The folate antagonist methotrexate diminishes replication of the coronavirus SARS-CoV-2 and enhances the antiviral efficacy of remdesivir in cell culture models.

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) and ~100-fold reductions in 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 100 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.

The MAPK-activated protein kinase 2 mediates gemcitabine sensitivity in pancreatic cancer cells.

Pancreatic carcinoma is the major clinical entity where the nucleoside analog gemcitabine is used for first-line therapy. Overcoming cellular resistance toward gemcitabine remains a major challenge in this context. This raises the need to identify factors that determine gemcitabine sensitivity in pancreatic carcinoma cells. We previously found the MAPK-activated protein kinase 2 (MK2), part of the p38/MK2 stress response pathway, to be required for DNA replication fork stalling when osteosarcoma-derived cells were treated with gemcitabine. As a consequence, inhibition or depletion of MK2 protects these cells from gemcitabine-induced death (Köpper, et al. Proc Natl Acad Sci USA 2013; 110:16856-61). Here, we addressed whether MK2 also determines the sensitivity of pancreatic cancer cells toward gemcitabine. We found that MK2 inhibition reduced the intensity of the DNA damage response and enhanced survival of the pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and Panc-1, which display a moderate to strong sensitivity to gemcitabine. In contrast, MK2 inhibition only weakly attenuated the DNA damage response intensity and did not enhance long-term survival in the gemcitabine-resistant cell line PaTu 8902. Importantly, in BxPC-3 and MIA PaCa-2 cells, inhibition of MK2 also rescued increased H2AX phosphorylation caused by inhibition of the checkpoint kinase Chk1 in the presence of gemcitabine. These results indicate that MK2 mediates gemcitabine efficacy in pancreatic cancer cells that respond to the drug, suggesting that the p38/MK2 pathway represents a determinant of the efficacy by that gemcitabine counteracts pancreatic cancer.

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity.

DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation. Moreover, MK2 activity was required for damage response, accumulation of ssDNA, and decreased survival when cells were treated with the nucleoside analogue gemcitabine or when the checkpoint kinase Chk1 was antagonized. By using DNA fiber assays, we found that MK2 inhibition or knockdown rescued DNA replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling.