LbDHODH Inhibitors 2i(University of São Paulo)

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 (Cys¹³¹) in its active site. From barbituric acid derivatives, we discovered 2i as a LbDHODH inhibitor with leishmanicidal activity, exhibiting an IC₅₀ of 0.5 ± 0.1 μM, a K_inact/KI of 767 M^-1s^-1, no inhibition of the human ortholog, and an EC₅₀ 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 Cys¹³¹ 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.