5 amino levulinic acid inhibits sars cov 2 infection in vitro CORD-Papers-2022-06-02 (Version 1)

Title: 5-amino levulinic acid inhibits SARS-CoV-2 infection in vitro
Abstract: 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. The antiviral effects could be detected in both human and non-human cells without significant cytotoxicity. Therefore 5-ALA is a candidate as an oral antiviral drug for COVID-19.
Published: 2020-10-28
Journal: bioRxiv
DOI: 10.1101/2020.10.28.355305
DOI_URL: http://doi.org/10.1101/2020.10.28.355305
Author Name: Sakurai Yasuteru
Author link: https://covid19-data.nist.gov/pid/rest/local/author/sakurai_yasuteru
Author Name: Ngwe Tun Mya Myat
Author link: https://covid19-data.nist.gov/pid/rest/local/author/ngwe_tun_mya_myat
Author Name: Kurosaki Yohei
Author link: https://covid19-data.nist.gov/pid/rest/local/author/kurosaki_yohei
Author Name: Sakura Takaya
Author link: https://covid19-data.nist.gov/pid/rest/local/author/sakura_takaya
Author Name: Inaoka Daniel Ken
Author link: https://covid19-data.nist.gov/pid/rest/local/author/inaoka_daniel_ken
Author Name: Fujine Kiyotaka
Author link: https://covid19-data.nist.gov/pid/rest/local/author/fujine_kiyotaka
Author Name: Kita Kiyoshi
Author link: https://covid19-data.nist.gov/pid/rest/local/author/kita_kiyoshi
Author Name: Morita Kouichi
Author link: https://covid19-data.nist.gov/pid/rest/local/author/morita_kouichi
Author Name: Yasuda Jiro
Author link: https://covid19-data.nist.gov/pid/rest/local/author/yasuda_jiro
sha: 0957281efdf8d4d7fbcb9d366495fb6a6ee7c621
license: biorxiv
license_url: https://www.biorxiv.org/about-biorxiv
source_x: BioRxiv; WHO
source_x_url: https://www.biorxiv.org/https://www.who.int/
url: https://doi.org/10.1101/2020.10.28.355305
has_full_text: TRUE
Keywords Extracted from Text Content: 5-amino levulinic acid oral SARS-CoV-2 human non-human cells amino acid anticancer 5-ALA COVID-19 coronavirus intestine nasal specimen SARS-CoV-2 sodium ferrous citrate plaques Hoechst33342 dye COVID-19 [19 cells penicillin/streptomycin PPIX 5-ALA patients influenza A virus Zika virus lung cytoplasm SARS-CoV-2 [9-13 Fig 2B SARS-CoV-2 N protein VeroE6 cells JPN/NGS/IA-1/2020 × SFC COVID-19 patient Caco-2 cells DNA 5-ALA [9] human colon-derived Caco-2 cells Fig. 2B protoporphyrin IX tissues organs/tissues host G4 5-amino levulinic acid heme ferrous progeny virions FBS patient multiple host cell anti-SARS-CoV-2 5-ALA [16] nonstructural protein 3 SUD angiotensin-converting enzyme 2 trachea cancers COVID-19 iron Figure 3A Nsp3 Fig. 1A human cells coronaviruses oral Heme Dengue virus human Gquadruplex Fig. 2D B ACE2 human health [7 Fluor 488 goat anti-rabbit IgG host cells cytochromes
Extracted Text Content in Record: First 5000 Characters: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. The antiviral effects could be detected in both human and non-human cells, without significant cytotoxicity. Therefore, 5-ALA is a candidate as an oral antiviral drug 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 also been 3 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 [9-13]. 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. Fluor 488 goat anti-rabbit IgG (ThermoFisher Scientific) and 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 VeroE6 cells. Culture supernatants were collected 4 days after infection, clarified by centrifugation at 2,000 × g for 15 min and stored at -80 o C until use. Virus titer was determined by a plaque assay using VeroE6 cells. After 1 hour infection, 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. 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 VeroE6 cells and showed a strong cytopathic effect (CPE). Next, using an antibody for SARS-CoV-2 N protein (Cayman Chemical), we developed an immunofluorescencebased assay to efficiently quantify SARS-CoV-2 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 useful to test antiviral 6 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 ( F
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