Cell-Based Ligand Discovery for the ENL YEATS Domain

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• The ENL protein acts as a transcriptional coactivator, essential for recruiting cellular machinery to active gene regulatory elements.
• ENL's YEATS domain, a chromatin reader module, binds to acylated lysine residues, playing a significant role in acute leukemia.
• Existing cell-based assays are insufficient for high-throughput screening and validation of ENL YEATS domain inhibitors.

Purpose of the Study:

• To develop a novel, high-throughput cell-based assay for discovering ligands targeting the ENL YEATS domain.
• To identify small-molecule inhibitors of the ENL/AF9 YEATS domain interaction.

Main Methods:

• Development of a modified cellular thermal shift assay (CETSA) that detects target engagement at 37 °C without temperature shifts.
• Miniaturization of the assay to a 1536-well format for high-throughput screening.
• Screening of 37,120 small molecules to identify ENL YEATS domain inhibitors.

Main Results:

• A simplified, two-step target engagement assay (drug treatment and luminescence detection) was established.
• The assay was successfully miniaturized and applied to screen a large chemical library.
• An acyl-lysine-competitive inhibitor of the ENL/AF9 YEATS domain was identified.

Conclusions:

• The developed assay enables high-throughput ligand discovery for the ENL YEATS domain in living cells.
• This method overcomes limitations of existing assays and facilitates hit validation.
• The identified inhibitor provides a starting point for developing novel therapeutics for ENL-driven cancers, particularly acute leukemia.