Natural products as inhibitors of purine nucleoside phosphorylase, a target against Plasmodium spp

Malaria is a parasitic disease killing over one million people every year, mainly in poor regions of the globe where access to diagnostics and conventional therapy is restricted. The use of traditional medicines with proved clinical efficacy has recently been considered a strategy in the early treatment of uncomplicated falciparum malaria. Such a strategy encourages the clinical assessment of traditional plant preparations based on their ethnopharmacology and in vitro results. Indeed, a large number of plant extracts and isolated natural products have shown in vitro and in vivo antimalarial activity so far. However, their mechanisms of action have rarely been identified. This work presents the validation of an assay measuring the inhibition of purine nucleoside phosphorylase (PNP), a target against Plasmodium falciparum, for the screening of natural products. Enzyme kinetic parameters were determined in a 96-well plate spectrophotometric assay. Cladribine, a known PNP inhibitor, was selected as a positive control for its IC₅₀ (154µM), K_i (76µM) and mode of inhibition (non-competitive mixed). The validated assay was then applied to a library of ca. 40 natural products of various classes, known or predicted to present an in vitro antiplasmodial activity. Two compounds, plumbagin and rhein, inhibited PNP with IC₅₀ values of 114 and 158µM, respectively, comparable to cladribine. Previous studies showed the in vitro activity of plumbagin against P. falciparum with an IC₅₀ of 0.4µM and with no effect on the inhibition of β-hematin. Our results suggest a novel mechanism of action for this compound, and show the application of the PNP screening in searching for natural antimalarials and in elucidating mechanisms of action.