Strategic Collaborations Mgr Promega Corporation San Marcos, CA, United States
Abstract: NanoBRET™ Target Engagement is a real-time biophysical technique that enables quantitative assessment of compound-target protein binding in live cells, without disruption of cellular membrane integrity. Specifically, cellular compound affinity, occupancy, and residence time can be measured in a multi-well plate format. The method utilizes energy transfer from a NanoLuc® luciferase-tagged target protein and a cell-permeable fluorescent tracer that reversibly engages the target protein of interest. The quantitative capability is achieved for select target kinases by simply adjusting the concentration of the NanoBRET tracer to an appropriate level based on its predetermined target affinity.
This technique has now been broadly applied to the kinome. Cellular assays for >300 full-length protein kinases are available, including integral membrane receptors and over 30 specific CDK-cyclin pairings. Assay conditions that enable quantitative binding measurements for each kinase have been determined. Quantitative cellular affinity for various types of kinase inhibitors will be demonstrated, including type I, II, and allosteric compounds. We will show how this cellular method can also be applied to quantitative measurement of compound selectivity against numerous kinases.
Time-dependent target-compound occupancy (or residence time) can also be obtained with using the NanoBRET method. An assessment of kinetic and equilibrium selectivity of various kinase inhibitors revealed different residence times for compounds with similar equilibrium affinity. By monitoring both cellular affinity and residence time for a compound, unique inhibitor development opportunities can be revealed.