Item – Theses Canada

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Brewer, Dyanne Patricia,1971-
The design and synthesis of antifungal peptides based on the structure of salivary histatins.
Ph. D. -- University of Waterloo, 2000
Ottawa :National Library of Canada = Bibliothèque nationale du Canada,[2004]
3 microfiches.
Includes bibliographical references.
Histatins are a family of histidine-rich peptides found in human saliva. They have potent antibacterial and antifungal activity thus contributing to the host non-immune defense system. This thesis presents an examination of the histatin antifungal mode of action through the design of analogs based on the conformational analysis of two of the naturally occurring histatins: histatin 3 (H3) and histatin 5 (H5). Research into the mode of action of these peptides can provide insight into innate immunity as well as new targets for antifungal therapy. The conformation of H3 and H5 were examined by circular dichroism (CD) and proton nuclear magnetic resonance (NMR) spectroscopy in various environments. H5 had no defined structure in aqueous solution but adopted a more helical conformation in dimethylsulfoxide (DMSO) and trifluoroethanol. The longer H3 peptide had less conformational mobility in aqueous solution. It showed characteristics of a turn or loop structure in the middle of the sequence as well as a C-terminal helix in 50% DMSO. In membrane mimicking environments (sodium dodecyl sulfate and L-[alpha]-phosphatidylcholine dimyristoyl), H3 showed long-range interactions that indicated an extended loop from residues 10 to 20. H3 and H5, although similar in sequence, behave conformationally very differently in various environments. Both peptides demonstrate a high degree of flexibility in all environments examined. Electrospray mass spectrometry was used to analyze the interactions of H3 and H5 with metal ions. These two peptides show potential for interacting with Cu2+ and Ni2+ and to a lesser extent Zn 2+. Metal interaction with either peptide did not result in regular secondary structure formation by CD analysis. Some subtle conformational changes were observed in the peptides in the presence of some metals. Cyclic analogs were designed to stabilize the loop or turn structure centered at Glu16-Lys17 that was observed in the conformational analysis of H3. The peptides were cyclized by the introduction of a disulfide bond. Several analogs were made by varying the placement of the two cysteine replacement residues within the sequence. All analogs were tested for improved antifungal activity in a fungistatic assay comparing them to both H5 and the antifungal drug ketoconazole. The most active analog db2-121 had the sequence RHHCYKRKFHEKHHCHRGY and an activity of 0.30-0.60 [mu]M against 'Saccharomyces cerevisiae '. This activity is approximately 100 times more potent than H5. The introduction of further constraint with a second disulfide bond resulted in a loss of activity. The conformation of db2-121 revealed a looped structure with charged residues protruding externally while three aromatic residues and four to six histidines pack into an internal core. It is proposed that this structure is protein-like and may play an important role in the biological activity of db2-121.