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Molecular Basis of Phospholipase A 2 Inhibition by Petrosaspongiolide M
Author(s) -
Dal Piaz Fabrizio,
Casapullo Agostino,
Randazzo Antonio,
Riccio Raffaele,
Pucci Piero,
Marino Gennaro,
GomezPaloma Luigi
Publication year - 2002
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/1439-7633(20020703)3:7<664::aid-cbic664>3.0.co;2-9
Subject(s) - adduct , chemistry , stereochemistry , residue (chemistry) , covalent bond , active site , aldehyde , phospholipase a2 , enzyme , schiff base , biochemistry , organic chemistry , catalysis
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 γ ‐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 γ ‐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 α ‐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 ε ‐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 γ ‐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.