Abstract:
The 2014 report from the World Health Organization (WHO) on antimicrobial resistance revealed an alarming rise in antibiotic resistance all around the world and advocated that: “without action, common infections and minor injuries can once again kill”. Endogenous antimicrobial proteins (AMPs) exist widely throughout nature and protect organisms from infection by destroying a broad range of pathogens. Unlike classical antibiotics, with the exception of a few species, no acquired resistance towards AMPs has been reported. Therefore, AMPs represent leads for the development of novel antibiotics. Caenopore-5 (Cp-5) is constitutively expressed in the intestine of the nematode Caenorhabditis elegans. The threedimensional solution structure reveals that this AMP, composed of 5 α-helices and 3 disulfide bonds, is a member of the lipid binding saposin-like-protein (SAPLIP) family. Like other SAPLIP members, Cp-5 is decorated with charged amino acids that are postulated to be functionally important in targeting particular types of lipid head groups. The first aim of the research was to characterize the interaction of caenopore-5 with membrane mimetics using nuclear magnetic resonance (NMR) spectroscopy. 15N-labeled caenopore-5 was over-expressed in E. coli and purified to homogeneity using affinity and size-exclusion chromatography procedures. NMR spectroscopy was used to quantitatively characterize the binding properties of caenopore-5 in the presence of large unilamellar vesicles (LUVs). Caenopore-5 was observed to bind LUVs composed of anionic lipids and this interaction increased as the percentage of anionic lipids present in the LUVs increased. The binding of caenopore-5 to phospholipid vesicles was found to be a pH-controlled reversible process, in which, lower pH values enhanced the binding event due to the neutralization of the sidechains of the acidic amino acids. Binding to the negatively charged LUVs was inhibited when NaCl was included in the reaction, therefore indicating that the interaction between protein and lipids involves charge-charge interactions. The second aim of the research focused on the design of a robust and efficient method for the chemical synthesis of Cp-5, which was amenable to the preparation of analogues. The 82 residue protein caenopore-5 was successfully synthesized by native chemical ligation of two smaller polypeptide fragment, which were prepared using Boc SPPS technique. The strategy adopted used a single NCL site at a native cysteine residue and, although it required larger fragments (35 and 47 amino acids in length), it was shown to be a better strategy for the synthesis of Cp-5 than using a three fragments approach with two ligations. The synthetic protein was successfully folded and the synthetic material was found to have identical structural features as the recombinant Cp-5, as determined by 1H NMR spectroscopy and circular dichroism experiments. The last aim of the research was to generate a Cp-5 analogue with higher conformational stability to increase resistance to degradation by proteases; hence the cysteine residues were successfully replaced by selenocysteine to form isosteric and non-reducible diselenide bonds. With the aim of assembling a structure-activity relationship profile, 5 individual α-helical synthetic peptides, each comprising 15-22 residues in Cp-5, were synthesized. The bioactivity of each peptide was evaluated by measuring their permeabilization activity. The selenocysteine analogue was found to be more active than the native protein. Interestingly, two analogues of the N-terminus region (one with an incorporated selenocysteine) were found to be promising antimicrobial agent as they both exhibit a high cell permeability activity and are shorter than the native protein (35 amino acids compared to 82). Future work will focus on the design of more peptidomimetics from these model peptides that could be used as cell-penetrating peptides (CPPs) to deliver large molecules and even small particles inside the cell (such as antibodies, contrast imaging agents, toxins, and nanoparticular drug carriers).