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NanoBRET Tracer Development for Class I Bromodomain Target Engagement in Live Cells
Molly S Sneddon1, Chun-Ju Tsou1, Xiang Fu2
1Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, Minnesota 55455, United States.
Abstract:
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 inhouse inhibitors to demonstrate tracer versatility for future target engagement studies and inhibitor development.
Insights
A new NanoBRET tracer, MS-1, enables live-cell assessment of non-BET bromodomain inhibitors, specifically targeting BPTF and other class I bromodomains for disease research.
Area of Science:
- Chemical Biology
- Epigenetics
- Drug Discovery
Background:
- Bromodomain proteins are implicated in diseases like cancer and inflammation.
- BET bromodomain inhibitors are well-studied, but non-BET bromodomains remain underexplored.
- Limited high-throughput assays exist for assessing bromodomain inhibitor activity.
Purpose of the Study:
- To develop a novel NanoBRET tracer for live-cell target engagement assays.
- To assess the functional activity of inhibitors against the BPTF bromodomain.
- To investigate the tracer's applicability to other class I bromodomains (PCAF, GCN5, CECR2).
Main Methods:
- Design of a NanoBRET tracer (MS-1) based on a BPTF inhibitor analog.
- Utilizing live-cell NanoBRET assays for target engagement studies.
- Testing MS-1 against BPTF, PCAF, GCN5, and CECR2 in HEK293T cells.
Main Results:
- MS-1 effectively engages BPTF, PCAF, GCN5, and CECR2 in live cells.
- Low to sub-micromolar concentrations were sufficient for tracer binding.
- EC50 values for various inhibitors were determined, demonstrating tracer utility.
Conclusions:
- MS-1 is a versatile NanoBRET tracer for class I bromodomains.
- This tracer facilitates live-cell target engagement studies for inhibitor development.
- The findings support future research into non-BET bromodomain inhibitors for therapeutic applications.
