Assay development and inhibitor discovery targeting ADP- ribosyl hydrolysing macrodomains

ADP-ribosylation is a reversible post-translational modification in which ADP-ribose units are attached to target substrates. Macrodomains are ADP-ribosyl binding and/or hydrolysing modules that interact with ADP-ribosylated substrates, regulate downstream signalling pathways, and control the turnover of ADP-ribosylation. Macrodomains are widely distributed across all kingdoms of life and their biological functions have been implicated in several diseases and pathological processes, such as cancer, viral infections, and bacterial infections. From drug discovery perspective, macrodomains therefore represent interesting targets. However, their study is still hindered by the lack of selective inhibitors that can be used as chemical probes to support target validation and future therapeutic exploration.

This thesis focuses on the development of macrodomain inhibitors in the context of early-stage drug discovery. The work focuses on three macrodomain targets and is divided into three corresponding subprojects: Mycobacterium tuberculosis DNA ADP-ribosylglycohydrolase DarG, an antitoxin hydrolase in the DarTG toxin-antitoxin system; the chikungunya virus (CHIKV) macrodomain, which is important for viral replication and virulence; and human PARP9 macrodomain 1, which has been implicated in androgen receptor-related transcriptional regulation in prostate cancer.

In the first subproject, a biochemical screening assay based on fluorescence resonance energy transfer was developed for DarG, enabling high-throughput screening for DarG inhibitors. In the second subproject, a combination of high-throughput screening and structure-activity relationship studies led to the identification of CHIKV macrodomain inhibitors. In the third subproject, PARP9 macrodomain 1 inhibitors were identified by utilising previously developed SARS-CoV-2 macrodomain inhibitor scaffolds. The inhibitors identified in this work provide proof of concept for targeting macrodomains with small molecules and establish a foundation for futher hit optimisation.