A nonradiometric, high-throughput assay for poly(ADP-ribose) glycohydrolase (PARG): application to inhibitor identification and evaluation

doi:10.1016/j.ab.2004.04.032

Analytical Biochemistry

Volume 333, Issue 2, 15 October 2004, Pages 256-264

https://doi.org/10.1016/j.ab.2004.04.032 Get rights and content

Abstract

The enzyme poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of glycosidic bonds of ADP-ribose polymers, producing monomeric ADP-ribose units. Thus, in conjunction with poly(ADP-ribose) polymerase (PARP), PARG activity regulates the extent of in vivo poly(ADP-ribosyl)ation. Small molecule inhibitors of PARP and PARG have shown considerable promise in cellular models of ischemia–reperfusion injury and oxidative neuronal cell death. However, currently available PARG inhibitors are not ideal due to cell permeability, size, and/or toxicity concerns; therefore, new small molecule inhibitors of this important enzyme are sorely needed. Existing methodologies for in vitro assessment of PARG enzymatic activity do not lend themselves to high-throughput screening applications, as they typically use a radiolabeled substrate and determine product quantities through TLC analysis. This article describes a method whereby the ADP-ribose product of the PARG-catalyzed reaction is converted into a fluorescent dye. This highly sensitive and reproducible method is demonstrated by identifying two known PARG inhibitors in a 384-well plate assay and by subsequently determining IC₅₀ values for these compounds. Thus, this high-throughput, nonradioactive PARG assay should find widespread use in experiments directed toward identification of novel PARG inhibitors.

Section snippets

Reagents

PARG isolated from bovine thymus was purchased from Biomol (Plymouth Meeting, PA). ADP-HPD was purchased from Calbiochem (San Diego, CA). Ethacridine (6,9-diamino-2-ethoxyacridine) and ADP-ribose were purchased from Sigma (St. Louis, MO). XL1-Blue Escherichia coli was purchased from Stratagene (La Jolla, CA). A plasmid containing the full-length human PARP-1 gene (pSD6.3) [26] was the kind gift of Serge Desnoyers (University of Laval, Quebec, Canada). The 96- and 384-well fluorescence plates

Results and discussion

Through the action of PARG, the PAR polymer is catabolized into ADP-ribose monomer units. Thus, the immediate products of the PARG-catalyzed reaction are the ADP-ribose hemiacetal and the remaining polymer chain. Given that the standard PARG enzymatic assay employs a radioactive substrate and a TLC-based quantitation, we speculated that analysis of the ADP-ribose-reducing sugar might offer a practical, convenient, sensitive, nonradioactive, and high-throughput alternative for assessment of PARG

Acknowledgements

We thank Serge Desnoyers (University of Laval, Quebec, Canada) for the gift of the plasmid pSD6.3. This work was supported by the University of Illinois and a grant from the National Science Foundation (NSF-CAREER Award to Paul J. Hergenrother).

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Poly(ADP-ribose) glycohydrolase (PARG) plays an essential role in poly(ADP-ribose) (PAR) turnover, and thereby regulating DNA transactions, such as DNA repair, replication, transcription and recombination. Here, we examined the inhibitory activities of 6-hydroxy-3H-xanthene-3-one (HXO) derivatives and analyzed their binding modes in the active site of PARG by in silico docking study. Among the derivatives, Rose Bengal was found to be the most potent inhibitor of PARG and its halogen groups were revealed to cooperatively potentiate the inhibitory activity. Importantly, the binding mode of Rose Bengal occupied the active site of PARG revealed the presence of unique “Sandwich” residues of Asn869 and Tyr792, which enable the inhibitor to bind tightly with the active pocket. This sandwich interaction could stabilize the π-π interactions of HXO scaffold with Phe902 and Tyr795. In addition, to increase the binding affinity, the iodine and chlorine atoms of this inhibitor could contribute to the inducing of favorable disorders, which promote an entropy boost on the active site of PARG for structural plasticity, and making the stable configuration of HXO scaffold in the active site, respectively, as judged by the analysis of binding free energy. These results provide new insights into the active site of PARG and an additional opportunity for designing selective PARG inhibitors.
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Compared with PARP activity in somatic cells, PARG catalyzes the hydrolysis of glycosidic bonds of ADP-ribose polymers and produces monomeric ADP-ribose units, while PARP polymerizes monomeric ADP-ribose units. It is thought that the activity of PARG-mediated hydrolysis is stronger than that of PARP-mediated polymerization, and the inhibition of PARG activity is necessary for the measurement of PARP activity [16,17]. We determined the optimal ADP-HPD concentration by testing different concentrations in PARP activity assays.
Many studies have reported that human-induced pluripotent stem (hiPS)/embryonic stem (hES) cells have an exceptional ability to repair damaged DNA. Moreover, unlike differentiated cells, hES cells have features and mechanisms such as apoptosis-prone mitochondria, which prevent any changes in genetic information caused by DNA damage to be transmitted to their descendants. Type-A (dark) spermatogonia and cancer stem cells are thought to be dormant. However, hiPS/hES cells, the so-called stem cells used in regenerative medicine, generally have a high proliferative capacity. This suggests that in these cells, oxidative DNA damage associated with vigorous proliferation and DNA scission associated with replication occur frequently. Although pluripotency according to change of genomic structure is well studied, the change of DNA repair through reprogramming has not been well studied.
We analyzed the expression of DNA repair-related genes in hiPS cells using microarray and western blotting analyses and assessed changes in PARP activity through reprogramming.
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Poly(ADP-ribosyl)ation is a unique post-translational modification of proteins. The metabolism of poly(ADP-ribose) (PAR) is tightly regulated mainly by poly(ADP-ribose) polymerases (PARP) and poly(ADP-ribose) glycohydrolase (PARG). Accumulating evidence has suggested the biological functions of PAR metabolism in control of many cellular processes, such as cell proliferation, differentiation and death by remodeling chromatin structure and regulation of DNA transaction, including DNA repair, replication, recombination and transcription. However, the physiological roles of the catabolism of PAR catalyzed by PARG remain less understood than those of PAR synthesis by PARP. Noteworthy biochemical studies have revealed the importance of PAR catabolic pathway generating nuclear ATP via the coordinated actions of PARG and ADP-ribose pyrophosphorylase (ADPRPPL) for the driving of DNA repair and the maintenance of DNA replication apparatus while repairing DNA damage. Furthermore, genetic studies have shown the value of PARG as a therapeutic molecular target for PAR-mediated diseases, such as cancer, inflammation and many pathological conditions. In this review, we present the current knowledge of de-poly(ADP-ribosyl)ation catalyzed by PARG focusing on its role in DNA repair, replication and apoptosis. Furthermore, the induction of apoptosis code of DNA replication catastrophe by synthetic lethality of PARG inhibition and the recent progresses regarding the development of small molecule PARG inhibitors and their therapeutic potentials in cancer chemotherapy are highlighted in this review.
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There has been a lack of sensitive high-throughput biochemical assays available to measure PARG activity, and this may be associated with the failure to identify good quality small molecule inhibitors (tool compounds) that can be used to help explore the hypotheses surrounding PARG. Until now, options for PARG assays have involved radioactivity [14], surface plasmon resonance (SPR) [9], or heating to 110 °C to generate a measurable product [13], all of which are not amenable to HTS. Low-throughput enzyme-linked immunosorbent assay (ELISA)-based commercial kits that are set up in 96-well formats are too expensive and of insufficient sensitivity to support hit or lead identification for a drug discovery program.
Poly(ADP-ribose) (PAR) polymers are transient post-translational modifications, and their formation is catalyzed by poly(ADP-ribose) polymerase (PARP) enzymes. A number of PARP inhibitors are in advanced clinical development for BRCA-mutated breast cancer, and olaparib has recently been approved for BRCA-mutant ovarian cancer; however, there has already been evidence of developed resistance mechanisms. Poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of the endo- and exo-glycosidic bonds within the PAR polymers. As an alternative strategy, PARG is a potentially attractive therapeutic target. There is only one PARG gene, compared with 17 known PARP family members, and therefore a PARG inhibitor may have wider application with fewer compensatory mechanisms. Prior to the initiation of this project, there were no known existing cell-permeable small molecule PARG inhibitors for use as tool compounds to assess these hypotheses and no suitable high-throughput screening (HTS)-compatible biochemical assays available to identify start points for a drug discovery project. The development of this newly described high-throughput homogeneous time-resolved fluorescence (HTRF) assay has allowed HTS to proceed and, from this, the identification and advancement of multiple validated series of tool compounds for PARG inhibition.
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Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme that catalyzes the hydrolysis of poly(ADP-ribose) (PAR), an essential biopolymer that is synthesized by poly(ADP-ribose) polymerases (PARPs) in the cell. By regulating the hydrolytic arm of poly(ADP-ribosyl)ation, PARG participates in a number of biological processes, including the repair of DNA damage, chromatin dynamics, transcriptional regulation, and cell death. Collectively, the research investigating the roles of PARG in the cell has identified the importance of PARG and its value as a therapeutic target. However, the biological role of PARG remains less understood than the role of PAR synthesis by the PARPs. Further complicating the study of PARG is the existence of multiple PARG isoforms in the cell, the lack of optimal PARG inhibitors, and the lack of viable PARG-null animals. This review will present our current knowledge of PARG, with a focus on its roles in DNA-damage repair and cell death.

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A nonradiometric, high-throughput assay for poly(ADP-ribose) glycohydrolase (PARG): application to inhibitor identification and evaluation

Abstract

Section snippets

Reagents

Results and discussion

Acknowledgements

Trends Biochem. Sci

Biochim. Biophys. Acta

Exp. Cell Res

Exper. Cell Res

J. Biol. Chem

J. Biol. Chem

Mutat. Res

Pharmacol. Res

Biochim. Biophys. Acta

J. Biol. Chem

Biochem. Biophys. Res. Commun

Biochim. Biophys. Acta

Brain Res

Anal. Biochem

Anal. Biochem

J. Biochem. Biophys. Meth

Anal. Biochem

Anal. Biochem

Anal. Biochem

Anal. Biochem

Anal. Chim. Acta

Anal. Biochem