NanoBRET™ Live-Cell Kinase Selectivity Profiling Adapted for High-Throughput Screening

Amanda N Nieman; Kaitlin K Dunn Hoffman; Elizabeth R Dominguez; Jennifer Wilkinson; James D Vasta; Matthew B Robers; Ngan Lam

doi:10.1007/978-1-0716-3397-7_8

NanoBRET™ Live-Cell Kinase Selectivity Profiling Adapted for High-Throughput Screening

Methods Mol Biol. 2023:2706:97-124. doi: 10.1007/978-1-0716-3397-7_8.

Authors

Amanda N Nieman¹, Kaitlin K Dunn Hoffman¹, Elizabeth R Dominguez¹, Jennifer Wilkinson¹, James D Vasta¹, Matthew B Robers¹, Ngan Lam²

Affiliations

¹ Promega Corporation, Madison, WI, USA.
² Promega Corporation, Madison, WI, USA. ngan.lam@promega.com.

PMID: 37558944
DOI: 10.1007/978-1-0716-3397-7_8

Abstract

Kinases represent one of the most therapeutically tractable targets for drug discovery in the twenty-first century. However, confirming engagement and achieving intracellular kinase selectivity for small-molecule kinase inhibitors can represent noteworthy challenges. The NanoBRET^TM platform enables broad-spectrum live-cell kinase selectivity profiling in most laboratory settings, without advanced instrumentation or expertise. However, the prototype workflow for this selectivity profiling is currently limited to manual liquid handling and 96-well plates. Herein, we describe a scalable workflow with automation and acoustic dispensing, thus dramatically improving the throughput. Such adaptations enable profiling of larger compound sets against 192 full-length protein kinases in live cells, with statistical robustness supporting quantitative analysis.

Keywords: BRET; High-Throughput Screening (HTS); Kinase profiling; Live-cell target engagement; NanoBRETTM.

MeSH terms

Drug Discovery
High-Throughput Screening Assays*
Protein Kinases* / metabolism

Substances

Protein Kinases