From Biochemical TR-FRET Assay to Live Cell NanoBRET Assay: A Case Study with the BRD4 Protein

This is the authors' abstract. We don't add key points for this paper.

Fluorescent probe-based TR-FRET biochemical assays are widely utilized in characterizing ligands’ binding activities, whereas fluorescent probe-based NanoBRET target engagement (TE) assays are gaining popularity in characterizing ligands for their cellular activities. Compared to TR-FRET assays, few fluorophores were explored in NanoBRET assays. In this study, we investigated five (+)-JQ1-based fluorescent probes with BODIPY FL, FITC, TAMRA, NanoBRET 590 and Cy5 as the corresponding fluorophores and tested them as the probes in both TR-FRET and NanoBRET assays with BRD4 as the target. All five probes functioned as suitable TR-FRET probes with the BODIPY FL-(+)-JQ1 having the highest sensitivity and affinity. However, only BODIPY FL-(+)-JQ1 and NanoBRET 590-(+)-JQ1 were compatible with live-cell NanoBRET TE assays, with the NanoBRET 590-(+)-JQ1 demonstrating superior sensitivity, but the other 3 probes: (+)-JQ1-FITC, TAMRA-(+)-JQ1 and Cy5-(+)-JQ1, were functional only under digitonin-permeabilized conditions. By testing a panel of BRD4 ligands, we found that the combination of TR-FRET binding and NanoBRET TE assays reveal not only ligands’ binding activities but also their cellular permeability.