A High-Throughput BRET Cellular Target Engagement Assay Links Biochemical to Cellular Activity for Bruton’s Tyrosine Kinase

Abstract

Protein kinases are intensely studied mediators of cellular signaling. While traditional biochemical screens are capable of identifying compounds that modulate kinase activity, these assays are limited in their capability of predicting compound behavior in a cellular environment. Here, we aim to bridge target engagement and compound-cellular phenotypic behavior by utilizing a bioluminescence resonance energy transfer (BRET) assay to characterize target occupancy within living cells for Bruton’s tyrosine kinase (BTK). Using a diverse chemical set of BTK inhibitors, we determine intracellular engagement affinity profiles and successfully correlate these measurements with BTK cellular functional readouts. In addition, we leveraged the kinetic capability of this technology to gain insight into in-cell target residence time and the duration of target engagement, and to explore a structural hypothesis.

In Vitro and Cellular Probes to Study PARP Enzyme Target Engagement

“…Traditionally the ability to do this has mainly been limited to in vitro techniques (Copeland et al, 2011;Renaud et al, 2016). Recently, NanoBRET probe displacement has been applied to other target classes to understand residence time in the complex cellular milieu where proteins may exist in complexes, have diverse post-translational modifications, and are interacting with substrates whose concentrations may be fluctuating (Bouzo-Lorenzo et al, 2019; Ong et al, 2020;Robers et al, 2015). We used slow-off and fast-off inhibitors of PARP7 and PARP14 to show that cellular residence time can be measured for the PARP enzymes using NanoBRET and that, for these enzyme-inhibitor pairs, the residence time measured by SPR aligns closely with the NanoBRET value.…”

Development of a NanoBRET assay to validate inhibitors of Sirt2-mediated lysine deacetylation and defatty-acylation that block prostate cancer cell migration

“…* For the calculation we used the tracer concentration in the medium as this is a common procedure in literature. [73][74][75] After having shown that the binding of our fluorescent tracer (13) to N-terminally labeled Nluc-Sirt2 can be monitored via NanoBRET, we were curious, if we could use our method in order to study cellular target engagement of Sirt2 inhibitors. For our displacement setup, HEK293T cells were transiently transfected with the N-terminal fusion protein and treated with the fluorescent tracer 13 (2 µM) in the presence of varying concentrations of the unlabeled competitors.…”

Luminescence Energy Transfer–Based Screening and Target Engagement Approaches for Chemical Biology and Drug Discovery

“…NanoBRET is adaptable to a high-throughput format and has been used to aid in the development of inhibitors of Bruton’s tyrosine kinase (BTK), 31 bromodomains, 32 the adenosine A3 receptor, 33 and salt-inducible kinases. 34 Some other examples include a study by Pavlinov et al, who used an NLuc/HaloTag strategy to assess Beclin 1–ATG14L interactions.…”