Design, synthesis and study of the biological and biophysical activity of antimicrobial peptides

• Autores: Ariadna Grau Campistany
• Directores de la Tesis: Francesc Rabanal Anglada (dir. tes.), Yolanda Cajal Visa (dir. tes.)
• Lectura: En la Universitat de Barcelona ( España ) en 2014
• Idioma: inglés
• Tribunal Calificador de la Tesis: Luis Ignacio Rivas López (presid.), Vicente Marchan Sancho (secret.), Stephan L. Grage (voc.)
• Materias:
  • Química
    • Bioquímica
      • Péptidos
    • Química orgánica
  • Ciencias de la vida
    • Microbiología
      • Antibióticos
• Enlaces
  • Tesis en acceso abierto en:  TDX  Dipòsit Digital de la UB 
• Resumen

  • The global emergence and spread of multidrug-resistant bacteria is an important public health issue. However, the antimicrobial pipeline remains unacceptably lean, in fact over the last 25 years, the number of antimicrobial agents that reach the market has sharply decreased. In this context, there is now a renewed interest in the search for drugs that have more than one target on the bacterial cell, rather than a specific chiral receptor or enzyme. Antimicrobial peptides (AMPs) are a class of antibiotics that have attracted great interest in the last few years because they rarely spur the development of resistant organisms as their mechanism of action involves disruption of the bacterial membrane. In this thesis we report the design, preparation and activity of new compounds based on the sequence of the polymyxins, a class of antibiotics highly active against Gramnegative bacteria, and used clinically as last resort treatment for multidrug-resistant pathogens. The compounds synthesized proved to be highly active against both Grampositive and Gram-negative bacteria, including several strains of resistant bacteria. Furthermore, biophysical experiments using liposomes and monolayers as model membranes and flow cytometry and transmission electron microscopy using bacteria were carried out to study the mechanism of action of these compounds. The results of the most active candidate indicate that an alternative, non-membrane dependent mechanism of action might be involved. In the second part of the thesis, a series of 9 peptides were designed and synthesized from repeated KIAGKIA motifs, based in the sequence of the antimicrobial peptide PGLa from the magainin family, with lengths between 14 and 28 amino acids. Circular dichroism spectroscopy showed that they all formed alpha-helices when found in a lipid environment. Biological assays for haemolysis and antimicrobial activity, as well as fluorescence vesicle leakage and solid-state NMR spectroscopy, were used to correlate peptide length with membrane activity. These data are fully consistent with the formation of transmembrane pores. Only peptides that are long enough to span the hydrophobic bilayer core can induce vesicle leakage, haemolysis, and inhibit bacterial growth. The shorter peptides do not show these effects. Solid-state NMR analysis in oriented bilayers with different thickness also demonstrated the need for a minimum peptide length to flip from a surface-bound alignment into a more inserted, possibly even transmembrane state. With increasing length the peptides start to tilt and perturb the bilayer. Since the threshold behaviour seen for biological activity closely matches the biophysical results, the peptides could be used as molecular rulers to determine the thickness of bacterial membranes, showing that E. coli (? 27 Å) < S. aureus and P. aeruginosa (? 30 Å) < E. faecalis (? 34 Å).