CRT0044876

AP site cleavage assay

BER reaction buffer comprised 40 mM HEPES-KOH (pH 7.8), 5 mM MgCl2, 0.5 mM DTT and 0.1 mM EDTA. A 10 μL AP site cleavage reaction comprised of BER buffer mix, purified protein (3.3 nM final concentration of APE1) and 0.75 ng reduced AP site double-stranded oligonucleotide. The mixture was incubated at 37 °C for 1 hr. A total of 1 μL of stop buffer (50% glycerol, 10 mM Tris–HCl, 1 mM EDTA, 0.1% bromophenol blue and 0.1% Xylene cyanol) was added, and the sample mixture was denatured at 90 ~ 100 °C for 2 mins. The sample was then loaded on a 15% TBE Criterion Pre-Cast Gel, with electrophoresis at a constant current of 30 mA for 30 mins, and the radiolabeled substrate and reaction products were visualized using a phosphorImager. The inhibitory activity of potential APE1-targeting compounds was analyzed at drug concentrations ranging from 0.1 to 100 μM. The resolved radiolabeled bands were quantified using ImageQuant software analysis, and IC50 values were calculated.

Cell lines

Human HT1080 fibrosarcoma cells

Preparation method

The solubility of this compound in DMSO is > 55.6 mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 °C for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 °C for several months.

Reacting condition

1 ~ 2 hrs

Applications

In Human HT1080 fibrosarcoma cells, CRT0044876 significantly increased apurinic/apyrimidinic (AP) site accumulation. HT1080 cells treated with methylmethane sulfonate (MMS) also elevated the level of AP sites. The combination of CRT0044876 and MMS caused a synergistic increase in the level of AP sites.

References:

[1]. Madhusudan S, Smart F, Shrimpton P, et al. Isolation of a small molecule inhibitor of DNA base excision repair. Nucleic Acids Res, 2005, 33(15): 4711-4724.