Exploring bicyclic peptide and fragment-based inhibitors for targeting ALT-positive cancers

Abstract

Telomeres are repetitive nucleoprotein structures shielding the end of chromosomes from degradation with its length controlling the survival capacity of a cell. Cancer cells exploit mechanisms such as alternative lengthening of telomeres (ALT) to maintain long telomeres with ALT-positive cancers specifically relying on a DNA recombination-based pathway for their progression, where the PPI between FANCM and RMI1/2 via the FANCM-MM2 domain is reported for being essential for ALT activity. As of now, there are currently no reported inhibitors of this interaction and thus, the overall aim of this thesis is to identify and characterise two classes of PPI inhibitors – small molecules and peptides – for the FANCM-RMI1/2 interaction.

Chapter 2 discusses a structure-based rational design and synthesis of bicyclic peptide mimics based on the native FANCM-MM2 peptide using a novel asymmetrical 1,2,3-substituted linker. A FBDD campaign against RMI1/2 identified two promising fragments hits – F4 and F5 – on which follow up SAR studies are described in Chapter 3. Through a collaborative effort, eighty analogues of these fragments were synthesised, and binding affinities were evaluated in an SPR assay with an optimised peptide control. In Chapter 4, the production and screening of two phage displayed peptide libraries with closely spaced cysteines against RMI1/2 is described. Six unique libraries were panned, and hits were obtained for two libraries. An attempt to validate the peptides using FP indicated that two peptide hits bound with micromolar affinity at the desired MM2 pocket.

The experiments in this thesis lay the groundwork for the development of inhibitors for the PPI between FANCM and RMI1/2. The identified fragment and peptide hits provide a solid foundation for optimisation into more potent leads, paving the way for novel therapeutics candidates against ALT-positive cancers.