Activity-Based Screening Assay for Mono-ADP-Ribosylhydrolases

Abstract

ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes. This posttranslational modification is carried out by ADP-ribosyltransferases converting β-NAD+ into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR. The reverse reaction, release of ADP-ribose from the acceptor molecule, is catalyzed by ADP-ribosylhydrolases. Several hydrolases contain a macrodomain fold, and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation. Macrodomains have been linked to diseases such as cancer, making them potential drug targets. Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases, and here we describe an inhibitor screening assay against mono-ADP-ribosylhydrolyzing enzymes. The activity-based assay uses an α-NAD+, anomer of β-NAD+, which is accepted as a substrate by MacroD1, MacroD2, and ARH3 due to its resemblance to the protein-linked ADP-ribose. The amount of α-NAD+ present after hydrolysis is measured by chemically converting it on a microtiter plate to a fluorescent compound. We optimized the assay for MacroD2 and performed a proof-of-concept compound screening. Three compounds were identified as screening hits with micromolar potency. However, further characterization of the compounds identified them as protein destabilizers, excluding further follow-up studies. Validation and screening demonstrated the usability of the in vitro assay for MacroD2, and we also demonstrate the applicability of the assay as a tool for other human ADP-ribosylhydrolases.

Discovery of Drug-Like Ligands for the Mac1 Domain of SARS-CoV-2 Nsp3

“…3 The most attractive alternative to a DSF binding assay would be an enzyme assay that monitors the ability of Mac1 to hydrolyze ADPr-based substrates, which are presently under development. 24 The most intriguing hits in DSF screens were the steroids and telmisartan (Fig. 3).…”

Multiple crystal forms of human MacroD2

“…Immediately after screening, we tried to obtain a complex structure of MacroD2 with a hit compound, Bay 11-7085, by soaking the MacroD2 crystals with the compound. When we subsequently performed quality analysis for the compound by mass spectrometry we did not detect the intact compound, which was likely to be owing to degradation (Wazir et al, 2020). We also did not see any complete electron density for Bay 11-7085 in the crystal structures.…”

“…The crystals were soaked in the cryoprotectant for 2-3 min before flash-cooling with liquid nitrogen. We also supplemented the cryoprotectant with 1-3 mM Bay 11-7085, a small-molecule compound obtained as a screening hit from our recent study (Wazir et al, 2020). X-ray diffraction data were collected from the crystals on beamlines ID23-1 at ESRF, Grenoble, France and I04-1 at Diamond Light Source (DLS), Oxfordshire, UK.…”

“…We used the same MacroD2 construct for both crystallization and inhibitor screening (Wazir et al, 2020). Immediately after screening, we tried to obtain a complex structure of MacroD2 with a hit compound, Bay 11-7085, by soaking the MacroD2 crystals with the compound.…”

“…In addition, they can hydrolyze O-acetyl-ADP-ribose (OAADPr) and other nicotinamide adenine dinucleotide (NAD + ) metabolites to generate free ADPr (Chen et al, 2011). Subsequently, it has been shown that MacroD2 can also hydrolyze -NAD + as a substrate (Stevens et al, 2019;Wazir et al, 2020). In addition to hydrolyzing MAR from the target proteins, it has been reported that ADP-ribosylation of RNA and DNA can also be reversed by MacroD1, MacroD2, TARG1, poly-ADP-riboseglycohydrolase (PARG) and ADP-ribosyl-acceptor-hydrolases (ARHs) (Sakthianandeswaren et al, 2018;Munnur et al, 2019;Munnur & Ahel, 2017).…”