Human poly(ADP-ribose) Glycohydrolase Research Articles

Abstract 3366: FoRx-06-428 is a novel PARG inhibitor with potent anti-tumor efficacy in preclinical cancer models

Abstract The formation of poly(ADP-ribose) (PAR) chains from NAD+ precursors by PARP enzymes is a characteristic posttranslational modification during DNA damage repair. PAR chains function as docking platforms for DNA repair proteins that are required to resolve DNA lesions. Equally important is the subsequent removal of PAR chains from protein substrates to conclude the repair process and restore genomic integrity. Poly(ADP-ribose) glycohydrolase (PARG) is the key enzyme in this process and has recently garnered significant attention as a novel cancer target. FoRx-06-428 is a novel, orally bioavailable and highly potent, low molecular weight PARG inhibitor. It binds to the catalytic domain of human PARG with high affinity, as evidenced by surface plasmon resonance (SPR), and displays potent inhibition of enzymatic activity. FoRx-06-428 shows selective reduction of viability in a panel of cancer cell lines highlighting therapeutic potential in multiple cancer indications. Pharmacological inhibition of PARG leads to accumulation of PAR chains and is indicative of target engagement, accompanied by modulation of DNA damage response markers, such as phosphorylation of replication protein A (pRPA) and phosphorylation of H2AX (γH2AX). Compelling anti-tumor efficacy as single agent was observed in several xenograft models (breast and gastric cancer) with excellent tolerability. Dose-dependent accumulation of PAR chains was demonstrated in vivo and constitutes a tractable target engagement biomarker for clinical exploration. Sensitivity to PARG inhibition is partly driven by distinct genomic instabilities, such as defects in homologous recombination (HR) repair, however the activity of PARG inhibitors is also influenced by aberrations in other cancer-relevant pathways. This report represents the first disclosure of the pharmacological characterization of FoRx-06-428 and highlights the value of PARG inhibition as a novel targeted approach to treat DNA repair-deficient cancers. Citation Format: Olivier Querolle, Ulrich Lücking, Luca Iacovino, Alena Freudenmann, Giacomo Rossetti, Marina Gysin, Serena Bologna, Vasilis Dionellis, Marta Malattia, Alessandro Potenza, Nicolas Bocquet, Irena Konstantinova, Andreas Goutopoulos, Sotirios Sotiriou, Thanos Halazonetis, Tarig Bashir, Frank Zenke. FoRx-06-428 is a novel PARG inhibitor with potent anti-tumor efficacy in preclinical cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3366.

Purification of Recombinant Human PARG and Activity Assays.

The purification of poly(ADP-ribose) glycohydrolase (PARG) from overexpressing bacteria Escherichia coli is described here as a fast and reproducible one chromatographic step protocol. After cell lysis, GST-PARG-fusion proteins from the crude extract are affinity purified by a glutathione 4B sepharose chromatographic step. The PARG proteins are then freed from their GST-fusion by overnight enzymatic cleavage using the preScission protease. As described in the protocol, more than 500μg of highly active human PARG can be obtained from 1.5L of E. coli culture.

Cell-Active Small Molecule Inhibitors of the DNA-Damage Repair Enzyme Poly(ADP-ribose) Glycohydrolase (PARG): Discovery and Optimization of Orally Bioavailable Quinazolinedione Sulfonamides

DNA damage repair enzymes are promising targets in the development of new therapeutic agents for a wide range of cancers and potentially other diseases. The enzyme poly(ADP-ribose) glycohydrolase (PARG) plays a pivotal role in the regulation of DNA repair mechanisms; however, the lack of potent drug-like inhibitors for use in cellular and in vivo models has limited the investigation of its potential as a novel therapeutic target. Using the crystal structure of human PARG in complex with the weakly active and cytotoxic anthraquinone 8a, novel quinazolinedione sulfonamides PARG inhibitors have been identified by means of structure-based virtual screening and library design. 1-Oxetan-3-ylmethyl derivatives 33d and 35d were selected for preliminary investigations in vivo. X-ray crystal structures help rationalize the observed structure-activity relationships of these novel inhibitors.

Synthesis of dimeric ADP-ribose and its structure with human poly(ADP-ribose) glycohydrolase.

Poly(ADP-ribosyl)ation is a common post-translational modification that mediates a wide variety of cellular processes including DNA damage repair, chromatin regulation, transcription, and apoptosis. The difficulty associated with accessing poly(ADP-ribose) (PAR) in a homogeneous form has been an impediment to understanding the interactions of PAR with poly(ADP-ribose) glycohydrolase (PARG) and other binding proteins. Here we describe the chemical synthesis of the ADP-ribose dimer, and we use this compound to obtain the first human PARG substrate-enzyme cocrystal structure. Chemical synthesis of PAR is an attractive alternative to traditional enzymatic synthesis and fractionation, allowing access to products such as dimeric ADP-ribose, which has been detected but never isolated from natural sources. Additionally, we describe the synthesis of an alkynylated dimer and demonstrate that this compound can be used to synthesize PAR probes including biotin and fluorophore-labeled compounds. The fluorescently labeled ADP-ribose dimer was then utilized in a general fluorescence polarization-based PAR-protein binding assay. Finally, we use intermediates of our synthesis to access various PAR fragments, and evaluation of these compounds as substrates for PARG reveals the minimal features for substrate recognition and enzymatic cleavage. Homogeneous PAR oligomers and unnatural variants produced from chemical synthesis will allow for further detailed structural and biochemical studies on the interaction of PAR with its many protein binding partners.

Structures of the Human Poly (ADP-Ribose) Glycohydrolase Catalytic Domain Confirm Catalytic Mechanism and Explain Inhibition by ADP-HPD Derivatives

Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG). Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR), adenosine 5′-diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors.

ADP-ribosylhydrolase 3 (ARH3), Not Poly(ADP-ribose) Glycohydrolase (PARG) Isoforms, Is Responsible for Degradation of Mitochondrial Matrix-associated Poly(ADP-ribose)

Important cellular processes are regulated by poly(ADP-ribosyl)ation. This protein modification is catalyzed mainly by nuclear poly(ADP-ribose) polymerase (PARP) 1 in response to DNA damage. Cytosolic PARP isoforms have been described, whereas the presence of poly(ADP-ribose) (PAR) metabolism in mitochondria is controversial. PAR is degraded by poly(ADP-ribose) glycohydrolase (PARG). Recently, ADP-ribosylhydrolase 3 (ARH3) was also shown to catalyze PAR-degradation in vitro. PARG is encoded by a single, essential gene. One nuclear and three cytosolic isoforms result from alternative splicing. The presence and origin of a mitochondrial PARG is still unresolved. We establish here the genetic background of a human mitochondrial PARG isoform and investigate the molecular basis for mitochondrial poly(ADP-ribose) degradation. In common with a cytosolic 60-kDa human PARG isoform, the mitochondrial protein did not catalyze PAR degradation because of the absence of exon 5-encoded residues. In mice, we identified a transcript encoding an inactive cytosolic 52-kDa PARG lacking the mitochondrial targeting sequence and a substantial portion of exon 5. Thus, mammalian PARG genes encode isoforms that do not catalyze PAR degradation. On the other hand, embryonic fibroblasts from ARH3(-/-) mice lack most of the mitochondrial PAR degrading activity detected in wild-type cells, demonstrating a potential involvement of ARH3 in PAR metabolism.

ARH3 catalyzes degradation of mitochondrial matrixaccumulated Poly (ADP‐ribose)

Poly (ADP‐ribose) (PAR) metabolism, which is mainly mediated by members of the Poly (ADP‐ribose) polymerase (PARP) and Poly (ADP‐ribose) glycohydrolase (PARG) families, is involved in several cellular pathways including DNA stability, apoptosis, and mitosis. PARG, initially identified as a PAR degrading‐enzyme, exists as multiple isoforms with different subcellular localization, resulting from alternative splicing of the single gene. ADP‐ribosylhydrolase 3 (ARH3) also has PAR‐degrading activity. The enzymes participating in a putative PAR metabolism in mitochondria are still unknown.An alternatively spliced, small human PARG isoform (PARG55) and ARH3 are located in mitochondria owing to a mitochondrial targeting sequence at their N‐terminus. Using a modified PARP1 construct to produce PAR selectively in mitochondria, ARH3‐deficient cells were shown to have more mitochondrial PAR accumulation than wild‐type cells. Overexpression of ARH3 decreased mitochondrial PAR content, while overexpression of PARG55 had no effect. PARG55 was shown to lack exon5, which is essential for PAR‐degrading activity. These data suggest that ARH3 rather than PARG55 is the primary enzyme involved in catalyzing the degradation of PAR in mitochondria.

PARG: A Macrodomain in Disguise

Our understanding of poly-ADP-ribosylation as a posttranslational modification was limited by the lack of structural information on poly-ADP-ribose (PAR) hydrolysing enzymes. A recent study in Nature (Slade etal., 2011) reports the structure of PAR glycohydrolase (PARG), revealing unexpected similarity to the ubiquitous ADP-ribose-binding macrodomains.

Bacterial production of recombinant human poly(ADP-ribose) glycohydrolase

Poly(ADP-ribosyl)ation, which is mainly involved in DNA repair and replication, is catalyzed mainly by poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Although recombinant human PARP-1 (hPARP-1) is commercially available, there are no reports on the preparation of recombinant human PARG (hPARG). Here, we report the efficient expression and purification of a recombinant hPARG-catalytic domain (hPARG-CD) from Escherichia coli ( E. coli). hPARG-CD was expressed as a fusion protein with a glutathione S-transferase (GST) tag at the N-terminus and a hexahistidine (6His) tag at the C-terminus. Both high cell density and low temperature culture conditions were important for the maximum production of soluble recombinant hPARG-CD. After sequential affinity chromatography using immobilized metal affinity resin and glutathione-Sepharose (GSH-Sephasrose), more than 95% pure recombinant hPARG-CD was obtained with a yield of approximately 2 mg per 1 L of E. coli culture medium. The km and Vmax values of purified recombinant hPARG-CD were 9.0 μM and 35.6 μmol/min/mg protein, respectively. These kinetic values were similar to those of purified endogenous hPARG reported previously. Furthermore, the recombinant hPARG-CD was inhibited by known PARG inhibitors such as adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), eosin Y, and phloxine B. These results show that the recombinant hPARG-CD is useful to search for specific inhibitors and to elucidate the regulatory mechanisms of hPARG.

Characterization of the promoter region of the human PARG gene and its response to PU.1 during differentiation of HL‐60 cells

The metabolism of poly(ADP-ribose) plays important roles in the nuclear function of mammalian cells. Previously, we analyzed expression of the poly(ADP-ribose) glycohydrolase (PARG) gene during HL-60 cell differentiation and found that expression was greatly reduced by 4 h after 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment and returned to the initial level within 20 h. In the present study, a 2.1-kb fragment of the 5'-flanking (promoter) region of the human PARG gene was isolated from the HL-60 genome by polymerase chain reaction and ligated into a luciferase-expression vector, pGL3, to generate the pPARG-Luc#2 reporter plasmid. Deletion analysis revealed that a 75-nt sequence is required for basal promoter activity and TPA responsiveness. Mutations in this 75-nt sequence reduced promoter activity and the TPA response of HL-60 cells. TFSEARCH analysis revealed that Ets family binding motifs are located in the 75-nt sequence. Chromatin immunoprecipitation assay, electrophoretic mobility shift assay and competition analysis indicated that PU.1 (Spi-1) binds to the 75-nt sequence. Moreover, co-transfection of HL-60 cells with a PU.1 expression plasmid and pPARG-Luc indicated that PU.1 down-regulate the PARG promoter. These results suggest that PARG gene expression is modulated by PU.1 during TPA-induced differentiation of HL-60 cells.

Two small enzyme isoforms mediate mammalian mitochondrial poly(ADP-ribose) glycohydrolase (PARG) activity

Poly(ADP-ribose)glycohydrolase (PARG) is the major enzyme capable of rapidly hydrolyzing poly(ADP-ribose) (PAR) formed by the diverse members of the PARP enzyme family. This study presents an alternative splice mechanism by which two novel PARG protein isoforms of 60 kDa and 55 kDa are expressed from the human PARG gene, termed hPARG60 and hPARG55, respectively. Homologous forms were found in the mouse (mPARG63 and mPARG58) supporting the hypothesis that expression of small PARG isoforms is conserved among mammals. A PARG protein of ∼60 kDa has been described for decades but with its genetic basis unknown, it was hypothesized to be a product of posttranslational cleavage of larger PARG isoforms. While this is not excluded entirely, isolation and expression of cDNA clones from different sources of RNA indicate that alternative splicing leads to expression of a catalytically active hPARG60 in multiple cell compartments. A second enzyme, hPARG55, that can be expressed through alternative translation initiation from hPARG60 transcripts is strictly targeted to the mitochondria. Functional studies of a mitochondrial targeting signal (MTS) in PARG exon IV suggest that hPARG60 may be capable of shuttling between nucleus and mitochondria, which would be in line with a proposed function of PAR in genotoxic stress-dependent, nuclear–mitochondrial crosstalk.

Altered DNA damage response in Caenorhabditis elegans with impaired poly(ADP-ribose) glycohydrolases genes expression

Poly(ADP-ribosyl)ation is one of the first cellular responses induced by DNA damage. Poly(ADP-ribose) is rapidly synthesized by nick-sensor poly(ADP-ribose) polymerases, which facilitate DNA repair enzymes to process DNA damage. ADP-ribose polymers are rapidly catabolized into free ADP-ribose units by poly(ADP-ribose) glycohydrolase (PARG). The metabolism of poly(ADP-ribose) is a well-defined biochemical process for which a physiological role in animals is just beginning to emerge. Two Caenorhabditis elegans PARGs, PME-3 and PME-4, have been cloned by our group. The pme-3 gene encodes an enzyme of 89 kDa having less than 18% overall identity with human PARG but 42% identity with the PARG signature motif. The pme-4 gene codes for a PARG of 55 kDa with approximately 22% overall identity with human PARG and 40% identity with the PARG signature motif. Two alternatively spliced forms of PME-3 were identified with an SL1 splice leader on both forms of the mRNA and were found to be expressed throughout the worm's life cycle. Similarly, pme-4 was shown to be expressed in all developmental stages of the worm. Recombinant enzymes that were expressed in bacteria displayed a PARG activity that may partly account for the PARG activity measured in the total worm extract. Reporter gene analysis of pme-3 and pme-4 using a GFP fusion construct showed that pme-3 and pme-4 are mainly expressed in nerve cells. PME-3 was shown to be nuclear while PME-4 localized to the cytoplasm. Worms with pme-3 and pme-4 expression knocked-down by RNAi showed a significant sensitivity toward ionizing radiations. Taken together, these data provide evidence for a physiological role for PARGs in DNA damage response and survival. It also shows that PARGs are evolutionarily conserved enzymes and that they are part of an ancient cellular response to DNA damage.

Human poly(ADP-ribose) glycohydrolase is expressed in alternative splice variants yielding isoforms that localize to different cell compartments

Poly(ADP-ribose) glycohydrolase (PARG) is the only protein known to catalyze hydrolysis of ADP-ribose (ADPR) polymers to free ADP-ribose. While numerous genes encode different poly(ADP-ribose) polymerases (PARPs) that all synthesize ADP-ribose polymer, only a single gene coding for PARG has been detected in mammalian cells. Here, we describe two splice variants of human PARG mRNA, which lead to expression of PARG isoforms of 102 kDa (hPARG102) and 99 kDa (hPARG99) in addition to the full-length PARG protein (hPARG111). These splice variants differ from hPARG111 by the lack of exon 1 (hPARG102) or exons 1 and 2 (hPARG99). They are generated by the utilization of ambiguous splice donor sites in the PARG gene 5′ untranslated region. The hPARG111 isoform localizes to the nucleus, whereas hPARG102 and hPARG99 are cytoplasmic proteins. The nuclear targeting of hPARG111 is due to a nuclear localization signal (NLS) in exon 1 that was mapped to the amino acids (aa) 10CTKRPRW16. Immunocytochemistry, immunoblotting, and PARG enzyme activity measurements show that the cytoplasmic isoforms of PARG account for most of the PARG activity in cells in the absence and presence of genotoxic stress. The predominantly cytoplasmic location of cellular PARG is intriguing as most known cellular PARPs have a nuclear localization.

Tannins elevate the level of poly(ADP–ribose) in HeLa cell extracts

Phenolic phytochemicals such as tannins, which are natural constituents of green tea, red wine, and other plant products, are considered to have cancer-preventive properties. An important endogenous mediator of tumorigenesis is the nuclear enzyme poly(ADP–ribose) polymerase 1 (PARP-1). PARP-1 synthesizes polymers of ADP–ribose (PAR), which, in turn, are degraded by the catabolic enzyme poly(ADP–ribose) glycohydrolase (PARG). In the present study, we investigated the effects of tannins on the level of PAR in HeLa nuclear extracts. The addition of tannins to nuclear extracts led to a 40-fold elevation of PAR-levels. The observed increased PAR-levels resulted from inhibition of the catalytic activity of PARG. Additionally, the human PARG cDNA was cloned and the recombinant enzyme was overexpressed and isolated. Recombinant PARG was immobilized using an affinity column composed of tannins covalently linked to Sepharose beads. Finally, an interaction between immobilized PARG and endogenous PARP-1 from HeLa cell extracts is demonstrated.

Human poly(ADP-ribose) glycohydrolase ( PARG) gene and the common promoter sequence it shares with inner mitochondrial membrane translocase 23 ( TIM23)

Poly(ADP-ribosyl)ation is a posttranslational protein modification mediated by members of the poly(ADP-ribose) polymerase (PARP) family. The ADP-ribose polymers, synthesized by the diverse PARP enzymes by cleavage of NAD +, are involved in the regulation of multiple cellular functions. At present, only a single enzyme, poly (ADP-ribose) glycohydrolase (PARG), has been identified to catalyze ADP-ribose polymer hydrolysis in the cell causing a rapid turnover of the biopolymer which may ultimately result in lethal depletion of cellular NAD + pools. In this study, we describe the construction of the first human PARG cDNA clone by reverse transcription of CF3 human fibroblast RNA. Using the NCBI “Genome BLAST” program, the human PARG gene was mapped to chromosome 10 (10q11.23) in agreement to earlier results obtained by in situ hybridization. In vitro coupled transcription and translation of the cDNA yielded several specific bands in the range of 111–85 kDa, indicating possible usage of alternative translation initiation sites. The gene structure was characterized by further detailed computational analyses. The open reading frame consists of 18 exons and 17 introns with exons 9 to 14 forming the catalytic center of the enzyme and exons 1 to 3 encoding the putative regulatory domain. We show that the human PARG gene shares a 470-bp common promoter region with the inner mitochondrial membrane translocase 23 ( TIM23). The human bidirectional promoter region was cloned and expression studies in transiently transfected HEK293 cells was performed using an EGFP-luciferase reporter fusion gene ( GFL) to quantify transcription activation in both directions. The activity of the promoter was found to be 3.7 fold higher for TIM23 than for PARG, indicating that the two genes are expressed at different levels, although coregulation of the two genes remains an interesting possibility.

Subcellular localization of poly(ADP-ribose) glycohydrolase in mammalian cells

Posttranslational modification plays important roles in a range of cellular functions. Poly(ADP-ribosyl)ation influences DNA repair, transcription, centrosome duplication, and chromosome stability. Poly(ADP-ribose) attached to acceptor proteins should be properly hydrolyzed by poly(ADP-ribose) glycohydrolase (PARG). However the subcellular localization and the role of PARG have not been well characterized. Here, we transiently expressed GFP- or Myc-tagged human PARG in mammalian cells and revealed that the subcellular distribution of human PARG changes dramatically during the cell cycle. GFP-hPARG is found almost exclusively in the nucleus during interphase. During mitosis, most GFP-hPARG protein localizes to the cytoplasm and hardly any GFP-hPARG protein is found associated with the chromosomes. Furthermore, we found that GFP-hPARG localizes to the centrosomes during mitosis. Our findings suggest that shuttling of PARG between nucleus and cytoplasm and proper control of poly(ADP-ribose) metabolism throughout the cell cycle may play an important role in regulating cell cycle progression and centrosome duplication.