Dihydroorotate Dehydrogenase Inhibitors are Potent Antiviral Agents against Henipaviruses and Arenaviruses.

Hendra virus (HeV) and Nipah virus (NiV) are zoonotic henipaviruses associated with high mortality and are classified as Biosafety Level 4 (BSL-4) agents. To identify small molecule inhibitors, we performed a cell-based high-throughput screening (HTS) of an antiviral compound library using recombinant Cedar virus (rCedV) as a non-pathogenic BSL-2 henipavirus surrogate. Hit compounds were evaluated by cytotoxicity and antiviral assays to determine CC50 and EC50 values. Among eleven primary hits, five compounds-Farudodstat, Atovaquone, PTC299, hDHODH-IN-7, and Brequinar-were prioritized based on antiviral potency. These compounds are known inhibitors of dihydroorotate dehydrogenase (DHODH), a host enzyme essential for de novo pyrimidine biosynthesis and viral RNA replication. To validate their mechanism of action, metabolite rescue assays were performed using intermediates from the pyrimidine biosynthesis pathway, demonstrating that pyrimidine metabolite supplementation reversed the antiviral activity of PTC299. In addition, the enantiomer of PTC299, a much weaker inhibitor of DHODH, was 100-fold less potent in the antiviral assay. Both experiments confirm that the antiviral effects of these compounds against rCedar virus are mediated through inhibition of pyrimidine biosynthesis. In contrast, the antiviral activity of mycophenolic acid, a purine biosynthesis inhibitor, was not reversed by pyrimidine metabolite supplementation. Four pyrimidine biosynthesis inhibitors demonstrated broad-spectrum antiviral activity across multiple virus families. Notably, PTC299 exhibited a selectivity index exceeding 500. Together, these in vitro findings indicate that DHODH inhibitors have potential as potent broad-spectrum host-directed antivirals, especially against high-consequence and emerging viruses.