Homogeneous Assay for Target Engagement Utilizing Bioluminescent Thermal Shift.

Protein thermal shift assays (TSAs) provide a means for characterizing target engagement through ligand-induced thermal stabilization. Although these assays are widely utilized for screening libraries and validating hits in drug discovery programs, they can impose encumbering operational requirements, such as the availability of purified proteins or selective antibodies. Appending the target protein with a small luciferase (NanoLuc) allows coupling of thermal denaturation with luminescent output, providing a rapid and sensitive means for assessing target engagement in compositionally complex environments such as permeabilized cells.

Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement.

For kinase inhibitors, intracellular target selectivity is fundamental to pharmacological mechanism. Although a number of acellular techniques have been developed to measure kinase binding or enzymatic inhibition, such approaches can fail to accurately predict engagement in cells. Here we report the application of an energy transfer technique that enabled the first broad-spectrum, equilibrium-based approach to quantitatively profile target occupancy and compound affinity in live cells.

Target engagement and drug residence time can be observed in living cells with BRET.

The therapeutic action of drugs is predicated on their physical engagement with cellular targets. Here we describe a broadly applicable method using bioluminescence resonance energy transfer (BRET) to reveal the binding characteristics of a drug with selected targets within intact cells. Cell-permeable fluorescent tracers are used in a competitive binding format to quantify drug engagement with the target proteins fused to Nanoluc luciferase.