JultikaUniversity of Oulu repository

Diphtheria toxin-like human ADP-ribosyltransferase 3 (ARTD3) is a poorly characterized member of the ARTD superfamily of enzymes. ARTDs are enzymes that catalyze ADP-ribosylation, a reversible post-translational modification whereby growing chains of ADP-ribose is attached onto target proteins. ARTDs are involved in a wide variety of roles within the cell, including DNA damage repair and maintenance of genomic stability. ARTD3 is activated by damaged DNA and believed to play a role in double-strand break repair (DSBR) in the non-homologous end-joining (NHEJ) pathway, and has been implicated as a drug target in the treatment of cancer. In this study, an activity assay previously reported for other ARTDs was adopted for ARTD3 in order to screen the enzyme with two compound libraries consisting of 918 compounds in order to discover new potential chemical probes for the protein. The assay was validated through statistical criteria and its performance tested in a preliminary screen with 32 known ARTD inhibitors and their analogs. From all the compounds screened, 12 were ordered and 8 verified as hits in a counter-screen at 10 µM compound concentration. These 8 compounds were then further characterized with thermofluor to determine their ability to bind ARTD3 catalytic domain, fluorescence polarization (FP) to determine their effects on ARTD3’s binding affinity with DNA and the compounds had their IC50 values were measured. 6 compounds that exhibited interesting results in these experiments were classified according to their proposed mechanism of inhibition or compound class. These compounds consisted of two DNA chelating topoisomerase inhibitors, one insecticide, one heavy-metal containing polycyclic hydrocarbon (PHC) and two unclassified tumor suppressors. None of the compounds stabilized full-length ARTD3 or the catalytic fragment, but the DNA chelating agents lowered the binding affinity of ARTD3 to DNA. While no traditional ARTD inhibitor with potential for use as a chemical probe was discovered, the effects of DNA chelating agents on ARTD3’s binding affinity with DNA could be further investigated via binding affinity studies such as isothermal titration calorimetry (ITC). One of the unclassified tumor suppressors with decent potency (IC50: 2.7 µM) could also be tested against other ARTDs and if it was found to be selective against ARTD3, its binding to ARTD3 could be determined with co-crystallization and x-ray crystallography.