Keywords
merimepodib, remdesivir, SARS-CoV-2, COVID-19
This article is included in the Emerging Diseases and Outbreaks gateway.
This article is included in the Coronavirus collection.
merimepodib, remdesivir, SARS-CoV-2, COVID-19
The novel coronavirus SARS-CoV-2 is responsible for the current coronavirus disease 2019 (COVID-19) pandemic that originated in Wuhan, China in December of 2019. Since its emergence, the virus has infected 2.5 million people and is responsible for 170,000 deaths at the time of writing. In addition to the significant health burden, the pandemic is causing a worldwide economic crisis as countries attempt to limit the spread of SARS-CoV-2. There is a dire need for efficacious treatments, as the United States has yet to approve a vaccine or therapeutic to combat COVID-19. The most efficient means of approval involves drugs with a history of clinical testing. Two of these drugs, remdesivir (REM) and merimepodib (MMPD), have already been tested in patients. REM is currently in clinical trials as a COVID-19 therapeutic, while MMPD has previously been tested against chronic hepatitis C and psoriasis with an excellent safety profile1,2.
REM is an adenosine analogue that displays broad-spectrum antiviral activity against RNA viruses, and has been developed for the treatment of Ebola virus disease3. The drug causes a delayed chain termination of RNA synthesis roughly five nucleotides downstream of the incorporated nucleotide analogue4. REM has demonstrated antiviral effects against both Ebola and Nipah viruses in nonhuman primates (NHPs)5,6, and has recently shown efficacy as a COVID-19 therapeutic in NHPs7. However, the drug must be administered intravenously limiting the ability for use outside of a hospital setting.
MMPD is a selective and potent inhibitor of inosine-5’-monophosphate dehydrogenase (IMPDH), an enzyme involved in de novo guanine nucleotide synthesis8, which manipulates cellular nucleoside triphosphate levels to control the rapid replication of viruses. The drug has been tested for in vitro inhibition of many RNA viruses including Ebola, Junin, Lassa, Zika, and Chikungunya viruses9. We recently determined that MMPD suppresses the replication of SARS-CoV-2 by 3-log when Vero cells were pre-treated 4 hours prior to infection10.
In this study, we aimed to compare the antiviral effect of REM to MMPD in a tissue culture model in which cells were pre-treated with either agent prior to SARS-CoV-2 infection, and to determine whether pre-treatment with both REM and MMPD could further reduce the replication of SARS-CoV-2 in vitro.
Vero cells (ATCC Cat# CCL-81, RRID:CVCL_0059) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (HyClone). SARS-CoV-2 USA-WA1/2020 was obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA), which obtained the original isolate from the Centers for Disease Control and Prevention (CDC). Upon obtaining the isolate, virus titration was performed. A subsequent low multiplicity of infection (MOI) passage was performed in Vero cells (DMEM supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin), and the resulting stock was titrated prior to use. All experiments were conducted within approved biosafety level three (BSL-3) laboratories at the University of Texas Medical Branch (UTMB). All personnel undergo routine medical surveillance.
MMPD clinical lot #2 (VX-497 Bulk Drug Substance # 300445), which is supplied as a white solid, was obtained from the PCI (Newburyport, MA). 10 mM stock of MMPD was prepared in DMSO and stored at -80°C. REM (Synonym GS-5734), Cat. # HY-104077, which is supplied as 10 mM solution in DMSO, was obtained from MedChemExpress (Monmouth Junction, NJ) and stored at -80°C. Both MMPD and REM then were diluted in compound dilution media to reach the assay target concentrations and the final DMSO concentration was equal or below 0.1%. Vero cells were pre-treated 4 hours prior to infection with either REM or MMPD diluted to the indicated concentration in DMEM, or a combination of the two drugs at the indicated concentrations diluted into DMEM (12-well plates, 1 mL per well). The media was removed at the time of infection, and the cells were inoculated with SARS-CoV-2 (MOI = 0.05) in 0.1 mL of inoculum, which was pre-mixed with the appropriate drug compound(s). The cells were incubated for 1 hour at 37°C to allow for infection. The cells were subsequently rinsed three times with 1 mL of DMEM, and then 1 mL of DMEM containing the appropriate drug compound(s) at the indicated concentration was added to each appropriate well. The cells were incubated for 0, 16, or 24 hours at 37°C before collecting a portion of each supernatant for titration. Upon collection, an equal amount of DMEM containing the compound was added back to each well. The infectious titer of SARS-CoV-2 for each sample was determined by TCID50 assay in Vero cells. The collected samples were serially diluted ten-fold a total of six times in DMEM, with dilutions performed in quadruplicate. The resulting dilutions were used to inoculate Vero cells in 96-well plates, and the plates were allowed to incubate for 4 days at 37°C. Cells were then fixed by addition of 10% formalin to each well for 30 minutes, and subsequently stained for 5 minutes with crystal violet solution. TCID50 values were obtained by counting the lowest dilutions in which infection was visible after crystal violet stain for two of four replicates. Each experiment was performed in biological triplicate, and the results were analyzed using a one-tailed t-test conducted in the GraphPad Prism 7 built-in statistical analysis software.
We have previously observed a peak in SARS-CoV-2 titer at 24 hours post-infection, which plateaus through 48 hours post-infection11. During the exponential growth phase, SARS-CoV-2 replication can be inhibited by 3-log with an overnight pre-treatment of MMPD at a concentration of 10µM, or 1.5-log with 4 hour pre-treatment at 5µM10.
Both REM and MMPD exhibit a potent antiviral effect on SARS-CoV-2 at concentrations of 2.5µM and 5µM after a 4 hour pre-treatment12. REM pre-treatment at a 5µM concentration provides a 2.1-log decrease in titer at 16 hours-post infection (p=0.001) and a 3.9-log decrease in titer at 24 hours post-infection (p<0.001). MMPD causes a similar antiviral effect as REM at the 5µM concentration, exhibiting a 2.1-log decrease in titer at 16 hours-post infection (p=0.001) and a 2.7-log decrease in titer at 24 hours post-infection (p<0.001). The effect of REM was dose-dependent, with the 2.5µM concentration of REM decreasing the titer by 1-log at 16 hours post-infection (p=0.103) and 1.5-log at 24 hours post-infection (p=0.002). However, MMPD antiviral effects appeared to be less dose-dependent, maintaining a 1.9-log decrease in titer at 16 hours post-infection (p=0.003) and a 2.5-log decrease in titer at 24 hours post-infection (p=0.001) at a concentration of 2.5µM (Figure 1). At sixteen hours post-infection 2.5µM REM does not have a statistically significant (p<0.05) difference between REM and untreated samples, whereas MMPD significantly reduced the infectious titer at this time point. When compared directly, MMPD causes a significantly greater reduction in SARS-CoV-2 infectious particles at both the 16-hour (p=0.022) and 24-hour (p=0.014) than REM. Therefore, MMPD provides a stronger inhibitory effect than REM at the 2.5µM concentration.
Since REM and MMPD inhibit virus replication via separate mechanisms, we tested the inhibitory effect of both drugs in combination. We tested combinations ranging from 5µM + 5µM to 0.31µM + 0.31µM (Figure 2), performing the same 4 hour drug pre-treatment. Inhibition of SARS-CoV-2 was dose-dependent, and the virus titer was reduced to undetectable levels at all time points (p<0.001) for concentrations of 1.25µM +2.5µM, 2.5µM + 1.25µM, 2.5µM +2.5µM, 2.5µM + 5µM, 5µM + 2.5 µM, and 5µM + 5µM concentrations of REM and MMPD, respectively (Figure 2A, 2B). The lowest concentrations tested were 0.31µM REM + 0.31µM MMPD, which reduced the titer by 0.2-log at 16 hours post-infection (p=0.371) and 1.3-log at 24 hours post-infection (p=0.015). All combinations of concentrations between 0.31µM + 0.31µM and 1.25µM + 1.25µM produced a dose-dependent reduction in titer between 0.2-log and 2.5-log at 16 hours post-infection and a dose-dependent reduction between 1.3-log and 3.7-log at 24 hours post-infection (Figure 2C).
We noticed a more robust inhibitory effect when the MMPD was provided at a lower concentration as opposed to REM being provided at a lower concentration (p<0.001, p=0.01), as illustrated 24 hours post-infection with the 0.31µM REM + 0.62µM MMPD and 0.62µM REM + 1.25µM MMPD to the 0.62µM REM + 0.31µM MMPD and 1.25µM REM + 0.62µM MMPD combinations (Figure 2C). This further supports that MMPD provides more potent SARS-CoV-2 inhibition than REM within the tested concentration ranges. More importantly, the data provides evidence that a treatment of REM + MMPD at a concentration as low as 2.5µM + 1.25µM reduces the production of infectious SARS-CoV-2 progeny to undetectable levels in cell culture.
Our results provide evidence that MMPD potently inhibits SARS-CoV-2 replication in vitro at concentrations as low as 2.5µM, which was the lowest concentration tested. Further work will determine the EC50 and EC90 for MMPD. Previous work established that cellular cytotoxicity for MMPD is greater than 10 μM so the activity against the virus cannot be attributed to the drug being cytotoxic.
The inhibitory effect was similar for both MMPD and REM at 2.5µM at 24 hours post-infection. However, MMPD was the only drug to significantly inhibit SARS-CoV-2 at the 16 hour time point using a 2.5µM concentration. REM also appears to have a slightly higher range of dose-dependent efficacy in the 1–10µM range than MMPD. Here, we show a 2.4-log difference in antiviral activity for REM between 2.5µM and 5µM concentrations at 24 hours post-infection and only a difference of 0.2-log for MMPD using the same conditions. Previously, we determined that only a 1.5-log difference exists in MMPD inhibitory effect between 10µM and 3.3µM concentrations10.
The concentration of MMPD used in this study is well below the clinically-achievable concentration based on human PK trials. Oral administration of 50mg MMPD achieves a plasma concentration of around 2500ng/mL (5.5 μM) which makes it highly attractive for antiviral therapy2. Intravenous administration of a 10mg/kg dose of REM results in blood concentrations of 10µM in NHPs13. REM and MMPD affect the replication of RNA viruses through different mechanisms. REM is an adenosine analogue that causes a delayed chain termination of RNA synthesis upon being incorporated into viral RNA4; whereas MMPD is a selective IMPDH inhibitor that manipulates the nucleoside triphosphate levels within cells, acting indirectly as a host-directed therapy. Therefore, we also tested whether the combination treatment using both, direct antiviral REM and host targeted drug MMPD could possibly have synergistic anti-COVID-19 activity. Our data demonstrate that this combination can completely abolish production of infectious virus in our cell culture model. Neither agent by itself completely abrogates viral production. While we are now determining the dose response of MMPD in this model system, REM data demonstrates that a combination of both drugs still gives significant reduction of viral productions with low concentrations of both agent.
Our study indicates that MMPD alone, and/or in combination with REM, could be a viable treatment option for COVID-19 based on the potent inhibitory effect in cells culture at an easily achievable dose in the clinical setting. The drug could be quickly tested in a clinical setting based on its clinical trial history, and might be a useful prophylactic treatment to prevent spread of SARS-CoV-2 to the lower respiratory system10.
Harvard Dataverse: Remdesivir and Merimepodib co-treatment for SARS-CoV-2. https://doi.org/10.7910/DVN/HE6ZYJ12.
This project contains the following underlying data:
Raw Data REM + MMPD co-treatment for SARS-CoV-2.xlsx (SARS-CoV-2 inhibition data for remdesivir and merimepodib co-treatment in Vero cells)
Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).
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Is the work clearly and accurately presented and does it cite the current literature?
Partly
Is the study design appropriate and is the work technically sound?
Partly
Are sufficient details of methods and analysis provided to allow replication by others?
Partly
If applicable, is the statistical analysis and its interpretation appropriate?
No
Are all the source data underlying the results available to ensure full reproducibility?
No
Are the conclusions drawn adequately supported by the results?
Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Pharmacology and drug discovery
Is the work clearly and accurately presented and does it cite the current literature?
Yes
Is the study design appropriate and is the work technically sound?
Partly
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Partly
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Virology and immunology.
Alongside their report, reviewers assign a status to the article:
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Version 1 13 May 20 |
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