Optimization of Ligands Using Focused DNA-Encoded Libraries To Develop a Selective, Cell-Permeable CBX8 Chromodomain Inhibitor

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.