NanoBRET Tracer Development for Class I Bromodomain Target Engagement in Live Cells Article Swipe

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Molly S Sneddon , Chun-Ju Tsou , Xiang Fu , Anang A. Shelat , William C. K. Pomerantz , Molly S Sneddon , Chun-Ju Tsou , Xiang Fu , Anang A. Shelat , William C. K. Pomerantz ·

YOU? · · 2025 · Open Access · · DOI: https://doi.org/10.1101/2025.11.25.689777

Epigenetic reader proteins, such as bromodomains, are often associated with diseases such as cancer and inflammation. BET bromodomain inhibitors have been studied extensively; however, non-BET bromodomains are understudied. Moreover, available high-throughput biological assays to assess inhibitors are limited. One non-BET bromodomain-containing protein, BPTF, has a recently reported inhibitor, BZ1, with an in vitro affinity of 6.3 nM. Additionally, BZ1 is known to be non-selective towards other class I bromodomains PCAF, GCN5, and CECR2. Here, we use a BZ1 analog, BZ1-THQ, to design a small molecule NanoBRET tracer, MS-1, for assessing inhibitor functional activity through live-cell target engagement against the BPTF bromodomain. Further, we investigate the versatility of MS-1 against PCAF, GCN5, and CECR2. We observe that MS-1 is a broadly applicable NanoBRET tracer for class I bromodomains, effectively binding BPTF, PCAF, GCN5, and CECR2 in HEK293T cells at low to sub-micromolar concentrations. We report EC50 values of commercially available and in-house inhibitors to demonstrate tracer versatility for future target engagement studies and inhibitor development.

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Computational Biology

Protein–Protein Interaction

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Bromodomain Computational biology Chemistry Class (philosophy) In vitro Small molecule Biology In vivo Cancer Plasma protein binding Cell biology Binding site HEK 293 cells BRD4 Cancer research Biological activity Protein–protein interaction Biochemistry Ligand binding assay Cancer treatment Pharmacology Computer science Cancer cell Cancer therapy In vitro toxicology Bioinformatics Genetics

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Landing Page: https://doi.org/10.1101/2025.11.25.689777
PDF: https://www.biorxiv.org/content/biorxiv/early/2025/11/26/2025.11.25.689777.full.pdf
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DOI: https://doi.org/10.1101/2025.11.25.689777

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Title: NanoBRET Tracer Development for Class I Bromodomain Target Engagement in Live Cells

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Publication year: 2025

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Publication date: 2025-11-26

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Authors: Molly S Sneddon, Chun-Ju Tsou, Xiang Fu, Anang A. Shelat, William C. K. Pomerantz, Molly S Sneddon, Chun-Ju Tsou, Xiang Fu, Anang A. Shelat, William C. K. Pomerantz

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Landing page: https://doi.org/10.1101/2025.11.25.689777

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Concepts: Bromodomain, Computational biology, Chemistry, Class (philosophy), In vitro, Small molecule, Biology, In vivo, Cancer, Plasma protein binding, Cell biology, Binding site, HEK 293 cells, BRD4, Cancer research, Biological activity, Protein–protein interaction, Biochemistry, Ligand binding assay, Cancer treatment, Pharmacology, Computer science, Cancer cell, Cancer therapy, In vitro toxicology, Bioinformatics, Genetics

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abstract_inverted_index.Moreover,	28
abstract_inverted_index.assessing	89
abstract_inverted_index.available	29, 148
abstract_inverted_index.inhibitor	90, 162
abstract_inverted_index.live-cell	94
abstract_inverted_index.proteins,	2
abstract_inverted_index.Epigenetic	0
abstract_inverted_index.applicable	120
abstract_inverted_index.associated	8
abstract_inverted_index.biological	31
abstract_inverted_index.engagement	96, 159
abstract_inverted_index.functional	91
abstract_inverted_index.inhibitor,	47
abstract_inverted_index.inhibitors	18, 35, 151
abstract_inverted_index.bromodomain	17
abstract_inverted_index.demonstrate	153
abstract_inverted_index.effectively	127
abstract_inverted_index.investigate	103
abstract_inverted_index.versatility	105, 155
abstract_inverted_index.bromodomain.	100
abstract_inverted_index.bromodomains	25, 68
abstract_inverted_index.commercially	147
abstract_inverted_index.development.	163
abstract_inverted_index.extensively;	22
abstract_inverted_index.Additionally,	57
abstract_inverted_index.bromodomains,	5, 126
abstract_inverted_index.inflammation.	15
abstract_inverted_index.non-selective	63
abstract_inverted_index.understudied.	27
abstract_inverted_index.sub-micromolar	140
abstract_inverted_index.concentrations.	141
abstract_inverted_index.high-throughput	30
abstract_inverted_index.bromodomain-containing	40
cited_by_percentile_year
countries_distinct_count	0
institutions_distinct_count	10
citation_normalized_percentile