Selective Small Molecule Inhibition of Poly(ADP-Ribose) Glycohydrolase (PARG)

Finch, Kristin E.; Knezevic, Claire E.; Nottbohm, Amanda C.; Partlow, Kathryn C.; Hergenrother, Paul J.

doi:10.1021/cb200506t

Cited by 48 publications

(50 citation statements)

References 68 publications

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“…Inhibition of mammalian PARG enzymes was recently shown for several related rhodanine-containing compounds 16 . Not unexpectedly, we were able to show that TTPARG is also inhibited by this class of inhibitors ( Supplementary Fig.…”

Section: Structure Of a Mammalian Parg Inhibitor:ttparg Complexmentioning

confidence: 94%

“…For crystallization studies, the T. thermophila PARG2 was purified by FPLC on a HisTrap HP column followed by gel filtration, using HiLoad 16/60 Superdex 200 column (GE Healthcare). Rhodanine-containing PARG inhibitor RBPI-3 was synthetized, as described 16 .…”

Section: Methodsmentioning

confidence: 99%

“…Inhibition of TTPARG by RBPI-3 was determined, as previously reported 16 , with the following modifications. Purified TTPARG was employed at a final concentration of 50 nM and reactions were incubated at 37 °C for 60 min before stopping by the addition of 1.0 µl 1% SDS.…”

Section: Parg Inhibition Assaymentioning

confidence: 99%

See 2 more Smart Citations

Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase

Dunstan¹,

Barkauskaite

Lafite

et al. 2012

Nat Commun

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Poly(ADP-ribosyl)ation is a reversible post-translational protein modification involved in the regulation of a number of cellular processes including DnA repair, chromatin structure, mitosis, transcription, checkpoint activation, apoptosis and asexual development. The reversion of poly(ADP-ribosyl)ation is catalysed by poly(ADP-ribose) (PAR) glycohydrolase (PARG), which specifically targets the unique PAR (1′′-2′) ribose-ribose bonds. Here we report the structure and mechanism of the first canonical PARG from the protozoan Tetrahymena thermophila. In addition, we reveal the structure of T. thermophila PARG in a complex with a novel rhodaninecontaining mammalian PARG inhibitor RBPI-3. our data demonstrate that the protozoan PARG represents a good model for human PARG and is therefore likely to prove useful in guiding structure-based discovery of new classes of PARG inhibitors.

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Section: Structure Of a Mammalian Parg Inhibitor:ttparg Complexmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase

Dunstan¹,

Barkauskaite

Lafite

et al. 2012

Nat Commun

Self Cite

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“…anti-oxidant properties 4 ). Other compounds, such as APD-HPD and rhodamine-based PARG inhibitors (RBPIs), have shown good specificity for eukaryotic PARG but are either not cell permeable or have only been tested in biochemical assays 5– 7 . Attempts to discover new synthetic PARG inhibitors have resulted in compounds that also inhibit PARP or have low potency 8– 10 .…”

Section: Introductionmentioning

confidence: 99%

An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells

et al. 2016

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After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.

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“…This single method was able to characterize the NADase and polymerase activity of PARP as well as the pADPr hydrolyzing activities of PARG and phosphodiesterase. This method is more sensitive than gel shift assays, more quantitative than western blotting, and does not require the use of radioactivity [42; 58; 59]. …”

Section: Discussionmentioning

confidence: 99%

Large-scale preparation and characterization of poly(ADP-ribose) and defined length polymers

Tan¹,

Krukenberg²,

Mitchison³

2012

Analytical Biochemistry

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Poly(ADP-ribose) [pADPr] is a large, structurally complex polymer of repeating ADP-ribose units. It is biosynthesized from NAD+ by poly(ADP-ribose) polymerases [PARPs] and degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. PADPr is involved in many cellular processes and exerts biological function through covalent modification and non-covalent binding to specific proteins. Very little is known about molecular recognition and structure activity relationships for non-covalent interaction between pADPr and its binding proteins, in part because of lack of access to the polymer on large scale and to units of defined lengths. We prepared polydisperse pADPr from PARP1 and tankyrase 1 at hundreds of milligram scale by optimizing enzymatic synthesis and scaling up chromatographic purification methods. We developed and calibrated an anion exchange chromatography method to assign pADPr size and scaled it up to purify defined length polymers on the milligram scale. Furthermore, we present a pADPr profiling method to characterize the polydispersity of pADPr produced by PARPs under different reaction conditions and find that substrate proteins affect the pADPr size distribution. These methods will facilitate structural and biochemical studies of pADPr and its binding proteins.

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Selective Small Molecule Inhibition of Poly(ADP-Ribose) Glycohydrolase (PARG)

Cited by 48 publications

References 68 publications

Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase

Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase

An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells

Large-scale preparation and characterization of poly(ADP-ribose) and defined length polymers

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