An ultrasensitive high throughput screen for DNA methyltransferase 1-targeted molecular probes

Rebecca L Fagan; Meng Wu; Frédéric Chédin; Charles Brenner

doi:10.1371/journal.pone.0078752

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An ultrasensitive high throughput screen for DNA methyltransferase 1-targeted molecular probes

Journal article

Open access

Peer reviewed

An ultrasensitive high throughput screen for DNA methyltransferase 1-targeted molecular probes

Rebecca L Fagan, Meng Wu, Frédéric Chédin and Charles Brenner

PloS one, Vol.8(11), e78752

2013

DOI: 10.1371/journal.pone.0078752

PMCID: PMC3827244

PMID: 24236046

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Abstract

DNA methyltransferase 1 (DNMT1) is the enzyme most responsible for epigenetic modification of human DNA and the intended target of approved cancer drugs such as 5-aza-cytidine and 5-aza-2'-deoxycytidine. 5-aza nucleosides have complex mechanisms of action that require incorporation into DNA, and covalent trapping and proteolysis of DNMT isozymes. Direct DNMT inhibitors are needed to refine understanding of the role of specific DNMT isozymes in cancer etiology and, potentially, to improve cancer prevention and treatment. Here, we developed a high throughput pipeline for identification of direct DNMT1 inhibitors. The components of this screen include an activated form of DNMT1, a restriction enzyme-coupled fluorigenic assay performed in 384 well plates with a z-factor of 0.66, a counter screen against the restriction enzyme, a screen to eliminate DNA intercalators, and a differential scanning fluorimetry assay to validate direct binders. Using the Microsource Spectrum collection of 2320 compounds, this screen identified nine compounds with dose responses ranging from 300 nM to 11 µM, representing at least two different pharmacophores with DNMT1 inhibitory activity. Seven of nine inhibitors identified exhibited two to four-fold selectivity for DNMT1 versus DNMT3A.

Drug Evaluation, Preclinical - methods

Molecular Probe Techniques

DNA (Cytosine-5-)-Methyltransferase 1

Escherichia coli

Humans

Recombinant Proteins - chemistry

Enzyme Assays

DNA (Cytosine-5-)-Methyltransferases - antagonists & inhibitors

DNA (Cytosine-5-)-Methyltransferases - chemistry

DNA - chemistry

DNA Methylation

High-Throughput Screening Assays

Enzyme Inhibitors - chemistry

Sensitivity and Specificity

Details

Title: Subtitle: An ultrasensitive high throughput screen for DNA methyltransferase 1-targeted molecular probes
Creators: Rebecca L Fagan - Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States of America
Meng Wu
Frédéric Chédin
Charles Brenner
Resource Type: Journal article
Publication Details: PloS one, Vol.8(11), e78752
DOI: 10.1371/journal.pone.0078752
PMID: 24236046
PMCID: PMC3827244
NLM abbreviation: PLoS One
ISSN: 1932-6203
eISSN: 1932-6203
Publisher: United States
Grant note: R01 GM094299 / NIGMS NIH HHS R01 CA75954 / NCI NIH HHS GM094299 / NIGMS NIH HHS HHSN261200433000C / PHS HHS
Language: English
Date published: 2013
Academic Unit: Pharmacy; Core Research Facilities; Biochemistry and Molecular Biology; Internal Medicine
Record Identifier: 9983788432902771

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