5 amino levulinic acid inhibits sars cov 2 infection in vitro CORD-Papers-2021-10-25 (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, 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.
Published: 3/19/2021
Journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2021.01.091
DOI_URL: http://doi.org/10.1016/j.bbrc.2021.01.091
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: 346eb7ad61ce726fcf4b2aa82c4a73f8f064076b
license: no-cc
license_url: [no creative commons license associated]
source_x: Elsevier; Medline; PMC
source_x_url: https://www.elsevier.com/https://www.medline.com/https://www.ncbi.nlm.nih.gov/pubmed/
pubmed_id: 33571909
pubmed_id_url: https://www.ncbi.nlm.nih.gov/pubmed/33571909
pmcid: PMC7846235
pmcid_url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846235
url: https://www.ncbi.nlm.nih.gov/pubmed/33571909/ https://www.sciencedirect.com/science/article/pii/S0006291X2100156X?v=s5 https://api.elsevier.com/content/article/pii/S0006291X2100156X https://doi.org/10.1016/j.bbrc.2021.01.091
has_full_text: TRUE
Keywords Extracted from Text Content: cell human colon-derived Caco-2 cells 5-ALA amino acid COVID-19 SARS-CoV-2 J. Yasuda 5-amino levulinic acid VeroE6 cells human J o u r n a l P r e -p r o o f 2 J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f anticancer non-human cells 5-ALA 5-ALA [16] SUD PPIX anti-SARS-CoV-2 DNA Caco-2 cells COVID-19 SARS-CoV-2 5-amino levulinic acid organs/tissues SARS-CoV-2 [9 multiple host cell coronaviruses influenza A virus cancers human health [7 ACE2 cells Hoechst33342 dye trachea Heme coronavirus tissues Fig. 2C oral Zika virus iron J o u r n a l P r e -p r o o f SARS-CoV-2 nasal specimen sodium ferrous citrate COVID-19 patient nonstructural protein 3 lung Dengue virus Nsp3 Figure 3A SFC C progeny virions host cells heme human human cells Fig. 2B 5-ALA [9] human colon-derived Caco-2 cells angiotensin-converting enzyme 2 patient cytoplasm cytochromes ferrous patients intestine VeroE6 cells Fig 2B COVID-19 [19 protoporphyrin IX host G4 B
Extracted Text Content in Record: First 5000 Characters:J. Yasuda). J o u r n a l P r e -p r o o f 2 J o u r n a l P r e -p r o o f 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, 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. Highlights 5-amino levulinic acid (5-ALA) inhibited SARS-CoV-2 infection in cell culture. Antiviral activity of 5-ALA was more potent in human colon-derived Caco-2 cells than VeroE6 cells. The antiviral activity of 5-ALA was dose-dependent without significant cytotoxicity. J o u r n a l P r e -p r o o f 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
Keywords Extracted from PMC Text: human cells penicillin/streptomycin nuclear human intestine nonstructural protein 3 iron COVID-19 [19,26,27 cell TritonX-100 MA Dulbecco's modified host cells COVID-19 ACE2 multiple host cell Caco-2 Cell 5-amino levulinic acid host G4 COVID-19 patient patients 5-amino leuvulinic acid bovine serum human colon-derived Caco-2 cells coronaviruses Heme protoporphyrin IX Sodium ferrous citrate Dengue virus Fig. 3 5-ALA [9] SUD sodium ferrous citrate Alexa Fluor 488 1 M HCl SFC ferrous VeroE6 cells serum Fig. 2B cell nuclei patient lung tissues cytochromes oral influenza A virus rabbit anti-SARS-CoV N antibody Alexa Fluor FBS SARS-CoV-2 [[9 cytoplasm cancers SARS-CoV-2 N protein trachea anti-SARS-CoV-2 Nsp3 progeny virions NOVUS heme IgG Caco-2 cells PPIX human health [7 Zika virus 5-ALA SARS-CoV-2 plaques DMEM −80 °C B 5-ALA [16] cells × Fig. 3D–F angiotensin-converting enzyme 2 Hoechst33342 dye organs/tissues DNA human Caco-2 cells rabbit nasal specimen Fig. 2 A JPN/NGS/IA-1/2020 coronavirus
Extracted PMC Text Content in Record: First 5000 Characters: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 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. 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 water with 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. 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 °C until use. Virus titer was determined by a plaque assay using VeroE6 cells. After 1 h 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. To evaluate antiviral activity of compounds, VeroE6 cells or Caco-2 were plated in 96-well plates and incubated with each compound 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 the presence of compounds at 37 °C. After 2 days (VeroE6 cells) or 3 days (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
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