Novel antibiotic peptides

Abstract

During bacterial replication DNA lesions might occur and lead to DNA damage. Some lesions are repaired by DNA repair pathways, but this is not always successful. In these cases, instead of repairing the damage, the DNA lesions are bypassed by translesion synthesis (TLS). Replicative polymerases are then replaced with TLS polymerases that are able to bypass the lesion. However, TLS polymerases are error-prone, leading to increased mutation levels in the newly synthesized DNA strand. Antibiotic resistance and infections by multi-resistant bacteria is an increasing health and economic problem in today´s society. It has been shown that TLS is important for development of antibiotic resistance and it is therefore of interest to find a new antibacterial drug that can inhibit TLS polymerases. Unpublished data has shown that the APIM-peptide (101) has antibacterial and antimutagenic activities, likely mediated via interaction with the β-clamp, and thereby inhibition of interaction between the β-clamp and the TLS polymerases. The goal in this thesis was to investigate different variants of the APIM-peptide with different linkers and cell penetrating peptides (CPP) to find a good candidate for use as an antibacterial drug. The antibacterial activity was studied by overexpressing and adding the APIM-variants to E. coli BL21. Rif-assay was performed to study the mutation frequency of the APIM-variants and a mutation spectra of the rpoB gene from the Rif(R) colonies were further studied to investigate the affinity between the β-clamp and the peptides, and thereby the peptides ability to inhibit TLS polymerases. The peptides were also added to HEK 293 and U2OS cells to examine the toxicity of the peptides, and to isolated monocytes stimulated with LPS to study their effect on the cytokine secretion. The results presented in this thesis suggest that 101-Y and 101-prot are equally good antibacterial drug candidates as 101. 101-Y is a W4Y 101 variant, while 101-prot has a HIVTAT CPP. These peptides have a high antibacterial activity without having any toxic effect on HEK 293 and U2OS. Further, these peptides are also observed to reduce the cytokine secretion in monocytes stimulated with LPS.