Biology Reference
In-Depth Information
FIGURE 3.4
High through-put screens (HTS) to discover APE1 DNA repair inhibitors. (A) Schematic of the high through-put screen format
that has been utilized to discover APE1 endonuclease inhibitors. The AP site mimic is represented by a red square. This assay can identify
compounds that block APE1 endonuclease activity by monitoring its cutting ability on oligonucleotides that contain an AP site mimic (THF,
tetrohydrofuran residue). When APE1 is active, there is cleavage at the THF residue. This cut in the oligonucleotide causes the dissociation of the
two strands and the quencher ( ) is not in close proximity to stop the fluorescence ( ) of the complimentary strand. In the presence of
a potential inhibitor, the increase in fluorescence will not be observed. This compound can then be used in other assays to validate that it is
inhibiting APE1 and not binding to DNA. (B) Current APE1 inhibitors block the AP endonuclease activity in two ways. Inhibitors like
methoxyamine (MX) bind to the AP site in the DNA blocking the downstream members of the BER pathway. HTS assays as in Panel A can
identify compounds (
) that bind directly to APE and block its activity directly.
Several studies also test the putative APE1 repair inhib-
itor for the ability to enhance the cytotoxicity of MMS
or TMZ and/or the persistence of AP sites following
treatment with the APE1 inhibitor and these alkylating
agents.
230
Lucanthone or Miracil D affects APE1's DNA repair
activity but is also a topoisomerase II inhibitor.
231
Many years ago this compound was shown to sensitize
HeLa cells to radiation through an unknown mecha-
nism.
232
Further characterization of
the compound
