Structure-Guided DOT1L Probe Optimization by Label-Free Ligand Displacement

The
DOT1L lysine methyltransferase has emerged as a validated therapeutic
target in MLL-rearranged (MLLr) acute leukemias.
Although S-adenosylmethionine competitive inhibitors have demonstrated
pharmacological proof-of-principle in MLLr-leukemia,
these compounds require further optimization to improve cellular potency
and pharmacokinetic stability. Limiting DOT1L inhibitor discovery
and ligand optimization have been complex biochemical methods often
using radionucleotides and cellular methods requiring prolonged culture.
We therefore developed a new suite of assay technologies that allows
comparative assessment of chemical tools for DOT1L in a miniaturized
format. Coupling these assays with structural information, we developed
new insights into DOT1L ligand binding and identified several functionalized
probes with increased cellular potency (IC50 values ∼10
nM) and excellent selectivity for DOT1L. Together these assay technologies
define a platform capability for discovery and optimization of small-molecule
DOT1L inhibitors.