A Simple Method to Study ADP-Ribosylation Reversal: From Function to Drug Discovery

Chemoenzymatic and Synthetic Approaches To Investigate Aspartate- and Glutamate-ADP-Ribosylation

“…Human ADP-ribosylhydrolases are known to selectively hydrolyze certain linkage types, with the MacroD1/D2 enzymes being responsible for reversing Asp/Glu-ADPr . We sought to demonstrate the utility of our Asp/Glu-ADPr-containing peptides in high-throughput ADP-ribosylhydrolase activity assays, which included the enzymes ARH1, ARH3, MacroD1, MacroD2, and PARG. , As expected, ARH3 and MacroD1/D2 exhibited potent Asp/Glu-ADPr hydrolysis activity (Figure a). We note that PARG, while known to be selective for ribose-ribose bonds, is able to hydrolyze the Asp/Glu-ADPr linkage in the context of the peptide substrates at the concentration tested (1 μM).…”

“…Future kinetic and structural analysis with homogenously ADP-ribosylated substrates will be critical to determine the preferred Asp/Glu-ADPr linkage isomer for MacroD1/D2 and ARH3 activity. Overall, integration of homogenously modified peptides into these assay platforms may help to accelerate ongoing ADP-ribosylhydrolase inhibitor discovery efforts. , …”

4-Thioribose Analogues of Adenosine Diphosphate Ribose (ADPr) Peptides

“…Organic synthesis has been used to procure ADPr fragments, as well as analogues and mimics equipped with a tag or fluorescent label, for biological testing . The glycosidic linkage in serine ADPr fragments represents a labile functionality that is potentially vulnerable to cleavage or anomerization reactions . We reasoned that close mimetics, in which the ribosyl serine bond is stabilized, can present valuable tool compounds for both functional and structural studies.…”

“…With the required building block 1 in hand, the solid phase assembly of mono-4- S -ADP-ribosylated peptide 19 , derived from the N-terminus of histone H2B, was undertaken. , The standard Fmoc-based methodology was combined with amino acid building blocks carrying acid sensitive protecting groups on the side chains (Mtt for lysine and Trt for serine and threonine). As depicted in Scheme , the acid labile TentaGel S AC resin, preloaded with glycine, was elongated using the selected protected amino acid building blocks to generate full-length ribosylated immobilized peptide 14 .…”

“…ARH3 is known to hydrolyze the O-glycosidic linkage of Ser-ADPr and indeed could convert the Ser-ADPr control peptide efficiently (Figure ). In contrast, our preliminary screen using a panel of human hydrolases utilizing an established luminescence reporter assay showed only marginal activity of ARH3 against Ser-thio-ADPr. While a complete hydrolysis of native Ser-ADPr was achieved in 45 min, only approximately 3% of the Ser-thio-ADPr counterpart was removed in the same amount of time.…”