Biochemical and Biophysical Research Communications 545 (2021) 203e207

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

5-amino levulinic acid inhibits SARS-CoV-2 infection in vitro
Yasuteru Sakurai a, b, *, Mya Myat Ngwe Tun c, Yohei Kurosaki a, b, Takaya Sakura d,Daniel Ken Inaoka d, f, Kiyotaka Fujine e, Kiyoshi Kita f, g, **, Kouichi Morita c, ***,Jiro Yasuda a, b, ****a

Department of Emerging Infectious Diseases, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, 852-8523, Japan
National Research Center for the Control and Prevention of Infectious Diseases, Nagasaki University, Nagasaki, 852-8521, Japanc
Department of Virology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, 852-8523, Japand
Department of Molecular Infection Dynamics, Shionogi Global Infectious Diseases Division, Institute of Tropical Medicine (NEKKEN), Nagasaki University,
Nagasaki, 852-8523, Japane
Pharmaceutical Research Department, Global Pharmaceutical R&D Division, Neopharma Japan Co., Ltd, Tokyo, 102-0071, JapanfSchool of Tropical Medicine and Global Health, Nagasaki University, Nagasaki, 852-8523, Japang
Department of Host - Defense Biochemistry, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, 852-8523, Japanba r t i c l e i n f o

a b s t r a c t

Article history:
Received 13 January 2021
Accepted 25 January 2021
Available online 29 January 2021

The current COVID-19 pandemic requires urgent development of effective therapeutics. 5-amino levulinic acid (5-ALA) is a naturally synthesized amino acid and has been used for multiple purposes
including as an anticancer therapy and as a dietary supplement due to its high bioavailability. In thisstudy, we demonstrated that 5-ALA treatment potently inhibited infection of SARS-CoV-2, a causative
agent of COVID-19, in cell culture. The antiviral effects could be detected in both human and non-humancells, without signiﬁcant cytotoxicity. Therefore, 5-ALA is worth to be further investigated as an antiviraldrug candidate for COVID-19.
© 2021 Elsevier Inc. All rights reserved.

Keywords:
COVID-19
SARS-CoV-2
5-ALA
Antiviral drug

1. Introduction
COVID-19 is an emerging infectious disease, which quickly
became a global public health emergency after the ﬁrst reports ofthe disease in December 2019 [1]. The pandemic has resulted in
more than 41.5 million cases and 1,100,000 deaths in 218 affected
countries (as of 23 October 2020, WHO). The infection is caused bya novel coronavirus, SARS-CoV-2, which is an enveloped virus
possessing a positive strand RNA genome. The virus enters into hostcells using angiotensin-converting enzyme 2 (ACE2) as the receptor[2]. Then, replication/transcription of the viral genome occurs in* Corresponding author. Department of Emerging Infectious Diseases, Institute ofTropical Medicine (NEKKEN), Nagasaki University, Nagasaki, 852-8523, Japan.
** Corresponding author. School of Tropical Medicine and Global Health, Nagasaki
University, Nagasaki, 852-8523, Japan.
*** Corresponding author.
**** Corresponding author. National Research Center for the Control and Prevention of Infectious Diseases, Nagasaki University, Nagasaki, 852-8521, Japan.
E-mail addresses: ysakurai@nagasaki-u.ac.jp (Y. Sakurai), kitak@nagasaki-u.ac.jp(K. Kita), moritak@nagasaki-u.ac.jp (K. Morita), j-yasuda@nagasaki-u.ac.jp(J. Yasuda).
https://doi.org/10.1016/j.bbrc.2021.01.091
0006-291X/© 2021 Elsevier Inc. All rights reserved.

the cytoplasm of infected cells, followed by assembly and release ofprogeny virions using multiple host cell machineries [3]. SARSCoV-2 mainly replicates in the respiratory organs/tissues
including lung and trachea, while viral antigens/RNA have also
been detected in other multiple tissues, suggesting a complicatedpathology [4]. Currently several drugs, which were developed forother purposes, have been approved for COVID-19. However, they
are mainly administrated to severe cases with only partial effectiveness and concerns of side effects. Therefore, development of
more effective and safe therapeutics, which can be prescribed to abroad range of patients, is required.
5-amino levulinic acid (5-ALA) is a natural amino acid andubiquitously exists in animals, plants, fungi and bacteria. Conjugation of eight molecules of 5-ALA produces protoporphyrin IX(PPIX), which generates heme by the insertion of ferrous ion [5].
Heme functions in various kinds of physiological processes bycomposing protein complexes such as cytochromes. As 5-ALA enhances aerobic energy metabolism, it has been clinically used formetabolic improvement in human diseases including diabetes [6].
Moreover, utilizing a photosensitive feature of PPIX, 5-ALA has also
been used for diagnosis and therapy for various cancers, suggesting
the beneﬁts of 5-ALA in many ﬁelds of human health [7]. Currently

Y. Sakurai, M.M. Ngwe Tun, Y. Kurosaki et al.

Biochemical and Biophysical Research Communications 545 (2021) 203e207

Fig. 1. A developed assay conﬁrms the antiviral effect of remdesivir on SARS-CoV-2 infection. (A and B) VeroE6 cells (A) and Caco-2 cells (B) were pretreated with the indicateddoses of remdesivir for 1 h and then challenged with SARS-CoV-2. Infectivity and cell viability were calculated by counting the number of infected cells and total nuclei number,respectively, and normalizing them to untreated cells. Dose-dependent curves (left) and immunoﬂuorescence images (right) are shown for each cell line. Each data set is representative of at least two independent experiments.

the inoculum was washed out and the cells were incubated with
0.7% agarose gel in Minimum Essential Medium (MEM) supplemented with 2% FBS and 1% penicillin/streptomycin solution for 3
days. After virus inactivation using 4% paraformaldehyde (PFA)
overnight, the cells were stained with crystal violet solution. The
plaques were counted manually to calculate the virus titer. All experiments with replication competent SARS-CoV-2 were performed in a biosafety level 3 (BSL3) laboratory at Nagasaki
University.

we are developing its application to infectious diseases such as
malaria [8]. In addition, recent ﬁndings revealed that PPIX had
antiviral effects against a broad range of viruses including humanpathogens such as Dengue virus, Zika virus, inﬂuenza A virus and
SARS-CoV-2 [9e13]. However, bioavailability of PPIX is poor due to
inefﬁcient uptake in intestine and incorporation to cells and its
practical use as a medicine is not realistic [14]. Therefore, this study
addressed the potential of 5-ALA as an anti-SARS-CoV-2 drug.
2. Materials and methods

2.3. Infection assay with immunoﬂuorescence
2.1. Cells and reagentsTo evaluate antiviral activity of compounds, VeroE6 cells or
Caco-2 were plated in 96-well plates and incubated with eachcompound in appropriate concentration for indicated time. Then,the cells were challenged with SARS-CoV-2 at an MOI of 0.002 for
VeroE6 or an MOI of 0.02 for Caco-2 cells, and incubated in thepresence of compounds at 37  C. After 2 days (VeroE6 cells) or 3days (Caco-2 cells), the cells were ﬁxed using 4% paraformaldehyde(PFA) overnight. Virus infectivity was determined by immunoﬂuorescence using 0.2% TritonX-100 for permeabilization, 10% goat
serum for blocking, rabbit anti-SARS-CoV N antibody as a primary
antibody, Alexa Fluor 488 goat anti-rabbit IgG as a secondary
antibody and Hoechst33342 dye for nuclear staining. The cells wereimaged by a Cytation 5 imaging plate reader (BioTek Instruments,
Winooski, VT) with a 4 lens. Counting of the cell nuclei andinfected cells was performed using Cell Proﬁler image analysis
software (Broad Institute, MIT, Boston, MA) and customized analysis pipeline (available on request).

VeroE6 cells (donated by Dr. Ayato Takada, Hokkaido University,
Japan) and Caco-2 cells (donated by Dr. Tetsuya Iida, Osaka University, Japan) were maintained in Dulbecco’s modiﬁed Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum (FBS)and 1% penicillin/streptomycin solution. 5-amino leuvulinic acid
(5-ALA) was donated by Neopharma Japan (Tokyo, Japan) and was
dissolved to 100 mM in water. Sodium ferrous citrate (SFC) was also
donated by Neopharma Japan and was dissolved to 25 mM in waterwith 1 M HCl. Remdesivir (Cayman Chemical, Ann Arbor, MI) was
dissolved to 10 mM in DMSO. For immunostaining, goat serum(ThermoFisher Scientiﬁc, Waltham, MA), rabbit anti-SARS-CoV N
antibody (NOVUS, Centennial, CO), Alexa Fluor 488 goat anti-rabbit
IgG (ThermoFisher Scientiﬁc) and Hoechst33342 dye (ThermoFisher Scientiﬁc) was used.
2.2. Virus propagation
A JPN/NGS/IA-1/2020 strain of SARS-CoV-2 (GISAID accession
no. EPI-ISL-481251), which was isolated from a Japanese patient,
was propagated in VeroE6 cells. Culture supernatants werecollected 4 days after infection, clariﬁed by centrifugation at
2,000  g for 15 min and stored at 80  C until use. Virus titer wasdetermined by a plaque assay using VeroE6 cells. After 1 h infection,

3. Results
In order to identify the candidate compounds which are usefulas therapeutics for COVID-19, at ﬁrst, we isolated SARS-CoV-2 fromthe nasal specimen of a COVID-19 patient in Japan (a JPN/NGS/IA-1/
2020 strain). It could be efﬁciently propagated in VeroE6 cells and204

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Biochemical and Biophysical Research Communications 545 (2021) 203e207

for metabolism of exogenously supplied 5-ALA [16]. 5-ALA pretreatment for 72 h potently inhibited SARS-CoV-2 infection eitherwith or without SFC (Fig. 2D). In contrast to VeroE6 cells, Caco2 cells were resistant to SARS-CoV-2 infection even after 48-hour
pretreatment of 5-ALA with and without SFC (Fig. 2E). This ispossibly due to more efﬁcient metabolism of 5-ALA and accumulation of the metabolites in Caco-2 cells. These results indicate that
5-ALA has antiviral effects in human cells.
To further address the speciﬁcity of 5-ALA effects on SARS-CoV2 infection, the dose-dependency was investigated. As shown in
Fig. 3A, 5-ALA treatment of VeroE6 cells inhibited SARS-CoV-2
infection in a dose dependent manner with an IC50 of 570 mM.
Cotreatment of 5-ALA and SFC, but not SFC only, also inhibited thevirus infection in a dose dependent manner with an IC50 of 695 mM,
suggesting the speciﬁc antiviral effect (Fig. 3B and C). In humanCaco-2 cells, 5-ALA with or without SFC, but not SFC only, inhibited
SARS-CoV-2 infection with IC50 of 39 mM and 63 mM, respectively,
indicating that the antiviral activity is more potent in human Caco2 cells than VeroE6 cells (Fig. 3DeF). No signiﬁcant cytotoxicity wasshowed a strong cytopathic effect (CPE). Next, using an antibody for
SARS-CoV-2 N protein (Cayman Chemical), we developed an
immunoﬂuorescence-based assay to efﬁciently quantify SARS-CoV2 infection. We conﬁrmed the previously reported antiviral effects
of remdesivir in VeroE6 cells and human colon-derived Caco-2 cells
using this assay (Fig. 1A and B), suggesting that our method is usefulto test antiviral candidates [15].
Then, the antiviral effect of 5-ALA was tested using this assay(Fig. 2A). We found that 72-hour pretreatment of VeroE6 cells with
5-ALA blocked SARS-CoV-2 infection (Fig. 2B). Cotreatment of 5ALA with sodium ferrous citrate (SFC), which supplies divalentiron for enhancing heme generation in combination with 5-ALA [9],also inhibited the infection in the similar efﬁcacy. However, 48hour pretreatment with 5-ALA with and without SFC did not
signiﬁcantly affect SARS-CoV-2 infection (Fig. 2C), suggesting that alonger incubation time for 5-ALA treatment is required to makehost cells resistant to the infection. To conﬁrm the antiviral effects
in human cells, 5-ALA was also tested with SARS-CoV-2 infection inhuman colon-derived Caco-2 cells, which have been characterized

Fig. 2. 5-ALA inhibits SARS-CoV-2 infection in both VeroE6 cells and Caco-2 cells. (A) A chemical structure of 5-ALA. (B and C) To address the effects of compounds on virus
infection, VeroE6 cells were treated with 1000 mM of 5-ALA with and without 25 mM of SFC for 72 h (B) or 48 h (C) and challenged with SARS-CoV-2. Virus infectivity was calculated
by counting the number of infected cells and normalizing it to untreated cells (mean ± SD, n ¼ 3). (D and E) Infectivity of SARS-CoV-2 in Caco-2 cells with pretreatment for 72 h (D)or 48 h (E) were determined as (B) and (C). Each data set is representative of at least two independent experiments.
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Biochemical and Biophysical Research Communications 545 (2021) 203e207

Fig. 3. 5-ALA with/without SFC inhibits SARS-CoV-2 infection in dose dependent manners. (A to C) To address the dose-dependency of 5-ALA (A), 5-ALA with SFC (B) and SFC (C),VeroE6 cells were pretreated with the indicated doses of each compound for 72 h and then challenged with SARS-CoV-2. Infectivity and cell viability were calculated by counting
the number of infected cells and total nuclei number, respectively, and normalizing them to untreated cells (mean ± SD, n ¼ 3). (D to F) Infectivity of SARS-CoV-2 in Caco-2 cells andthe cell viability with pretreatment of 5-ALA (D), 5-ALA with SFC (E) and SFC (F) for 72 h were determined as (A) to (C). Each data set is representative of at least two independent
experiments.

In the current COVID-19 pandemic, most patients show mild tomoderate symptoms [1]. As these patients are the main sources of
disease transmission, development of therapeutics for such populations is important to control spread of the disease. 5-ALA is
synthesized in most animals and plants and we are continuously
consuming it in our food. Moreover, 5-ALA can be efﬁciently taken
by an oral route due to its high bioavailability [29]. Therefore, as
either a medicine or a supplement, it can be safely and easily prescribed to a wide range of populations including non-severe casesof COVID-19. Moreover, as 5-ALA was reported to show antiinﬂammation effects in human, it can be an effective therapeutic
to the severe cases due to the combination of the antiviral activity
and anti-inﬂammation effects [30]. Since 5-ALA is a different classof medicine from currently approved therapeutics and the leading
candidates, it may provide another option for treating patients aswell as preventing infection. Further analyses of the antiviral activity by mechanistic research and animal studies should beundertaken.

observed at all the tested concentration of the compounds with the
maximum being 2,000 mM of 5-ALA, which further conﬁrms the
speciﬁc antiviral effects. These results indicate that 5-ALA is apotent and speciﬁc inhibitor of SARS-CoV-2 infection in multiple
cell types.

4. Discussion
Exogenously supplied 5-ALA has been reported to affect the hostthrough multiple mechanisms [17]. We observed that PPIX gradually accumulated inside VeroE6 cells treated with 5-ALA (data notshown), which correlated to our results showing time-dependent
increase of anti-SARS-CoV-2 activity of 5-ALA in VeroE6 cells(Fig. 2B and C). Therefore, 5-ALA metabolites such as PPIX and thedownstream metabolite, heme, appear to affect viral infection inside host cells. Among the molecular targets, a G-quadruplex (G4)structure is the potential target for its antiviral activity according toa recent report demonstrating that a G4-binding compound
inhibited SARS-CoV-2 replication [18,19]. G4s are tetrahelicalstructures formed by guanine-rich regions of DNA or RNA, regulating genome stability, gene expression and protein quality control[20,21]. G4 structures are also found in the genome of many viruses
including coronaviruses and can regulate viral replication cycles[22,23]. Moreover, several coronaviruses have a G4 binding domain
in their nonstructural protein 3 (Nsp3), which is so-called SARS
unique macrodomain (SUD) and plays a key role in the genome
replication/transcription [24,25]. Recent studies identiﬁed G4
structures in the SARS-CoV-2 RNA genome and also predicted an
SUD-like motif in the viral protein Nsp3, suggesting that interactionof G4 structures with the binding proteins are a potential antiviraltarget to combat COVID-19 [19,26,27]. Heme, one of the metabolitesof 5-ALA, is known to be a ligand of G4 structures [28]. Exogenous
5-ALA supplementation induces increased generation of PPIX andheme inside host cells, potentially interfering with interaction of G4
structures in the host or viral genome with viral protein Nsp3 or
host G4 binding proteins, which inhibits SARS-CoV-2 infection.

Declaration of competing interest
The authors declare no known competing ﬁnancial interests orno personal relationships that could have appeared to inﬂuence thework reported in this paper.
Acknowledgements
The authors are grateful to the members of Department ofEmerging Infectious Diseases (Nagasaki University) for helpful
discussions. The authors also thank to Dr. Chris Smith (Nagasaki
University) for supporting manuscript preparation, and Dr. Asuka
Nanbo (Nagasaki University) for coordinating the material transfer.
Critical discussion on G4 by Dr. Norifumi Shioda (Kumamoto University) is appreciated. This work was supported by grants fromJapan Agency for Medical Research and Development (grant
number JP20wm0125006).
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Biochemical and Biophysical Research Communications 545 (2021) 203e207

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