Discovery of inhibitors targeting mono-ADP-ribosylhydrolysing macrodomains

Post-translational modifications play significant roles in modulating functions of different proteins by adding covalent functional groups to the residues thereby, regulating vital cellular processes. ADP-ribosylation is one such modification carried out by PARP enzymes of the diphtheria toxin-like ADP-ribosyltransferases (ARTDs). PARPs catalyse the cleavage of β-NAD+ to nicotinamide and ADP-ribose (ADPr), and transfer ADPr to the target proteins, DNA or RNA. PARPs can either catalyse mono- or poly-ADP-ribosylation of these targets. The reverse reaction, removal of the modification from the modified macromolecules is catalysed by proteins called ADP-ribosylhydrolases, which often contain a macrodomain fold. In humans, there are 16 macrodomains that exist as a stand-alone domain or as part of a multidomain protein. These macrodomains vary in activities from reading to erasing mono- or poly-ADP-ribosylation. Dysregulation of function, overexpression and in some cases mutations link macrodomains directly to several diseases for instance: cancer, neurodegeneration, developmental disorders and viral infections. Due to that, macrodomains have been suggested to be potential drug targets. In the past, members of ARTD family enzymes have been extensively studied because of their involvement in DNA repair mechanisms. Notably, some of the ARTD family enzymes have been targeted by the approved drugs, but in contrast inhibitor development for macrodomains is clearly behind.
This thesis focuses on two human enzymes MacroD1, and MacroD2, and a viral macrodomain Mac1 of non-structural protein 3 from SARS-CoV-2 that has caused the recently emerged pandemic (COVID-19). The aim of the study was to discover selective compounds inhibiting these hydrolysing macrodomains by screening compound libraries with the developed activity and binding assays. In order to further validate the identified hit compounds, a range of biochemical and biophysical techniques were utilized that also assess their initial quality as a chemical probe. As a result, a promising hit compound MDOLL-0169 inhibiting SARS-CoV-2 Mac1 was discovered. MDOLL-0169 was profiled against all the human and a panel of viral macrodomains. It was confirmed to be selective towards SARS-CoV-2 Mac1 among tested viral proteins, but it also showed inhibition with a 5-fold lower potency for human PARP9 MD1. MDOLL-0169 was co-crystallized with SARS-CoV-2 Mac1 to determine its binding mode and interactions with the catalytic site of the enzyme. The structural studies helped in understanding the important parts of the compound scaffold, which can be further modified to develop selective and efficient inhibitors of SARS-CoV-2 Mac1.