The folate antagonist methotrexate diminishes replication of the coronavirus SARS-CoV-2 and enhances the antiviral efficacy of remdesivir in cell culture models
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Abstract
The search for successful therapies of infections with the coronavirus SARS-CoV-2 is ongoing. We tested inhibition of host cell nucleotide synthesis as a promising strategy to decrease the replication of SARS-CoV-2-RNA, thus diminishing the formation of virus progeny. Methotrexate (MTX) is an established drug for cancer therapy and to induce immunosuppression. The drug inhibits dihydrofolate reductase and other enzymes required for the synthesis of nucleotides. Strikingly, the replication of SARS-CoV-2 was inhibited by MTX in therapeutic concentrations around 1 μM, leading to more than 1000-fold reductions in virus progeny in Vero C1008 (Vero E6) as well as Calu-3 cells. Virus replication was more sensitive to equivalent concentrations of MTX than of the established antiviral agent remdesivir. MTX strongly diminished the synthesis of viral structural proteins and the amount of released virus RNA. Virus replication and protein synthesis were rescued by folinic acid (leucovorin) and also by inosine, indicating that purine depletion is the principal mechanism that allows MTX to reduce virus RNA synthesis. The combination of MTX with remdesivir led to synergistic impairment of virus replication, even at 300 nM MTX. The use of MTX in treating SARS-CoV-2 infections still awaits further evaluation regarding toxicity and efficacy in infected organisms, rather than cultured cells. Within the frame of these caveats, however, our results raise the perspective of a two-fold benefit from repurposing MTX for treating COVID-19. Firstly, its previously known ability to reduce aberrant inflammatory responses might dampen respiratory distress. In addition, its direct antiviral activity described here would limit the dissemination of the virus.
SIGNIFICANCE
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MTX is one of the earliest cancer drugs to be developed, giving rise to seven decades of clinical experience. It is on the World Health Organization’s List of Essential Medicines, can be administered orally or parenterally, and its costs are at single digit € or $ amounts/day for standard treatment. In case of its successful further preclinical evaluation for treating SARS-CoV-2 infections, its repurposing to treat COVID-19 would thus be feasible, especially under low-resource conditions.
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Additional drugs exist to interfere with the synthesis of nucleotides, e.g. additional folate antagonists, inhibitors of GMP synthetase, or inhibitors of dihydroorotate dehydrogenase (DHODH). Such inhibitors have been approved as drugs for different purposes and might represent further therapeutic options against infections with SARS-CoV-2
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Remdesivir is currently the most established drug for treating COVID-19. Our results argue that MTX and remdesivir, even at moderate concentrations, can act in a synergistic fashion to repress virus replication to a considerably greater extent than either drug alone.
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COVID-19, in its severe forms, is characterized by pneumonia and acute respiratory distress syndrome, and additional organ involvements. These manifestations are not necessarily a direct consequence of virus replication and cytopathic effects, but rather a result of an uncontrolled inflammatory and immune response. Anti-inflammatory drugs such as glucocorticoids are thus being evaluated for treating COVID-19. However, this bears the risk of re-activating virus spread by suppressing a sufficient and specific immune response. In this situation, it is tempting to speculate that MTX might suppress both excessive inflammation as well as virus replication at the same time, thus limiting both the pathogenesis of pneumonia and also the spread of virus within a patient.
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SciScore for 10.1101/2020.07.18.210013: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Experimental Model And Methods: Quantification And Statistical Analysis: Statistical testing was performed using Graph Pad Prism 6 (RRID:SCR_002798). Graph Pad Prismdetected: GraphPad Prism ( RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Having …
SciScore for 10.1101/2020.07.18.210013: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Experimental Model And Methods: Quantification And Statistical Analysis: Statistical testing was performed using Graph Pad Prism 6 (RRID:SCR_002798). Graph Pad Prismdetected: GraphPad Prism ( RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Having said this, a number of caveats still apply. Firstly, the dose of MTX that would be needed for antiviral treatment is probably higher than the typical dose taken by patients for the purpose of long-term anti-inflammation treatment. Plasma concentrations in patients treated for rheumatoid arthritis, typically taking 7.5 mg/week for months or even years, are only around 25 nM (Hornung et al., 2008), which is unlikely to interfere with virus replication. On the other hand, when applying high-dose therapies, up to 1000 μM plasma concentrations are achievable (Comandone et al., 2005), i.e. 1000-fold more than what would be required for the antiviral effect in our study. Under such circumstances, a limiting toxicity would consist in mucositis, which is often observed when treating leukemia with high-dose MTX in a pediatric setting (den Hoed et al., 2015). Antiviral activities of MTX would presumably require an intermediate shortterm dose for a week or two. The antiviral concentration of MTX (> 1 μM) is achieved, for instance, by single intramuscular application of 50 mg MTX, which is a common procedure to induce abortions at early stages of pregnancy (Creinin and Vittinghoff, 1994). In this setting, tolerable side effects were reported, e.g. transient nausea, diarrhea, and headache (Creinin and Krohn, 1997). This comparison, on the other hand, makes it very clear that antiviral doses of MTX are contraindicated in pregnant women. A clinical study using nanoparticle-coupled MTX...
Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04425252 Completed Safety and Anti-coronavirus Response of Suppression of Host … NCT04352465 Not yet recruiting Efficacy and Safety of MTX-loaded Nanoparticles to Treat Sev… Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 33. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- Thank you for including a protocol registration statement.
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