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

Rebecca L. Fagan, Meng Wu, Frederic Chedin, Charles Brenner

Research output: Contribution to journalArticle

14 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere78752
JournalPLoS One
Volume8
Issue number11
DOIs
StatePublished - Nov 13 2013

Fingerprint

Molecular Probes
methyltransferases
Methyltransferases
probes (equipment)
Throughput
DNA
decitabine
Isoenzymes
neoplasms
isozymes
Assays
Enzymes
enzymes
Proteolysis
Intercalating Agents
cytidine
Cytidine
Fluorometry
Neoplasms
fluorometry

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

An ultrasensitive high throughput screen for DNA methyltransferase 1-targeted molecular probes. / Fagan, Rebecca L.; Wu, Meng; Chedin, Frederic; Brenner, Charles.

In: PLoS One, Vol. 8, No. 11, e78752, 13.11.2013.

Research output: Contribution to journalArticle

Fagan, Rebecca L. ; Wu, Meng ; Chedin, Frederic ; Brenner, Charles. / An ultrasensitive high throughput screen for DNA methyltransferase 1-targeted molecular probes. In: PLoS One. 2013 ; Vol. 8, No. 11.
@article{7b6a64c0f155444c8d56680a722a6efc,
title = "An ultrasensitive high throughput screen for DNA methyltransferase 1-targeted molecular probes",
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.",
author = "Fagan, {Rebecca L.} and Meng Wu and Frederic Chedin and Charles Brenner",
year = "2013",
month = "11",
day = "13",
doi = "10.1371/journal.pone.0078752",
language = "English (US)",
volume = "8",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "11",

}

TY - JOUR

T1 - An ultrasensitive high throughput screen for DNA methyltransferase 1-targeted molecular probes

AU - Fagan, Rebecca L.

AU - Wu, Meng

AU - Chedin, Frederic

AU - Brenner, Charles

PY - 2013/11/13

Y1 - 2013/11/13

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84893529302&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84893529302&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0078752

DO - 10.1371/journal.pone.0078752

M3 - Article

C2 - 24236046

AN - SCOPUS:84893529302

VL - 8

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 11

M1 - e78752

ER -