COVID-19 Drugs: 5-Amino Levulinic Acid And Ethacridine Identified As Potential SARS-CoV-2 Inhibitors In Two Separate Studies

COVID-19 Drugs: With the current COVID-19 pandemic fast escalating and no effective drugs to date, researchers around the world are desperately trying to find suitable drug candidates to further explore their efficacy against the SARS-CoV-2 coronavirus. In two separate studies, two possible drug candidates that demonstrated to inhibit the SARS-CoV-2 virus were identified, one being 5-Amino Levulinic Acid and the other Ethacridine.


 
In the first study, Japanese researchers from Nagasaki University tested 5-amino levulinic acid in vitro in human-derived cells and found it prevents SARS-CoV-2 infection. Because it is safe and ubiquitous in plants and animals, it could be a potential therapeutic against SARS-CoV-2.
 
The study findings were published on a preprint server and have yet to be peer reviewed. https://www.biorxiv.org/content/10.1101/2020.10.28.355305v1
 
5-amino levulinic acid (5-ALA) is a natural amino acid,  found in plants, animals, bacteria, and fungi,  and it forms protoporphyrin IX (PPIX) when eight molecules come together. PPIX can generate heme when combined with a ferrous ion. 5-ALA has been used in various therapies, such as metabolic improvement, because of its ability to improve aerobic energy metabolism and cancer diagnosis and therapy using a photosensitive feature of PPIX.
Recent studies suggest 5-ALA also has antiviral properties against human pathogens like dengue virus, Zika virus, and SARS-CoV-2.
 
In order to test the potency of 5-ALA compounds against SARS-CoV-2, the study team isolated the virus from a nasal swab of a patient in Japan and propagated it in VeroE6 cells. They quantified the infection using an immunofluorescence assay they developed using an antibody for the SARS-CoV-2 N protein.
 
The team found that when VeroE6 cells were treated with 5-ALA for 72 hours before infection, it prevented SARS-CoV-2 infection. However, a shorter pretreatment time of 48 hours did not prevent the infection. The authors found that PPIX gradually accumulated inside the cells. This could be why there is a time-dependent effect on virus inhibition.
 
Interestingly adding sodium ferrous citrate (SFC) along with 5-ALA also helped prevent infection, but only with 72 hours of pretreatment. This is likely because SFC supplies iron for generating heme, along with 5-ALA.
 
The study team next tested the antiviral effect of 5-ALA in Caco-2 cells derived from the human colon, which can metabolize 5-ALA.
 
The researchers found that both 72 and 48-hour pretreatment, with and without SFC was effective in preventing treatment. This indicates 5-ALA has antiviral effects in humans.
 
The study team found that the IC50, which is the amount of a drug needed to inhibit a biological process by 50%, for inhibiting SARS-CoV-2 infection in VeroE6 cells using 5-ALA was 570 mm without SFC and 695 mm with SFC.
 
Significantly, the antiviral effect was much more powerful in human cells, with the IC50 being 39 mm and 63 mm with and without SFC, respectively. The authors also tested if the compound was toxic, and they found 5-ALA was not toxic to cells up to a concentration of 2000 mm.
 
The study team suggests a G-quadruplex (G4) structure could be a potential target of the antivirals, and compounds binding to it could inhibit SARS-CoV-2 infection. G4 is a tetrahelical structure formed by guanine-rich regions of DNA or RNA. It is also found in coronaviruses and can affect virus replication. https://pubmed.ncbi.nlm.nih.gov/29554280/
https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1006675
 
Many coronaviruses have a G4 binding domain in the non-structural protein 3 (Nsp3), which is essential in genome transcription.
 
From past studies it was found that the SARS-CoV-2 genome also has G4 structures with a SARS unique macrodomain (SUD)-like motif in its Nsp3 protein. Hence, it is likely that G4 interactions with binding proteins could be a potential target for antiviral drugs. https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1000428
 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4567502/
 
Furthermore, heme, a 5-ALA metabolite, is known to complex with G4 structures. https://pubmed.ncbi.nlm.nih.gov/32329781/
 

5-amino levulinic acid inhibits SARS-CoV-2 infection in vitro

Lead cum corresponding author, Professsor Dr Yasuteru Sakurai from the department of Emerging Infectious Diseases, Institute of Tropical Medicine (NEKKEN), Nagasaki University told Thailand Medical News, “Exogenous 5-ALA supplementation induces increased generation of PPIX and heme inside host cells, potentially interfering with the 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.”
 
As 5-ALA is made in most plants and animals, and humans are routinely consuming it in food, it is safe to be used by the general population. Because of its high bioavailability, it can easily be taken orally. 5-ALA also shows anti-inflammation effects, which could be another advantage of the compound.
 
Also the use of 5-ALA could provide another option for treatment and prevention of SARS-CoV-2 infection, being a different class of drugs from those currently being used or tested. Further testing in animal models could help advance the use of 5-ALA in treatments.
 
Ethacridine Identified As Potential SARS-CoV-2 Inhibitor
 
In the second study, scientists from the University of California-San Francisco, and Stanford University-California  reported a safe and potent antiseptic for use in humans named ethacridine, which effectively inhibits SARS-CoV-2 even at very low concentrations of EC50 ~ 0.08 µM.
 
The study findings were also published on a preprint server and have yet to be peer reviewed. https://www.biorxiv.org/content/10.1101/2020.10.28.359042v1
 
The drug candidate ethacridine was identified by high-throughput screening of an FDA-approved drug library in living cells with the help of a fluorescent assay.
 
So as to identify drugs that inhibit the SARS-CoV-2 virus, the study team redesigned green fluorescent protein (GFP) into an activity reporter of Mpro that turns fluorescent on cleavage by the active Mpro. Using this fluorescent assay, they identified many drugs that inhibit Mpro activity, and the most effective of them was ethacridine, which inhibits the production of SARS-CoV-2 through inactivation of the viral particles.


Demonstration of a GFP-based activity reporter of SARS-CoV2 main protease Mpro. (a) Schematic of the reporter. (b) Sequence of the flipped GFP10-11. (c) Construct of the reporter FlipGFPMpro. (d) Fluorescence images (left) and quantitative analysis (right) of SARS-CoV-2 or mock-infected HEK293T cells that co-expressed hACE2. The images in the FlipGFP channel were brightened 30-fold compared to those in (e). (e) Fluorescence images of HEK293T cells expressing FlipGFPMpro and mCherry, together with the inactive Mpro mutant C145A (upper panels) or wild type Mpro (lower panels). (f) Normalized FlipGFP fluorescence by mCherry. The ratio of FlipGFP/mCherry for the Mpro/C145A is normalized to 1. Data are mean ± SD (n = 5). FlipGFPTEV is a TEV activity reporter containing TEV cleavage sequence in FlipGFP. Scale bar: 5 μm (d); 10 μm (f).

It was found that ethacridine blocks SARS-CoV-2 in human cell lines and is not cytotoxic in animal models.
 
The research findings show a novel approach against SARS-CoV-2 that inactivates viral particles, and the efficacy of this approach is expected to be independent of the cell type. Ethacridine was shown to block SARS-CoV-2 in human cell line A549ACE2 cells and in primary HNE cells. Moreover, it was not toxic in animal models, including mice, rats, and rabbits.
 
Interestingly, it was found that ethacridine has stronger antiviral potency compared to remdesivir and also has very little cell toxicity. Other drugs identified by this study show a similar IC50 range in inhibiting Mpro and EC50 against the SARS-CoV-2 virus.
 
Ethacridine however showed much higher antiviral potency of EC50 ~ 0.08 μM than Mpro-inhibiting activity (IC50 ~ 3.5 μM). This shows that ethacridine's main action is not inhibiting Mpro.
 
Corresponding author, Dr Raul Andino from the department of microbiology and immunology, University of California-San Francisco, told Thailand Medical News, "Our findings reveal a new approach against SARS-CoV-2 via inactivating viral particles, of which the efficacy is expected to be cell type-independent."
 
It was found that ethacridine inactivates viral particles without affecting replication of the virus.
 
The study team claims that their work has identified a powerful drug with a clear mode of action against the SARS-CoV-2 virus as ethacridine inactivates viral particles and thus prevents binding to the host cells. The work also provides direct evidence of SARS-CoV-2 particles losing their infectivity and ability to bind host cells after ethacridine treatment.
 
Furthermore qRT-PCR data shows ethacridine does not impact viral RNA replication. Thus, the results suggest that ethacridine blocks the SARS-CoV-2 virus mainly by inactivating the viral particles without affecting the virus's replication. The further investigation focused on ultrastructural changes in viral particles and ethacridine binding on viral RNA or protein is needed to understand how ethacridine inactivates the virus.
 
Dr Andino added, "The detailed mechanisms of how ethacridine inactivates the viral particles will require further investigation, such as potential ultrastructural changes of viral particles and binding of ethacridine to viral RNA or protein."
 
Presently, ethacridine is used as a topical disinfectant, but it has already been used to treat patients with puerperal sepsis via intravenous injection. Thus, its antiviral effect can be easily validated in animal models as well as COVID-19 patients. Also, since it directly inactivates SARS-CoV-2 virus particles, ethacridine can be combined with drugs such as remdesivir, the replicase inhibitor, to treat COVID-19 patients and improve their clinical outcome.
 
The study team commented, "While currently ethacridine is mainly used as a topical wound disinfectant, it has been applied to patients for treating puerperal sepsis via intravenous injection."
 
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