Abstract

Cyclin-dependent kinases (CDK’s) play key roles in cell cycle, cell proliferation and transcriptional regulation. CDK activity is tightly regulated by interactions with intracellular cyclins.
NanoBRET target engagement (TE) technology was used to develop a comprehensive panel of >30 specific CDK/cyclin pairings in live-cell assay formats. NanoBRET TE is the first real-time biophysical method to enable quantitative assessment of kinase-inhibitor binding and residence time under physiological ‘live cell’ conditions. Quantitative BRET-based capability is achieved via energy transfer from cell-permeable fluorescence tracers reversibly engaged to Nanoluc luciferase-tagged CDK proteins. Untagged cyclins are simultaneously co-expressed with the Nanoluc-CDK, allowing for the interrogation of specific CDK-cyclin pairings.
Time-dependent target-compound occupancy (or residence time) was measured using the NanoBRET TE assays in combination with electrospun scaffold material. Cells expressing specific CDK/cyclin pairingswere adhered to scaffold material to form a ‘colony’ that was physically moved between wells, allowing pre-established assay reagent cocktails to be set up prior to initiating residency experiments by adding cells on scaffolds to wells and immediately detecting NanoBRET. An assessment of kinetic selectivity of compounds with similar equilibrium affinity for CDK9/cyclin T1 was examined. Despite the similar cellular equilibrium affinity, the compounds displayed different intracellular residence times. The residency time methodology has been applied to other NanoBRET kinase assays to demonstrate the high throughput utility of this residency time approach.