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The serotonin reuptake inhibitor Fluoxetine inhibits SARS-CoV-2
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The serotonin reuptake inhibitor Fluoxetine inhibits SARS-CoV-2
Melissa Zimniak 1
, Luisa Kirschner1 , Helen Hilpert 1
, Jürgen Seibel 2 , and Jochen Bodem 1*
1 Julius-Maximilians-Universität Würzburg, Institut für Virologie und Immunbiologie
2 Julius-Maximilians-Universität Würzburg, Institute of Organic Chemistry
*Contact:
Jochen Bodem
Institute of Virology and Immunobiology
Versbacher Str. 7
97078 Würzburg
Germany
Jochen.Bodem@vim.uni-wuerzburg.de
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprintthis version posted June 14, 2020..https://doi.org/10.1101/2020.06.14.150490doi:bioRxiv preprint
Melissa Zimniak 1
, Luisa Kirschner1 , Helen Hilpert 1
, Jürgen Seibel 2 , and Jochen Bodem 1*
1 Julius-Maximilians-Universität Würzburg, Institut für Virologie und Immunbiologie
2 Julius-Maximilians-Universität Würzburg, Institute of Organic Chemistry
*Contact:
Jochen Bodem
Institute of Virology and Immunobiology
Versbacher Str. 7
97078 Würzburg
Germany
Jochen.Bodem@vim.uni-wuerzburg.de
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprintthis version posted June 14, 2020..https://doi.org/10.1101/2020.06.14.150490doi:bioRxiv preprint
Abstract:
To circumvent time-consuming clinical trials, testing whether existing drugs are effective
inhibitors of SARS-CoV-2, has led to the discovery of Remdesivir. We decided to follow this
path and screened approved medications “off-label” against SARS-CoV-2. In these
screenings, Fluoxetine inhibited SARS-CoV-2 at a concentration of 0.8µg/ml significantly,
and the EC50 was determined with 2.5ng/ml. Fluoxetine is a racemate consisting of both
stereoisomers, while the S-form is the dominant serotonin reuptake inhibitor. We found that
both isomers show similar activity on the virus. Fluoxetine treatment resulted in a decrease in
viral protein expression. Furthermore, Fluoxetine inhibited neither Rabies virus, human
respiratory syncytial virus replication nor the Human Herpesvirus 8 or Herpes simplex virus
type 1 gene expression, indicating that it acts virus-specific. We see the role of Fluoxetine in
the early treatment of SARS-CoV-2 infected patients of risk groups.
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprintthis version posted June 14, 2020..https://doi.org/10.1101/2020.06.14.150490doi:bioRxiv preprint
To circumvent time-consuming clinical trials, testing whether existing drugs are effective
inhibitors of SARS-CoV-2, has led to the discovery of Remdesivir. We decided to follow this
path and screened approved medications “off-label” against SARS-CoV-2. In these
screenings, Fluoxetine inhibited SARS-CoV-2 at a concentration of 0.8µg/ml significantly,
and the EC50 was determined with 2.5ng/ml. Fluoxetine is a racemate consisting of both
stereoisomers, while the S-form is the dominant serotonin reuptake inhibitor. We found that
both isomers show similar activity on the virus. Fluoxetine treatment resulted in a decrease in
viral protein expression. Furthermore, Fluoxetine inhibited neither Rabies virus, human
respiratory syncytial virus replication nor the Human Herpesvirus 8 or Herpes simplex virus
type 1 gene expression, indicating that it acts virus-specific. We see the role of Fluoxetine in
the early treatment of SARS-CoV-2 infected patients of risk groups.
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprintthis version posted June 14, 2020..https://doi.org/10.1101/2020.06.14.150490doi:bioRxiv preprint
Starting in December 2019, SARS-CoV-2 originated in central China became a pandemic
thread with more than 7.000.000 cases worldwide and more than 400.000 deaths so far.
Despite these high case rates Remdesivir,1 the only effective treatment is still not available
for most of the patients. At the same time, other promising substances like Lopinavir2 and
Chloroquine 3 showed little effect or led to severe adverse side effects. Furthermore, some
drugs, such as ribavirin and interferon, 4 had no significant impact on patient survival rates.
To circumvent time-consuming clinical trials, testing whether existing drugs are effective
inhibitors of SARS-CoV-2, has led to the discovery of Remdesivir.5 We decided to follow this
path and screened approved medications “off-label”6 against SARS-CoV-2. In such a trial, we
investigated the effect of the serotonin reuptake receptor inhibitors (SRRI) Fluoxetine,
Escitalopram, and Paroxetine on viral replication. For that, cytotoxicity and viral replication
rates of a patient-derived virus isolate were measured. To investigate cytotoxicity, Vero cells
were incubated with the compounds for three days, and cell growth was determined using a
Perkin Elmer Ensight reader. The Vero cells were incubated with the compounds at
increasing concentrations and subsequently infected with SARS-CoV-2 at an MOI of approx.
0.5. The concentrations were selected near the concentration used for the treatment of
depression (e.g., 0.8 µg/ml for Fluoxetine). DMSO was used as solvent control. After three
days, viral replication supernatants were collected and viral RNA was extracted using a
MagNA Pure 24 system (Roche). Viral replication was quantified by real time RTqPCR with
the LightMix Assay SARS-CoV-2 RdRP RTqPCR assay kit (TIB MOLBIOL, Germany) and
the RNA Process Control kit (Roche) (Figure 1A). The PCR was pipetted with a pipette robot
device to ensure quality (BRAND, Germany). All infections were performed in triplicates and
reaped twice. Fluoxetine inhibited SARS-CoV-2 at a concentration of 0.8µg/ml significantly,
and the EC50 was determined with 2.5ng/ml (Figure 1A). However, it is unlikely that the
direct inhibition of the serotonin reuptake transporter is responsible for this suppression since
neither Paroxetine nor Escitalopram interfere with viral replication. Furthermore, since most
of the cellular off-target factors of the different compounds are similar, Fluoxetine presumably
targets the virus directly. Fluoxetine is a racemate consisting of both stereoisomers, while
the S-form is the dominant SSRI.7 Thus, we analyzed which stereoisomer is inhibiting SARS-
CoV-2 replication. We found that both isomers show similar activity on the virus (Figure 1A)
underlining that the antiviral effect is unrelated to the serotonin reuptake receptor. To get
further insights into the mechanism of inhibition, we sought to visualize viral protein
expression by immunofluorescence with a patient-derived antiserum. Fluoxetine treatment
resulted in a decrease in protein expression, showing that Fluoxetine acts upstream of gene
expression (Figure 1B). To further analyze the specificity of Fluoxetine, the inhibition of other
viruses was determined. Fluoxetine inhibited neither Rabies virus, human respiratory
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprintthis version posted June 14, 2020..https://doi.org/10.1101/2020.06.14.150490doi:bioRxiv preprint
thread with more than 7.000.000 cases worldwide and more than 400.000 deaths so far.
Despite these high case rates Remdesivir,1 the only effective treatment is still not available
for most of the patients. At the same time, other promising substances like Lopinavir2 and
Chloroquine 3 showed little effect or led to severe adverse side effects. Furthermore, some
drugs, such as ribavirin and interferon, 4 had no significant impact on patient survival rates.
To circumvent time-consuming clinical trials, testing whether existing drugs are effective
inhibitors of SARS-CoV-2, has led to the discovery of Remdesivir.5 We decided to follow this
path and screened approved medications “off-label”6 against SARS-CoV-2. In such a trial, we
investigated the effect of the serotonin reuptake receptor inhibitors (SRRI) Fluoxetine,
Escitalopram, and Paroxetine on viral replication. For that, cytotoxicity and viral replication
rates of a patient-derived virus isolate were measured. To investigate cytotoxicity, Vero cells
were incubated with the compounds for three days, and cell growth was determined using a
Perkin Elmer Ensight reader. The Vero cells were incubated with the compounds at
increasing concentrations and subsequently infected with SARS-CoV-2 at an MOI of approx.
0.5. The concentrations were selected near the concentration used for the treatment of
depression (e.g., 0.8 µg/ml for Fluoxetine). DMSO was used as solvent control. After three
days, viral replication supernatants were collected and viral RNA was extracted using a
MagNA Pure 24 system (Roche). Viral replication was quantified by real time RTqPCR with
the LightMix Assay SARS-CoV-2 RdRP RTqPCR assay kit (TIB MOLBIOL, Germany) and
the RNA Process Control kit (Roche) (Figure 1A). The PCR was pipetted with a pipette robot
device to ensure quality (BRAND, Germany). All infections were performed in triplicates and
reaped twice. Fluoxetine inhibited SARS-CoV-2 at a concentration of 0.8µg/ml significantly,
and the EC50 was determined with 2.5ng/ml (Figure 1A). However, it is unlikely that the
direct inhibition of the serotonin reuptake transporter is responsible for this suppression since
neither Paroxetine nor Escitalopram interfere with viral replication. Furthermore, since most
of the cellular off-target factors of the different compounds are similar, Fluoxetine presumably
targets the virus directly. Fluoxetine is a racemate consisting of both stereoisomers, while
the S-form is the dominant SSRI.7 Thus, we analyzed which stereoisomer is inhibiting SARS-
CoV-2 replication. We found that both isomers show similar activity on the virus (Figure 1A)
underlining that the antiviral effect is unrelated to the serotonin reuptake receptor. To get
further insights into the mechanism of inhibition, we sought to visualize viral protein
expression by immunofluorescence with a patient-derived antiserum. Fluoxetine treatment
resulted in a decrease in protein expression, showing that Fluoxetine acts upstream of gene
expression (Figure 1B). To further analyze the specificity of Fluoxetine, the inhibition of other
viruses was determined. Fluoxetine inhibited neither Rabies virus, human respiratory
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprintthis version posted June 14, 2020..https://doi.org/10.1101/2020.06.14.150490doi:bioRxiv preprint
syncytial virus (RSV) replication nor the Human Herpesvirus 8 or Herpes simplex virus type 1
gene expression, indicating that it acts virus-specific (Figure 1C).
Fluoxetine was introduced in clinics during the seventies and is a well-studied drug since it
has been used in humans for almost four decades. Furthermore, the patent of Fluoxetine has
long expired. Thus, it is available from different companies, and relatively cheap. We see the
role of Fluoxetine in the early treatment of SARS-CoV-2 infected patients of risk groups.
References
1 Goldman JD, Lye DCB, Hui DS et al. Remdesivir for 5 or 10 Days in Patients with Severe
Covid-19. N Engl J Med 2020.
2 Cao B, Wang Y, Wen D et al. A Trial of Lopinavir-Ritonavir in Adults Hospitalized with
Severe Covid-19. N Engl J Med 2020; 382:1787-1799.
3 Ferner RE, Aronson JK. Chloroquine and hydroxychloroquine in covid-19. Bmj 2020;
369:m1432.
4 Hung IF-N, Lung K-C, Tso EY-K et al. 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; 395:1695-1704.
5 Wang M, Cao R, Zhang L et al. Remdesivir and chloroquine effectively inhibit the recently
emerged novel coronavirus (2019-nCoV) in vitro. Cell Res 2020; 30:269-271.
6 Gerard A, Romani S, Fresse A et al. "Off-label" use of hydroxychloroquine, azithromycin,
lopinavir-ritonavir and chloroquine in COVID-19: A survey of cardiac adverse drug reactions
by the French Network of Pharmacovigilance Centers. Therapie 2020.
7 Gram L. Fluoxetine. N Engl J Med 1994; 331:1354-1361.
Figure legend
Figure 1: Fluoxetine inhibits SARS-CoV-2 replication. A. Vero cells were incubated with
the compounds and subsequently infected with SARS-CoV-2 (S: S-stereoisomer; R: R-
stereoisomer). Cellular supernatants were collected three days after infection, and viral titers
were determined with RTqPCR. B. Vero cells were infected with SARS-CoV-2 for 72h and
viral proteins were detected with a SARS-CoV-2 specific antiserum (1:100) and a TexasRed-
labeled donkey anti-human antibody (1:500, Dianova). The nuclei were stained with DAPI. C.
BHK21 and HepG2 cells were infected with either gfp-encoding HSV-1, HHV8, with a Rabies
vaccine strain or with a patient derived RSV. Viral titers were determined by RTqPCR (RSV,
Rabies) or infected gfp-expressing cells were counted with an Ensight device (PerkinElmer).
Acknowledgement: We would like to thank Novartis Germany and Jörg Vogel for support.
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprintthis version posted June 14, 2020..https://doi.org/10.1101/2020.06.14.150490doi:bioRxiv preprint
gene expression, indicating that it acts virus-specific (Figure 1C).
Fluoxetine was introduced in clinics during the seventies and is a well-studied drug since it
has been used in humans for almost four decades. Furthermore, the patent of Fluoxetine has
long expired. Thus, it is available from different companies, and relatively cheap. We see the
role of Fluoxetine in the early treatment of SARS-CoV-2 infected patients of risk groups.
References
1 Goldman JD, Lye DCB, Hui DS et al. Remdesivir for 5 or 10 Days in Patients with Severe
Covid-19. N Engl J Med 2020.
2 Cao B, Wang Y, Wen D et al. A Trial of Lopinavir-Ritonavir in Adults Hospitalized with
Severe Covid-19. N Engl J Med 2020; 382:1787-1799.
3 Ferner RE, Aronson JK. Chloroquine and hydroxychloroquine in covid-19. Bmj 2020;
369:m1432.
4 Hung IF-N, Lung K-C, Tso EY-K et al. 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; 395:1695-1704.
5 Wang M, Cao R, Zhang L et al. Remdesivir and chloroquine effectively inhibit the recently
emerged novel coronavirus (2019-nCoV) in vitro. Cell Res 2020; 30:269-271.
6 Gerard A, Romani S, Fresse A et al. "Off-label" use of hydroxychloroquine, azithromycin,
lopinavir-ritonavir and chloroquine in COVID-19: A survey of cardiac adverse drug reactions
by the French Network of Pharmacovigilance Centers. Therapie 2020.
7 Gram L. Fluoxetine. N Engl J Med 1994; 331:1354-1361.
Figure legend
Figure 1: Fluoxetine inhibits SARS-CoV-2 replication. A. Vero cells were incubated with
the compounds and subsequently infected with SARS-CoV-2 (S: S-stereoisomer; R: R-
stereoisomer). Cellular supernatants were collected three days after infection, and viral titers
were determined with RTqPCR. B. Vero cells were infected with SARS-CoV-2 for 72h and
viral proteins were detected with a SARS-CoV-2 specific antiserum (1:100) and a TexasRed-
labeled donkey anti-human antibody (1:500, Dianova). The nuclei were stained with DAPI. C.
BHK21 and HepG2 cells were infected with either gfp-encoding HSV-1, HHV8, with a Rabies
vaccine strain or with a patient derived RSV. Viral titers were determined by RTqPCR (RSV,
Rabies) or infected gfp-expressing cells were counted with an Ensight device (PerkinElmer).
Acknowledgement: We would like to thank Novartis Germany and Jörg Vogel for support.
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprintthis version posted June 14, 2020..https://doi.org/10.1101/2020.06.14.150490doi:bioRxiv preprint
1,00E+05
1,00E+06
1,00E+07
1,00E+08
- 0.8 0.4
Fluoxetine
RSV
108
107
106
105
V
ira
l
lo
a
d
[1
/
m
l]
1,00E+05
1,00E+06
1,00E+07
1,00E+08
- 0.8 0.4
Fluoxetine
108
107
106
105
Rabies
V
ira
l
lo
a
d
[1
/
m
l]
[µg/ml]
100
1000
10000
100000
1.6 0.8 -
Fluoxetine
HHV8
In
fe
c
te
d
c
e
lls
105
104
103
102
V
ira
l
lo
a
d
[1
/
m
l]
100
1000
10000
100000
1.6 0.8 -
Fluoxetine
105
104
103
102
HSV-1
In
fe
c
te
d
c
e
lls
DAPI
-
1.6
0.8
αSARS-CoV-2
Fluoxetine
[µg/ml]
1,E+06
1,E+07
1,E+08
1,E+09
0.66 0.33 -
Paroxetine
109
108
107
106
v
ira
l
lo
a
d
[1
/
m
l]
1,E+06
1,E+07
1,E+08
1,E+09
0.52 0.26 0.13 -
Escitalopram
109
108
107
106
[µg/ml]
1,00E+04
1,00E+05
1,00E+06
1,00E+07
1,00E+08
1,00E+09
1.6 0.8 0.4 0.2 1.6 0.8 0.4 1.6 0.8 0.4
Fluoxtine R S -
109
104
108
107
106
105
V
ira
l
lo
a
d
[1
/
m
l]
A
B
C
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprintthis version posted June 14, 2020..https://doi.org/10.1101/2020.06.14.150490doi:bioRxiv preprint
1,00E+06
1,00E+07
1,00E+08
- 0.8 0.4
Fluoxetine
RSV
108
107
106
105
V
ira
l
lo
a
d
[1
/
m
l]
1,00E+05
1,00E+06
1,00E+07
1,00E+08
- 0.8 0.4
Fluoxetine
108
107
106
105
Rabies
V
ira
l
lo
a
d
[1
/
m
l]
[µg/ml]
100
1000
10000
100000
1.6 0.8 -
Fluoxetine
HHV8
In
fe
c
te
d
c
e
lls
105
104
103
102
V
ira
l
lo
a
d
[1
/
m
l]
100
1000
10000
100000
1.6 0.8 -
Fluoxetine
105
104
103
102
HSV-1
In
fe
c
te
d
c
e
lls
DAPI
-
1.6
0.8
αSARS-CoV-2
Fluoxetine
[µg/ml]
1,E+06
1,E+07
1,E+08
1,E+09
0.66 0.33 -
Paroxetine
109
108
107
106
v
ira
l
lo
a
d
[1
/
m
l]
1,E+06
1,E+07
1,E+08
1,E+09
0.52 0.26 0.13 -
Escitalopram
109
108
107
106
[µg/ml]
1,00E+04
1,00E+05
1,00E+06
1,00E+07
1,00E+08
1,00E+09
1.6 0.8 0.4 0.2 1.6 0.8 0.4 1.6 0.8 0.4
Fluoxtine R S -
109
104
108
107
106
105
V
ira
l
lo
a
d
[1
/
m
l]
A
B
C
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprintthis version posted June 14, 2020..https://doi.org/10.1101/2020.06.14.150490doi:bioRxiv preprint
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