title: The biflavonoid morelloflavone inhibits the enzymatic and biological activities of a snake venom phospholipase A2.
abstract: The biflavonoid morelloflavone has been reported as inhibitor of secretory PLA2s (phospholipases A2 from human synovial and bee venom sources); however, its capacity to interact and inhibit snake venom PLA2 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 PLA2 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 PLA2 enzymatic activity, when either aggregated or monodispersed substrates were used, respectively. In addition, morelloflavone inhibited in a time-dependent manner and irreversibly the PLA2 enzymatic activity. When mice were injected with PLA2 preincubated (preincubation assay) with 0.13, 0.63 and 1.26 mM of the biflavonoid, the myotoxic activity induced by the PLA2 was inhibited up to 63%. Nevertheless, these values decreased up to 38% when the morelloflavone was injected into muscle after PLA2. Moreover, morelloflavone inhibited, in a concentration-dependent manner, edema-forming activity of the PLA2 in the footpad. Morelloflavone also inhibited the anticoagulant activities of the PLA2 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 PLA2. 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 π-π 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 PLA2. Morelloflavone is a candidate for the development of inhibitors to be used in snakebite envenomation.
authors.affiliation: Programa de Ofidismo/Escorpionismo, Facultad de Química Farmacéutica, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia. Electronic address: andrespj20@yahoo.es.
authors.forename: Jaime Andrés
authors.initials: JA
authors.lastname: Pereañez
authors.affiliation: Programa de Ofidismo/Escorpionismo, Facultad de Química Farmacéutica, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia.
authors.forename: Arley Camilo
authors.initials: AC
authors.lastname: Patiño
authors.affiliation: Programa de Ofidismo/Escorpionismo, Facultad de Química Farmacéutica, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia; Escuela de Microbiología Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia.
authors.forename: Vitelbina
authors.initials: V
authors.lastname: Núñez
authors.affiliation: Grupo de Investigación en Sustancias Bioactivas, Facultad de Química Farmacéutica, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia.
authors.forename: Edison
authors.initials: E
authors.lastname: Osorio
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000494:pharmacology; D044946:Biflavonoids / Q000737:chemistry / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D020128:Inhibitory Concentration 50; D062105:Molecular Docking Simulation; D015394:Molecular Structure; D064801:Phospholipase A2 Inhibitors / Q000494:pharmacology; D054467:Phospholipases A2 / Q000378:metabolism*; D010944:Plants / Q000737:chemistry; D012910:Snake Venoms / Q000201:enzymology*; D013997:Time Factors
chemical_list: D000925:Anticoagulants; D044946:Biflavonoids; D064801:Phospholipase A2 Inhibitors; D012910:Snake Venoms; C105620:morelloflavone; D054467:Phospholipases A2
keywords: Docking; Local tissue damage; Morelloflavone; Phospholipase A(2); Snakebite