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.

中文翻译：

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ENL YEATS域的基于单元的配体发现。

ENL是一种转录共激活因子，在其YEATS域（一种染色质阅读器模块）与酰化赖氨酸侧链结合后，募集延伸机制来激活顺式调控元件。ENL YEATS域的发现化学受到其在急性白血病病理生理学中的重要性的强烈推动，但是目前尚不存在能够支持大规模筛选或命中验证工作的基于细胞的测定法。在这里，我们报告的目标参与测定的发现，允许在活细胞中发现高通量配体。该测定基于细胞热位移测定（CETSA），但不需要将细胞暴露于高温下，因为小分子配体能够在37°C的温度下稳定ENL YEATS结构域。通过消除温度变化，我们开发了一种简化的目标参与分析，仅需两个步骤：药物处理和发光检测。为了证明其对更高通量应用的价值，我们将测定法小型化为1536孔形式，并筛选了37120个小分子，最终鉴定出了具有赖氨酸竞争性的ENL / AF9 YEATS结构域抑制剂。