Description
Quantifying E3 Ligase Target Engagement and Intracellular Availability using NanoBRET® TE E3 Ligase Assays
Shown below is an intracellular availability workflow example for the NanoBRET® TE Intracellular E3 Ligase Assays. It involves:
Performing the live-cell assay mode to determine intracellular compound affinity
Performing the permeabilized-cell assay mode to determine compound affinity in the absence of the cell membrane
Calculating the intracellular availability for a compound using live-cell and permeabilized-cell mode data
Intracellular Affinity in Live-Cell Mode
The NanoBRET® TE E3 Ligase Assays can be used to quantify the intracellular affinity of ligands for a target E3 ligase in live cells. Shown here, the NanoBRET® TE E3 Ligase CRBN Assay was used to quantify the intracellular affinity of two PROTACs (dBET1 and dBET6) and two small-molecule CRBN molecular glues (iberdomide and lenalidomide). The NanoLuc®-CRBN fusion protein was transiently expressed in HEK293 cells. Both PROTACs have weaker affinity for CRBN compared to iberdomide or lenalidomide; the live cell affinities are tabularized below.
Affinity in Permeabilized-Cell Mode
Using the permeabilized-cell mode for the NanoBRET® TE E3 Ligase Assays, the intrinsic affinity of ligands for a target E3 ligase can be quantified in the absence of the cell membrane. Shown here, the NanoBRET® TE E3 Ligase CRBN Assay was used to quantify the intrinsic affinity for the same two PROTACs (dBET1 and dBET6) and small-molecule CRBN molecular glues (iberdomide and lenalidomide). The NanoLuc®-CRBN fusion protein was transiently expressed in HEK293 cells. The permeabilized cell affinities are tabularized below. The affinities of both PROTACs in permeabilized cells were stronger relative to that measured in live cells, while the affinities of the molecular glues were only minimally changed between permeabilized- and live-cell modes.
Calculating Intracellular Availability
Using the results from the live-cell and permeabilized-cell modes of the NanoBRET® TE E3 Ligase Assay, the relative binding affinity (RBA) and intracellular availability index (AI) can be calculated. The RBA for a compound is the ratio of its affinity live / affinity perm . Shown here, the RBA of the four compounds tested above were calculated. Calculating the intracellular availability index involves normalizing the RBA of a compound to the RBA a control compound (RBA compound /RBA control ). This enables comparison of intracellular availability across compounds. In this example:
Lenalidomide was used as the control compound, yielding an AI equal to 1. An AI >1 would indicate intracellular availability that is less than the control compound, while an AI <1 would indicate greater intracellular availability compared to the control.
The PROTAC dBET1 had the highest AI value and therefore the least intracellular availability among the tested compounds.
dBET6 had approximately tenfold lower AI value and greater intracellular availability compared to dBET1.
Iberdomide demonstrated high cellular availability, comparable to that of the control lenalidomide.
