title: Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A2 from Bothrops pirajai venom.
abstract: BACKGROUND
  Harpalycin 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.
  
  
  METHODS
  HP-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.
  
  
  RESULTS
  HP-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.
  
  
  CONCLUSIONS
  HP-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.
authors.affiliation: Department of Physiology and Pharmacology, Federal University of Ceará, UFC, Rua Coronel Nunes de Melo 1127, Fortaleza, CE 60430-270, Brazil.
authors.forename: Rafael M
authors.initials: RM
authors.lastname: Ximenes
authors.forename: Renata S
authors.initials: RS
authors.lastname: Alves
authors.forename: Ticiana P
authors.initials: TP
authors.lastname: Pereira
authors.forename: Renata M
authors.initials: RM
authors.lastname: Araújo
authors.forename: Edilberto R
authors.initials: ER
authors.lastname: Silveira
authors.forename: Marcelo M
authors.initials: MM
authors.lastname: Rabello
authors.forename: Marcelo Z
authors.initials: MZ
authors.lastname: Hernandes
authors.forename: Veronica C G
authors.initials: VC
authors.lastname: Soares
authors.forename: Daniel
authors.initials: D
authors.lastname: Bristot
authors.forename: Camila L
authors.initials: CL
authors.lastname: Pires
authors.forename: Daniela O
authors.initials: DO
authors.lastname: Toyama
authors.forename: Henrique H
authors.initials: HH
authors.lastname: Gaeta
authors.forename: Helena S A
authors.initials: HS
authors.lastname: Monteiro
authors.forename: Marcos H
authors.initials: MH
authors.lastname: Toyama
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D034341:Aristolochic Acids / Q000494:pharmacology; D052117:Benzodioxoles / Q000302:isolation & purification / Q000494:pharmacology* / Q000627:therapeutic use; D017837:Bothrops; D001938:Brazil; D003435:Crotalid Venoms / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology* / Q000627:therapeutic use; D007887:Fabaceae / Q000737:chemistry*; D054501:Group II Phospholipases A2 / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006801:Humans; D007529:Isoflavones / Q000302:isolation & purification / Q000494:pharmacology* / Q000627:therapeutic use; D009579:Nitrobenzoates / Q000378:metabolism; D008517:Phytotherapy; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use; D018515:Plant Leaves; D010974:Platelet Aggregation / Q000187:drug effects*; D030162:Reptilian Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D012909:Snake Bites / Q000188:drug therapy / Q000201:enzymology
chemical_list: C104459:4-nitro-3-(octanoyloxy)benzoic acid; D000098:Acetophenones; D034341:Aristolochic Acids; D052117:Benzodioxoles; D003435:Crotalid Venoms; D004791:Enzyme Inhibitors; D007529:Isoflavones; D009579:Nitrobenzoates; D010936:Plant Extracts; D030162:Reptilian Proteins; C581153:harpalycin 2; C000228:aristolochic acid I; D054501:Group II Phospholipases A2; C120516:piratoxin III; C006821:4-bromophenacyl bromide