Repurposed dihydroorotate dehydrogenase inhibitors with efficacy against drug-resistant
 Acinetobacter baumannii


 New antimicrobials are needed for the treatment of extensively drug-resistant
 Acinetobacter baumannii
 . The de novo pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is a validated drug target for malaria and human autoimmune diseases. We provide genetic evidence that
 A. baumannii
 DHODH (
 Ab
 DHODH) is essential for bacterial survival in rodent infection models. We chemically validate the target by repurposing a unique library of ~450 triazolopyrimidine/imidazopyrimidine analogs developed for our malaria DHODH program to identify 21 compounds with submicromolar activity on
 Ab
 DHODH. The most potent (DSM186, DHODH IC
 50
 28 nM) had a minimal inhibitory concentration of ≤1 µg/ml against geographically diverse
 A. baumannii
 strains, including meropenem-resistant isolates. A structurally related analog (DSM161) with a long in vivo half-life conferred significant protection in the neutropenic mouse thigh infection model. Encouragingly, the development of resistance to these compounds was not identified in vitro or in vivo. Lastly, the X-ray structure of
 Ab
 DHODH bound to DSM186 was solved to 1.4 Å resolution. These data support the potential of
 Ab
 DHODH as a drug target for the development of antimicrobials for the treatment of
 A. baumannii
 and potentially other high-risk bacterial infections.
