the homology model. The homology model was
built with the Amber12:EHT force field 59-60. Automatic detection of disulfide bridges was disabled. Ten
intermediate models were generated and refined using a medium refinement by molecular mechanics (highly
tethered minimization to relieve steric strains). The final model was calculated using Coulomb and Generalized
Born / Volume Integral (GB/VI) interaction energies 61 and was not further refined.
After the generation of the homology model the structure was revised using the Structure Preparation function
in MOE. In order to further evaluate the quality of the homology model for future studies the phi/psi angles
were analyzed in the Ramachandran plot using the Protein Geometry tool of MOE. Identified outliers were
investigated and, if relevant, corrected manually. Then the validation of the model was carried out using
RAMPAGE Ramachandran plot analysis (accessed at mordred.bioc.cam.ac.uk/~rapper/rampage.php). Amino
acid
environment
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3.8
Site Finder
The site identification tool Site Finder, which is comprised in the software suite MOE, was run on the prepared
CVB3 homology model to identify possible active sites for the known 2C targeting compounds. Two binding
pockets (termed site A and site B) were selected on the basis of their vicinity to known mutations in the
224AGSINA229 loop that convey resistance against several known 2C inhibitors including fluoxetine 29, 34-35
.
For both pockets, a set of dummy atoms was created in the positions of the alpha spheres that are used to
determine pockets in Site Finder.
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Docking
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After the identification of site A and site B, both the compounds and the protein were prepared for the docking
with Glide. The homology model of CVB3 was prepared with the Protein Preparation Wizard embedded in
Maestro. For each binding site a grid box for the positioning of the molecules during the docking was generated
setting the centers of the boxes to the coordinates of representative dummy atoms generated by Site Finder.
The stereochemistry on the chiral center of fluoxetine was defined using the molecule builder in MOE and the
two enantiomers were saved in separate .sdf files. Both of them were subjected to the ligand preparation
protocol (ligprep) in Maestro creating up to 32 conformations each. Then, all conformations obtained for (R)-
and (S)-fluoxetine, were docked with Maestro Glide in standard precision (SP) mode into each binding site.
The poses were inspected for their fit within the pockets and their interactions with the protein. The best
protein-ligand complexes for each site and each enantiomer were saved and prepared for molecular dynamics
(MD) simulations.
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3.10
Molecular dynamics simulations
All MD simulations were performed using Desmond, part of the Maestro v11.4 simulation package
(Schrödinger LLC, New York, NY, 2017). OPLS3 was used as the force field. The complexes of (R)-fluoxetine
and (S)-fluoxetine docked to 2C of CVB3 were placed in a cubic box (buffer 10Å) using the TIP3P water
model. The negative charges on the protein were neutralized adding Na+ atoms to the system. Magnesium
chloride (10 mM) was added to the box to simulate physiological conditions. Before the MD simulation the
system was first equilibrated for 112ps at 10 K in an NVT ensemble and then simulated for 48 ps at constant
pressure of 1 atm using the NPT ensemble. All MD simulations were performed for 100 ns at constant
temperature (300 K) and pressure recording snapshots every 160 ps.
The estimated ΔGbinding was calculated using the Desmond command-line script thermal_mmgbsa.py. After
splitting the trajectory file of the MD simulation into snapshots the script is calculating the average computed
binding energy of the ligand (Table 2 and Supplementary Figure 2).
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Chemistry
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All solvents and reagents used were obtained from commercial sources unless otherwise indicated. All
reactions were performed under a nitrogen atmosphere. 1H and 13C NMR spectra were recorded with a Bruker
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standard. Deuterated dimethyl sulfoxide (DMSO) was used as the solvent for NMR experiments. 1H chemical
shifts values (δ) are referenced to the residual non-deuterated components of the NMR solvents (δ = 2.50 ppm
for DMSO). The 13C chemical shifts (δ) are referenced to DMSO (central peak, δ = 39.5 ppm). Thin layer
chromatography (TLC) was performed on Silica gel plates (Merck Kieselgel 60 F254), which were developed
by the ascending method. Column chromatography was performed on an Isolera Biotage system. Purity of
synthesized compounds was determined by UPLC-UV-MS analysis (Waters UPLC system with both Diode
Array detection and Electrospray (+’ve and –‘ve ion) MS detection). The purity of all compounds was
determined to be >95% by UPLC using the eluents H2O containing 0.1% Trifluoroacetic acid (eluent A) and
Acetonitrile containing 0.1% Trifluoroacetic acid (eluent B) at the following conditions: Waters Acquity
UPLC BEH C18 1.7 µm 2.1x50 mm column, 0.5 mL/min, column temperature 40°C. Sample diluent:
Acetonitrile. Sample concentration 10 µg/mL. Injection volume 2 µL, gradient 90% eluent A (0.1 min), 90%-
0% eluent A (1.5 min), 0% eluent A (1.4 min), 90% eluent A (0.1 min) (method 1).
3.12
Synthesis of 1-(3-bromopropoxy)-4-(trifluoromethyl)benzene (intermediate)
To a solution of 1,3-dibromopropane (6.17 mmol) and potassium carbonate (4.63 mmol) in DMF (3 mL), 4-
(trifluoromethyl)phenol (3.08 mmol) in DMF (1.2 mL) was added dropwise, the reaction mixture was stirred
at room temperature for two hours and then heated to 70ºC for two hours. The mixture was filtrated, diluted
with ethyl acetate (10 mL) and washed with water (3x10 mL). The organic layer was dried over sodium
sulphate and evaporated under reduced pressure. The residue was purified by flash column chromatography
eluting with n-hexane/EtOAc 100:0 v/v increasing to n-hexane/EtOAc 70:30 v/v. 207 mg of 1-(3-
bromopropoxy)-4-(trifluoromethyl)benzene were obtained in 47% yield as a yellow oil. 1H NMR (DMSO) δ
7.65 (d, J = 8.7 Hz, 2H), 7.14 (d, J = 8.7 Hz, 2H), 4.17 (t, J = 6.0 Hz, 2H), 3.68 (t, J = 6.6 Hz, 2H), 2.28 (p, J
= 6.3 Hz, 2H). 19F NMR (DMSO) δ -59.83 (s, 3F). 13C NMR (DMSO) δ 161.67, 127.44 (m), 125.02 (q, J =
271.0 Hz), 121.73 (q, J = 32.1 Hz), 115.44, 32.08, 31.50.
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