Cell-based ligand discovery for the ENL YEATS domain.

ENL is a transcriptional co-activator that recruits elongation machinery to active cis-regulatory elements upon binding of its YEATS domain - a chromatin reader module - to 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 1,536-well format and screened 37,120 small molecules, ultimately identifying an acyl-lysine-competitive ENL/AF9 YEATS domain inhibitor.