Selective small molecule inhibition of poly(ADP-ribose) glycohydrolase (PARG) - PubMed (original) (raw)

. 2012 Mar 16;7(3):563-70.

doi: 10.1021/cb200506t. Epub 2012 Jan 26.

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Selective small molecule inhibition of poly(ADP-ribose) glycohydrolase (PARG)

Kristin E Finch et al. ACS Chem Biol. 2012.

Abstract

The poly(ADP-ribose) (PAR) post-translational modification is essential for diverse cellular functions, including regulation of transcription, response to DNA damage, and mitosis. Cellular PAR is predominantly synthesized by the enzyme poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 is a critical node in the DNA damage response pathway, and multiple potent PARP-1 inhibitors have been described, some of which show considerable promise in the clinic for the treatment of certain cancers. Cellular PAR is efficiently degraded by poly(ADP-ribose) glycohydrolase (PARG), an enzyme for which no potent, readily accessible, and specific inhibitors exist. Herein we report the discovery of small molecules that effectively inhibit PARG in vitro and in cellular lysates. These potent PARG inhibitors can be produced in two chemical steps from commercial starting materials and have complete specificity for PARG over the other known PAR glycohydrolase (ADP-ribosylhydrolase 3, ARH3) and over PARP-1 and thus will be useful tools for studying the biochemistry of PAR signaling.

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Figures

Figure 1

Figure 1

(A) Screening of 224 rhodanine-containing compounds reveals RBPI-1 as a PARG inhibitor, and a collection of >70 compounds was then synthesized based on this scaffold and evaluated for PARG inhibition. From this work RBPI-2, -3, -4, -5, and -6 were identified as potent PARG inhibitors. Inactive-1 and -2 are structurally-related compounds that do not inhibit PARG. (B) Representative dose-response PARG inhibition curves for each compound shown in A. For IC50 determination, compounds were incubated with PARG for 10 min, then 32P-PAR was added and incubated for 2 h at 37°C, at which point the extent of PAR degradation was assessed by separation on TLC plates and phosphorimaging. For TLC images and triplicate IC50 curves please see Supplementary Figure S7.

Figure 2

Figure 2

RBPI inhibition of PARG is insensitive to the presence of detergent. Compounds (50 μM) were incubated with PARG in the presence or absence of 0.1% Triton X-100 for 10 min, 32P-PAR was added and incubated for 2 h at 37°C, and the extent of PAR degradation was assessed by separation on TLC plates and phosphorimaging. (A) Representative TLC plate from this experiment; (B) Quantitation of the experiment described above by densitometry, n=3, error bars indicate standard error of the mean.

Figure 3

Figure 3

RBPIs do not inhibit β-lactamase. RBPIs and Congo Red (5-50 μM) were incubated with ß-lactamase (A) in the absence of detergent and (B) in the presence of 0.1% Triton X-100. After addition of the colorimetric substrate, enzyme activity was measured by recording absorbance at 405 nm, n=3, error bars indicate standard error of the mean.

Figure 4

Figure 4

Inhibition of PARG by RBPIs persists even after 2 h incubation with substrate. RBPI-3, -6, and ADP-HPD were incubated with PARG for 10 min, then 32P-PAR was added and incubated at 37°C for (A) 0.5 h or (B) 2 h. The extent of PAR degradation was assessed by separation on TLC plates and phosphorimaging. (C) Quantitation of data shown in A and B, n=3, error bars indicate standard error of the mean.

Figure 5

Figure 5

ADP-HPD, but not RBPIs, inhibits ARH3 PAR glycohydrolase activity. Compounds were incubated with (A) ARH3 (256 nM) or (B) PARG (0.24 nM) for 10 min, and then 32P-PAR was added and incubated at 37°C for 2 h. The extent of PAR degradation was visualized by separation on a TLC plate and phosphorimaging. (C) Quantitation of data from part A and B, n=3, error bars indicate standard error of the mean. See Supplementary Figure S10 for IC50 data of ADP-HPD inhibition of ARH3.

Figure 6

Figure 6

Processing of 32P-PAR by cellular lysate (MEF, 1.5 μg) over 60 min in the presence of DMSO, 25 μM RBPI-4, or 25 μM ADP-HPD. Compounds were incubated with lysate for 10 min, then 32P-PAR was added and incubated at 37°C for 0-60 min. The extent of PAR degradation was visualized by separation on a TLC plate and phosphorimaging. Graph is representative of three independent experiments, see Supplementary Figure S13 for triplicate data.

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