Abstract
Covalent drug design applied to parasite proteins enables selective therapies by targeting nucleophilic residues of macromolecules. We present the first covalent inhibitors of Leishmania braziliensis dihydroorotate dehydrogenase (LbDHODH), a key enzyme in pyrimidine biosynthesis with a reactive cysteine (Cys(131)) in its active site. From barbituric acid derivatives, we discovered 2i as a LbDHODH inhibitor with leishmanicidal activity, exhibiting an IC(5)(0) of 0.5 ± 0.1 μM, a K(inact)/KI of 767 M(-1)s(-1), no inhibition of the human ortholog, and an EC(5)(0) of 11 ± 5 μM in L. braziliensis promastigotes, with no cytotoxicity in THP-1 cells and good passive permeability. X-ray crystallography confirms covalent bond formation with Cys(131) and reveals active-site rearrangements. These findings support the proposed covalent inhibition mechanism and provide structural insights for further optimization. Our study validates LbDHODH as a promising target for leishmaniasis therapy and highlights the potential of covalent inhibition in antiparasitic drug discovery.