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. 2022 Feb 17;29(2):287-299.e8.
doi: 10.1016/j.chembiol.2021.07.018. Epub 2021 Sep 13.

Development of an intracellular quantitative assay to measure compound binding kinetics

Charles S Lay  1 , Daniel A Thomas  2 , John P Evans  3 , Matthew Campbell  3 , Kristopher McCombe  4 , Alexander N Phillipou  3 , Laurie J Gordon  3 , Emma J Jones  5 , Kristin Riching  6 , Mahnoor Mahmood  3 , Cassie Messenger  3 , Charlotte E Carver  3 , Kelly M Gatfield  3 , Peter D Craggs  7
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Free article

Development of an intracellular quantitative assay to measure compound binding kinetics

Charles S Lay et al. Cell Chem Biol. .
Free article

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Abstract

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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Conflict of interest statement

Declaration of interests K.R. is an employee of Promega Corporation, which holds the proprietary rights to the NanoBRET assay technology. A.N.P., M.C., L.J.G., E.J.J., C.M., K.M.G., and P.D.C. are employees and shareholders of GSK. D.A.T. and J.P.E. are former employees of GSK and remain shareholders. C.S.L. formerly worked at GSK as an undergraduate student and is now working on a part-funded GSK PhD studentship. C.S.L. is not a shareholder in GSK and declares no competing interests. C.E.C., K.M., and M.M. formerly worked at GSK as undergraduate students and declare no competing interests.

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