Optimization of Ligands Using Focused DNA-Encoded Libraries To Develop a Selective, Cell-Permeable CBX8 Chromodomain
Sijie Wang1, Kyle E Denton1, Kathryn F Hobbs2
1Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy , Purdue University and Purdue University Center for Cancer Research , 575 Stadium Mall Drive , West Lafayette , Indiana 47906 , United States.
Abstract:
Polycomb repressive complex 1 (PRC1) is critical for mediating gene expression during development. Five chromobox (CBX) homolog proteins, CBX2, CBX4, CBX6, CBX7, and CBX8, are incorporated into PRC1 complexes, where they mediate targeting to trimethylated lysine 27 of histone H3 (H3K27me3) via the N-terminal chromodomain (ChD). Individual CBX paralogs have been implicated as drug targets in cancer; however, high similarities in sequence and structure among the CBX ChDs provide a major obstacle in developing selective CBX ChD inhibitors. Here we report the selection of small, focused, DNA-encoded libraries (DELs) against multiple homologous ChDs to identify modifications to a parental ligand that confer both selectivity and potency for the ChD of CBX8. This on-DNA, medicinal chemistry approach enabled the development of SW2_110A, a selective, cell-permeable inhibitor of the CBX8 ChD. SW2_110A binds CBX8 ChD with a Kd of 800 nM, with minimal 5-fold selectivity for CBX8 ChD over all other CBX paralogs in vitro. SW2_110A specifically inhibits the association of CBX8 with chromatin in cells and inhibits the proliferation of THP1 leukemia cells driven by the MLL-AF9 translocation. In THP1 cells, SW2_110A treatment results in a significant decrease in the expression of MLL-AF9 target genes, including HOXA9, validating the previously established role for CBX8 in MLL-AF9 transcriptional activation, and defining the ChD as necessary for this function. The success of SW2_110A provides great promise for the development of highly selective and cell-permeable probes for the full CBX family. In addition, the approach taken provides a proof-of-principle demonstration of how DELs can be used iteratively for optimization of both ligand potency and selectivity.
Insights
Researchers developed a selective inhibitor, SW2_110A, targeting the CBX8 chromodomain (ChD) using DNA-encoded libraries. This breakthrough offers potential for developing targeted cancer therapies by inhibiting gene expression in leukemia cells.
Area of Science:
- Epigenetics and Gene Regulation
- Medicinal Chemistry
- Cancer Biology
Background:
- Polycomb repressive complex 1 (PRC1) regulates gene expression during development.
- Chromobox (CBX) proteins are key components of PRC1, targeting histone modifications like H3K27me3 via their chromodomains (ChDs).
- CBX paralogs are cancer drug targets, but their similar ChD structures hinder selective inhibitor development.
Purpose of the Study:
- To develop selective inhibitors for CBX chromodomains (ChDs), specifically targeting CBX8.
- To utilize DNA-encoded libraries (DELs) for iterative optimization of ligand potency and selectivity.
- To validate the functional role of CBX8 ChD in MLL-AF9 driven leukemia.
Main Methods:
- Selection of focused DNA-encoded libraries (DELs) against multiple homologous CBX ChDs.
- Medicinal chemistry approach for ligand modification and optimization.
- In vitro binding assays (Kd determination) and cellular assays to assess target engagement and functional effects.
Main Results:
- Development of SW2_110A, a selective and cell-permeable inhibitor of the CBX8 ChD with a Kd of 800 nM.
- SW2_110A demonstrated minimal 5-fold selectivity over other CBX paralogs in vitro.
- SW2_110A inhibited CBX8 chromatin association, THP1 leukemia cell proliferation, and MLL-AF9 target gene expression (e.g., HOXA9).
Conclusions:
- The CBX8 ChD is essential for MLL-AF9 transcriptional activation in leukemia.
- SW2_110A serves as a potent and selective chemical probe for studying CBX8 function.
- The DEL approach provides a successful strategy for developing selective inhibitors against homologous protein targets.
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