Antifolate agent aminopterin demonstrates potent anti-monkeypox virus activity in vitro and in vivo

The ongoing spread of the monkeypox outbreak underscores the urgent need for the development of effective therapeutics against the monkeypox virus (MPXV). Aminopterin is a classical antifolate agent known for its anti-tumor activity through the inhibition of folate metabolism. Here, aminopterin was identified as an effective anti-MPXV agent. It significantly inhibited MPXV replication, unlike other antifolate agents such as methotrexate, pemetrexed, and LSN3213218. In Vero E6 cells, the half-maximal inhibitory concentration (IC₅₀) was 36.10 nM, the half-cytotoxic concentration (CC₅₀) was 3,310 nM, and the selectivity index (SI) was 91.690. In human foreskin fibroblast cells, the IC50 was 32.73 nM, CC₅₀ exceeded 10 μM, and SI was greater than 305.530. In vivo, aminopterin not only inhibited MPXV infection but also mitigated high-dose viral infection-associated inflammation. Furthermore, the data indicated that aminopterin reduced viral load by interfering with viral attachment and replication. Together, these findings identify aminopterin as a promising candidate for anti-MPXV drug development.