Lapachol, a dihydroorotate dehydrogenase inhibitor, demonstrates antiviral activity against feline calicivirus in vitro and in vivo.

Feline calicivirus (FCV) infection poses a significant threat to domestic cats, causing a spectrum of clinical symptoms ranging from mild respiratory issues to severe systemic diseases. Although FCV vaccines are available, their protective efficacy is limited by the extensive genetic diversity and antigenic variability of FCV. To date, no approved antiviral drug is available, highlighting the urgent need for effective therapeutics. Here, we screened a library of 431 small-molecule compounds to identify novel antiviral agents against FCV, leading to the discovery of ten new inhibitors, including five naphthoquinones. Among these, lapachol demonstrated the most potent anti-FCV activity, with a half-maximal effective concentration (EC50) of 1.87 μM and a selectivity index (SI) of 677. Mechanistic studies revealed that lapachol exerts its antiviral effects by inhibiting feline dihydroorotate dehydrogenase (DHODH), thereby disrupting the synthesis of pyrimidine nucleotides, which are essential for viral replication. Further investigations showed that silencing DHODH enhanced lapachol's antiviral activity, while supplementation with pyrimidine nucleotides or DHODH overexpression reversed its effects. Importantly, oral administration of lapachol at 5 mg/kg significantly reduced virus shedding from the oral and nasal cavities in FCV-infected cats, promoted recovery from weight loss, and alleviated oral ulceration and pulmonary lesions, without inducing observable hepatic or renal toxicity. These findings demonstrate that lapachol is a promising candidate for the treatment of FCV infections, and underscore the potential of DHODH as a viable therapeutic target for antiviral drug development.