A molecular toolbox for ADP-ribosyl binding proteins

Assay technologies facilitating drug discovery for ADP‐ribosyl writers, readers and erasers

“…Nanoluciferase coupled macrodomains produced in E. coli; some hydrolysis activity present; no sample separation; quantification requires optimization of the detection buffer; blots prepared with an acoustic dispenser allow high density formats [22] and product before the measurement. Suitability for inhibitor screening was also tested and IC 50 values comparable to the literature for selected compounds were obtained.…”

“…[47] Alternatively, mono-or PAR groups can be detected with highly sensitive nanoluciferase linked macrodomains in HT dot blot format. [22]…”

“…TR-FRET 1536-well plates Activity-based; HTRF; biotinylated NAD + and PARP1 automodification; Streptavidin-europium and anti-His MAb-XL665 required; homogeneous [61] Luminescence Dot blots Activity-based; PAR-antibody needed; described for PARG enzyme degrading PAR; not demonstrated for HT [62] Fluorescence 384-well plates Activity-based; chemical conversion of α-NAD + ; can be applied for enzymes consuming α-NAD + ; inexpensive generally available reagents; protein concentration needed may limit sensitivity; requires a fume hood for chemical conversion; homogeneous [63] AlphaScreen 384-well plates Activity-based; amplified luminescence; enzymes must accept biotin-ADP-ribosyl as a substrate; commercial beads needed; requires a plate reader with an AlphaScreen module; homogeneous [64] AlphaScreen 384-well plates Binding assay; amplified luminescence; PARP catalytic domain sequences in pNIC-Bio3 vector co-expressed with BirA; commercial beads needed; requires a plate reader with an AlphaScreen module; homogeneous [65] AlphaScreen 384-well plates Binding assay; amplified luminescence; synthesized biotinylated peptide with a ring-opened ADP-ribose required; commercial beads needed; requires a plate reader with an AlphaScreen module; homogeneous; applicable to readers and erasers [56] Fluorescence polarization 384-well plates Binding assay; synthesized fluorescent ADP-ribose dimer required; for readers or inactivated eraser mutants; homogeneous [66] FRET, multiple techniques 384-well plates Binding assay; GAP-tag attached to proteins and a cysteine labeled with ADP-ribose using a toxin; applicable to readers and erasers. [22] Multiple techniques Not defined Enzymatic labelling of MAR and PAR; OAS1 enzyme and labelled dATP needed [67] A HT activity assay for PARG and ARH3 reported by Stowell et al [61] uses biotinylated NAD + to automodify His-tagged PARP1. PARG activity on degrading the PAR chains subsequently causes a loss of the timeresolved FRET signal generated by anti-his antibody coupled to a longlived fluorescent dye XL665 and biotin-europium cryptate.…”

“…[3] Later, an assay method to detect PARP protein and PARP activity directly from nitrocellulose blots using radiolabeled substrate [ 32 P]NAD + was developed by Simonin et al [15] The low-throughput Western blot or dot blot methods have been used with other detection methods including biotinylated NAD + analogs [16] and streptavidin coupled HRP, PAR H10, or MAR/PAR antibodies [17][18][19] and various ADP-ribosylation affinity reagents developed recently. [20][21][22][23] It is noteworthy that when using purified proteins, it is possible to see the disappearance of a protein band upon addition of NAD + with a simultaneous appearance of a high molecular weight smear on a coomassie stained SDS-PAGE. This is a simple and widely available qualitative method of assessing poly-ART activity of enzymes capable of forming PAR chains [24] and can similarly be used to measure PAR hydrolysis activity.…”

“…Toxins however are sequence specific and modify typically only certain residues in the target proteins and therefore MS spectra is easy to interpret. [22] Similarly, MS has been used to detect hydrolysis of MAR and PAR as well as metabolites like O-acetyl-ADP-ribose. [27,28] Most of the methods described above are low-throughput and measurement of each sample requires more preparation and analysis time in contrast to if the samples could be measured in a highly parallel fashion.…”