Antimyotoxic Activity of Synthetic Peptides Derived from Bothrops atrox Snake Gamma Phospholipase A2 Inhibitor Selected by Virtual Screening

Author(s): J.C. Sobrinho, A.F. Francisco, R. Simões-Silva, A.M. Kayano, J.J. Alfonso Ruiz Diaz, A.F. Gomez Garay, A. Arruda, A.S. Ferreira, A.P.A. Santos, M.B. Luiz, C.B.G. Teles, S.S. Pereira, F.B. Zanchi, L.A. Calderon, J.P. Zuliani, A.M. Soares*

Journal Name: Current Topics in Medicinal Chemistry

Volume 19 , Issue 22 , 2019

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Graphical Abstract:


Background: Several studies have aimed to identify molecules that inhibit the toxic actions of snake venom phospholipases A2 (PLA2s). Studies carried out with PLA2 inhibitors (PLIs) have been shown to be efficient in this assignment.

Objective: This work aimed to analyze the interaction of peptides derived from Bothrops atrox PLIγ (atPLIγ) with a PLA2 and to evaluate the ability of these peptides to reduce phospholipase and myotoxic activities.

Methods: Peptides were subjected to molecular docking with a homologous Lys49 PLA2 from B. atrox venom modeled by homology. Phospholipase activity neutralization assay was performed with BthTX-II and different ratios of the peptides. A catalytically active and an inactive PLA2 were purified from the B. atrox venom and used together in the in vitro myotoxic activity neutralization experiments with the peptides.

Results: The peptides interacted with amino acids near the PLA2 hydrophobic channel and the loop that would be bound to calcium in Asp49 PLA2. They were able to reduce phospholipase activity and peptides DFCHNV and ATHEE reached the highest reduction levels, being these two peptides the best that also interacted in the in silico experiments. The peptides reduced the myotubes cell damage with a highlight for the DFCHNV peptide, which reduced by about 65%. It has been suggested that myotoxic activity reduction is related to the sites occupied in the PLA2 structure, which could corroborate the results observed in molecular docking.

Conclusion: This study should contribute to the investigation of the potential of PLIs to inhibit the toxic effects of PLA2s.

Keywords: Snake envenomation, PLIγ inhibitor, Coupling phospholipase A2-inhibitor, Peptides derived, Antimyotoxic activity, Virtual screening.

Lomonte, B.; Gutiérrez, J.M. A new muscle damaging toxin, myotoxin II, from the venom of the snake Bothrops asper (terciopelo). Toxicon, 1989, 27(7), 725-733.
[] [PMID: 2781572]
Soares, A.M.; Giglio, J.R. Chemical modifications of phospholipases A2 from snake venoms: effects on catalytic and pharmacological properties. Toxicon, 2003, 42(8), 855-868.
[] [PMID: 15019487]
Soares, A.M.; Fontes, M.R.M.; Giglio, J.R. Phospholipase A2 myotoxins from Bothrops snake venoms: structure function relationship. Curr. Org. Chem., 2004, 8(17), 1677-1690.
Burke, J.E.; Dennis, E.A. Phospholipase A2 structure/function, mechanism, and signaling. J. Lipid Res., 2009, 50(Suppl.), S237-S242.
[] [PMID: 19011112]
Burke, J.E.; Dennis, E.A. Phospholipase A2 biochemistry. Cardiovasc. Drugs Ther., 2009, 23(1), 49-59.
[] [PMID: 18931897]
Six, D.A.; Dennis, E.A. The expanding superfamily of phospholipase A(2) enzymes: classification and characterization. Biochim. Biophys. Acta, 2000, 1488(1-2), 1-19.
[] [PMID: 11080672]
Lizano, S.; Domont, G.; Perales, J. Natural phospholipase A(2) myotoxin inhibitor proteins from snakes, mammals and plants. Toxicon, 2003, 42(8), 963-977.
[] [PMID: 15019494]
Campos, P.C.; de Melo, L.A.; Dias, G.L.F.; Fortes-Dias, C.L. Endogenous phospholipase A2 inhibitors in snakes: a brief overview. J. Venom. Anim. Toxins Incl. Trop. Dis., 2016, 22, 37.
[] [PMID: 28031735]
Dunn, R.D.; Broady, K.W. Snake inhibitors of phospholipase A(2) enzymes. Biochim. Biophys. Acta, 2001, 1533(1), 29-37.
[] [PMID: 11514233]
Soares, A.M.; Marcussi, S.; Stábeli, R.G.; França, S.C.; Giglio, J.R.; Ward, R.J.; Arantes, E.C. Structural and functional analysis of BmjMIP, a phospholipase A2 myotoxin inhibitor protein from Bothrops moojeni snake plasma. Biochem. Biophys. Res. Commun., 2003, 302(2), 193-200.
[] [PMID: 12604331]
Ohkura, N.; Okuhara, H.; Inoue, S.; Ikeda, K.; Hayashi, K. Purification and characterization of three distinct types of phospholipase A2 inhibitors from the blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus. Biochem. J., 1997, 325(Pt 2), 527-531.
[] [PMID: 9230137]
Picelli, C.G.; Borges, R.J.; Fernandes, C.A.H.; Matioli, F.M.; Fernandes, C.F.C.; Sobrinho, J.C.; Holanda, R.J.; Ozaki, L.S.; Kayano, A.M.; Calderon, L.A.; Fontes, M.R.M.; Stábeli, R.G.; Soares, A.M. Molecular cloning and structural modelling of gamma-phospholipase A2 inhibitors from Bothrops atrox and Micrurus lemniscatus snakes. Int. J. Biol. Macromol., 2017, 103, 525-532.
[] [PMID: 28527998]
Eswar, N.; Webb, B.; Marti-Renom, M.A.; Madhusudhan, M.S.; Eramian, D.; Shen, M.Y.; Pieper, U.; Sali, A. Comparative protein structure modeling using Modeller. Curr. Protoc. Bioinformatics, John Wiley and Sons, New York, 2006; pp 5, 6.
[PMID: 18428767]
Laskowski, R.a.; MacArthur, M.W.; Moss, D.S.; Thornton, J.M. PROCHECK: A program to check the stereochemical quality of protein structures. J. Appl. Cryst., 1993, 26, 283-291.
Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Greenblatt, D.M.; Meng, E.C.; Ferrin, T.E. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem., 2004, 25(13), 1605-1612.
[] [PMID: 15264254]
Morris, G.M.; Ruth, H.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; Olson, A.J. Software News and updates autodock4 and autodock tools4: automated docking with selective receptor flexibility. J. Comput. Chem., 2009, 30, 2785-2791.
[] [PMID: 19399780]
Goodford, P.J. A computational procedure for determining energetically favorable binding sites on biologically important macromolecules. J. Med. Chem., 1985, 28(7), 849-857.
[] [PMID: 3892003]
Holzer, M.; Mackessy, S.P. An aqueous endpoint assay of snake venom phospholipase A2. Toxicon, 1996, 34(10), 1149-1155.
[] [PMID: 8931255]
Homsi-Brandeburgo, M.I.; Queiroz, L.S.; Santo-Neto, H.; Rodrigues-Simioni, L.; Giglio, J.R. Fractionation of Bothrops jararacussu snake venom: partial chemical characterization and biological activity of bothropstoxin. Toxicon, 1988, 26(7), 615-627.
[] [PMID: 3176051]
Lomonte, B.; Angulo, Y.; Rufini, S.; Cho, W.; Giglio, J.R.; Ohno, M.; Daniele, J.J.; Geoghegan, P.; Gutiérrez, J.M. Comparative study of the cytolytic activity of myotoxic phospholipases A2 on mouse endothelial (tEnd) and skeletal muscle (C2C12) cells in vitro. Toxicon, 1999, 37(1), 145-158.
[] [PMID: 9920486]
Mora-Obando, D.; Fernández, J.; Montecucco, C.; Gutiérrez, J.M.; Lomonte, B. Synergism between basic Asp49 and Lys49 phospholipase A2 myotoxins of viperid snake venom in vitro and in vivo. PLoS One, 2014, 9(10)e109846
[] [PMID: 25290688]
Bastos, V.A.; Gomes-Neto, F.; Perales, J.; Neves-Ferreira, A.G.C.; Valente, R.H. Natural inhibitors of snake venom metalloendopeptidases: history and current challenges. Toxins (Basel), 2016, 8(9), 8.
[] [PMID: 27571103]
Borges, R.J.; Cardoso, F.F.; Fernandes, C.A.H.; Dreyer, T.R.; de Moraes, D.S.; Floriano, R.S.; Rodrigues-Simioni, L.; Fontes, M.R.M. Functional and structural studies of a Phospholipase A2-like protein complexed to zinc ions: Insights on its myotoxicity and inhibition mechanism. Biochim. Biophys. Acta, Gen. Subj., 2017, 1861(1 Pt A), 3199-3209.
[] [PMID: 27531710]
Dos Santos, J.I.; Marchi-Salvador, D.P.; Fernandes, C.A.H.; Silveira, L.B.; Soares, A.M.; Fontes, M.R.M. Preliminary X-ray crystallographic studies of a Lys49-phospholipase A2 homologue from Bothrops pirajai venom complexed with p-bromo-phenacyl bromide and α-tocopherol inhibitors. Protein Pept. Lett., 2007, 14(7), 698-701.
[] [PMID: 17897096]
Chandra, V.; Jasti, J.; Kaur, P.; Dey, S.; Perbandt, M.; Srinivasan, A.; Betzel, Ch.; Singh, T.P. Crystal structure of a complex formed between a snake venom phospholipase A(2) and a potent peptide inhibitor Phe-Leu-Ser-Tyr-Lys at 1.8 A resolution. J. Biol. Chem., 2002, 277(43), 41079-41085.
[] [PMID: 12186870]
Dos Santos, J.I.; Cardoso, F.F.; Soares, A.M.; dal Pai Silva, M.; Gallacci, M.; Fontes, M.R.M. Structural and functional studies of a bothropic myotoxin complexed to rosmarinic acid: new insights into Lys49-PLA2 inhibition. PLoS One, 2011, 6, 1-10.
Nobuhisa, I.; Inamasu, S.; Nakai, M.I. A.T.; Mimori, T.; Ogawa, T.; Shimohigashi, Ya.; Fukumaki, Y.; Hattori, S.; Kihara, H.; Ohno, M. Characterization and evolution of a gene encoding a Trimeresurus flavoviridis serum protein that inhibits basic phospholipase A, isozymes in the snake’s venom. Eur. J. Biochem., 1997, 845, 838-845.
[] [PMID: 9395334]
Soares, A.M.; Sestito, W.P.; Marcussi, S.; Stábeli, R.G.; Andrião-Escarso, S.H.; Cunha, O.A.B.; Vieira, C.A.; Giglio, J.R. Alkylation of myotoxic phospholipases A2 in Bothrops moojeni venom: a promising approach to an enhanced antivenom production. Int. J. Biochem. Cell Biol., 2004, 36(2), 258-270.
[] [PMID: 14643891]
Cintra-Francischinelli, M.; Pizzo, P.; Rodrigues-Simioni, L.; Ponce-Soto, L.A.; Rossetto, O.; Lomonte, B.; Gutiérrez, J.M.; Pozzan, T.; Montecucco, C. Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors. Cell. Mol. Life Sci., 2009, 66(10), 1718-1728.
[] [PMID: 19370311]
Fernandes, C.A.H.; Comparetti, E.J.; Borges, R.J.; Huancahuire-Vega, S.; Ponce-Soto, L.A.; Marangoni, S.; Soares, A.M.; Fontes, M.R.M. Structural bases for a complete myotoxic mechanism: crystal structures of two non-catalytic phospholipases A2-like from Bothrops brazili venom. Biochim. Biophys. Acta, 2013, 1834(12), 2772-2781.
[] [PMID: 24145104]
Ambrosio, A.L.B.; Nonato, M.C.; de Araújo, H.S.; Arni, R.; Ward, R.J.; Ownby, C.L.; de Souza, D.H.F.; Garratt, R.C. A molecular mechanism for Lys49-phospholipase A2 activity based on ligand-induced conformational change. J. Biol. Chem., 2005, 280(8), 7326-7335.
[] [PMID: 15596433]
Marcussi, S.; Sant’Ana, C.D.; Oliveira, C.Z.; Rueda, A.Q.; Menaldo, D.L.; Beleboni, R.O.; Stabeli, R.G.; Giglio, J.R.; Fontes, M.R.M.; Soares, A.M. Snake venom phospholipase A2 inhibitors: medicinal chemistry and therapeutic potential. Curr. Top. Med. Chem., 2007, 7(8), 743-756.
[] [PMID: 17456038]
Oliveira, C.Z.; Santos-Filho, N.A.; Menaldo, D.L.; Boldrini-Franca, J.; Giglio, J.R.; Calderon, L.A.; Stabeli, R.G.; Rodrigues, F.H.S.; Tasic, L.; da Silva, S.L.; Soares, A.M. Structural and functional characterization of a γ-type phospholipase A2 inhibitor from bothrops jararacussu snake plasma. Curr. Top. Med. Chem., 2011, 11(20), 2509-2519.
[] [PMID: 21682685]
Gimenes, S.N.C.; Ferreira, F.B.; Silveira, A.C.P.; Rodrigues, R.S.; Yoneyama, K.A.G.; Izabel Dos Santos, J.; Fontes, M.R.D.M.; de Campos Brites, V.L.; Santos, A.L.Q.; Borges, M.H.; Lopes, D.S.; Rodrigues, V.M. Isolation and biochemical characterization of a γ-type phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum. Toxicon, 2014, 81, 58-66.
[] [PMID: 24513130]
Oliveira, C.Z.; Menaldo, D.L.; Marcussi, S.; Santos-Filho, N.A.; Silveira, L.B.; Boldrini-França, J.; Rodrigues, V.M.; Soares, A.M. An α-type phospholipase A(2) inhibitor from Bothrops jararacussu snake plasma: structural and functional characterization. Biochimie, 2008, 90(10), 1506-1514.
[] [PMID: 18549822]
Xiong, S.; Luo, Y.; Zhong, L.; Xiao, H.; Pan, H.; Liao, K.; Yang, M.; Huang, C. Investigation of the inhibitory potential of phospholipase A2 inhibitor gamma from Sinonatrix annularis to snake envenomation. Toxicon, 2017, 137, 83-91.
[] [PMID: 28746861]

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Year: 2019
Published on: 23 October, 2019
Page: [1952 - 1961]
Pages: 10
DOI: 10.2174/1568026619666190725102812
Price: $65

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