posted on 2020-03-19, 15:03authored byJoshua
N. Asiaban, Natalia Milosevich, Emily Chen, Timothy R. Bishop, Justin Wang, Yuxiang Zhang, Christopher J. Ackerman, Eric N. Hampton, Travis S. Young, Mitchell V. Hull, Benjamin F. Cravatt, Michael A. Erb
ENL
is a transcriptional coactivator that recruits elongation machinery
to active cis-regulatory elements upon binding of
its YEATS domaina chromatin reader moduleto acylated
lysine side chains. Discovery chemistry for the ENL YEATS domain is
highly motivated by its significance in acute leukemia pathophysiology,
but cell-based assays able to support large-scale screening or hit
validation efforts do not presently exist. Here, we report on the
discovery of a target engagement assay that allows for high-throughput
ligand discovery in living cells. This assay is based on the cellular
thermal shift assay (CETSA) but does not require exposing cells to
elevated temperatures, as small-molecule ligands are able to stabilize
the ENL YEATS domain at 37 °C. By eliminating temperature shifts,
we developed a simplified target engagement assay that requires just
two steps: drug treatment and luminescence detection. To demonstrate
its value for higher throughput applications, we miniaturized the
assay to a 1536-well format and screened 37 120 small molecules,
ultimately identifying an acyl-lysine-competitive ENL/AF9 YEATS domain
inhibitor.