The IMPDH inhibitor merimepodib provided in combination with the adenosine analogue remdesivir reduces SARS-CoV-2 replication to undetectable levels in vitro [version 1; peer review: 2 approved with reservations]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the novel coronavirus responsible for the ongoing COVID-19 pandemic, which has resulted in over 2.5 million confirmed cases and 170,000 deaths worldwide as of late April 2020. The pandemic currently presents major public health and economic burdens worldwide. No vaccines or therapeutics have been approved for use to treat COVID19 cases in the United States despite the growing disease burden, thus creating an urgent need for effective treatments. The adenosine analogue remdesivir (REM) has recently been investigated as a potential treatment option, and has shown some activity in limiting SARS-CoV-2 replication. We previously reported that the IMPDH inhibitor merimepodib (MMPD) provides a dose-dependent suppression of SARS-CoV-2 replication in vitro. Here, we report that a 4-hour pre-treatment of Vero cells with 2.5μM MMPD reduces the infectious titer of SARS-CoV-2 more effectively than REM at the same concentration. Additionally, pre-treatment of Vero cells with both REM and MMPD in combination reduces the infectious titer of SARS-CoV-2 to values below the detectable limit of our TCID50 assay. This result was achieved with concentrations as small as 1.25 μM MMPD and 2.5 μM REM. At concentrations of each agent as low as 0.31 μM, significant reduction of viral production occurred. This study provides evidence that REM and MMPD administered in combination might be an effective treatment for COVID-19 cases.


Introduction
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 profile 1,2 .
REM is an adenosine analogue that displays broad-spectrum antiviral activity against RNA viruses, and has been developed for the treatment of Ebola virus disease 3 . The drug causes a delayed chain termination of RNA synthesis roughly five nucleotides downstream of the incorporated nucleotide analogue 4 . 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 NHPs 7 . 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 synthesis 8 , 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 viruses 9 . 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 infection 10 .
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.

Cells and viruses
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.
Compound treatment and infection of Vero cells 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 TCID 50 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.

Results
We have previously observed a peak in SARS-CoV-2 titer at 24 hours post-infection, which plateaus through 48 hours post-infection 11 . 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 5µM 10 .
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-treatment 12 . 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 dosedependent, 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.

Discussion
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 EC 50 and EC 90 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 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 therapy 2 . Intravenous administration of a 10mg/kg dose of REM results in blood concentrations of 10µM in NHPs 13 . 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 RNA 4 ; 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 system 10 .
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). Despite the provided piece of experiments and results of this manuscript, we still unable to precisely know the potency of these two drugs. The description of results was using the two concentrations of the two drugs without further important details. The potency of the drugs must be presented in terms of IC50. In this regard, several compounds were proved to be effective on SARS CoV-2 replication in the low nanomolar range and could be at least several folds more potent than REM and MMPD. In this case, the potency of REM and MMPD can be related to other anti-SARS CoV-2 drugs.

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The relation of the two drugs combination must be discussed in terms of additive effect, synergism or antagonism. For this purpose software as CompuSyn or similar can be used to calculate the combination index, which will scientifically assess the nature of these two drugs combination effect.
○ "Oral administration of 50mg MMPD achieves a plasma concentration of around 2500ng/mL (5.5 μM) which makes it highly attractive for antiviral therapy" recheck this sentence ○ For assay reproducibility, another cell type is required e,g, Calu-3 cells ○ The raw data of crystal violet stain pictures and plaques count could be provided as supplementary file.

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The two drugs seems to act on late stages of virus replication, at least not in the attachment ○ and fusion steps, the 4-hr pretreatment needs clarification. The time of addition experiment must be provided during different hours before and after infection.

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

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 observation, along with the observation on additive effects of remdesivir and merimepomid in combination. The authors demonstrate robust antiviral activity of the two substances in the low micromolar concentration and a robust antiviral activity when both substances are administered in combination at a concentration of 1,25 µM. The major shortcoming of the study is that all results were generated on VeroE6, a green monkey cell line that is not necessarily representative of the natural sites of infection. This issue is particularly important because merimepodib acts by inhibiting inosine-5'-monophosphate dehydrogenase (IMPDH), a cellular enzyme, and potential polymorphisms between this gene in non-human primates and humans may result in a different efficacy of the drug in clinical settings. Therefore, the study would be strengthened by expanding the assay to human epithelial cells, ideally of lung origin.
Another smaller shortcoming of the study is that all statistical analysis was performed by t-tests, but the data are for the most part much more complex and require ANOVA analysis, and due to the low n in the experimental groups should be performed by Kruskal-Wallis. This in itself is not a major point of contention in my eyes. Reproducibility in experimental settings trumps statistical analysis by definition, the results in this study are clear already by eyeballing them, and the core results have been reproduced in several experiments shown in the two figures. Nevertheless, if statistical significance is used as an argument to validate the results, then one should use statistical methods that fit the experimental design and the group sizes.
In sum, despite these shortcomings, this is an important set of data that brings us closer to the development of antiviral strategies against the COVID19 pandemics and a substantial contribution to the field.

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

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 significant reservations, as outlined above.
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