Non-steroidal anti-inflammatory drugs as potent inhibitors of phospholipase A₂: structure of the complex of phospholipase A₂ with niflumic acid at 2.5 Å resolution

Phospholipase A₂ (PLA₂; EC 3.1.3.4) catalyzes the first step of the production of proinflammatory compounds collectively known as eicosanoids. The binding of phospholipid substrates to PLA₂ occurs through a well formed hydrophobic channel. Surface plasmon resonance studies have shown that niflumic acid binds to Naja naja sagittifera PLA₂ with an affinity that corresponds to a dissociation constant (K_d) of 4.3 × 10^-5 M. Binding studies of PLA₂ with niflumic acid were also carried out using a standard PLA₂ kit that gave an approximate binding constant, K_i, of 1.26 ± 0.05 × 10^-6 M. Therefore, in order to establish the viability of PLA₂ as a potential target molecule for drug design against inflammation, arthritis and rheumatism, the three-dimensional structure of the complex of PLA₂ with the known anti-inflammatory agent niflumic acid {2-[3-(trifluoromethyl)anilino]nicotinic acid} has been determined at 2.5 Å resolution. The structure of the complex has been refined to an R factor of 0.187. The structure determination reveals the presence of one niflumic acid molecule at the substrate-binding site of PLA₂. It shows that niflumic acid interacts with the important active-site residues His48 and Asp49 through two water molecules. It is observed that the niflumic acid molecule is completely buried in the substrate-binding hydrophobic channel. The conformations of the binding site in PLA₂ as well as that of niflumic acid are not altered upon binding. However, the orientation of the side chain of Trp19, which is located at the entry of the substrate-binding site, has changed from that found in the native PLA₂, indicating its familiar role.