Researchers discovered that ethacridine is a potent drug against SARS-CoV-2

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Researchers from the University of California-San Francisco along with support from Stanford University are exploring the use of the abortion drug Ethacridine to inhibit SARS-CoV-2 by inactivating viral particles and to assess its potential as a prophylaxis and also therapeutic agent to treat COVId-19.

Their study findings were published in the peer reviewed journal: PLOS Pathogens. https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009898

The drug Ethacridine is commercially available as a US FDA approved drug in the form of Ethacridine lactate (Ethacridine monolactate monohydrate acrinol) and the trade name is Rivanol.

It is an aromatic organic compound based on acridine.) Its formal name is 2-ethoxy-6,9-diaminoacridine monolactate monohydrate. It forms orange-yellow crystals with a melting point of 226 °C and it has a stinging smell.

It is used primarily as an antiseptic in solutions of 0.1%. It is effective against mostly Gram-positive bacteria, such as Streptococci and Staphylococci, but ineffective against Gram-negative bacteria such as Pseudomonas aeruginosa.

Ethacridine is also commonly used as an abortion agent for second trimester abortion. Up to 150 ml of a 0.1% solution is instilled extra-amniotically using a foley catheter. After 20 to 40 hours, ‘mini labor’ ensues. It is used extensively in China via an intra-amniotic method.

Ethacridine as an abortifacient is found to be safer and better tolerated than 20% hypertonic saline.

The study team reports the discovery of ethacridine as a potent drug against SARS-CoV-2 (EC50 ~ 0.08 μM). Hence it could be repurposed as one of many prospective COVID-19 Drugs.

Ethacridine was identified via high-throughput screening of an FDA-approved drug library in living cells using a fluorescence assay. Plaque assays, RT-PCR and immunofluorescence imaging at various stages of viral infection demonstrate that the main mode of action of ethacridine is through inactivation of viral particles, preventing their binding to the host cells.

Consistently, ethacridine is effective in various cell types, including primary human nasal epithelial cells that are cultured in an air-liquid interface.

In order to determine how ethacridine inhibits SARS-CoV-2, the study team tested infectivity of the virus particles after ethacridine treatment with plaque assay, and they also measured viral RNA levels using qRT-PCR.

The study team examined the antiviral effect of ethacridine on different stages of the lifecycle of SARS-CoV-2, including virus-cell binding, RNA replication, and budding. To test overall effect of ethacridine on virus replication, the team pre-incubated SARS-CoV-2 particles with ethacridine (5 μM) or DMSO control for 1 hour. The mixture was then added to Vero E6 cells for viral adsorption at a multiplicity of infection (MOI) at 0.5.

Next, they removed the solution and added fresh medium containing ethacridine (5 μM) or DMSO control. Sixteen hours later, they collected supernatant and conducted plaque assay with overlaid agar without ethacridine or DMSO to measure viral titer.

The study team also conducted qRT-PCR and measured viral RNA levels in the supernatant and within cells. In this way, they developed three conditions: 1) Control (DMSO + DMSO): the virus and cells were exposed to DMSO and not the drug; 2) The virus and cells were exposed to the drug at all stages, including 1 hour before infection, during replication, and after viral budding (i.e. Eth. + Eth.); 3) The virus and cells were exposed to the drug only after viral entry, during replication, and after budding (i.e. DMSO + Eth.).

Lastly, the study team used a fourth condition: they conducted plaque assay right after pre-treatment of SARS-CoV-2 with ethacridine for 1 hr (i.e. Eth. [1 hr]), which determines direct effect of the drug on viral particles.

Interestingly when ethacridine was present continuous prior to plaque assay (Eth. + Eth.), viral titers were reduced 3–4 logs, compared with the control (DMSO + DMSO).

When ethacridine was added to the Vero cells after viral entry (DMSO + Eth.), similar level of reduced infectivity (3–4 logs reduction in viral titer) was observed.

Importantly, when SARS-CoV2 was pre-incubated with ethacridine for 1 hr (Eth. [1 hr]) and followed by plaque assay without drug, the team also observed 3–4 logs reduction in infectivity.

The study findings  suggests that the drug directly inactivates SARS-CoV2 viral particles. Because of similar-level reduction in infectivity in all of the three conditions, our data strongly suggests that ethacridine inhibits SARS-CoV-2 mainly by inactivating viral particles.

The study team also examined viral RNA accumulation in infected cells. qRT-PCR measurement revealed no change of viral RNA (vRNA) levels when the drug was added after viral binding and cell entry (DMSO + Eth.) in both the supernatant and the cells, compared with the control (DMSO + DMSO).

This indicates that the drug has no effect on vRNA replication. As the infectivity of supernatant from “DMSO + Eth” treated samples showed 3–4 logs reduction in plaque assay, these data suggests that ethacridine inhibits SARS-CoV-2 by inactivating the viral particles without effect on vRNA replication.

This is consistent with the results of plaque assays for the supernatant samples with 3 different treatments in that showed 3~4 log reduction in infectivity after virions in the supernatant were exposed to the drug before plaque assay.

When ethacridine was present continuously (i.e. Eth. + Eth.), 4–5 fold reductions were observed in vRNA copies in the supernatant and within cells. Because plaque assay-based measurement of the same conditioned sample (Eth. + Eth.) showed 2400-fold reduction in viral titer, the effect of ethacridine on viral replication (4–5 fold reduction) is about 500-fold smaller than its effect on viral infectivity.

This further supports the conclusion that ethacridine inhibits SARS-CoV-2 by inactivating the viral particles. The 4–5 fold reduction of vRNA copies is likely due to reduced viral copy numbers that may bind to the cells, because here the additional step is that the virus was pre-incubated with the drug.

Hence the plaque assay and qRT-PCR data suggests that ethacridine inhibits SARS-CoV-2 mainly by inactivating viral particles, including the virus before binding to cells and in the supernatant after budding from host cells, with no or little effect on vRNA replication.

The study team also conducted immunofluorescence staining and imaging to determine whether the ethacridine-treated SARS-CoV-2 can bind to the cells.

They treated SARS-CoV-2 with ethacridine (5 μM) or DMSO for 1 hour at 37°C. Then the virus was added to cells for adsorption (4°C, 1 hour) at a MOI = 100. Cells were then quickly washed and fixed with 4% PFA . Immunostaining with antibodies against the nucleocapsid protein (N) of SARS-CoV-2 showed strong anti-N fluorescence on the plasma membrane of the cells infected with control virus, but little anti-N fluorescence in cells exposed to ethacridine treated virus. Immunostaining against the Spike protein (S) of SARS-CoV-2 showed the same results.

Furthermore, to examine whether ethacridine blocks viral binding to cells by perturbing the cellular factors for viral binding such as cellular receptors, the study team pre-treated cells with ethacridine for 3 hours. This was followed by washing and drug removal, immediately prior to addition of SARS-CoV-2.

The data showed that viral infection was not affected by these procedures (S6 Fig), suggesting that the main effect of the drug is not on the cells, but on the viral particles. These results indicate that ethacridine-treated SARS-CoV-2 cannot bind cells to initiate infection.

To further support their hypothesis model, the study team conducted an additional experiment. They mixed ethacridine with SARS-CoV-2 and immediately added the drug/virus mixture to the Vero cells (i.e. no preincubation of the drug with the virus). After adsorption for 1 hour (37°C), they overlayed the cells with agar and media for plaque assay. Under this condition, first, the drug will be able to inhibit potential cellular factors since the drug is not removed after the adsorption step.

Second, the drug is not preincubated with the virus, and thus according to their model, they expect much smaller effect of the drug on the virus than when the drug was preincubated with the virus. Indeed, they observed dramatically smaller effect of the drug: ~2.7-fold inhibition versus 3–4 logs inhibition when the drug was preincubated with the virus.

The study findings further support the model that ethacridine inhibits SARS-CoV-2 by mainly inactivating the viral particles.

The study team also tested the dependency of the viral-inactivation effect of ethacridine on dose, incubation time and incubation temperature with a plaque assay. For the conditions tested, the viral-inactivation effect showed dose-dependency but was comparable to a 1- or 2-hour incubation at room temperature or at 37°C.

Taken together, the study findings identify a promising, potent, and new use of the old drug via a distinct mode of action for inhibiting SARS-CoV-2.

Ethacridine is highly soluble and non-toxic in various animal models including rat, mice and rabbits. For example, mice treated with 20 mg/kg ethacridine by i.p. injection showed no toxicity. Ethacridine has also been administered into patients for treating puerperal sepsis via intravenous injection.

Ethacridine (also known as rivanol) has been used in the oral treatment of enteric diseases such as traveller’s diarrhoea and shigellosis. Thus, it is plausible to validate its antiviral effect in animal models and COVID-19 patients. https://pubmed.ncbi.nlm.nih.gov/26624983/

https://www.sciencedirect.com/science/article/abs/pii/S000296102990444X

The study findings herein reveal a new approach against SARS-CoV-2 through the inactivation of viral particles, for which the efficacy is expected to be cell type-independent. Indeed, ethacridine blocks SARS-CoV-2 infection of human cell line A549ACE2 cells and, importantly, in primary human nasal epithelial cells.

Because ethacridine has been safely used as a topical drug, one application is as a prophylactic agent to block SARS-CoV-2 infection. For example, ethacridine has been used in postoperative nasal infusion-irrigation for disinfection, after sinusotomy in patients with fungal infection of the sinuses. https://www.jstage.jst.go.jp/article/jibiinkoka1947/84/9/84_9_945/_article/-char/ja/

Hence, ethacridine may be applied to nasal epithelia as a prophylactic treatment to prevent SARS-CoV-2 infection.


Ethacridine inhibits SARS-CoV-2 by inactivating viral particles

To determine how ethacridine inhibits SARS-CoV-2, we tested infectivity of the virus particles after ethacridine treatment using plaque assay, and we also measured viral RNA levels using qRT-PCR. We examined the antiviral effect of ethacridine on different stages of the viral lifecycle of SARS-CoV-2, including before virus-cell binding, after cell entry, and after budding. In particular, we pre-incubated SARS-CoV-2 particles with ethacridine (5 μM) or DMSO for 1 hour. The mixture was then added to Vero E6 cells for viral adsorption at a multiplicity of infection (MOI) at 0.5.

Next, we removed the solution and added fresh medium containing ethacridine (5 μM) or DMSO. Sixteen hours later, we collected supernatant and conducted plaque assay with overlaid agar without ethacridine or DMSO, measured viral titer of the supernatant. We also conducted qRT-PCR and measured viral RNA levels in the supernatant and within cells. In this way, we developed three conditions (Fig. 4a): 1) Control (DMSO + DMSO): the virus and cells were not exposed to DMSO and not the drug; 2)

The virus and cells were exposed to the drug at all stages, including 1 hour before infection, during replication, and after viral budding (i.e. Eth. + Eth.); 3) The virus and cells were exposed to the drug only after viral entry, during replication, and after budding (i.e. DMSO + Eth.). Lastly, we used a fourth condition (Fig. 4b): we conducted plaque assay right after pre-treatment of SARS-CoV-2 with ethacridine for 1 hr (i.e. Eth. [1 hr]), which determines antiviral activity of the drug on the viral particles (equivalent to addition of the drug after viral budding).

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Fig. 4.
Ethacridine bocks SARS-CoV-2 by inactivating viral particles.
(a) Upper panel: schematic showing the experimental design for plaque assay and qRT-PCR. The virus was pre-incubated with ethacridine or DMSO for 1 hr. The mixture was added to Vero cells for adsorption at 4°C for 1 hr. Details can be found in the text. Lower panel: quantitative analysis of viral titer from plaque assay (left), and viral RNA (vRNA) copies by qRT-PCR in Vero cells (middle) and supernatant (right). (b) Quantitative analysis of viral titer by plaque assay. (c) Proposed mode of action of ethacridine by mainly inactivating viral particles of the coronavirus with no or little effect on viral RNA replication. (d) Schematic showing the experimental design for immunostaining. (e, f) Representative images of immunostaining against nucleocapsid protein (N) and spike (S) in Vero E6 cells after infection with the virus that was pre-treated with ethacridine (5 μM) or DMSO (control), or no infection (mock). (g) Quantitative analysis of the immunofluorescence. Data are mean ± SEM (n = 3 or 4 biological replicas). **: p value < 0.01. *: p value < 0.05. Scale bar, 20 μm.

When ethacridine was present continuous prior to plaque assay (Eth. + Eth.), viral titers were reduced 3 – 4 logs, compared with the control (DMSO + DMSO) (Fig. 4a, lower left panel). When ethacridine was added to the Vero cells after viral entry (DMSO + Eth.), similar level of reduced infectivity (3 – 4 logs reduction in viral titer) was observed (Fig. 4a). Interestingly, when SARS-CoV2 was pre-incubated with ethacridine for 1 hr (Eth. [1 hr]) and followed by plaque assay without drug, we also observed 3 – 4 logs reduction in infectivity (Fig. 4b). This result suggests that the drug directly inactivates SARS-CoV2 viral particles. Because of similar-level reduction in infectivity in all of the three conditions, our data strongly suggests that ethacridine inhibits SARS-CoV-2 mainly by inactivation of viral particles.

We next examined viral RNA accumulation in infected cells. qRT-PCR measurement revealed no change of viral RNA (vRNA) levels when the drug was added after viral binding and cell entry (DMSO + Eth.) in both the supernatant and the cells (Fig. 4a, lower middle and right panels), compared with the control (DMSO + DMSO). This indicates that the drug has no effect on vRNA replication. Because plaque assay-based measurement of the same-conditioned sample (DMSO + Eth.) showed 3 – 4 logs reduction in infectivity, our data suggests that ethacridine inhibits SARS-CoV-2 by inactivating the viral particles without effect on vRNA replication. This is consistent with the results of plaque assays for the supernatant samples with 3 different conditions that showed similar level of reduction in infectivity. Here the virions in the supernatant were exposed to the drug before plaque assay-based measurement of viral titer.

Next, when ethacridine was present continuously (i.e. Eth. + Eth.), 4 – 5 fold reductions were observed in vRNA copies in the supernatant and wthin the cells (Fig. 4a, middle and right panels). Because plaque assay-based measurement of the same conditioned sample (Eth. + Eth.) showed 2400-fold reduction in viral titer, the effect of ethacridine on viral replication (4 – 5 fold reduction) is about 500-fold smaller than its effect on viral infectivity. This further supports the conclusion that ethacridine inhibits SARS-CoV-2 by inactivating the viral particles. The 4 – 5 fold reduction of vRNA copies is likely due to reduced viral copy numbers that may bind to the cells (see below), because here the additional step is that the viruses were pre-incubated with the drug.

Thus, our data based on plaque assay and qRT-PCR of different conditioned samples suggests that ethacridine inhibits SARS-CoV-2 by mainly inactivating viral particles, including the virus before binding to Vero cells, as well as virions in the supernatant after budding from host cells, with no or little effect on vRNA replication (Fig. 4c).

To further investigate the mechanism of ethacridine-based inactivation of the viral particles, we conducted immunofluorescence staining and imaging to determine whether the ethacridine-treated SARS-CoV-2 can bind to and enter the cells. We treated SARS-CoV-2 with ethacridine (5 μM) or DMSO for 1 hour. Then the virus was added to cells for adsorption (37°C, 1 hour) at a MOI = 100. Cells were then quickly washed and fixed with 4% PFA (Fig. 4d).

Immunostaining with antibodies against the nucleocapsid protein (N) of SARS-CoV-2 showed strong anti-N fluorescence on the plasma membrane of the cells infected with virus+DMSO mixture, but little anti-N fluorescence in cells treated with virus+ethacridine mixture (Fig. 4e). Immunostaining against the Spike protein (S) of SARS-CoV-2 also revealed fluorescence signals on control cells, but minimal fluorescence on cells infected with the virus+ethacridine mixture (Fig. 4f). Quantification of fluorescence revealed that ethacridine treatment led to a dramatic reduction in detectable anti-N and anti-S fluorescence (Fig. 4g). These results indicate that ethacridine-treated SARS-CoV-2 cannot bind cells to initiate infection.

We also tested the dependency of the viral-inactivation effect of ethacridine on dose, incubation time and incubation temperature with a plaque assay. For the conditions tested, the viral-inactivation effect showed dose-dependency but was comparable to a 1- or 2-hour incubation at room temperature or 37°C (Supporting Fig. S5).

reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605555/

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