Barbituric Acid Derivatives as Covalent Inhibitors of <i>Leishmania braziliensis</i> Dihydroorotate Dehydrogenase

Covalent drug design applied to parasite proteins enables selective therapies by targeting nucleophilic residues of macromolecules. We present the first covalent inhibitors of <i><i>Leishmania braziliensis</i></i> dihydroorotate dehydrogenase (<i>Lb</i>DHODH), a key enzyme in pyrimidine biosynthesis with a reactive cysteine (Cys¹³¹) in its active site. From barbituric acid derivatives, we discovered <b>2i</b> as a <i>Lb</i>DHODH inhibitor with leishmanicidal activity, exhibiting an IC₅₀ of 0.5 ± 0.1 μM, a K_inact/K<i>I</i> of 767 M^-1s^-1, no inhibition of the human ortholog, and an EC₅₀ of 11 ± 5 μM in <i><i>L. braziliensis</i></i> promastigotes, with no cytotoxicity in THP-1 cells and good passive permeability. X-ray crystallography confirms covalent bond formation with Cys¹³¹ and reveals active-site rearrangements. These findings support the proposed covalent inhibition mechanism and provide structural insights for further optimization. Our study validates <i>Lb</i>DHODH as a promising target for leishmaniasis therapy and highlights the potential of covalent inhibition in antiparasitic drug discovery.