- Email: [email protected]
46. Biological Features of an Enantiomeric Antimicrobial Peptide from Scorpion Venom
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Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
46. Biological Features of an Enantiomeric Antimicrobial Peptide from Scorpion Venom Daniel Juarez Lopez 1, Gerardo Corzo 2, Alexis Rodriguez 2, Elba Villegas 1 1
Centro de Investigacion en Biotecnologia, Laboratorio de Estructura, Funcion, e Ingenieria de Proteinas, UAEM, Cuernavaca, Morelos, Mexico 2 Instituto de Biotecnologia, Departamento de Medicina Molecular y Bioprocesos, UNAM, Cuernavaca, Morelos, Mexico E-mail address: [email protected] (E. Villegas).
Background: Pin2 is an antimicrobial peptide (AMP) originally isolated and purified from the venom of the scorpion Pandinus imperator. It has been proved to be highly active on disrupting bacterial and eukaryotic cell membranes. Pin2 could be an original drug to be used as pioneering peptide antibiotic for topical infections; however, it is easily cleaved in the presence of proteolytic enzymes. Therefore, to explore innovative derivatives of Pin2 an enantiomeric form was chemically synthesized. In this work we investigated some biological characteristics of D-Pin2 in the presence of a clinical isolated pathogenic strain of Pseudomonas aeruginosa. Methods: P. aeruginosa was clinically isolated from an infected wound. It was microbiologically verified by morphological, biochemical and molecular assays. P. aeruginosa samples were grown on King B liquid media for 2 days to allow production of proteases. The supernatants were separated by centrifugation at 4oC and filtered. The protein was precipitated using acetone. The proteolytic degradation of D-Pin2 in the presence of trypsin, human serum and protein extracts from P.aeruginosa was assayed for 3, 12, and 24h at 37oC. Native L-Pin2 was used as control. The content of D- or L- Pin2 was followed by HPLC. Results: D-Pin2 was found to be less hemolytic and more efficient against Gram – and þ strains. However, the minimal inhibitory concentration in the presence of P. aeruginosa was 50% higher than that of native L-Pin2, but D-Pin2 was resistant to proteolytic activity of trypsin and enzymes from human serum. It is known that P. aeruginosa secretes several proteases including protease IV and alkaline protease, among others. Therefore, when D-Pin2 samples were incubated with precipitated extracts of P. aeruginosa, it was found a decrease of 20, 50 and 100 % of D-Pin2 at 3, 12 and 24h, respectively, indicating that P. aeruginosa may contain enantio-selective proteases. Discussion: It is assumed a possible degradation of D-Pin2 by the activity of D-aminoacid dehydrogenase or racemase reported in P.aeruginosa. D-aminoacid dehydrogenase from P.aeruginosa is highly active on D-alanine and some D-amino acids. P.aeruginosa extracts with D-aminoacid activity are not reported. Could there be the presence of a protease degrading D-aminoacids? Conclusion: D-Pin2 has less hemolytic activity and it is more resistant to protease degradation; therefore, it could be a drug candidate for topical infections but will be less effective towards P. aeruginosa. Acknowledgements This work was supported by grants from CONACyT CB2010-153606, CONACyT CB-2008-01-106949 and RedFarmed. Keywords: Pin-2, degradation, P. aeruginosa 10.1016/j.toxicon.2012.04.047
47. Rational Development of Novel Leads from Animal Secretion Based on Coagulation and Cell Targets Ana Marisa Chudzinski-Tavassi, Kerly Fernanda Mesquita Pasqualoto, Linda Christian Carrijo-Carvalho Biochemistry and Biophysics Laboratory, Butantan Institute, Av. Vital Brasil. São Paulo, SP, Brazil E-mail address: [email protected] (A.M. Chudzinski-Tavassi).
Review: Animal venoms and secretions are sources of novel pharmacologically active molecules of potential therapeutic value. We have screened venoms aiming to discover, identify and isolate peptide molecules active in the mammalian haemostatic system. This research initiative yielded a portfolio of promising drug candidates comprising Lopap from bristles of the Lonomia obliqua caterpillar and Amblyomin-X from saliva of the Amblyomma cajennense tick. These novel recombinant proteins and synthetic peptides turned out to be multifunctional molecules, which are presently under different phases of development processes. Amblyomin-X is a potential candidate to treat cancer and metastasis. Lopap is a prothrombin activator which belongs to the lipocalin family and displays serine protease-like activity with procoagulant effect. It also induces cytokine secretion and antiapoptotic pathways in human cultured endothelial cells. A Lopap-derived peptide is capable of inducing collagen synthesis in fibroblast culture and in the animal dermis. In this context is crucial to elucidate the multifunctional properties of those molecules and provide a further data integration allowing the identification of potential targets as well as possible mechanisms of action, and then the prediction of novel leads (new chemical entities, NCEs) as drug candidates optimizing their therapeutic effects considering the system biology. In vitro and in vivo models reinforce therapeutic applications. The in silico methods (docking, molecular dynamics simulations, protein homology modeling, quantitative structure-activity and/or structure-property relationships, chemometric analysis) will generate molecular and mathematical models to establish the mechanisms of action hypothesis and support the rational design of novel leads with improved pharmacokinetic and pharmacodynamic profiles. Lopap and Amblyomin-X have patents licensed to pharmaceutical industries and are subjects of efforts to reach firm proofs of concept. The non-clinical phase studies are currently underway for Amblyomin-X. Financial support: FAPESP, INCTTOX-CNPq. Keywords: lonomia, amblyomma, coagulation 10.1016/j.toxicon.2012.04.048
48. Use of Connectivity Maps and Platform Technologies for Drug Lead/Biosimilar Discovery in Venoms Jay W. Fox, Aramadhaka Lavakumar Reddy, Alyson Prorock, Bojan Dragulev, Yongde Bao University of Virginia School of Medicine, Department of Microbiology, Immunology and Cancer Biology, Charlottesville, VA, USA E-mail address: [email protected] (J.W. Fox).
Background: Venoms have long been mined as resources for new drugs or drug leads. Traditionally, drug discovery has
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
46. Biological Features of an Enantiomeric Antimicrobial Peptide from Scorpion Venom Daniel Juarez Lopez 1, Gerardo Corzo 2, Alexis Rodriguez 2, Elba Villegas 1 1
Centro de Investigacion en Biotecnologia, Laboratorio de Estructura, Funcion, e Ingenieria de Proteinas, UAEM, Cuernavaca, Morelos, Mexico 2 Instituto de Biotecnologia, Departamento de Medicina Molecular y Bioprocesos, UNAM, Cuernavaca, Morelos, Mexico E-mail address: [email protected] (E. Villegas).
Background: Pin2 is an antimicrobial peptide (AMP) originally isolated and purified from the venom of the scorpion Pandinus imperator. It has been proved to be highly active on disrupting bacterial and eukaryotic cell membranes. Pin2 could be an original drug to be used as pioneering peptide antibiotic for topical infections; however, it is easily cleaved in the presence of proteolytic enzymes. Therefore, to explore innovative derivatives of Pin2 an enantiomeric form was chemically synthesized. In this work we investigated some biological characteristics of D-Pin2 in the presence of a clinical isolated pathogenic strain of Pseudomonas aeruginosa. Methods: P. aeruginosa was clinically isolated from an infected wound. It was microbiologically verified by morphological, biochemical and molecular assays. P. aeruginosa samples were grown on King B liquid media for 2 days to allow production of proteases. The supernatants were separated by centrifugation at 4oC and filtered. The protein was precipitated using acetone. The proteolytic degradation of D-Pin2 in the presence of trypsin, human serum and protein extracts from P.aeruginosa was assayed for 3, 12, and 24h at 37oC. Native L-Pin2 was used as control. The content of D- or L- Pin2 was followed by HPLC. Results: D-Pin2 was found to be less hemolytic and more efficient against Gram – and þ strains. However, the minimal inhibitory concentration in the presence of P. aeruginosa was 50% higher than that of native L-Pin2, but D-Pin2 was resistant to proteolytic activity of trypsin and enzymes from human serum. It is known that P. aeruginosa secretes several proteases including protease IV and alkaline protease, among others. Therefore, when D-Pin2 samples were incubated with precipitated extracts of P. aeruginosa, it was found a decrease of 20, 50 and 100 % of D-Pin2 at 3, 12 and 24h, respectively, indicating that P. aeruginosa may contain enantio-selective proteases. Discussion: It is assumed a possible degradation of D-Pin2 by the activity of D-aminoacid dehydrogenase or racemase reported in P.aeruginosa. D-aminoacid dehydrogenase from P.aeruginosa is highly active on D-alanine and some D-amino acids. P.aeruginosa extracts with D-aminoacid activity are not reported. Could there be the presence of a protease degrading D-aminoacids? Conclusion: D-Pin2 has less hemolytic activity and it is more resistant to protease degradation; therefore, it could be a drug candidate for topical infections but will be less effective towards P. aeruginosa. Acknowledgements This work was supported by grants from CONACyT CB2010-153606, CONACyT CB-2008-01-106949 and RedFarmed. Keywords: Pin-2, degradation, P. aeruginosa 10.1016/j.toxicon.2012.04.047
47. Rational Development of Novel Leads from Animal Secretion Based on Coagulation and Cell Targets Ana Marisa Chudzinski-Tavassi, Kerly Fernanda Mesquita Pasqualoto, Linda Christian Carrijo-Carvalho Biochemistry and Biophysics Laboratory, Butantan Institute, Av. Vital Brasil. São Paulo, SP, Brazil E-mail address: [email protected] (A.M. Chudzinski-Tavassi).
Review: Animal venoms and secretions are sources of novel pharmacologically active molecules of potential therapeutic value. We have screened venoms aiming to discover, identify and isolate peptide molecules active in the mammalian haemostatic system. This research initiative yielded a portfolio of promising drug candidates comprising Lopap from bristles of the Lonomia obliqua caterpillar and Amblyomin-X from saliva of the Amblyomma cajennense tick. These novel recombinant proteins and synthetic peptides turned out to be multifunctional molecules, which are presently under different phases of development processes. Amblyomin-X is a potential candidate to treat cancer and metastasis. Lopap is a prothrombin activator which belongs to the lipocalin family and displays serine protease-like activity with procoagulant effect. It also induces cytokine secretion and antiapoptotic pathways in human cultured endothelial cells. A Lopap-derived peptide is capable of inducing collagen synthesis in fibroblast culture and in the animal dermis. In this context is crucial to elucidate the multifunctional properties of those molecules and provide a further data integration allowing the identification of potential targets as well as possible mechanisms of action, and then the prediction of novel leads (new chemical entities, NCEs) as drug candidates optimizing their therapeutic effects considering the system biology. In vitro and in vivo models reinforce therapeutic applications. The in silico methods (docking, molecular dynamics simulations, protein homology modeling, quantitative structure-activity and/or structure-property relationships, chemometric analysis) will generate molecular and mathematical models to establish the mechanisms of action hypothesis and support the rational design of novel leads with improved pharmacokinetic and pharmacodynamic profiles. Lopap and Amblyomin-X have patents licensed to pharmaceutical industries and are subjects of efforts to reach firm proofs of concept. The non-clinical phase studies are currently underway for Amblyomin-X. Financial support: FAPESP, INCTTOX-CNPq. Keywords: lonomia, amblyomma, coagulation 10.1016/j.toxicon.2012.04.048
48. Use of Connectivity Maps and Platform Technologies for Drug Lead/Biosimilar Discovery in Venoms Jay W. Fox, Aramadhaka Lavakumar Reddy, Alyson Prorock, Bojan Dragulev, Yongde Bao University of Virginia School of Medicine, Department of Microbiology, Immunology and Cancer Biology, Charlottesville, VA, USA E-mail address: [email protected] (J.W. Fox).
Background: Venoms have long been mined as resources for new drugs or drug leads. Traditionally, drug discovery has