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

UNLABELLED: 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.

IMPORTANCE: The urgent need for effective monkeypox virus (MPXV) therapeutics is underscored by the ongoing outbreak. This study identifies aminopterin as a potent anti‑MPXV agent among four antifolate agents. Crucially, aminopterin suppresses viral infection and mitigates infection-associated inflammation in vivo. These findings position aminopterin as a potential candidate for the development of an antiviral agent against MPXV.