Protein post-translational modifications (PTMs) are important for regulating protein function. One such modification, known as ADP-ribosylation, has been shown to be involved in the regulation of various biological processes, and to play a crucial role in the development of pathological conditions and diseased phenotypes, such as cancer. However, the lack of appropriate tools for identifying the protein targets this PTM is found on, in a cellular context and in vivo, limits the identification of novel, biologically relevant therapeutic targets.
This thesis focuses on the development, synthesis and biological validation of novel chemical probes for the identification of intracellular physiological ADP-ribosylation targets. Specifically, cell-permeable clickable NAD+ precursor probes were designed and synthesised. Their utilisation in the cellular NAD+ biosynthesis pathway, for intracellular metabolic conversion into the corresponding NAD+ analogues, was investigated through a combination of metabolomics and proteomics methods in several cell lines. Metabolomics analysis revealed that the precursor probes 6Yn-Ad (25) and 6Yn-Pro (47) were successfully converted intracellularly into the corresponding 6Yn-NAD+ (3) analogue in multiple cell lines. Subsequent analysis by proteomics demonstrated the utilisation of 6Yn-Pro (47) for labelling and enrichment of ADP-ribosylated proteins through its metabolic conversion into 6Yn-NAD+ (3), which resulted in the identification of automodified PARP1 and of multiple known ADP-ribosylated proteins in HEK293T cells.
This work therefore provides novel chemical probes for identification of intracellular ADP-ribosylation targets. Ultimately, their use will provide fundamental insights into the role of ADP-ribosylation in biological processes and its contribution to the development of diseased states.