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 A(2) (PLA(2)) enzymes. The details of the mechanism, which leads to a covalent adduct between PLA(2) and gamma-hydroxybutenolide-containing molecules such as PM, are still a matter of debate. In this paper the covalent binding of PM to bee venom PLA(2) has been investigated by mass spectrometry and molecular modeling. The mass increment observed for the PM-PLA(2) adduct is consistent with the formation of a Schiff base by reaction of a PLA(2) amino group with the hemiacetal function (masked aldehyde) at the C-25 atom of the PM gamma-hydroxybutenolide ring. Proteolysis of the modified PLA(2) by the endoprotease LysC followed by HPLC MS analysis allowed us to establish that the PLA(2) alpha-amino terminal group of the Ile-1 residue was the only covalent binding site for PM. The stoichiometry of the reaction between PM and PLA(2) 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 epsilon -amino group of the Lys-85 residue. Similar results in terms of the reaction profile, mass increments, and location of the PLA(2) binding site were obtained for manoalide, a paradigm for irreversible PLA(2) inhibitors, which suggests that the present results may be considered of more general interest within the field of anti-inflammatory sesterterpenes that contain the gamma-hydroxybutenolide pharmacophore. Finally, a 3D model, constrained by the above experimental results, was obtained by docking the inhibitor molecule into the PLA(2) binding site through AFFINITY calculations. The model provides an interesting insight into the PM-PLA(2) inhibition process and may prove useful in the design of new anti-inflammatory agents that target PLA(2) secretory enzymes.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D001514:Bee Venoms / Q000201:enzymology*; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000737:chemistry; D054467:Phospholipases A2; D013729:Terpenes / Q000737:chemistry / Q000494:pharmacology
D000596:Amino Acids; D000894:Anti-Inflammatory Agents, Non-Steroidal; D001514:Bee Venoms; D004791:Enzyme Inhibitors; D013729:Terpenes; C118309:petrosaspongiolide m; D009828:Oleanolic Acid; C045673:manoalide; D010741:Phospholipases A; D054467:Phospholipases A2