The inhibition of phospholipase A 2 by manoalide and manoalide analogues

• Published in: Biochimica et biophysica acta. Lipids and lipid metabolism Vol. 917; no. 2; pp. 258 - 268
• Main Authors: Deems, Raymond A., Lombardo, Dominique, Morgan, Bradley P., Mihelich, Edward D., Dennis, Edward A.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 1987
• Subjects: Enzyme inhibitor; Manoalide; Phospholipase A 2; γ-Lactone; diacylPC, 1,2-diacyl- sn-glycero-3-phosphorylcholine; HDHB, 3( cis,cis-7,10)-hexadecadienyl-4-hydroxy-2-butenolide; Manoalide; γ-Lactone; Phospholipase A 2; DNPH, dinitrophenylhydrazine; cis-HDHB, the open form of HDHB with a cis conformation of the 2 double bond, is ( Z, Z, Z)-3-formyl-2,10,13-nonadecatrienoic acid; Enzyme inhibitor; lysoPC, 1-palmitoyl- sn-glycero-3-phosphorylcholine; trans-HDHB, the open form of HDHB with a trans conformation of the 2 double bond, is ( E, Z, Z)-3-formyl-2,10,13-nonadecatrienoic acid thioPC, 1,2-bis(decanoylthio)-1,2-dideoxy- sn-glycerol-3-phosphorylcholine
• ISSN: 0005-2760; 1879-145X
• DOI: 10.1016/0005-2760(87)90130-5

Manoalide, a natural product from sponge, displays anti-inflammatory activity in vivo. Previous work has shown that manoalide is also a potent covalent inhibitor of the extracellular phospholipase A 2 from cobra venom and that the inhibition correlated with a pH-dependent change in manoalide (Lombardo and Dennis (1985) J. Biol. Chem. 260, 7234–7240). Manoalide contains two rings and the opening of either would produce an α,β-unsaturated aldehyde. The cobra venom phospholipase A 2 may be able to catalyze the opening or isomerization of one of these rings, raising the possibility that manoalide is acting as a suicide substrate. To ascertain the role of the γ-lactone ring in the inhibition, we have now investigated a synthetic manoalide analogue, 3( cis,cis-7,10)-hexadecadienyl-4-hydroxy-2-butenolide (HDHB) which contains only the α,β-unsaturated γ-lactone ring. We have found that the closed and open forms are in rapid equilibrium between pH 4 and 9 with the cyclic form being preferred at acidic pH values and the open cis form preferred at pH 9.5. When the pH is raised above 12, the α,β double bond isomerizes to form trans-HDHB. Once the trans compound is formed, it is stable at all pH values and does not recyclize to the γ-lactone ring. The observed pK a of 7.7 found for the inhibition of manoalide agrees well with the transition of the closed to the cis form of the γ-lactone ring. Kinetic experiments with the HDHB compound show that under conditions in which the cis and closed form of the inhibitor are present in equal molar ratios, HDHB is not an irreversible inhibitor, but reversibly competes with substrate. However, the kinetics of this inhibition are complex and do not follow either pure competitive or non-competitive inhibition. The trans-HDHB exhibits similar complex kinetics but is several times more potent. The distinct differences between the behavior of manoalide and HDHB clearly indicate that while the γ-lactone ring may play an important role in manoalide inhibition, it alone does not produce irreversible inhibition.