AG14361 

The activity of full-length recombinant human PARP-1 is measured in a reaction mixture containing 20 nM PARP-1, 500 μM NAD⁺ plus [³²P]NAD⁺ (0.1-0.3 μCi per reaction mixture), and activated calf thymus DNA (10 μg/mL) at 25°C; the reaction is terminated after 4 minutes by adding ice-cold 10% (wt/vol) trichloroacetic acid. The reaction product [³²P]ADP-ribose incorporated into acid-insoluble material is deposited onto Whatman GF/C glass fiber filters with a microfiltration apparatus and quantified with a PhosphorImager. Inhibition of PARP-1 activity by AG14361 at 0-600 nM is measured, and the K_i for AG14361 is calculated by nonlinear regression analysis^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Cell viability assay is performed using a luciferase-coupled ATP quantization assay of metabolically active cells in a 96-well plate and MTT. For MTT, 1 to 2 × 10⁴ cells are plated per one well of a 24-well plate. Target drugs (AG14361) at various concentrations are dissolved in DMSO and then added to the cells in 10% fetal bovine serum-containing Dulbecco’s modified Eagle’s medium (DMEM), IC₅₀ concentration of AG14361 are also added to each well. The final DMSO concentration is kept at 0.1% after the addition to medium. After 48 hr medium is removed and 0.3 mL of 0.1% MTT in phosphate-buffered saline (PBS) is added in each well. After incubation for 30 min in a 37°C CO₂ incubator, MTT solution is removed and 0.8 mL of 2-propanol is added. After shaking for 30 min, OD560 is measured using a plate reader. Plating for each time point is done in triplicate^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

CD-1 nude mice bearing palpable, subcutaneous SW620 or LoVo xenografts are treated intraperitoneally with normal saline (control animals) or AG14361 (at 5 or 15 mg/kg) alone daily for 5 days (five mice per group). For drug combinations, AG14361 is administered intraperitoneally daily for 5 days immediately before administering the cytotoxic drug (NSC 362856 at 68 mg/kg orally or CPT-11 at 2.5 mg/kg intraperitoneally) or 30 minutes before applying 2 Gy of x-irradiation locally to the tumor daily for 5 days. Tumor volumes, determined from two-dimensional caliper measurements and the equation a² × b/2 (where a is the width and b is the length of the tumor), are presented as median relative tumor volume (RTV). That is, RTV1 is the tumor volume on the initial day of treatment (day 0), and RTV4 is the tumor volume 4 times that on the initial day of treatment. Tumor growth delay is defined as the time to RTV4 in drug-treated or irradiated mice compared with the time to RTV4 in control (vehicle alone) mice^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Calabrese CR, et al. Anticancer chemosensitization and radiosensitization by the novel poly(ADP-ribose) polymerase-1 inhibitor AG14361. J Natl Cancer Inst. 2004 Jan 7;96(1):56-67.  [Content Brief]

  [2]. Vazquez-Ortiz G, et al. Drug repurposing screen identifies lestaurtinib amplifies the ability of the poly (ADP-ribose) polymerase 1 inhibitor AG14361 to kill breast cancer associated gene-1 mutant and wild type breast cancer cells. Breast Cancer Res. 2014 Jun 24;16(3):R67.  [Content Brief]

  [3]. Smith LM, et al. The novel poly(ADP-Ribose) polymerase inhibitor, AG14361, sensitizes cells to topoisomerase I poisons by increasing the persistence of DNA strand breaks. Clin Cancer Res. 2005 Dec 1;11(23):8449-57.  [Content Brief]