The biflavonoid morelloflavone inhibits the enzymatic and biological activities of a snake venom phospholipase A2

The biflavonoid morelloflavone has been reported as inhibitor of secretory PLA(2)s (phospholipases A(2) from human synovial and bee venom sources); however, its capacity to interact and inhibit snake venom PLA(2) activities has not been described. In this work we tested the inhibitory ability of morelloflavone on the enzymatic, anticoagulant, myotoxic and edema-inducing activities of a PLA(2) isolated from Crotalus durissus cumanensis venom. The biflavonoid displayed IC50 values of 0.48 mM (95% Confidence intervals: 0.45-0.51) and 0.38 mM (95% Confidence intervals: 0.36-0.40) on the PLA(2) enzymatic activity, when either aggregated or monodispersed substrates were used, respectively. In addition, morelloflavone inhibited in a time-dependent manner and irreversibly the PLA(2) enzymatic activity. When mice were injected with PLA(2) preincubated (preincubation assay) with 0.13, 0.63 and 1.26 mM of the biflavonoid, the myotoxic activity induced by the PLA(2) was inhibited up to 63%. Nevertheless, these values decreased up to 38% when the morelloflavone was injected into muscle after PLA(2). Moreover, morelloflavone inhibited, in a concentration-dependent manner, edema-forming activity of the PLA(2) in the footpad. Morelloflavone also inhibited the anticoagulant activities of the PLA(2) in concentration-dependent mode. In order to have insights on the mode of action of morelloflavone, intrinsic fluorescence studies were performed. Results of these assays suggest that morelloflavone interacts directly with the PLA(2). These findings were supported by molecular docking results, which suggested that morelloflavone forms hydrogen bonds with residues Gly33, Asp49, Gly53 and Thr68 of the enzyme. In addition, our results suggested a pi-pi stacking interaction between rings A of morelloflavone with that of the residue Tyr52, and Van der Waals interactions with Gly32, His48 and Ala56. Our molecular modeling results suggest that morelloflavone may occupy part of substrate binding cleft of the PLA(2). Morelloflavone is a candidate for the development of inhibitors to be used in snakebite envenomation. (C) 2014 Elsevier Ireland Ltd. All rights reserved.