Structural and Functional Studies of a Bothropic Myotoxin Complexed to Rosmarinic Acid: New Insights into Lys49-PLA2 Inhibition

Abstract

Snakebite envenoming is an important public health problem in many tropical and subtropical countries, and is considered a neglected tropical disease by the World Health Organization. Most severe cases are inflicted by species of the families Elapidae and Viperidae, and lead to a number of systemic and local effects in the victim. One of the main problems regarding viperidic accidents is prominent local tissue damage whose pathogenesis is complex and involves the combined actions of a variety of venom components. Phospholipases A2 (PLA2s) are the most abundant muscle-damaging components of these venoms. Herein, we report functional and structural studies of PrTX-I, a Lys49-PLA2 from Bothops pirajai snake venom, and the influence of rosmarinic acid (RA) upon this toxin's activities. RA is a known active component of some plant extracts and has been reported as presenting anti-myotoxic properties related to bothopic envenomation. The myotoxic activity of Lys49-PLA2s is well established in the literature and although no in vivo neurotoxicity has been observed among these toxins, in vitro neuromuscular blockade has been reported for some of these proteins. Our in vitro studies show that RA drastically reduces both the muscle damage and the neuromuscular blockade exerted by PrTX-I on mice neuromuscular preparations (by ∼80% and ∼90%, respectively). These results support the hypothesis that the two effects are closely related and lead us to suggest that they are consequences of the muscle membrane-destabilizing activity of the Lys49-PLA2. Although the C-terminal region of these proteins has been reported to comprise the myotoxic site, we demonstrate by X-ray crystallographic studies that RA interacts with PrTX-I in a different region. Consequently, a new mode of Lys49-PLA2 inhibition is proposed. Comparison of our results with others in the literature suggests possible new ways to inhibit bothropic snake venom myotoxins and improve serum therapy.

Snake Venom PLA2s Inhibitors Isolated from Brazilian Plants: Synthetic and Natural Molecules

“…This compound demonstrates antimyotoxic properties and inhibits edema induced by crude B. jararacussu snake venom and its basic PLA 2 s [36, 55]. The three-dimensional structure of the PrTX-I, rosmarinic acid complex, was elucidated by Santos and collaborators [68], where rosmarinic acid was observed located at the entrance of the hydrophobic channel monomer A of the PrTX-I dimer via an interaction between hydrogen bonds and hydrophobic contacts in the same monomer. Interactions were also observed between rosmarinic acid and a residue of the C-terminal region of the monomer B.…”

“…Interactions were also observed between rosmarinic acid and a residue of the C-terminal region of the monomer B. The interaction between the rosmarinic acid molecule with the hydrophobic channel (monomer A) and the C-terminal region (myotoxic site, monomer B) suggests two mechanisms of myotoxicity inhibition [68]. …”

Structural and functional studies with mytoxin II from Bothrops moojeni reveal remarkable similarities and differences compared to other catalytically inactive phospholipases A2-like

“…Molecular comparisons of the structures were performed using the Coot v.0.7 program (Emsley and Cowtan, 2004) with only C a coordinates. The structures of MjTX-II/ stearic acid (PDB ID 1XXS) (Watanabe et al, 2005), BaspTX-II (PDB ID 1CLP) in its native form (Arni et al, 1995) and complexed to suramin (PDB ID 1Y4L) (Murakami et al, 2005), BthTX-I (PDB ID 3HZD), BthTX-I/PEG4000 (PDB ID 3IQ3), BthTX-I/BPB (PDB ID 3HZW), PrTX-I/BPB (PDB ID 2OK9) (Fernandes et al, 2010), BthTX-I/atocopherol (PDB ID 3CXI), PrTX-I (PDB ID 2Q2J), PrTX-I/atocopherol (PDB ID 3CYL) (dos Santos et al, 2009), PrTX-I/Rosmarinic acid (PDB ID 3QNL) (dos Santos et al, 2011a), PrTX-II/fatty acid (PDB ID 1QLL) (Lee et al, 2001), BnSP-6 and BnSP-7 (PDB ID 1PC9 and 1PA0 respectively) (Magro et al, 2003) and BnIV/ Myristic acid (PDB ID 3MLM) (Delatorre et al, 2011) were used in the comparative analysis.…”

CHARACTERIZATION OF THE MOST PROMISING FRACTION OF Swietenia macrophylla ACTIVE AGAINST MYOTOXIC PHOSPHOLIPASES A2 : IDENTIFICATION OF CATECHIN AS ONE OF THE ACTIVE COMPOUNDS

“…The polyphenol rosmarinic acid isolated from diverse species of the Boraginaceae and Lamiaceae, reduced 80% and 90 % the muscle damage and the neuromuscular blockade induced by PrTX-I (a PLA 2 Lys 49 from the venom of Bothrops pirajai) on mice neuromuscular (phrenic-diaphragm) preparations. X-ray crystallographic studies between two molecules demonstrated that rosmarinic acid obstruct the entrance of the hydrophobic channel in PrTX-I affecting the interaction with membrane [37].…”