Cell-Based Ligand Discovery for the ENL YEATS Domain

• Published in: ACS chemical biology Vol. 15; no. 4; pp. 895 - 903
• Main Authors: Asiaban, Joshua N, Milosevich, Natalia, Chen, Emily, Bishop, Timothy R, Wang, Justin, Zhang, Yuxiang, Ackerman, Christopher J, Hampton, Eric N, Young, Travis S, Hull, Mitchell V, Cravatt, Benjamin F, Erb, Michael A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 17.04.2020
• Subjects: Biological Assay - methods; Cell Line, Tumor; Drug Discovery; HEK293 Cells; High-Throughput Screening Assays - methods; Humans; Ligands; Protein Binding; Protein Domains - drug effects; Small Molecule Libraries - analysis; Small Molecule Libraries - metabolism; Small Molecule Libraries - pharmacology; Transcriptional Elongation Factors - antagonists & inhibitors; Transcriptional Elongation Factors - metabolism
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/acschembio.0c00124

ENL is a transcriptional coactivator 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 1536-well format and screened 37 120 small molecules, ultimately identifying an acyl-lysine-competitive ENL/AF9 YEATS domain inhibitor.