Development and validation of reagents and assays for EZH2 peptide and nucleosome high-throughput screens.
Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [(3)H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.
Platform Technology and Science, GlaxoSmithKline, Collegeville, PA 19426, USA.
Elsie
E
Diaz
Carl A
CA
Machutta
Stephanie
S
Chen
Yong
Y
Jiang
Christopher
C
Nixon
Glenn
G
Hofmann
Danielle
D
Key
Sharon
S
Sweitzer
Mehul
M
Patel
Zining
Z
Wu
Caretha L
CL
Creasy
Ryan G
RG
Kruger
Louis
L
LaFrance
Sharad K
SK
Verma
Melissa B
MB
Pappalardi
Baochau
B
Le
Glenn S
GS
Van Aller
Michael T
MT
McCabe
Peter J
PJ
Tummino
Andrew J
AJ
Pope
Sara H
SH
Thrall
Benjamin
B
Schwartz
Martin
M
Brandt
D004354:Drug Screening Assays, Antitumor / Q000379:methods; D000071221:Enhancer of Zeste Homolog 2 Protein; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D009707:Nucleosomes / Q000378:metabolism*; D010455:Peptides / Q000037:antagonists & inhibitors / Q000378:metabolism*; D063151:Polycomb Repressive Complex 2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results
D007202:Indicators and Reagents; D009707:Nucleosomes; D010455:Peptides; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2