The Binding Mode of Cladocoran A to the Human Group IIA Phospholipase A₂

Abstract

The molecular basis for human group IIA phospholipase A(2) inactivation by the marine natural product cladocoran A (CLD A) has been studied in order to elucidate its relevant anti-inflammatory properties. Indeed, secretory phospholipases A(2) are well-known to be implicated in the pathogenesis of inflammation, such as rheumatoid arthritis, septic shock, psoriasis and asthma, thus the understanding of their inactivation mechanism could be useful for the development of new chemical classes of selective inhibitors. Our results, collected by a combination of biochemical approaches, advanced mass spectrometry and molecular modeling, suggest a competitive inhibition mechanism guided by a noncovalent molecular recognition event, and disclose the key role of the CLD A γ-hydroxybutenolide ring in the chelation of the catalytic calcium ion inside the enzyme active site. Moreover, CLD A is able to react selectively with Ser82, although this covalent event seems to play a secondary role in terms of enzyme inhibition.

The Inactivation Mechanism of Human Group IIA Phospholipase A2 by Scalaradial

“…As in our previous studies, in which both bee venom and human synovial PLA 2 were shown to be affected by covalent and noncovalent interactions with bioactive natural inhibitors, [20][21][22][23][24] we applied a mass spectrometric approach in the analysis of the sPLA 2 -IIA-SLD adduct. First, we used of p-nitrophenacyl bromide (pNBr, M W 244 Da), a well-known specific ligand of the His47 of several PLA 2 s, the residue acting as Brønsted base in the catalytic process.…”

“…First, we used of p-nitrophenacyl bromide (pNBr, M W 244 Da), a well-known specific ligand of the His47 of several PLA 2 s, the residue acting as Brønsted base in the catalytic process. [20][21][22][23][24] Comparative pNBr modification experiments in the presence and absence of a given inhibitor provide valuable evidence on the type of inhibition mechanism (whether competitive or not). Therefore, we incubated sPLA 2 -IIA with pNBr, and the mixture was monitored by LC-ESI-MS.…”

“…By means of molecular docking, [33] we obtained a putative three-dimensional model of the interaction between SLD and sPLA 2 -IIA (PDB ID: 1POE). [34] For the docking calculations, we used our optimized model [21,24] of sPLA 2 -IIA, in which the active site of the macromolecule had been refined at the quantum mechanical (QM) level. In particular, the charge of the amino acids of the catalytic site (His27, Gly29, Gly30, Asp48, and the calcium ion) were calculated at density functional theory (DFT) B3LYP level by using the 6-31+G(d) basis set and ChelpG method for population analysis (see Computational Methods).…”

“…[ [17][18][19][20][21][22][23][24] Scalaradial (SLD), a marine tetracyclic sesterterpenoid with a scalarane skeleton, was isolated from the sponge Cacospongia mollior by Cimino and co-workers, [25] together with its congener 12-episcalaradial (eSLD). eSLD is a selective inhibitor of Secretory phospholipases A 2 (sPLA 2 s) are implicated in the pathogenesis of several inflammation diseases, such as rheumatoid arthritis, septic shock, psoriasis, and asthma.…”

“…The crystal structure of inhibited sPLA 2 -IIA from inflammatory exudates solved by Sigler and co-workers (PDB ID: 1POE) [34] was used as molecular target for docking calculations. As reported, [21,24] the charges of the sPLA 2 -IIA chain A (Ca 2 + ion and Gly29, Gly31, His27, and Asp48) in the catalytic center were calculated at the DFT B3LYP level by using the 6-31+G(d) basis set and ChelpG method for population analysis. [41] For the docking studies, we used a grid box (126 126 126) centered at 23.912 (x), 1.212 (y), 67.788 (z), with 0.375 spacing between the grid points, covering the active site of sPLA 2 -IIA.…”