Cantharidin impedes the activity of protein serine/threonine phosphatase in Plutella xylostella

Abstract

Cantharidin, a natural toxin produced by the blister beetle, was reported to be toxic to some pests, but the mechanism of its toxicity in insects remains undefined. We found that cantharidin exerted in vivo and in vitro inhibitory effects on protein serine/threonine phosphatases (PSPs) of Plutella xylostella. Five PSP genes, PP1, PP2A, PP4, PP5, and PP6, were cloned from P. xylostella. Phosphatase domain alignment showed a high similarity. Recombinant PxPP5 (rPxPP5) was expressed in Escherichia coli and purified. Cantharidin and its 11 analogs were used to perform the rPxPP5 activity inhibition assay in vitro. Cantharidin strongly inhibited rPxPP5 activity competitively, with an IC₅₀ of 0.38 μM. All analogs also showed inhibitory activity, with an IC₅₀ of 7.42–538.38 μM. The rank of IC₅₀ values was found to be consistent with their toxicities in P. xylostella larvae with a correlation coefficient (R²) of 0.87. 3D models of the phosphatase domains of these five PxPSPs were constructed which superimposed well indicating a high structural similarity demonstrating that the chemicals used may be inhibitors of the other four PxPSPs. Binding model analysis of cantharidin and its analogs which interacted with PxPP5 showed that the cantharidin-derived moiety was anchored to the active site, explaining their inhibitory effect on rPxPP5 in vitro. Results of binding free energy calculations are also well in line with their inhibition effects on rPxPP5, with a correlation coefficient (R²) of 0.72. In light of the above results we argue that protein serine/threonine phosphatases are the targets of cantharidin and its analogs acting on P. xylostella.