Molecular basis of phospholipase A2 inhibition by petrosaspongiolide M.

Petrosaspongiolide M (PM) is an anti-inflammatory marine metabolite that displays a potent inhibitory activity toward group II and III secretory phospholipase A2 (PLA2) enzymes. The details of the mechanism, which leads to a covalent adduct between PLA2 and γ-hydroxybutenolide-containing molecules such as PM, are still a matter of debate. In this paper the covalent binding of PM to bee venom PLA2 has been investigated by mass spectrometry and molecular modeling. The mass increment observed for the PM-PLA2 adduct is consistent with the formation of a Schiff base by reaction of a PLA2 amino group with the hemiacetal function (masked aldehyde) at the C-25 atom of the PM γ-hydroxybutenolide ring. Proteolysis of the modified PLA2 by the endoprotease LysC followed by HPLC MS analysis allowed us to establish that the PLA2 α-amino terminal group of the lle-1 residue was the only covalent binding site for PM. The stoichiometry of the reaction between PM and PLA2 was also monitored and results showed that even with excess inhibitor, the prevalent product is a 1:1 (inhibitor:enzyme) adduct, although a 2:1 adduct is present as a minor component. The 2:1 adduct was also characterized, which showed that the second site of reaction is located at the ε-amino group of the Lys-85 residue. Similar results in terms of the reaction profile, mass increments, and location of the PLA2 binding site were obtained for manoalide, a paradigm for irreversible PLA2 inhibitors, which suggests that the present results may be considered of more general interest within the field of anti-inflammatory sesterterpenes that contain the γ-hydroxybutenolide pharmacophore. Finally, a 3D model, constrained by the above experimental results, was obtained by docking the inhibitor molecule into the PLA2 binding site through AFFINITY calculations. The model provides an interesting insight into the PM-PLA2 inhibition process and may prove useful in the design of new anti-inflammatory agents that target PLA2 secretory enzymes