Methotrexate inhibits SARS‐CoV‐2 virus replication “in vitro”

Abstract In early 2020 the new respiratory syndrome COVID‐19 (caused by the zoonotic SARS‐CoV‐2 virus) spread like a pandemic, starting from Wuhan, China, causing severe economic depression. Despite some advances in drug treatments of medical complications in the later stages of the disease, the pandemic's death toll is tragic, as no vaccine or specific antiviral treatment is currently available. By using a systems approach, we identify the host‐encoded pathway, which provides ribonucleotides to viral RNA synthesis, as a possible target. We show that methotrexate, an FDA‐approved inhibitor of purine biosynthesis, potently inhibits viral RNA replication, viral protein synthesis, and virus release. The effective antiviral methotrexate concentrations are similar to those used for established human therapies using the same drug. Methotrexate should be most effective in patients at the earliest appearance of symptoms to effectively prevent viral replication, diffusion of the infection, and possibly fatal complications.

potentially lethal complications. [5][6][7] This more dangerous stage is characterized by a dysfunctional immune response that leads to a cytokine storm, which predisposes to hemostatic abnormalities. [5][6][7] High levels of interleukin-6 (IL-6), together with significant levels of SARS-CoV-2 RNA, are an index of a high lethality risk. In the most severe cases, acute respiratory distress syndrome (ARDS), often complicated by pulmonary thromboembolism, occurs finally, leading to death. 8,9 Extension of the viral infection to extrapulmonary districts 10 may have a role in the diffused post-COVID syndrome. 11 Patients with mild symptoms receive mostly symptomatic treatments. 6 Patients in the severe stage receive anti-inflammatory drugs, including low doses of steroids or drugs acting on the IL-6 axis. 12 As the COVID-19 pandemic would be substantially constrained by a drug blocking the reproduction of viral particles, several studies are ongoing 12 to reposition available drugs looking for their ability to interfere with essential viral functions.
The novelty of the present report is provided by considering, in a system biology approach, not only the events of viral entrance and reproduction into lung type II pneumocytes but also their necessary collaboration with the human host cellular pathways, among which the one providing nucleotides required for the synthesis of viral RNA. The synthesis of purines (a component of nucleotides) is inhibited by methotrexate (4-amino-10-methyl folic acid, MTX), 13 an FDA approved drug, used to treat several cancer types and, at lower doses, autoimmune diseases. Herein, we present the first round of experiments "in vitro," which clearly show that MTX blocks, with high efficiency, SARS-CoV-2 replication in the post-entry phase.

| Cells
African green monkey kidney Vero E6 cell line was obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo-Fisher Scientific) at 37°C in a humidified atmosphere of 5% CO 2 .

| Virus
We successfully isolated SARS-CoV-2 in Vero E6 cells from the na-    | 1781 (G-9; Santa Cruz Biotechnology). The antigen-antibody complexes were detected using peroxidase-conjugated goat anti-human or goat antimouse IgG (Sigma) and revealed using the enhanced chemiluminescence (ECL) system (Santa Cruz Biotechnology).

| Statistical analysis
Data were analyzed for statistical significance using the one-way analysis of variance, and the Bonferroni post test was used to compare data. Differences were considered significant when p < .05.
Statistical tests were performed using GraphPad Prism 8.   In rheumatoid arthritis and other inflammatory syndromes, the molecular target of MTX is not DHFR. 13 MTX decreases the levels of interleukin 6 (IL-6) and soluble IL-2 receptor, the reduction in cytokine levels being paralleled by an improvement in clinical indices. 20 Therefore, in COVID-19 patients in a more advanced stage, the treatment with MTX is expected to decrease virion production and down-regulate the IL-6 pathway, a strategy currently in use in different clinical trials. 12

CONFLICT OF INTERESTS
The authors declare that there is no conflict of interest.

AUTHOR CONTRIBUTIONS
Lilia Alberghina conceived the idea that led to the systems iden-

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.