Pralatrexate is a potent pan-serotype human adenovirus inhibitor through suppression of dihydrofolate reductase.

Human adenovirus (HAdV) is one of the most infectious pathogens that can cause diseases affecting multiple organ systems, posing a significant threat to public health. Currently, there are no specific antiviral therapies available for HAdV infection. In this study, three folate antagonists aminopterin (AMT), pralatrexate (PDX), and methotrexate (MTX) with potent antiviral activity against HAdV infection have been identified through a drug repurposing screening strategy. The three drugs showed broad and potent antiviral efficiency against multiple clinically prevalent HAdV serotypes, including HAdV-B3, HAdV-B7, HAdV-B55, HAdV-C2, and HAdV-C5 with half-maximal effective concentration (EC50) values ranging from 0.31 to 79.74 nM, and PDX further showed similar antiviral efficiency against HAdV-B55 in a 3D lung organoid model (EC50 = 0.51 nM). Mechanistic studies revealed that PDX achieved its antiviral activity through suppression of dihydrofolate reductase (DHFR). These findings support the potential of folate antagonists as repurposed therapeutic agents against HAdV infection and provide a rationale for the development of host-directed antiviral strategies.