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 ADME 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 problem by omitting the purification step and submitting reaction mixtures directly to bioassay. Here, we explore nanoscale hit-to-lead optimization through the multi-step 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 were 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.