Configuration of a high-content imaging platform for hit identification and pharmacological assessment of JMJD3 demethylase enzyme inhibitors.
The biological complexity associated with the regulation of histone demethylases makes it desirable to configure a cellular mechanistic assay format that simultaneously encompasses as many of the relevant cellular processes as possible. In this report, the authors describe the configuration of a JMJD3 high-content cellular mechanistic imaging assay that uses single-cell multiparameter measurements to accurately assess cellular viability and the enzyme-dependent demethylation of the H3K27(Me)3 mark by exogenously expressed JMJD3. This approach couples robust statistical analyses with the spatial resolving power of cellular imaging. This enables segregation of expressing and nonexpressing cells into discrete subpopulations and consequently pharmacological quantification of compounds of interest in the expressing population at varying JMJD3 expression levels. Moreover, the authors demonstrate the utility of this hit identification strategy through the successful prosecution of a medium-throughput focused campaign of an 87 500-compound file, which has enabled the identification of JMJD3 cellular-active chemotypes. This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme.
Platform Technology and Sciences, Medicines Research Centre, GlaxoSmithKline R&D, Stevenage, UK.
Alpa
A
Mulji
Carl
C
Haslam
Fiona
F
Brown
Rebecca
R
Randle
Bhumika
B
Karamshi
Julia
J
Smith
Robert
R
Eagle
Jordi
J
Munoz-Muriedas
Joanna
J
Taylor
Arshad
A
Sheikh
Angela
A
Bridges
Kirsty
K
Gill
Rob
R
Jepras
Penny
P
Smee
Mike
M
Barker
Mike
M
Woodrow
John
J
Liddle
Pamela
P
Thomas
Emma
E
Jones
Laurie
L
Gordon
Rob
R
Tanner
Melanie
M
Leveridge
Sue
S
Hutchinson
Margaret
M
Martin
Murray
M
Brown
Laurens
L
Kruidenier
Roy
R
Katso
D000918:Antibody Specificity; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D006657:Histones / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D056484:Jumonji Domain-Containing Histone Demethylases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D010539:Permeability; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D054852:Small Molecule Libraries
D004791:Enzyme Inhibitors; D006657:Histones; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D056484:Jumonji Domain-Containing Histone Demethylases; C524220:KDM6B protein, human; D008239:Lysine