Abstract
Apurinic/apyrimidinic (AP) endonuclease 1 (Ape1) is an essential DNA repair protein that plays a critical role in repair of AP sites via base excision repair. Ape1 has received attention as a druggable oncotherapeutic target, especially for treating intractable cancers such as glioblastoma. The goal of this study was to identify small-molecule inhibitors of Ape1 AP endonuclease. For this purpose, a fluorescence-based high-throughput assay was used to screen a library of 60,000 small-molecule compounds for ability to inhibit Ape1 AP endonuclease activity. Four compounds with IC50 values less than 10 μM were identified, validated, and characterized. One of the most promising compounds, designated Ape1 repair inhibitor 03 [2,4,9-trimethylbenzo[b][1,8]-naphthyridin-5-amine; AR03), inhibited cleavage of AP sites in vivo in SF767 glioblastoma cells and in vitro in whole cell extracts and inhibited purified human Ape1 in vitro. AR03 has low affinity for double-stranded DNA and weakly inhibits the Escherichia coli endonuclease IV, requiring a 20-fold higher concentration than for inhibition of Ape1. AR03 also potentiates the cytotoxicity of methyl methanesulfonate and temozolomide in SF767 cells. AR03 is chemically distinct from the previously reported small-molecule inhibitors of Ape1. AR03 is a novel small-molecule inhibitor of Ape1, which may have potential as an oncotherapeutic drug for treating glioblastoma and other cancers.
Footnotes
This work was supported by the National Institutes of Health National Cancer Institute [Grants CA114571 (to M.M.G.), CA94025, CA106298, CA114571, CA121168 (to M.R.K.)], Indiana University Melvin and Bren Simon Cancer Center Translational Research Acceleration Collaboration pilot funding (to M.M.G. and M.R.K.)], and the Riley Children's Foundation (to M.R.K.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.169128.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- BER
- base excision repair
- AP
- apurinic/apyrimidinic
- Ape1
- apurinic/apyrimidinic endonuclease 1
- TMZ
- temozolomide
- MMS
- methyl methane sulfonate
- MX
- methoxyamine
- NCA
- 7-nitro indole 2-carboxylic acid
- HTS
- high-throughput screen
- AR
- Ape1 repair inhibitor
- AR02
- 4-(2,6,8-trimethylquinolin-4-ylamino)phenol
- MES
- 2-(N-morpholino)ethanesulfonic acid
- DTT
- dithiothreitol
- FAM
- 5-carboxyfluorescein
- THF
- tetrahydrofuran
- HEX
- hexa-chloro phosphoramidite fluorescein
- DMSO
- dimethyl sulfoxide
- FID
- fluorescence intercalator displacement
- EtBr
- ethidium bromide
- PBS
- phosphate-buffered saline
- TBS
- Tris-buffered saline
- ARP
- aldehyde reactive probe
- AR01
- 2-(4-(2,5-dimethyl-1H-prryol-1-yl)phenoxy acetic acid
- AR03
- 2,4,9-trimethylbenzo[b][1,8]-naphthyridin-5-amine
- AR06
- N-(3-chlorophenyl)-5,6-dihyro-4H-cyclopenta[d] isoxazole-3-carboxamide
- AR07
- C22H28N2O3S
- AR04
- C20H22N4O2
- AR05
- C18H13N3O3S
- Compound 18 (A1NI2-A3NI1)
- 4-((2-carboxyphenoxy)methyl)-2,5-dimethylfuran-3-carboxylic acid
- 13755
- C7H6NO7Sb
- 13793
- C12H17O5Sb.
- Received April 12, 2010.
- Accepted May 25, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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