Temperature effect in the inhibition of PLA₂ activity of Bothrops brazili venom by Rosmarinic and Chlorogenic acids, experimental and computational approaches

Abstract

Myotoxicity caused by snakebite envenoming emerges as one of the main problems of ophidic accidents as it is not well neutralized by the current serum therapy. A promising alternative is to search for efficient small molecule inhibitors that can act against multiple venom components. Phospholipase A₂ (PLA₂) is frequently found in snake venom and is usually associated with myotoxicity. Thus it represents an excellent target for the search of new treatments. This work reports the effect of temperature in the inhibition of catalytic properties of PLA₂ from Bothrops brazili venom by Rosmarinic (RSM) and Chlorogenic (CHL) acids through experimental and computational approaches. Three temperatures were evaluated (25, 37 and 50 °C). In the experimental section, enzymatic assays showed that RSM is a better inhibitor in all three temperatures. At 50 °C, the inhibition efficiency decayed significantly for both acids. Docking studies revealed that both ligands bind to the hydrophobic channel of the protein dimer where the phospholipid binds in the catalytic process, interacting with several functional residues. In this context, RSM presents better interaction energies due to stronger interactions with chain B of the dimer. Molecular dynamics simulations showed that RSM can establish selective interactions with ARG112B of PLA₂, which is located next to residues of the putative Membrane Disruption Site in PLA2-like structures. The affinity of RSM and CHL acids towards PLA₂ is mainly driven by electrostatic interactions, especially salt bridge interactions established with residues ARG33B (for CHL) and ARG112B (RSM) and hydrogen bonds with residue ASP89A. The inability of CHL to establish a stable interaction with ARG112B was identified as the reason for its lower inhibition efficiency compared to RSM at the three temperatures. Furthermore, extensive structural analysis was performed to explain the lower inhibition efficiency at 50 °C for both ligands. The analysis performed in this work provides important information for the future design of new inhibitors.

Communicated by Ramaswamy H. Sarma

Keywords:

• Molecular dynamics
• phospholipase A2
• chlorogenic acid
• rosmarinic acid
• enzymatic inhibition

Acknowledgments

We thank the High-Performance Computing Center at UFRN (NPAD/UFRN) for the computer cluster facility and Karla Patrícia de Oliveira Luna of the State University of Paraíba, Brazil for providing the Bothrops brazili Venom.

Authors contribution

DSV and MFF designed the research project. DPS and SSF performed all experimental assays. EASD performed the computational simulations. All authors contributed to the analysis of results and to the writing of the manuscript.

Data availability statement

The data that support the findings of this study are available from the corresponding author, DSV, upon reasonable request.

Disclosure statement

The authors declare no competing financial interests related to the research presented in this article.

Additional information

Funding

This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES)—Finance Code 001 and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Grant:473327/2013-9)