PDD 00017273

A PARG inhibitor

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5mg

￥1212.00

970.00

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10mg

￥2287.00

1830.00

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25mg

￥4850.00

3880.00

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Background

PDD00017273 is a poly(ADP-ribose) glycohydrolase (PARG) inhibitor (IC₅₀ = 26 nM in an enzyme assay).¹ It maintains persistence of nuclear PAR chains in HeLa cells induced by the DNA damaging agent methylmethanesulfonate (MMS; IC₅₀ = 37 nM). PDD00017273, in combination with MMS, increases nuclear phosphorylated H2AX (γH2AX) intensity, a marker of DNA double strand breaks, in MCF-7 cells in a concentration-dependent manner. It reduces clonogenic growth of ZR-75-1 and MDA-MB-436 cells but not HCC1937 cells (IC₅₀s = 0.2, 0.8, and >10 μM, respectively).

1.James, D.I., Smith, K.M., Jordan, A.M., et al.First-in-Class Chemical Probes against Poly(ADP-ribose) Glycohydrolase (PARG) Inhibit DNA Repair with Differential Pharmacology to OlaparibACS Chem Biol.11(11)3179-3190(2016)

Protocol

Kinase experiment:

Briefly, PARG in vitro assays are conducted in a total volume of 15 μL in a standard 384-well format. A total of 5 μL of human full length PARG used at a final reaction concentration of 65 pM, is added to 5 μL of Bt-NAD ribosylated PARP1 substrate at a final reaction concentration of 4.8 nM in assay buffer (50 mM Tris pH 7.4, 0.1 mg/mL BSA, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 0.01% Tween 20, 50 mM KCl). The reaction is incubated at RT for 10 min, and then 5 μL of detection reagent is added. Detection reagent consists of 42 nM mAb anti-6HIS XL665 and 2.25 nM streptavidin europium cryptate, both at 3× working stock concentrations (final concentrations of 14 nM and 0.75 nM, respectively), in detection buffer (50 mM Tris pH 7.4, 0.1 mg/mL BSA and 100 mM KF). Following incubation at RT for 60 min in the dark, TR-FRET signal is measured at λEx 340 nm and λEm 665 nm and λEm 620 nm using a PHERAstar FS plate reader. The ratio is calculated as [Em665/EM620] × 104 for each well and used to calculate percent inhibition for test compounds[1].

Cell experiment:

HeLa cells are seeded in 30 μL of media at 1 × 104 cells/mL in Greiner 384-well plates. A total of 16-24 h later, cells are treated with inhibitors (8 pt dose response, 0.01-30 μM, triplicates) or vehicle (DMSO) control. The outer wells are left undosed to account for edge effects. After 72 h, 50 μL of 3.7% formaldehyde/PBS is added to each well, and cells are fixed for 20 min. Cells are then rinsed twice with PBS and stained for 1 h with Hoechst 33342/PBS (1:2000) in the dark. After two further rinses with PBS, images are captured and nuclei counted on a CellInsight. The maximum number of fields are captured from each triplicate well, which approximated to at least 1000 nuclei in vehicle-dosed wells[1].

参考文献:

[1]. James DI, et al. First-in-Class Chemical Probes against Poly(ADP-ribose) Glycohydrolase (PARG) Inhibit DNA Repair with Differential Pharmacology to AZD2281. ACS Chem Biol. 2016 Nov 18;11(11):3179-3190. Epub 2016 Oct 12.
[2]. Gravells P, et al. Specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase. DNA Repair (Amst). 2017 Apr;52:81-91.