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Work Package 1

"Development of novel antibiotics"

Various promising novel antimicrobial candidates and approaches are currently under study by the partners in DanCARD.

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A. Novel peptidomimetic α-peptide/β-peptoid chimeras6,10

Novel peptidomimetic α-peptide/β-peptoid chimeras have been found to exhibit antimicrobial activities comparable to other types of optimized peptidomimetics2,7. Automated solid-phase microwave-assisted synthesis has improved efficiency of the  synthesis of peptidomimetics allowing for optimization of drug properties by investigation of  novel structural modifications. Also, inspiration for design of novel peptidomimetics will be sought in natural antimicrobial sequences. The influence of the ratio between natural and unnatural residues  on proteolytic stability, haemolysis and antimicrobial activity will be investigated. NMR-spectroscopic investigation of the secondary structure of these novel peptidomimetics may lead to a structure-based design approach.
Contributors: Henrik Franzyk, Rasmus Jahnsen, Reinhard WimmerLars Erik Uggerhøj 

B. Anoplin:  

The natural antimicrobial peptide anoplin is found in the venom of the solitary wasp Anoplius samariensis. Using structure-activity studies, this peptide family will be further developed to identify novel anoplin analogues with potent and selective anti-gram-negative activity and low toxicity. Anoplin-variants modified by i) non-proteinogenic building blocks (D-amino acids and peptoids), ii) fatty acids and other lipids, iii) N-terminal and C-terminal truncations and iv) cyclization (C->N, lactam, thioether) will be synthesized. Mathematical models will be used in the design process.
Contributors: Paul Robert HansenJens Kristian Munk 

C. Small circular peptides: 

Small circular peptides are rarely found in nature (except theta-defensins3) and they are typically generated by manipulation of protein splicing8,9,11 and have been given little attention as putative antimicrobial agents. Using a Synechocystis sp. derived split intein system for circular ligation of peptides, a combinatory library of about 106 random hexameric cyclic peptides have been constructed. We have initially chosen to pursue inhibitors of bacterial DNA replication - a poorly exploited target. We have currently isolated several peptides that interfere with DNA synthesis in S. aureus and B. subtilis. These peptides target various components of the replicative polymerase. We wish to expand our library of circular peptides, investigate the mechanism of interaction of antimicrobial peptides with bacteria and further develop and recombinantly produce cyclic peptides targeted on essential processes in bacteria. With a large pool of assayed peptides we may start optimization of new lead peptides.
Contributors: Anders Løbner-OlesenSusanne KjelstrupPaul Robert Hansen 

D. Characterization of peptides and enzymes isolated from maggots: 

Novel antimicrobial peptides and enzymes isolated from secretions from maggots by transposon–assisted signal trapping (TAST) will be characterized1. Subsequently alternative invertebrate sources will be investigated.
Contributors: Karen A. Krogfelt, Mette E. SkindersøHans-Henrik Kristensen 

E. Peptide library screening for compounds with antibacterial and/or antiadhesive effects: 

Peptides from available libraries will be screened in in vitro and in vivo models for antibacterial and antiadhesive effect.  Furthermore, bacterial mutant libraries are available within the consortium to assess the mechanism of action of the peptides. We will use two-hybrid based screens for compounds capable of disrupting interaction between selected protein partners and a C. elegans based infection model for activity. Successful variants will be tested further for stability, solubility, haemolytic and histamine-release ability, toxic behaviour in mice, pharmacokinetics and finally in vivo antibacterial effect. NMR-spectroscopy will be used to investigate the peptide structures and their interaction with micelles and other lipids4,12.
Contributors: Karen A. Krogfelt, Mette E. SkindersøAnders Løbner-OlesenSusanne Kjelstrup 

F. Novel plant-derived antimicrobials: 

Various plant extracts with strong antimicrobial action have been isolated.
Preliminary data show that cranberries can prevent urinary tract infections. We will further assess the active components involved to allow for enhancement of the activity of cranberry juice itself.
Contributors:  Karen A. Krogfelt, Mette E. Skindersø, Lotte JakobsenNiels Frimodt-Møller 

G. Novel antibiotics of other types:

One novel fluoroquinolone and four antibacterial solutions produced by fungi will be acquired from a Danish biotech company, and further characterized and screened for their effect in vitro and in vivo.
Contributors: Lotte JakobsenNiels Frimodt-Møller


Reference List:

  1. Andersen, A. S., D. Sandvang, K. M. Schnorr, T. Kruse, S. Neve, B. Joergensen, T. Karlsmark, and K. A. Krogfelt. 2010. A novel approach to the antimicrobial activity of maggot debridement therapy. J.Antimicrob.Chemother. 65:1646-1654.
  2. Chongsiriwatana, N. P., J. A. Patch, A. M. Czyzewski, M. T. Dohm, A. Ivankin, D. Gidalevitz, R. N. Zuckermann, and A. E. Barron. 2008. Peptoids that mimic the structure, function, and mechanism of helical antimicrobial peptides. Proc.Natl.Acad.Sci.U.S.A 105:2794-2799.
  3. Evans, T. C., Jr., D. Martin, R. Kolly, D. Panne, L. Sun, I. Ghosh, L. Chen, J. Benner, X. Q. Liu, and M. Q. Xu. 2000. Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803. J.Biol.Chem. 275:9091-9094.
  4. Franzmann, M., D. Otzen, and R. Wimmer. 2009. Quantitative use of paramagnetic relaxation enhancements for determining orientations and insertion depths of peptides in micelles. Chembiochem. 10:2339-2347.
  5. Jensen, H. D., K. A. Krogfelt, C. Cornett, S. H. Hansen, and S. B. Christensen. 2002. Hydrophilic carboxylic acids and iridoid glycosides in the juice of American and European cranberries (Vaccinium macrocarpon and V. oxycoccos), lingonberries (V. vitis-idaea), and blueberries (V. myrtillus). J.Agric.Food Chem. 50:6871-6874.
  6. Olsen, C. A., G. Bonke, L. Vedel, A. Adsersen, M. Witt, H. Franzyk, and J. W. Jaroszewski. 2007. Alpha-peptide/beta-peptoid chimeras. Org.Lett. 9:1549-1552.
  7. Schmitt, M. A., B. Weisblum, and S. H. Gellman. 2007. Interplay among folding, sequence, and lipophilicity in the antibacterial and hemolytic activities of alpha/beta-peptides. J.Am.Chem.Soc. 129:417-428.
  8. Scott, C. P., E. Abel-Santos, M. Wall, D. C. Wahnon, and S. J. Benkovic. 1999. Production of cyclic peptides and proteins in vivo. Proc.Natl.Acad.Sci.U.S.A 96:13638-13643.
  9. Tang, Y. Q., J. Yuan, G. Osapay, K. Osapay, D. Tran, C. J. Miller, A. J. Ouellette, and M. E. Selsted. 1999. A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins. Science 286:498-502.
  10. Vedel, L., G. Bonke, C. Foged, H. Ziegler, H. Franzyk, J. W. Jaroszewski, and C. A. Olsen. 2007. Antiplasmodial and prehemolytic activities of alpha-peptide-beta-peptoid chimeras. Chembiochem. 8:1781-1784.
  11. Williams, N. K., P. Prosselkov, E. Liepinsh, I. Line, A. Sharipo, D. R. Littler, P. M. Curmi, G. Otting, and N. E. Dixon. 2002. In vivo protein cyclization promoted by a circularly permuted Synechocystis sp. PCC6803 DnaB mini-intein. J.Biol.Chem. 277:7790-7798.
  12. Zangger, K., M. Respondek, C. Gobl, W. Hohlweg, K. Rasmussen, G. Grampp, and T. Madl. 2009. Positioning of micelle-bound peptides by paramagnetic relaxation enhancements. J.Phys.Chem.B 113:4400-4406.


 

Last revised 16 July 2014