Identification and Characterization of Inhibitors of Human Apurinic/apyrimidinic Endonuclease APE1
Open Access
- 1 June 2009
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 4 (6), e5740
- https://doi.org/10.1371/journal.pone.0005740
Abstract
APE1 is the major nuclease for excising abasic (AP) sites and particular 3′-obstructive termini from DNA, and is an integral participant in the base excision repair (BER) pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC1280), a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays – a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen – and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals.Keywords
This publication has 54 references indexed in Scilit:
- Going Ape as an Approach to Cancer TherapeuticsAntioxidants and Redox Signaling, 2009
- Participation of DNA repair in the response to 5-fluorouracilCellular and Molecular Life Sciences, 2008
- A Robotic Platform for Quantitative High-Throughput ScreeningASSAY and Drug Development Technologies, 2008
- Characterization of Abasic Endonuclease Activity of Human Ape1 on Alternative Substrates, as Well as Effects of ATP and Sequence Context on AP Site IncisionJournal of Molecular Biology, 2008
- Compound Management for Quantitative High-Throughput ScreeningSLAS Technology, 2008
- Potent Inhibition of Human Apurinic/Apyrimidinic Endonuclease 1 by Arylstibonic AcidsMolecular Pharmacology, 2007
- Hypersensitivity phenotypes associated with genetic and synthetic inhibitor-induced base excision repair deficiencyDNA Repair, 2007
- Methylating Agents and DNA Repair Responses: Methylated Bases and Sources of Strand BreaksChemical Research in Toxicology, 2006
- Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical librariesProceedings of the National Academy of Sciences of the United States of America, 2006
- Merck molecular force field. I. Basis, form, scope, parameterization, and performance of MMFF94Journal of Computational Chemistry, 1996