Quantitative target engagement (TE) via bíoluminescence r,esonance energy transfer (BRET) methodologies enables non-permeabilized c live-cell high throughput profiling of drug candidates. To assess the precision and repeatability of lhis technology, a selection of FDAapproved small molecules, and a set of published kinase inhibitors were screened in ciase-response titrations against a panel of 240 full length Nanoluc tagged kinases and complimentary BRET tracers. These experimenls were executed using an automalion workflow, across eleven 240-kinase panel screens. Results were analyzed to determine inter-run variance and trends of individual assay performance against multiple groups of accepta,nce criteria. Additionally, intermediate precision of rank order profiling and potency results from FDA-approved kinase inhibitors were evaluated far reproducibility of biological replicates. We successfully demonstrate that NanoBRET-based quantitation of fractional target occupancy and derived compound IC50s are highly consistent between experiments. Furthermore, compound profiling results reproduce commercial pharmaceutical selectivity found in published literature, while providing contemporaneous live-cell target engagement data spanning a representative portian of the kinome.
Abstract
Poster
