Assay development and inhibitor discovery for human and bacterial mono-ADP-ribosyltransferases

ADP-ribosylation is a biochemical modification generally thought to have evolved in an interspecies arms race in bacteria where both bacterial toxins and host defence mechanisms exploit ADP‑ribosylation to modify target molecules. The defensive features of these enzymes are still present in human mono-ADP-ribosyltransferases, which are strongly associated with antiviral activity and regulation of immune response. Increasing evidence has linked many of these enzymes to clinically relevant biological processes, including allergic responses, tumourigenesis and cancer-related immunosuppressive effects. These findings have raised interest in the development of potent and selective mono-ADP-ribosyltransferase inhibitors, which has culminated in the recent clinical candidates for the inhibition of PARP7 and PARP14.

In this work, a homogeneous, cost‑effective and easily accessible activity assay was developed to enable the discovery of potent human mono‑ADP‑ribosyltransferase inhibitors and the family‑wide assessment of their selectivity profiles. The assay was subsequently applied to develop PARP10 inhibitors with low nanomolar IC50 values and greatly improved selectivity over other mono-ADP-ribosyltransferases, representing a significant advance over previously reported submicromolar inhibitors. The target protein PARP10 has emerged as a potential drug target due to its roles in DNA repair mechanisms, tumour formation and upregulation in various cancers, including acute myeloid leukaemia where its high expression level is associated with poor clinical outcomes.

Furthermore, a focused library of human ADP-ribosyltransferase-targeted inhibitors was screened to discover the first inhibitors for several bacterial Arr enzymes that contribute to the emerging antibiotic resistance crisis by ADP-ribosylating and thereby inactivating rifamycin antibiotics. The discovered inhibitors, complemented by structural and cellular studies, provide a foundation for future strategies to overcome rifamycin resistance.