key: cord-0883034-5vvyd0wx
authors: Sakurai, Yasuteru; Ngwe Tun, Mya Myat; Kurosaki, Yohei; Sakura, Takaya; Inaoka, Daniel Ken; Fujine, Kiyotaka; Kita, Kiyoshi; Morita, Kouichi; Yasuda, Jiro
title: 5-amino levulinic acid inhibits SARS-CoV-2 infection in vitro
date: 2021-03-19
journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2021.01.091
sha: 346eb7ad61ce726fcf4b2aa82c4a73f8f064076b
doc_id: 883034
cord_uid: 5vvyd0wx

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 this study, 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-human cells, without significant cytotoxicity. Therefore, 5-ALA is worth to be further investigated as an antiviral drug candidate for COVID-19.

COVID-19 is an emerging infectious disease, which quickly became a global public health emergency after the first reports of the 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 by a novel coronavirus, SARS-CoV-2, which is an enveloped virus possessing a positive strand RNA genome. The virus enters into host cells using angiotensin-converting enzyme 2 (ACE2) as the receptor [2] . Then, replication/transcription of the viral genome occurs in the cytoplasm of infected cells, followed by assembly and release of progeny virions using multiple host cell machineries [3] . SARS-CoV-2 mainly replicates in the respiratory organs/tissues including lung and trachea, while viral antigens/RNA have J o u r n a l P r e -p r o o f also been detected in other multiple tissues, suggesting a complicated pathology [4] .

Currently several drugs, which were developed for other 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 a broad range of patients, is required.

5-amino levulinic acid (5-ALA) is a natural amino acid and ubiquitously 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 by composing protein complexes such as cytochromes. As 5-ALA enhances aerobic energy metabolism, it has been clinically used for metabolic 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 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 human pathogens such as Dengue virus, Zika virus, influenza A virus and SARS-CoV-2 [9] [10] [11] [12] [13] . 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. Hoechst33342 dye (ThermoFisher Scientific) was used.

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 

In order to identify the candidate compounds which are useful as therapeutics for COVID-19, at first, we isolated SARS-CoV-2 from the nasal specimen of a COVID-19 patient in Japan (a JPN/NGS/IA-1/2020 strain). It could be efficiently propagated in human colon-derived Caco-2 cells using this assay ( Fig. 1A and B) , suggesting that our method is useful to 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 5-ALA with sodium ferrous citrate (SFC), which supplies divalent iron for enhancing heme generation in combination with 5-ALA [9] , also inhibited the infection in the similar efficacy. However, 48-hour pretreatment with 5-ALA with and without SFC did not significantly affect SARS-CoV-2 infection (Fig.   2C ), suggesting that a longer incubation time for 5-ALA treatment is required to make host cells resistant to the infection. To confirm the antiviral effects in human cells,

5-ALA was also tested with SARS-CoV-2 infection in human colon-derived Caco-2 cells, which have been characterized for metabolism of exogenously supplied 5-ALA [16] . 5-ALA pretreatment for 72 hours potently inhibited SARS-CoV-2 infection either with or without SFC (Fig. 2D) . In contrast to VeroE6 cells, Caco-2 cells were resistant to SARS-CoV-2 infection even after 48-hour pretreatment of 5-ALA with and without SFC (Fig. 2E ). This is possibly 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-CoV-2 infection, the dose-dependency was investigated. As shown in Figure 3A 

Exogenously supplied 5-ALA has been reported to affect the host through multiple mechanisms [17] . We observed that PPIX gradually accumulated inside VeroE6 cells treated with 5-ALA (data not shown), 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 the downstream 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 to a recent report demonstrating that a G4-binding compound inhibited SARS-CoV-2 replication [18, 19] . G4s are tetrahelical structures 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 J o u r n a l P r e -p r o o f SARS-CoV-2 RNA genome and also predicted an SUD-like motif in the viral protein Nsp3, suggesting that interaction of G4 structures with the binding proteins are a potential antiviral target to combat COVID-19 [19, 26, 27] . Heme, one of the metabolites of 5-ALA, is known to be a ligand of G4 structures [28] . Exogenous 5-ALA supplementation induces increased generation of PPIX and heme 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.

In the current COVID-19 pandemic, most patients show mild to moderate 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 cases of COVID-19. Moreover, as 5-ALA was reported to show anti-inflammation 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] . Each data set is representative of at least two independent experiments. 

Characteristics of SARS-CoV-2 and COVID-19

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

The molecular virology of coronaviruses

Multiorgan and Renal Tropism of SARS-CoV-2

5-Aminolevulinic acid regulates the inflammatory response and alloimmune reaction

Safety and Mode of Action of Diabetes Medications in comparison with 5-Aminolevulinic Acid (5-ALA)

Aminolevulinic Acid-Based Tumor Detection and Therapy: Molecular Mechanisms and Strategies for Enhancement

In Vivo Curative and Protective Potential of Orally Administered 5-Aminolevulinic Acid plus Ferrous Ion against Malaria

Inactivation of Dengue and Yellow Fever viruses by heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX

Mechanisms of Vesicular Stomatitis Virus Inactivation by Protoporphyrin IX, Zinc-Protoporphyrin IX, and Mesoporphyrin IX

Co-protoporphyrin IX and Sn-protoporphyrin IX inactivate Zika, Chikungunya and other arboviruses by targeting the viral envelope

heme oxygenase-1 agonist CoPP suppresses influenza virus replication through IRF3-mediated generation of IFN-alpha/beta

Protoporphyrin IX and verteporfin prevent SARS-CoV-2 infection in vitro and in a mouse model expressing human ACE2. bioRxiv

Comprehensive pharmacokinetic studies and oral bioavailability of two Mn porphyrin-based SOD mimics, MnTE-2-PyP 5+

MnTnHex-2-PyP 5+ , Free Radic

Dynamics of absorption, metabolism, and excretion of 5-aminolevulinic acid in human intestinal Caco-2 cells

5-Aminolevulinic Acid (5-ALA): a precursor of heme-Fermentation, metabolism and usage

The G-Quadruplex/Helicase World as a Potential Antiviral Approach Against COVID-19

Targeting RNA G-quadruplex in SARS-CoV-2: A Promising Therapeutic Target for COVID-19?

The regulation and functions of DNA and RNA G-quadruplexes

G-Quadruplexes act as sequence-dependent protein chaperones

G-quadruplexes and G-quadruplex ligands: targets and tools in antiviral therapy

G-quadruplex forming sequences in the genome of all known human viruses: A comprehensive guide

The SARS-unique domain (SUD) of SARS coronavirus contains two macrodomains that bind G-quadruplexes

A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex

Discovery of G-quadruplex-forming sequences in SARS-CoV-2

Whole genome identification of potential G-quadruplexes and analysis of the Gquadruplex binding domain for SARS-CoV-2, bioRxiv

High specificity and tight spatial restriction of self-biotinylation by DNA and RNA G-Quadruplexes complexed in vitro and in vivo with Heme

Clinical pharmacokinetics of 5-aminolevulinic acid in healthy volunteers and patients at high risk for recurrent bladder cancer

Oral administration of 5-aminolevulinic acid induces heme oxygenase-1 expression in peripheral blood mononuclear cells of healthy human subjects in combination with ferrous iron

The authors are grateful to the members of Department of Emerging Infectious Diseases J o u r n a l P r e -p r o o f