The hepatitis C virus (HCV) NS5B is essential for viral RNA replication and is therefore a prime target for development of HCV replication inhibitors. Here, we report the identification of a new class of HCV NS5B inhibitors belonging to the coumestan family of phytoestrogens. Based on the in vitro NS5B RNA-dependent RNA polymerase (RdRp) inhibition in the low micromolar range by wedelolactone, a naturally occurring coumestan, we evaluated the anti-NS5B activity of four synthetic coumestan analogues bearing different patterns of substitutions in their A and D rings, and observed a good structure-activity correlation. Kinetic characterization of coumestans revealed a noncompetitive mode of inhibition with respect to nucleoside triphosphate (rNTP) substrate and a mixed mode of inhibition towards the nucleic acid template, with a major competitive component. The modified order of addition experiments with coumestans and nucleic acid substrates affected the potencies of the coumestan inhibitors. Coumestan interference at the step of NS5B–RNA binary complex formation was confirmed by cross-linking experiments. Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC ₅₀ values. Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors.

The pterocarpanquinone LQB-118, previously demonstrated to be effective in vivo via oral delivery, was investigated for its mechanism in selective parasite killing. Oxidative stress in Leishmania amazonensis was analysed by evaluating reactive oxygen species (ROS) production (2′,7′-dichlorodihydrofluorescein diacetate) and the loss of mitochondrial membrane potential (ΔΨ_m) using rhodamine, JC-1 and MitoCapture. Ultrastructural analysis was performed using transmission electron microscopy (TEM). DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). Treatment with LQB-118 induced ROS production in the promastigotes of L. amazonensis in a concentration-dependent manner for the first 4 h and was sustained for 24 h. TEM analysis revealed several alterations typical of apoptosis. Promastigotes presented a reduction of ΔΨ_m after 24 h of incubation with 2.5 μM (18.7%), 5 μM (63.7%) or 10 μM (70.7%) LQB-118. A sub-G₀/G₁ cell cycle phenotype was observed in 21%–83% of the promastigotes incubated with 1.25–10 μM LQB-118. Concentration-dependent DNA fragmentation was observed in promastigotes treated with 2.5–10 μM LQB-118, and selective DNA fragmentation was observed in intracellular amastigotes after 72 h with 2.5 μM treatment. Our results suggest that LQB-118 selectively induces ROS-triggered and mitochondria-dependent apoptosis in this parasite.