Abstact

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 G(0), consistent with Cdk2 inhibition. This miniaturized workflow allows the upper tiers of a typical optimization screening cascade to be performed in a single experiment.