Homogeneous Assay for Target Engagement Utilizing Bioluminescent Thermal Shift

Abstract

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. The intrinsic thermal stability of NanoLuc is greater than mammalian proteins, and our results indicate that the appended luciferase does not alter thermal denaturation of the target protein. We have successfully applied the NanoLuc luciferase thermal shift assay (NaLTSA) to several clinically relevant protein families, including kinases, bromodomains, and histone deacetylases. We have also demonstrated the suitability of this assay method for library screening and compound profiling.

Figure 1

NaLTSA principle and workflow. (a) Compounds that bind Nluc-POI fusion will lead to stabilization compared to controls resulting in differences in T_m as detected by luminescence. (b) Assay workflow.

Figure 2

NaLTSA validation in permeabilized HeLa cells transiently expressing Nluc-MAPK14. (a) Samples were exposed to compounds [50 μM] and subjected to NaLTSA. (b) ΔT_m results obtained from n = 10 independent NaLTSAs to demonstrate reproducibility. (c) Melting curves for each [AMG 548]. (d) Isothermal dose response curves were generated by exposing duplicate sample plates to increasing [compound] at isothermal temperatures of 40 or 54 °C. (a,c,d) Shown are mean ± SEM, n = 4 independent experiments.

Figure 3

Primary hit screening, hit validation, and relative rank affinity for inhibitors of MAPK14. (a,b) Primary hit screening using HeLa cells transfected with Nluc-MAPK14 or unfused Nluc control using the ScreenWell Kinase Inhibitor library at [10 μM] in isothermal NaLTSA of 40 or 54 °C. Red lines denotes >40% stability hit identification. Dots: red, DMSO control; blue, AMG 548; green, hits >40% stabilization. Representative data shown. (c,d) Hit validation using NaLTSA in temperature gradient or isothermal dose response mode. Shown are mean ± SEM, n = 3 independent experiments. (e) Biochemical MAPK14 ADP-Glo kinase activity assay. Shown are mean RLU values, n = 3, of a representative experiment, with variability expressed as SD.