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Membrane Active Peptides as a Potential Therapeutic Option for Enveloped Viruses
380a
Tuesday, February 14, 2017
that the antimicrobial activity of these peptides can be correlated to the 3D-hydrophobic moment and to a simple structure-based packing parameter. This suggests that, in principle, one could design antimicrobial peptides based on such parameters. Our study shows that the nature of histidine favors its interaction with anionic lipid headgroups, i.e., a location at one end of an AMP, instead of the middle, and enhances the aggregation of cationic AMPs around anionic lipids, leading to transmembrane pore formation. The latter mechanism of disruption of the membrane can be correlated with the increased antimicrobial activity of these AMPs. Hence, the position of the histidine within the peptide sequence can be linked with AMP’s mechanism of interaction with the membrane surface. Furthermore, the presence of histidine residue reduced the cytotoxic and hemolytic activity of the peptides, in some cases maintaining the same efficacy against bacteria. Some of these peptides have the potential to become good candidates to fight against bacteria. Acknowledgments: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNDI-UEFISCDI, project number PNII-123/2012, PNII-98/2012, PN-II-ID-PCCE-2011-2-0027, PN 09370301 and PN-II-RU-TE-2014-4-2418. 1875-Pos Board B195 Development of Cell-Wall Deficient Bacteria for the Characterization of Histone-Derived Antimicrobial Peptides through Confocal Microscopy Dania M. Figueroa1, Donald E. Elmore2. 1 Biochemistry Program, Wellesley College, Wellesley, MA, USA, 2 Department of Chemistry and Biochemistry Program, Wellesley College, Wellesley, MA, USA. Antimicrobial peptides (AMPs) are cationic, amphipathic proteins with an innate ability to kill a wide variety of pathogens, including viruses, fungi and bacteria. AMPs can be grouped into two general categories based on their mechanism of action. One group acts via permeabilization, or disruption of the cell membrane. The other group acts via translocation, or diffusion across the cell membrane and disruption of an intracellular process. Confocal microscopy is a technique readily used to identify AMP methods of action as it allows researchers to visualize peptide localization in bacteria by taking cross sections of cells. However, the small size and different orientations of bacteria can produce low-resolution images. To combat this problem our lab has used the cell-wall deficient spheroplast form of Escherichia coli to obtain higher quality images. Thesespheroplasts are both spherical and larger than typical E. coli, leading to improved imaging. Previously, we showed that several previously characterized AMPs have the same behavior against E. coli spheroplasts as normal cells. This project is an extension of that work aimed at developing protocols to consider the visualization of AMPs with cell-wall deficient forms of other bacterial strains. Characterizing AMPs against a variety of bacteria is important as research shows that AMPs antimicrobial properties differ against different bacteria strains. To this end, we are developing protocols to form protoplasts of the gram-positive bacteria Bacillus subtilis and Bacillus megaterium for imaging with well-characterized control peptides, such as buforin II and magainin. We have also investigated the membrane integrity of cell-wall deficient bacteria using a microscopy-based permeabilization assay. 1876-Pos Board B196 Membrane Active Peptides as a Potential Therapeutic Option for Enveloped Viruses Shantanu Guha. Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA, USA. Every year, influenza affects populations all across the world, and every year, new vaccines must be developed due to the intrinsically high rate of mutation and reassortment in influenza virus. Currently, there are two classes of antiviral drugs available to treat influenza virus infection, neuraminidase inhibitors and M2 inhibitors, but both classes have limited efficacy and resistant strains of virus are common. Due to the difficulty in treating influenza and other enveloped viruses, there is an urgent need for new classes of antiviral agents. One potentially useful, but underdeveloped type of inhibitor is the peptide entry inhibitor (PEI). PEIs interfere with the key early steps by which enveloped viruses bind, fuse, enter, and eventually infect their target cells. By testing a large number of membrane active peptides in an influenza infection model system, we identified a panel of peptides which have potent inhibitory activity against the virus. We have hypothesized that some of these peptides directly interact with viral and/or host cellular membrane lipids and disrupt viral binding and fusion. This hypothesis leads to the prediction that membrane-active peptides, in general, will inherently have inhibitory activity against enveloped virus due to the nature of virus-host entry mechanisms. We have used cell biology and biophysics
to compare the peptide activity against synthetic membrane vesicles with the antiviral activities of each peptide. Taken together, our results show that interfacially active PEIs have the potential to be useful therapeutics which can possibly be applied to different enveloped viruses. 1877-Pos Board B197 Membrane Binding and Antimicrobial Activity of a Catioinc, PorphyrinBinding Peptide David Shirley, Christina L. Chrom, Gregory A. Caputo. Chemistry and Biochemistry, Rowan University, Glassboro, NJ, USA. A designed catioinic, amphiphilic peptide was previously shown to bind porphyrin molecules and promote the formation of excitonically coupled J-aggregate structures (Caputo et. al. 2009). Due to the catioinic and amphiphilic nature, this peptide was investigated for antimicrobial activity. We synthesized 3 variants of the peptide which differed only in the position of a single Trp residue, used as a reporter of membrane binding interactions. All three variants showed similar levels of antimicrobial activity against Gramþ and G- strains (low micromolar efficacy). Using these 3 versions we characterized the binding and membrane orientation of the peptides using fluorescence spectroscopy and quenching, secondary structure by CD, and bacterial membrane permeabilization assays to investigate mechanism of antibacterial activity. All peptides bound to anionic bilayers with higher affinity compared to zwitterionic bilayers, but there was little difference between the three variants. Additionally, quenching experiments yielded little difference between the three Trp positions in the peptides, indicating that the peptide is likely not adopting a single, uniform orientation in the bilayer. The peptides adopted helical conformations when bound to the bilayers. However, none of the peptides exhibited significant permeabilization of the E.coli inner membrane, indicating activity may be localized to the outer membrane. Ongoing work is focused on the depth of peptide penetration into the bilayer. 1878-Pos Board B198 Membrane Insertion of a Dinuclear Ruthenium Complex and Implications for Antibacterial Activity Daniel K. Weber1, Marc-Antoine Sani2, Matthew T. Downton1, J. Grant Collins3, Frances Separovic2, F. Richard Keene4. 1 Computational Biophysics, IBM Research Australia, Melbourne, Australia, 2 School of Chemistry, University of Melbourne, Melbourne, Australia, 3 School of Physical, Environmental and Mathematical Sciences, University of New South Wales, Australian Defence Force Academy, Canberra, Australia, 4School of Physical Sciences, University of Adelaide, Adelaide, Australia. Ruthenium-based metal complexes have received considerable interest over the past 30 years for their DNA/RNA-binding and anticancer properties. More recently, however, they have been recognized as potential antibacterial agents. In particular, cationic dinuclear polypyridylruthenium(II) complexes, bridged by flexible methylene linkers, have shown enhanced antibacterial activity over their mononuclear counterparts, and generally maintain activity against antibiotic-resistant strains. Furthermore, dinuclear complexes are known to depolarize and increase permeability of bacterial membranes, while their activity can be directly modulated by varying the length of the methylene bridge. Recently, we have applied a combination of solid-state NMR and simulation to provide an initial biophysical characterization of a dinuclear Ru(II) complex and a biologically-inactive Ir(III) analogue in membrane environments. We highlight that direct membrane-permeating mechanisms and diffusion though bacterial cell membranes may hinge on an exclusive ability of dinuclear Ru(II) complexes to insert as transmembrane structures. 1879-Pos Board B199 2 H NMR Studies of Bacteria: How Does the Peptidoglycan Layer Modify the Interaction between Antimicrobial Peptides and the Lipid Membrane Nury P. Santisteban1, Michael R. Morrow1, Valerie K. Booth2. 1 Physics and Physical Oceanography, Memorial University of Newfoundland, St. John’s, NL, Canada, 2Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada. Antimicrobial peptides (AMPs) are a group of small peptides with antimicrobial effects against pathogens and have been well studied because of their promise to be part of the solution to the rising problem of antibiotic resistance. Biophysical studies with AMPs in model lipid systems are commonly used to study AMPs’ permeabilizing effect on lipid bilayers. However, it is not clear if this membrane-permeabilizing characteristic is the only mechanism of cell killing. Studies suggest that at least some AMPs have additional targets, different from the lipid bilayer. This studies lead to the suggestion that membrane permeabilization is just part of a multi-hit mechanism of AMPs, or
Tuesday, February 14, 2017
that the antimicrobial activity of these peptides can be correlated to the 3D-hydrophobic moment and to a simple structure-based packing parameter. This suggests that, in principle, one could design antimicrobial peptides based on such parameters. Our study shows that the nature of histidine favors its interaction with anionic lipid headgroups, i.e., a location at one end of an AMP, instead of the middle, and enhances the aggregation of cationic AMPs around anionic lipids, leading to transmembrane pore formation. The latter mechanism of disruption of the membrane can be correlated with the increased antimicrobial activity of these AMPs. Hence, the position of the histidine within the peptide sequence can be linked with AMP’s mechanism of interaction with the membrane surface. Furthermore, the presence of histidine residue reduced the cytotoxic and hemolytic activity of the peptides, in some cases maintaining the same efficacy against bacteria. Some of these peptides have the potential to become good candidates to fight against bacteria. Acknowledgments: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNDI-UEFISCDI, project number PNII-123/2012, PNII-98/2012, PN-II-ID-PCCE-2011-2-0027, PN 09370301 and PN-II-RU-TE-2014-4-2418. 1875-Pos Board B195 Development of Cell-Wall Deficient Bacteria for the Characterization of Histone-Derived Antimicrobial Peptides through Confocal Microscopy Dania M. Figueroa1, Donald E. Elmore2. 1 Biochemistry Program, Wellesley College, Wellesley, MA, USA, 2 Department of Chemistry and Biochemistry Program, Wellesley College, Wellesley, MA, USA. Antimicrobial peptides (AMPs) are cationic, amphipathic proteins with an innate ability to kill a wide variety of pathogens, including viruses, fungi and bacteria. AMPs can be grouped into two general categories based on their mechanism of action. One group acts via permeabilization, or disruption of the cell membrane. The other group acts via translocation, or diffusion across the cell membrane and disruption of an intracellular process. Confocal microscopy is a technique readily used to identify AMP methods of action as it allows researchers to visualize peptide localization in bacteria by taking cross sections of cells. However, the small size and different orientations of bacteria can produce low-resolution images. To combat this problem our lab has used the cell-wall deficient spheroplast form of Escherichia coli to obtain higher quality images. Thesespheroplasts are both spherical and larger than typical E. coli, leading to improved imaging. Previously, we showed that several previously characterized AMPs have the same behavior against E. coli spheroplasts as normal cells. This project is an extension of that work aimed at developing protocols to consider the visualization of AMPs with cell-wall deficient forms of other bacterial strains. Characterizing AMPs against a variety of bacteria is important as research shows that AMPs antimicrobial properties differ against different bacteria strains. To this end, we are developing protocols to form protoplasts of the gram-positive bacteria Bacillus subtilis and Bacillus megaterium for imaging with well-characterized control peptides, such as buforin II and magainin. We have also investigated the membrane integrity of cell-wall deficient bacteria using a microscopy-based permeabilization assay. 1876-Pos Board B196 Membrane Active Peptides as a Potential Therapeutic Option for Enveloped Viruses Shantanu Guha. Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA, USA. Every year, influenza affects populations all across the world, and every year, new vaccines must be developed due to the intrinsically high rate of mutation and reassortment in influenza virus. Currently, there are two classes of antiviral drugs available to treat influenza virus infection, neuraminidase inhibitors and M2 inhibitors, but both classes have limited efficacy and resistant strains of virus are common. Due to the difficulty in treating influenza and other enveloped viruses, there is an urgent need for new classes of antiviral agents. One potentially useful, but underdeveloped type of inhibitor is the peptide entry inhibitor (PEI). PEIs interfere with the key early steps by which enveloped viruses bind, fuse, enter, and eventually infect their target cells. By testing a large number of membrane active peptides in an influenza infection model system, we identified a panel of peptides which have potent inhibitory activity against the virus. We have hypothesized that some of these peptides directly interact with viral and/or host cellular membrane lipids and disrupt viral binding and fusion. This hypothesis leads to the prediction that membrane-active peptides, in general, will inherently have inhibitory activity against enveloped virus due to the nature of virus-host entry mechanisms. We have used cell biology and biophysics
to compare the peptide activity against synthetic membrane vesicles with the antiviral activities of each peptide. Taken together, our results show that interfacially active PEIs have the potential to be useful therapeutics which can possibly be applied to different enveloped viruses. 1877-Pos Board B197 Membrane Binding and Antimicrobial Activity of a Catioinc, PorphyrinBinding Peptide David Shirley, Christina L. Chrom, Gregory A. Caputo. Chemistry and Biochemistry, Rowan University, Glassboro, NJ, USA. A designed catioinic, amphiphilic peptide was previously shown to bind porphyrin molecules and promote the formation of excitonically coupled J-aggregate structures (Caputo et. al. 2009). Due to the catioinic and amphiphilic nature, this peptide was investigated for antimicrobial activity. We synthesized 3 variants of the peptide which differed only in the position of a single Trp residue, used as a reporter of membrane binding interactions. All three variants showed similar levels of antimicrobial activity against Gramþ and G- strains (low micromolar efficacy). Using these 3 versions we characterized the binding and membrane orientation of the peptides using fluorescence spectroscopy and quenching, secondary structure by CD, and bacterial membrane permeabilization assays to investigate mechanism of antibacterial activity. All peptides bound to anionic bilayers with higher affinity compared to zwitterionic bilayers, but there was little difference between the three variants. Additionally, quenching experiments yielded little difference between the three Trp positions in the peptides, indicating that the peptide is likely not adopting a single, uniform orientation in the bilayer. The peptides adopted helical conformations when bound to the bilayers. However, none of the peptides exhibited significant permeabilization of the E.coli inner membrane, indicating activity may be localized to the outer membrane. Ongoing work is focused on the depth of peptide penetration into the bilayer. 1878-Pos Board B198 Membrane Insertion of a Dinuclear Ruthenium Complex and Implications for Antibacterial Activity Daniel K. Weber1, Marc-Antoine Sani2, Matthew T. Downton1, J. Grant Collins3, Frances Separovic2, F. Richard Keene4. 1 Computational Biophysics, IBM Research Australia, Melbourne, Australia, 2 School of Chemistry, University of Melbourne, Melbourne, Australia, 3 School of Physical, Environmental and Mathematical Sciences, University of New South Wales, Australian Defence Force Academy, Canberra, Australia, 4School of Physical Sciences, University of Adelaide, Adelaide, Australia. Ruthenium-based metal complexes have received considerable interest over the past 30 years for their DNA/RNA-binding and anticancer properties. More recently, however, they have been recognized as potential antibacterial agents. In particular, cationic dinuclear polypyridylruthenium(II) complexes, bridged by flexible methylene linkers, have shown enhanced antibacterial activity over their mononuclear counterparts, and generally maintain activity against antibiotic-resistant strains. Furthermore, dinuclear complexes are known to depolarize and increase permeability of bacterial membranes, while their activity can be directly modulated by varying the length of the methylene bridge. Recently, we have applied a combination of solid-state NMR and simulation to provide an initial biophysical characterization of a dinuclear Ru(II) complex and a biologically-inactive Ir(III) analogue in membrane environments. We highlight that direct membrane-permeating mechanisms and diffusion though bacterial cell membranes may hinge on an exclusive ability of dinuclear Ru(II) complexes to insert as transmembrane structures. 1879-Pos Board B199 2 H NMR Studies of Bacteria: How Does the Peptidoglycan Layer Modify the Interaction between Antimicrobial Peptides and the Lipid Membrane Nury P. Santisteban1, Michael R. Morrow1, Valerie K. Booth2. 1 Physics and Physical Oceanography, Memorial University of Newfoundland, St. John’s, NL, Canada, 2Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada. Antimicrobial peptides (AMPs) are a group of small peptides with antimicrobial effects against pathogens and have been well studied because of their promise to be part of the solution to the rising problem of antibiotic resistance. Biophysical studies with AMPs in model lipid systems are commonly used to study AMPs’ permeabilizing effect on lipid bilayers. However, it is not clear if this membrane-permeabilizing characteristic is the only mechanism of cell killing. Studies suggest that at least some AMPs have additional targets, different from the lipid bilayer. This studies lead to the suggestion that membrane permeabilization is just part of a multi-hit mechanism of AMPs, or