Nanoscale Direct-to-Biology Optimization of Cdk2 Inhibitors

Modern hit-to-lead optimization winnows down vast chemical
spaces
of virtual compounds into a selection of potent and selective compounds
that can be further profiled with in vitro assays.
Today, miniaturized chemical synthesis can be performed in high-throughput,
shifting the bottleneck to compound purification. Direct-to-biology
(D2B) approaches seek to overcome this hurdle by omitting the purification
step and submitting reaction mixtures directly to bioassays. Here,
we explore nanoscale hit-to-lead optimization through the multistep
synthesis of a library of Cdk2/CycE inhibitors, utilizing ultrahigh-throughput
experimentation (ultraHTE) in 1,536-well plates. Library performance
is assessed by D2B in functional biochemical, bioaffinity, and X-ray
crystallographic assays. A selection of potent lead Cdk2/CycE inhibitors
identified by D2B was submitted to a phenotypic cell painting assay,
which showed cell cycle arrest at G0, consistent with Cdk2
inhibition. This miniaturized workflow allows the upper tiers of a
typical optimization screening cascade to be performed in a single
experiment.