Insights into Peptidomimetic Approaches for Inhibiting the DNA Sliding Clamps

Abstract

In 2020, there were 48,266 deaths from cancer in Australia, a bewildering assortment of diseases that’s caused from uncontrollable proliferation. One of the main problems of conventional cancer therapy is the low specificity of chemotherapeutic drugs for carcinogenic cells over healthy cells. This highlights the necessity for effective cancer treatments and therapeutics. Human proliferating cell nuclear antigen (hPCNA) is a DNA replication processivity factor, which acts as a docking platform, allowing proteins to have access to the replication fork, and increasing the affinity of these DNA interacting proteins. PCNA is a promising target for the inhibition of cancer cell growth as it is critical for cell survival. The trimer structure of PCNA forms a ring-shaped oligomer allowing DNA to pass through the middle and interacting proteins to dock on the outside of the ring. Without this structural formation, there is a loss of DNA replication and repair in the cell. Due to the location of subunit-subunit termini, the addition of a purification tag can hamper crystallography and biophysical experiments, as the trimer complex folding can be impeded. To avoid these complications, a tag-less, step-wise purification was implemented, allowing the further structural research of this protein for cancer treatments. The known regulator protein p21 has been used to investigate new approaches for targeting and inhibiting PCNA for the development of cancer therapeutics. The p21 protein has been shown in the human system to block binding to the docking platform and halt cell replication. A peptide of the binding domain of p21 has been shown to bind and maintain the affinity of the full protein. Structural modifications of peptides guided by rational design and molecular modelling have been established to develop novel synthetic approaches, this is peptidomimetic research. This can be done using the PIP-box of p21, the PCNA interacting protein box, a sequence which allows tight interaction with PCNA. A peptide using the PIP-box of p21 has been investigated in previous literature and research for a PCNA inhibitor. A mutagenesis approach has been used to identify what characteristics increase or decrease peptide affinity for PCNA. This is so the differences in PIP-boxes can be shown at a residue level, to identify structural points which form a tightly bound confirmation. This has been done to reveal a new peptide sequence with a stronger binding affinity than the native protein. This isn’t the full extent of the use of this peptidomimetic research, it can be used on other species, as PCNA has been shown to be present in almost all forms of life. Aspergillus fumigatus is abundant in the environment and the most common cause of invasive fungal infection. Central nervous system aspergillosis has mortality rates of ~90%. As PCNA has important roles in replication and cell survival, it is hypothesised that it is a potential target for antifungal treatments, illustrating the importance of investigating the crystal structure of A. fumigatus PCNA, to understand how interacting peptides bind to aid rational drug design. This was done to reveal a newly designed mimetic with a new secondary structure with high affinity binding which can be utilised in further fungal mimetic research. This thesis presents a number of peer-reviewed publications which shed light on peptidomimetic use on the protein PCNA for a number of therapeutic pathways. The result is critical developments in the beginning of the drug developmental pipeline. In addition, this thesis includes publications highlighting the merit of repurposing the peptidomimetics synthesised for hPCNA in the development for a fungal treatment for A. fumigatus.