The molecular mechanism of human group IIA phospholipase A2 inactivation by bolinaquinone

Abstract

The molecular basis of the human group IIA secretory phospholipase A(2) inactivation by bolinaquinone (BLQ), a hydroxyquinone marine terpenoid, has been investigated for the comprehension of its relevant antiinflammatory properties, through the combination of spectroscopic techniques, biosensors analysis, mass spectrometry (MS) and molecular docking. Indeed, sPLA(2)s are well known to be implicated in the pathogenesis of inflammation such as rheumatoid arthritis, septic shock, psoriasis and asthma. Our results suggest a mechanism of competitive inhibition guided by a non-covalent molecular recognition event, disclosing the key role of the BLQ hydroxyl-quinone moiety in the chelation of the catalytic Ca(2+) ion inside the enzyme active site.The understanding of the sPLA(2)-IIA inactivation mechanism by BLQ could be useful for the development of a new chemical class of PLA(2) inhibitors, able to specifically target the enzyme active site.

The Application of Rational Design on Phospholipase A2 Inhibitors

“…Such data were in agreement with the biological assays, where BLQ inhibits GIIA sPLA 2 and bee venom GIII sPLA 2 with IC 50 values of 0.2 and 0.1 GM, respectively [46,72,77]. It was suggested that the comprehension of the mechanism of the enzyme inhibition would allow a rational design of simplified analogues of natural products as novel lead compounds with anti-inflammatory properties.…”

“…7) is a sesquiterpene metabolite isolated from the marine Dysidea sponge. This metabolite inhibits human GIIA sPLA 2 with IC 50 value of 0.245 DM [46]. Based on docking calculations, it was found that BLQ participates in hydrophobic contacts with the hydrophobic region of GIIA sPLA 2 active site formed by the residues Leu2, Phe5, His6, Ala17, Ala18, Tyr21, Gly22, Gly25, Cys28, Gly29, Cys44, Phe98.…”

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

“…As already carried out in our previous studies on sPLA 2 , [13][14][15][16][17][21][22][23] we started with the analysis of sPLA 2 potential covalent modification by CLD A through liquid chromatography-nano-ESI MS (LC-nano-ESI-MS). Time-course analysis of the CLD A and sPLA 2 -IIA reaction mixture was performed, and the chromatograms revealed the presence of the unmodified sPLA 2 -IIA (M W 13 860.05(AE0.45) Da) and of a species corresponding to a 1:1 sPLA 2 -IIA-CLD A covalent adduct with a mass increment of approximately 384 Da ( Figure 1 and Table 1).…”

“…As we wanted to determine the role of the noncovalent interactions in sPLA 2 -IIA inactivation by CLD A, we resorted to the use of p-nitrophenacyl bromide (pNBr, M W = 244 Da), a well-known specific ligand of the catalytic His resi- [17,[22][23][24] Indeed, comparative pNBr modification experiments in the presence and absence of a given inhibitor provides valuable clues about the inhibition mechanism (if competitive or not). We incubated sPLA 2 -IIA in the presence of pNBr and detected by LC-MS a significant amount of sPLA 2 -IIA covalently modified by this agent (Figure 2, back).…”

“…[26] As reported in our previous work, [23] we used a target model refined at quantum mechanical level, in which the charge of the amino acids of the catalytic site (namely His27, Gly29, Gly30 and Asp48 and calcium ion) were calculated at density functional theory (DFT) B3LYP level (see the computational methods in the Experimental Section). On the other hand, we considered whether the CLD A g-hydroxybutenolide ring opened and deprotonated at physiological pH.…”