Parviainen, Tomi A OTomi A OParviainenDuong, Men Thi HoaiMen Thi HoaiDuongPääkkönen, JohanJohanPääkkönenBurdova, KamilaKamilaBurdovaKuttichova, BarboraBarboraKuttichovaHanzlikova, HanaHanaHanzlikovaLehtiö, LariLariLehtiöHeiskanen, Juha PJuha PHeiskanen2025-09-162025-09-162025-10-17https://boris-portal.unibe.ch/handle/20.500.12422/218885Poly-ADP-ribosylation at sites of DNA damage, catalyzed by PARP enzymes, activates the DNA damage response, chromatin remodeling, and DNA repair. The modification is reversed by two enzymes in humans: PARG, which efficiently hydrolyzes the poly-ADP-ribose chains, and ARH3, which is the key enzyme for removing the last proximal mono-ADP-ribose from serine residues. While inhibitor development has largely focused on PARPs and PARG, no potent and selective inhibitors for ARH3 are currently available. We optimized a FRET-based competition assay for ARH3 and carried out high-throughput screening of small-molecule inhibitors. One hit compound, 1, with a potency of 22 μM was discovered, and through structure-activity relationship studies and synthesis, we improved its potency 10-fold to 2 μM (compound 27, MDOLL-0286). We demonstrate that the compound inhibits ARH3's poly-ADP-ribose hydrolytic activity on cellular substrates. Intriguingly, it does not effectively inhibit the hydrolysis of mono-ADP-ribosylation from natural protein substrates. This is despite the fact that the cocrystal structure of compound 1 bound to ARH3 reveals its overlap with the enzyme's ADP-ribose binding site, agreeing with the competition in the FRET assay. The first experimental ARH3 inhibitor complex provides a valuable starting point for developing more potent chemical probes to study DNA damage response mechanisms in the future.en600 - Technology::630 - Agriculturearticle10.48620/913454095234210.1021/acschembio.5c00461