Extended Data Fig. 3: dHTC1 structure-activity relationship.
From: High-throughput ligand diversification to discover chemical inducers of proximity
a, Structure of dHTC1 analogs. b, HiBiT-based intracellular ENL YEATS target engagement assay (3 h). MLN4924 pre-treatment (1 µM, 1 h) was used to prevent the potential for compound-induced degradation. Signal is normalized to DMSO-treated cells (mean, 4 biological replicates ± S.E.M). Data in Extended Data Fig. 4d is taken from the same experiment (rac-dHTC1 data are repeated in both panels). c, Binding to immobilized ENL YEATS as measured by SPR (mean of 2 technical replicates.). Data in Extended Data Fig. 4c is taken from the same experiment (rac-dHTC1 data are repeated in both panels). d, HiBiT-based ENL degradation assay (24 h). HiBiT luminescence signal is normalized to DMSO-treated cells (mean, 4 biological replicates ± S.E.M.). e, CRBN FP assay for 7 in the presence of recombinant ENL YEATS domain (3 µM) or buffer (mean of 3 biological replicates ± S.E.M., background subtracted and normalized to DMSO). f, Urea analog 8 (same as panel e). g, SPR analysis of CRBN-DDB1 binding to immobilized ENL in the presence of a fixed concentration of compound (1 µM) or running buffer (mean of 2 technical replicates). Same experiment as Fig. 2c (rac-dHTC1 and buffer are repeated in both panels). h, Structure of two JQ1 analogs functionalized with the spirosuccinimide building block, BRD4 intracellular target engagement in MV4;11-BRD4-HiBiT cells (1 h, HiBiT luminescence normalized to DMSO, 3 biological replicates ± S.E.M.), and BRD4 degradation assay in MV4;11-BRD4-HiBiT cells (16 h, HiBiT luminescence normalized to DMSO, mean, 4 biological replicates ± S.E.M.). i, Structure of PARP1/2 inhibitor scaffolds functionalized with the spirosuccinimide building block, PARP1 intracellular target engagement in MV4;11-PARP1-HiBiT cells after 4 h treatment (HiBiT luminescence signal normalized to DMSO, mean of 3 biological replicates ± S.E.M.), and HiBiT degradation assay in MV4;11-PARP1-HiBiT cells after 16 h treatment (HiBiT luminescence signal normalized to DMSO, mean of 4 biological replicates ± S.E.M.). Compounds are compared to SK-575, a known PARP1 PROTAC degrader, or rucaparib and olaparib, which are known PARP1 ligands.
