Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A2 from Bothrops pirajai venom

Abstract

BackgroundHarpalycin 2 (HP-2) is an isoflavone isolated from the leaves of Harpalyce brasiliana Benth., a snakeroot found in northeast region of Brazil and used in folk medicine to treat snakebite. Its leaves are said to be anti-inflammatory. Secretory phospholipases A2 are important toxins found in snake venom and are structurally related to those found in inflammatory conditions in mammals, as in arthritis and atherosclerosis, and for this reason can be valuable tools for searching new anti-phospholipase A2 drugs.MethodsHP-2 and piratoxin-III (PrTX-III) were purified through chromatographic techniques. The effect of HP-2 in the enzymatic activity of PrTX-III was carried out using 4-nitro-3-octanoyloxy-benzoic acid as the substrate. PrTX-III induced platelet aggregation was inhibited by HP-2 when compared to aristolochic acid and p-bromophenacyl bromide (p-BPB). In an attempt to elucidate how HP-2 interacts with PrTX-III, mass spectrometry, circular dichroism and intrinsic fluorescence analysis were performed. Docking scores of the ligands (HP-2, aristolochic acid and p-BPB) using PrTX-III as target were also calculated.ResultsHP-2 inhibited the enzymatic activity of PrTX-III (IC50 11.34 ± 0.28 μg/mL) although it did not form a stable chemical complex in the active site, since mass spectrometry measurements showed no difference between native (13,837.34 Da) and HP-2 treated PrTX-III (13,856.12 Da). A structural analysis of PrTX-III after treatment with HP-2 showed a decrease in dimerization and a slight protein unfolding. In the platelet aggregation assay, HP-2 previously incubated with PrTX-III inhibited the aggregation when compared with untreated protein. PrTX-III chemical treated with aristolochic acid and p-BPB, two standard PLA2 inhibitors, showed low inhibitory effects when compared with the HP-2 treatment. Docking scores corroborated these results, showing higher affinity of HP-2 for the PrTX-III target (PDB code: 1GMZ) than aristolochic acid and p-BPB. HP-2 previous incubated with the platelets inhibits the aggregation induced by untreated PrTX-III as well as arachidonic acid.ConclusionHP-2 changes the structure of PrTX-III, inhibiting the enzymatic activity of this enzyme. In addition, PrTX-III platelet aggregant activity was inhibited by treatment with HP-2, p-BPB and aristolochic acid, and these results were corroborated by docking scores.

Effect of Chlorogenic Acid (5-Caffeoylquinic Acid) Isolated fromBaccharis oxyodontaon the Structure and Pharmacological Activities of Secretory Phospholipase A2 fromCrotalus durissus terrificus

“…As flavonoids can inhibit sPLA2 and consequently decrease its proinflammatory activity [ 26 , 28 , 29 ], the effect of 5CQA on sPLA2 was evaluated. Following subplantar injections of Swiss mice, sPLA2 had substantial potential to induce oedema after 60 min ( Figure 7(a) ).…”

“…A paw oedema assay was performed using a previously described protocol [ 8 ]. Male Swiss mice (21 g) were anaesthetised by inhaling halothane.…”

Edema Induced by sPLA2 from Crotalus durissus terrificus Involves PLC and PKC Signaling, Activation of cPLA2, and Oxidative Stress

“…On the other hand, several articles have reported that natural antioxidant compounds, such as flavonoids and related substances, when given prior to sPLA2 injection, have significant anti-inflammatory activities. This probably stems from the ability of many of these compounds to partially inhibit the enzymatic and pharmacological activities of sPLA2 from Crotalus durissus terrificus, as well as from their strong antioxidant activities [53][54][55][56]97]. Thus, the search for new natural compounds with anti-inflammatory properties remains an important area of research.…”

“…Snake venom sPLA2 shares similar mechanisms of action and the same pathways of action with mammalian sPLA2. Experimental evidence in the literature demonstrates that both sPLA2 isoforms are able to induce inflammation and other similar biological activities [10,[17][18][19]. It is noteworthy that literature data demonstrate sPLA2 can activate signaling events that cannot be explained simply by its catalytic activity, and this fact emphasizes that sPLA2 could act essentially as a ligand of a receptor, rather than as an enzyme [20].…”

Perspective on the Therapeutics of Anti-Snake Venom

“…Edunol from Brongniartia podalyrioides (Leguminosae) reversed the toxic effects of B. atrox envenomation in rodent models [45]. Harpalycin 2 (HP-2) isolated from Harpalyce brasiliana (Papilionoideae) inhibited the enzymatic acitivity of piratoxin-III isolated from the venom of Bothrops pirajai [108].…”