Contemporary drug discovery typically quantifies the effect of a molecule on a biological target using the equilibrium-derived measurements of IC50, EC50, or KD. Kinetic descriptors of drug binding are frequently linked with the effectiveness of a molecule in modulating a disease phenotype; however, these parameters are yet to be fully adopted in early drug discovery. Nanoluciferase bioluminescence resonance energy transfer (NanoBRET) can be used to measure interactions between fluorophore-conjugated probes and luciferase fused target proteins. Here, we describe an intracellular NanoBRET competition assay that can be used to quantify cellular kinetic rates of compound binding to nanoluciferase-fused bromodomain and extra-terminal (BET) proteins. Comparative rates are generated using a cell-free NanoBRET assay and by utilizing orthogonal recombinant protein-based methodologies. A screen of known pan-BET inhibitors is used to demonstrate the value of this approach in the investigation of kinetic selectivity between closely related proteins.
Keywords: BET proteins; BRD4; NanoBRET; SKR; bromodomain; k(off); k(on); kinetics; residence time; target engagement.
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