Isolation of a small molecule inhibitor of DNA base excision repair - PubMed (original) (raw)

. 2005 Aug 19;33(15):4711-24.

doi: 10.1093/nar/gki781. Print 2005.

Fiona Smart, Paul Shrimpton, Jason L Parsons, Laurence Gardiner, Sue Houlbrook, Denis C Talbot, Timothy Hammonds, Paul A Freemont, Michael J E Sternberg, Grigory L Dianov, Ian D Hickson

Affiliations

Isolation of a small molecule inhibitor of DNA base excision repair

Srinivasan Madhusudan et al. Nucleic Acids Res. 2005.

Abstract

The base excision repair (BER) pathway is essential for the removal of DNA bases damaged by alkylation or oxidation. A key step in BER is the processing of an apurinic/apyrimidinic (AP) site intermediate by an AP endonuclease. The major AP endonuclease in human cells (APE1, also termed HAP1 and Ref-1) accounts for >95% of the total AP endonuclease activity, and is essential for the protection of cells against the toxic effects of several classes of DNA damaging agents. Moreover, APE1 overexpression has been linked to radio- and chemo-resistance in human tumors. Using a newly developed high-throughput screen, several chemical inhibitors of APE1 have been isolated. Amongst these, CRT0044876 was identified as a potent and selective APE1 inhibitor. CRT0044876 inhibits the AP endonuclease, 3'-phosphodiesterase and 3'-phosphatase activities of APE1 at low micromolar concentrations, and is a specific inhibitor of the exonuclease III family of enzymes to which APE1 belongs. At non-cytotoxic concentrations, CRT0044876 potentiates the cytotoxicity of several DNA base-targeting compounds. This enhancement of cytotoxicity is associated with an accumulation of unrepaired AP sites. In silico modeling studies suggest that CRT0044876 binds to the active site of APE1. These studies provide both a novel reagent for probing APE1 function in human cells, and a rational basis for the development of APE1-targeting drugs for antitumor therapy.

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Figures

Figure 1

Figure 1

Screening for APE1 inhibitors. (a) Fluorescence-based assay for APE1 activity. A DNA 30mer duplex was dual tagged with a quench (DABCYL)—fluor (FAM) pair. Separation of the quench and fluor occurs following cleavage of the single abasic site (tetrahydrofuran). (b) Fluorescence signal measurements using the APE1 assay. Increasing amounts of APE1 were mixed with 100 nM oligonucleotide substrate under conditions described in the Materials and Methods. A concentration dependent rise in fluorescence indicated AP site cleavage activity by APE1. (c) Scheme for the gel-based AP site cleavage assay. (d) Example of the assay, using a bacterial cell extract expressing recombinant APE1. The 18mer uncut substrate (lane 1) and 8mer APE1 cleavage product (lane 3) are indicated. Immunodepletion of APE1 in the extract using protein A-sepharose beads abolished the cleavage activity of the extract (lane 2) confirming that cleavage was mediated by APE1.

Figure 2

Figure 2

Identification of CRT0044876 as an APE1 inhibitor. (a) Inhibitory profile of CRT0044876. DNA substrate (lane 1); substrate with APE1 (lane 2); substrate with APE1 and different concentrations of CRT0044876 (lanes 3–10). Lane 11 shows that the DMSO solvent did not inhibit APE1. (b) Quantification of the data from panel A showing the percentage inhibition of APE1 as a function of CRT0044876 concentration. The IC50 value for APE1 inhibition by CRT0044876 was calculated to be 3.06 μM. (c) CRT0044876 inhibited the AP site cleavage activity in HeLa whole cell extracts (WCE).

Figure 3

Figure 3

CRT0044876 is an inhibitor of the exonuclease III family of AP endonucleases. (a) CRT0044876 inhibits the 3′–5′-exonuclease activity of exonuclease III. DNA substrate (lane 1 as indicated on the right); exonuclease III alone, which generated digestion products from the substrate, as indicated on the right (lane 2); effects of increasing concentrations of CRT0044876 on the exonuclease activity (lanes 3–7). (b) CRT0044876 inhibits the AP endonuclease activity of exonuclease III. Lanes as per (a). (c) CRT0044876 does not inhibit the AP endonuclease activity of endonuclease IV. Lanes as per (a).

Figure 4

Figure 4

CRT0044876 inhibits the 3′-phosphodiesterase activity of APE1. (a) Assay design. The substrate contains an end-labeled 16mer oligonucleotide with a 3′-PG moiety. APE1 removes the 3′-PG residue to produce a 3′-OH residue, which has a faster mobility on a 20% polyacrylamide gel (panel b). Lanes 1–5 show an APE1 concentration-dependent increase in conversion of the 3′-PG to 3′-OH. Lanes 6–10 show that CRT00044876 inhibited the 3′-phosphodiesterase activity of APE1 (protein fixed at 150 fmol).

Figure 5

Figure 5

Accumulation of AP sites in CRT0044876 treated human HT1080 cells. Quantification of the number of AP sites per 100 000 bp of nuclear DNA in untreated cells and in cells treated with CRT0044876 alone, MMS alone, or the combination of CRT0044876 and MMS (as indicated in the key below the bars). Samples were taken between 1 and 2 h after drug exposure as indicated. Values represented the mean of three independent determinations. Error bars denote Standard Errors of the mean.

Figure 6

Figure 6

Clonogenic survival analysis. The percentage survival of HT1080 cells as a function of drug dose or radiation dose is indicated in (a–h). In each case the filled circles indicate the survival for cells exposed to the DNA damaging agent alone, and the open circles indicate the survival for cells exposed to the damaging agent in the presence of 200 μM CRT0044876.

Figure 6

Figure 6

Clonogenic survival analysis. The percentage survival of HT1080 cells as a function of drug dose or radiation dose is indicated in (a–h). In each case the filled circles indicate the survival for cells exposed to the DNA damaging agent alone, and the open circles indicate the survival for cells exposed to the damaging agent in the presence of 200 μM CRT0044876.

Figure 7

Figure 7

Molecular Modeling. CRT0044876 is a chemically known as 7-nitro-1H-indole-2-carboxylic acid (a). Through molecular modeling studies, CRT0044876 was found to dock on to the active site of APE1 in two different low energy conformations (b) designated cluster I (in yellow) and cluster II (in blue). APE1 is shown as a ribbon structure. Selected active site amino acid residues are indicated. See text for details.

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