Cell-Based Ligand Discovery for the ENL YEATS Domain

Abstract

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.

Figure 1.

Discovery of a simplified CETSA protocol for YEATS domains. (a) Schematic depiction of CETSA melt curves using the HiBiT tag to enable detection of soluble protein from homogenous cell lysates. (b) CETSA melt curve of ENL(YEATS)-HiBiT in HEK293T cells after 1-hour treatments with drug or vehicle (N = 4, mean ± standard error of the mean, s.e.m.). Luminescence is normalized to 37 °C. (c) Raw luminescence values from the experiment depicted in (b). (d) ITDR at 37 °C for HEK293T cells expressing wild-type or mutant ENL(YEATS)-HiBiT and treated for 1 hour with SGC-iMLLT (N = 2, mean ± s.e.m.). Luminescence is normalized to DMSO control. (e) ITDR at 37 °C for OCI/AML-2 cells stably expressing AF9(YEATS)-HiBiT and treated for 1 hour (N = 4, mean ± s.e.m.). Luminescence is normalized to DMSO control.

Figure 2.

Mechanism of ligand-dependent luminescence at physiological temperature. (a) Cycloheximide-enabled determination of ENL(YEATS)-HiBiT half-life when treated with vehicle or drug. The depicted HiBiT luminescence measures of protein abundance are normalized to pre-treatment levels (N = 3, mean ± s.e.m.). (b) Kinetics of ENL(YEATS)-HiBiT luminescence after SGC-iMLLT treatment with values normalized to 24-hour DMSO treatment (N = 10, mean ± s.e.m.). (c) Schematic depiction of HiBiT CETSA performed on pre-lysed cells. (d) Raw luminescence values for HiBiT CETSA performed on cell lysates treated with vehicle or SGC-iMLLT (N = 4, mean ± s.e.m.). (e) CETSA melt curve of SMARCA4(bromodomain)-HiBiT in OCI/AML-2 cells after 1-hour treatments with drug or vehicle (N = 3, mean ± s.e.m.). (f) CETSA melt curve for ENL(YEATS)-HiBiT performed by treating cell lysates with a peptide substrate, H3K27cr (N = 3, mean ± s.e.m.).

Figure 3.

High-throughput ligand discovery for ENL YEATS in living cells. (a) Optimization of cell density for 1536-well formatted assay. Luminescence values are shown for vehicle and drug-treated OCI/AML-2 cells stably expressing ENL(YEATS)-HiBiT at increasing cell concentrations (N = 15). (b) Summary of high-throughput screen to identify ENL YEATS domain inhibitors. Chemical structure of sCGT990 is depicted at bottom. (c) Dose-dependent inhibition of homogenous time-resolved FRET (HTRF) signal generated by ENL YEATS association with H3K27cr. SGC-iMLLT is included as a positive control (N = 2, mean ± s.e.m.). (d) Biolayer interferometry validation of sCGT990 binding to immobilized biotin-ENL(YEATS). (e) Dose-dependent stabilization of ENL(YEATS)-HiBiT by sCGT990 and positive control compound, SGC-iMLLT. Luminescence values are depicted relative to DMSO (N = 3, mean ± s.e.m.). (f) Immunoblot analysis of CETSA melt curve for endogenous ENL protein in OCI/AML-2 cells treated with sCGT990 (80 μM) or vehicle (DMSO) for 1 hr. (g) Dose-dependent inhibition of HTRF signal generated by AF9 YEATS association with H3K27cr. SGC-iMLLT is included as a positive control (N = 2, mean ± s.e.m.). (h) Biolayer interferometry measures of sCGT990 binding to immobilized biotin-YEATS4(YEATS). (i) Same as in (g), but for BRD4 bromodomain 1 and tetra-acetyl H4 (JQ1-S included as a positive control, (N = 2, mean ± s.e.m.).