Cellular Target Engagement Approaches to Monitor Epigenetic Reader Domain Interactions

Phillipou, Alexander; Lay, Charles S.; Carver, Charlotte E.; Messenger, Cassie; Evans, John P.; Lewis, Antonia J.; Gordon, Laurie J.; Mahmood, Mahnoor; Greenhough, Luke A; Sammon, Douglas; Cheng, Aaron T.; Chakraborty, Samarshi; Jones, Emma; Lucas, Simon; Gatfield, Kelly M; Brierley, David; Craggs, Peter D.

doi:10.1177/2472555219896278

Cited by 10 publications

(10 citation statements)

References 51 publications

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“…There are many ways to introduce NL-target DNA into the chosen cell line, including viral transduction, stable transfection and CRISPR-Cas9 knock-in. The protocol below will detail how to introduce NL-BRD4 expression plasmids into HEK293 cells via transient transfection, however an alternative CRISPR-Cas9 knock-in approach to insert a HiBit tag (part of a split NL) onto the N- terminus of BRD4 in HEK293 cells has also been successful for this target ( Phillipou et al., 2019 ). It is advisable to test that the addition of the NL tag has not affected the expected localization of your protein, which can be done by immune-cytochemical or luminescence imaging.…”

Section: Before You Beginmentioning

confidence: 99%

A kinetic intra-cellular assay (KICA) to measure quantitative compound binding kinetics within living cells

et al. 2022

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Summary The Kinetic Intra-Cellular Assay (KICA) is a recombinant cell-based technique that utilizes NanoBRET technology. KICA enables the measurement of intracellular binding kinetics. This protocol describes steps for cellular transfection and expression, followed by addition of a target specific fluorophore conjugated probe and a range of concentrations of competitor compounds, followed by the measurement of BRET in a 384 well format. Fitting the BRET data allows measurement of forward and reverse binding rates and the determination of K D . For complete details on the use and execution of this profile, please refer to Lay et al. (2021) .

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Section: Before You Beginmentioning

confidence: 99%

A kinetic intra-cellular assay (KICA) to measure quantitative compound binding kinetics within living cells

et al. 2022

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“…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.…”

Section: Nanobretmentioning

confidence: 99%

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

Cho

Dalby

2021

SLAS Discovery

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“…First of all, the high quantum yield of NanoLuc and the possibility to combine BRET assay with NanoLuc-derived split luciferase reporter system (NanoBiT, described above) ensures the development of BRET assay with increased sensitivity. It allows the monitoring of protein–protein interactions at lower and more physiologically-relevant levels [ 182 ] and analysis of interactions with low selectivity or affinity which have previously been challenging [ 183 , 184 ]. The NanoLuc bioluminescence spectrum (λ max = ~460 nm) is about 20% narrower compared with that of RLuc, enabling improved spectral separation of donor and acceptor emissions and the use of various fluorophores as acceptors.…”

Section: Ctz-dependent Luciferase Analytical Applicationmentioning

confidence: 99%

“…Comprehensive reviews on NanoBRET methodology and applications have been published recently [ 197 , 198 ]. NanoBRET is successfully used for studying protein–protein interactions [ 184 , 199 ], or investigations on receptor activation [ 190 , 199 , 200 ], oligomerization [ 201 ], conformational states [ 186 ], proximities [ 189 , 202 ], and receptor–ligand interactions [ 200 , 201 , 203 ]. NanoBRET technology has been also applied for in vivo imaging.…”

Section: Ctz-dependent Luciferase Analytical Applicationmentioning

confidence: 99%

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

Krasitskaya

Bashmakova

Frank

2020

IJMS

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: The functioning of bioluminescent systems in most of the known marine organisms is based on the oxidation reaction of the same substrate—coelenterazine (CTZ), catalyzed by luciferase. Despite the diversity in structures and the functioning mechanisms, these enzymes can be united into a common group called CTZ-dependent luciferases. Among these, there are two sharply different types of the system organization—Ca2+-regulated photoproteins and luciferases themselves that function in accordance with the classical enzyme–substrate kinetics. Along with deep and comprehensive fundamental research on these systems, approaches and methods of their practical use as highly sensitive reporters in analytics have been developed. The research aiming at the creation of artificial luciferases and synthetic CTZ analogues with new unique properties has led to the development of new experimental analytical methods based on them. The commercial availability of many ready-to-use assay systems based on CTZ-dependent luciferases is also important when choosing them by first-time-users. The development of analytical methods based on these bioluminescent systems is currently booming. The bioluminescent systems under consideration were successfully applied in various biological research areas, which confirms them to be a powerful analytical tool. In this review, we consider the main directions, results, and achievements in research involving these luciferases.

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Cellular Target Engagement Approaches to Monitor Epigenetic Reader Domain Interactions

Cited by 10 publications

References 51 publications

A kinetic intra-cellular assay (KICA) to measure quantitative compound binding kinetics within living cells

A kinetic intra-cellular assay (KICA) to measure quantitative compound binding kinetics within living cells

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

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

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