The identification and optimization of a N-hydroxy urea series of flap endonuclease 1 inhibitors

Tumey, L. Nathan; Bom, David; Huck, Bayard R.; Gleason, Elizabeth; Wang, Jianmin; Silver, Daniel P.; Brunden, Kurt R.; Boozer, Sherry; Rundlett, Stephen E.; Sherf, Bruce A.; Murphy, Steven M.; Dent, Tom; Leventhal, Christina; Bailey, Andrew L.; Harrington, John; Bennani, Youssef L.

doi:10.1016/j.bmcl.2004.10.086

Cited by 82 publications

(86 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…[6][7][8][9] None of the methods identified in the literature seemed optimal for delivering a simple, robust assay capable of delivering our HTS campaign. We therefore chose to exploit FP technology to deliver an alternative, miniaturized, homogenous assay to monitor FEN1 activity using a singly-labeled FEN1 DNA substrate.…”

Section: Discussionmentioning

confidence: 99%

“…6,7 A high-throughput screening (HTS) approach was therefore considered to be a key strategy in finding novel leads. Various technologies have been exploited in studies of FEN1 activity in the past.…”

Section: Introductionmentioning

confidence: 99%

“…8,9 These methods are too low-throughput to be used for a screening campaign, and this has led to the development of several high-throughput assays including fluorogenic donor/quencher assays and a chemiluminescence-based AlphaScreen assay. 6,7 As FEN1 is a structure-specific DNA endonuclease, physiologically relevant substrates should contain certain key features including a 5′ flap and a single-nucleotide 3′ flap. All substrates used in previously published high-throughput assays lack a single nucleotide 3′ flap, which has been shown to be critical for specificity of the FEN1 cleavage site.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Development of a High-Throughput Fluorescence Polarization DNA Cleavage Assay for the Identification of FEN1 Inhibitors

et al. 2013

View full text Add to dashboard Cite

Flap endonuclease-1 (FEN1) is a highly conserved metallonuclease and is the main human flap endonuclease involved in the recognition and cleavage of single-stranded 5′ overhangs from DNA flap structures. The involvement of FEN1 in multiple DNA metabolism pathways and the identification of FEN1 overexpression in a variety of cancers has led to interest in FEN1 as an oncology target. In this article, we describe the development of a 1536-well high-throughput screening assay based on the change in fluorescence polarization of a FEN1 DNA substrate labeled with Atto495 dye. The assay was subsequently used to screen 850 000 compounds from the AstraZeneca compound collection, with a Z′ factor of 0.66 ± 0.06. Hits were followed up by IC 50 determination in both a concentration-response assay and a technology artifact assay.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Development of a High-Throughput Fluorescence Polarization DNA Cleavage Assay for the Identification of FEN1 Inhibitors

et al. 2013

View full text Add to dashboard Cite

show abstract

“…The previously reported APE1 inhibitor, aurintricarboxylic acid (ATA), was also shown to be a potent inhibitor of FEN1 in the chemiluminescent assays. 113,114 The specificity of these agents to FEN1 needs further investigation; however, it was shown that these compounds potentiate treatment with chemotherapies that induce DNA damage repaired by the BER pathway. They did not potentiate treatment with bleomycin, which does not activate the BER pathway, therefore suggesting their specificity to BER proteins, although further work is necessary.…”

Section: Fen1 Inhibitorsmentioning

confidence: 99%

DNA Repair Endonucleases: Physiological Roles and Potential as Drug Targets

Doherty

Madhusudan

2015

SLAS Discovery

View full text Add to dashboard Cite

Genomic DNA is constantly exposed to endogenous and exogenous damaging agents. To overcome these damaging effects and maintain genomic stability, cells have robust coping mechanisms in place, including repair of the damaged DNA. There are a number of DNA repair pathways available to cells dependent on the type of damage induced. The removal of damaged DNA is essential to allow successful repair. Removal of DNA strands is achieved by nucleases. Exonucleases are those that progressively cut from DNA ends, and endonucleases make single incisions within strands of DNA. This review focuses on the group of endonucleases involved in DNA repair pathways, their mechanistic functions, roles in cancer development, and how targeting these enzymes is proving to be an exciting new strategy for personalized therapy in cancer.

show abstract

“…We next examined the antibody specificity by testing whether known ATM inhibitors can specifically decrease the ionizing radiation (IR)-induced Ser1981 phosphorylation of ATM and if this inhibition has an effect on the downstream ATM target proteins using Western blots. HT29 cells were treated for 1 h with ATM-specific inhibitors KU-55933, 14 KU-60019, 15 and CP466722 16 and a range of other DDR inhibitors (DNA-PK: KU-57788, ATR: AZ20, and FEN1) 3,17,18 at a concentration of 1 µM (Fig. 1B).…”

Section: Assay Developmentmentioning

confidence: 99%

Development of a High-Content High-Throughput Screening Assay for the Discovery of ATM Signaling Inhibitors

Bardelle

Boros

2012

SLAS Discovery

View full text Add to dashboard Cite

The genome is constantly exposed to DNA damage agents, leading up to as many as 1 million individual lesions per cell per day. Cells have developed a variety of DNA damage repair (DDR) mechanisms to respond to harmful effects of DNA damage. Failure to repair the damaged DNA causes genomic instability and, as a result, leads to cellular transformation. Indeed, deficiencies of DDR frequently occur in human cancers, thus providing a great opportunity for cancer therapy by developing anticancer agents that work by synthetic lethality-based mechanisms or enhancing the clinical efficacy of radiotherapy and existing chemotherapies. Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to DNA double-strand breaks. Ionizing radiation causes double-strand breaks and induces rapid ATM autophosphorylation on serine 1981 that initiates ATM kinase activity. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage-response pathways, most notably cell-cycle checkpoints. We describe here the development and validation of a high-throughput imaging assay measuring levels of phospho-ATM Ser1981 in HT29 cells after exposure to ionizing radiation. We also examined activation of downstream ATM effectors and checked specificity of the endpoint using known inhibitors of DNA repair pathways.

show abstract

The identification and optimization of a N-hydroxy urea series of flap endonuclease 1 inhibitors

Cited by 82 publications

References 22 publications

Development of a High-Throughput Fluorescence Polarization DNA Cleavage Assay for the Identification of FEN1 Inhibitors

Development of a High-Throughput Fluorescence Polarization DNA Cleavage Assay for the Identification of FEN1 Inhibitors

DNA Repair Endonucleases: Physiological Roles and Potential as Drug Targets

Development of a High-Content High-Throughput Screening Assay for the Discovery of ATM Signaling Inhibitors

Contact Info

Product

Resources

About