Nitrile-Based Leishmania Cysteine Protease B Inhibitors with In Vivo Efficacy against Visceral Leishmaniasis

Abstract Leishmaniasis remains a major neglected tropical disease with limited therapeutic options and increasing concerns regarding toxicity and drug resistance. Cysteine protease B (CPB), a key virulence factor in Leishmania spp., represents an attractive molecular target for the development of new antileishmanial agents. In this study, we designed and synthesized a series of dipeptidyl nitrile derivatives as reversible covalent CPB inhibitors and evaluated their biological activity in vitro and in vivo. Molecular docking performed in covalent mode demonstrated favorable accommodation of the compounds within the CPB active site, with the benzyl-containing derivatives showing the most favorable interactions. Among the series, compound 1 ((S)-tert-butyl (1-((cyanomethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate) exhibited potent inhibition of recombinant LmCPB2.8ΔCTE (IC50 = 4.3 ± 0.2 μM) and strong activity against intracellular amastigotes of Leishmania infantum (EC50 = 3.9 ± 0.5 μM; SI ≈ 77) and Leishmania amazonensis (EC50 = 4.6 ± 1.6 μM; SI ≈ 65), surpassing miltefosine in selectivity. Compound 1 showed no cytotoxicity toward murine macrophages, no hemolytic activity, and good tolerability in the Galleria mellonella model. Oral administration of compound 1 to L. infantum-infected BALB/c mice (0.77 mg/kg/day for 5 days) resulted in a significant reduction in parasite burden in the liver and spleen (≈ 40–55%), even at a 10-fold lower dose than miltefosine. Although alterations in hepatic transaminases were observed, no evidence of renal dysfunction was detected. Collectively, these findings identify dipeptidyl nitriles as promising CPB-targeting scaffolds and position compound 1 as a lead structure for further optimization aimed at enhancing antileishmanial efficacy while minimizing host toxicity.