AI26 inhibits the ADP-ribosylhydrolase ARH3 and suppresses DNA damage repair

Abstract

The ADP-ribosylhydrolase ARH3 plays a key role in DNA damage repair, digesting poly(ADP-ribose) and removing ADP-ribose (ADPR) from serine residues of the substrates. Specific inhibitors that selectively target ARH3 would be a useful tool to examine DNA damage repair as well as a possible strategy for tumor suppression. However, efforts to date have not identified any suitable compounds. Here, we used in silico and biochemistry screening to search for ARH3 inhibitors. We discovered a small molecule compound named ARH3 Inhibitor 26 (AI26) as, to our knowledge, the first ARH3 inhibitor. AI26 binds to the catalytic pocket of ARH3 and inhibits the enzymatic activity of ARH3 with an estimated IC50 ~ 2.41 μM in vitro. Moreover, hydrolysis of DNA damage-induced ADP-ribosylation was clearly inhibited when cells were pretreated with AI26, leading to defects in DNA damage repair. And tumor cells with DNA damage repair defects were hypersensitive to AI26 treatment as well as combinations of AI26 and other DNA-damaging agents such as camptothecin and doxorubicin. Collectively, these results reveal not only a chemical probe to study ARH3-mediated DNA damage repair but also a chemotherapeutic strategy for tumor suppression.

A molecular toolbox for ADP-ribosyl binding proteins

“…The high complexity of ADP-ribosyl associated pathways makes the involved proteins notoriously hard to study (Bonfiglio et al, 2020;Lüscher et al, 2018). While potent and specific inhibitors against many of the ADP-ribosyl transferases fundamentally helped to broaden our understanding of these enzymes (Durkacz et al, 1980;Huang et al, 2009;Kirby et al, 2018;Venkannagari et al, 2016;Wang et al, 2018), such inhibitors against ADP-ribosyl readers and erasers are still scarcely available or in early stages of development (Harrision et al, 2020;James et al, 2016;Liu et al, 2020;Palazzo and Ahel, 2018;Schuller et al, 2017). It was suggested that a possible bottleneck for the discovery of inhibitors is due to the lack of accessible high-throughput technologies (Schuller et al, 2017).…”

Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes

“…Our data clearly demonstrated that the NCAG assay can selectively detect nanomolar concentration of protein-free ADP-ribose in vitro and suggest this NCAG assay can be used as an effective ADP-ribose sensor both in vitro and in cell extracts. Finally, this NCAG assay was developed in a multi-well format and therefore is suitable for high-throughput screening of small-molecule probes that specifically target the biomedically important ADP-ribosylation reversal enzymes, such as PARG [ 51 ], ARH3 [ 35 , 52 ], ARH1 [ 26 , 27 ], MacroD1/D2 [ 23 , 24 ], TARG1 [ 49 ], and SARS-CoV-2 Mac1 [ 53 ].…”

“…developed in a multi-well format and therefore is suitable for high-throughput screening of small-molecule probes that specifically target the biomedically important ADP-ribosylation reversal enzymes, such as PARG [51], ARH3 [35,52], ARH1 [26,27], MacroD1/D2 [23,24], TARG1 [49], and SARS-CoV-2 Mac1 [53].…”

The Making and Breaking of Serine-ADP-Ribosylation in the DNA Damage Response

“…ARH3 deficiency is therefore a potential novel PARP1 inhibitor resistance mechanism, similar to what has been described for loss of PARG, which causes PARP inhibitor resistance in cancer cells due to stabilization of the PARylation signal ( Gogola et al, 2018 ). Moreover, pharmacological inhibition of ARH3 appears to negatively impact DNA damage repair ( Liu et al, 2020 ). With several lines of evidence pointing at a protective role of ARH3 against neurodegeneration there exists a further pathway to therapeutic application of ARH3 antagonists that can be explored in the future ( Danhauser et al, 2018 ; Ghosh et al, 2018 ; Mashimo et al, 2019 ).…”