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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. Bromodomain and extra-terminal (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 nonselective toward 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 IC50 values of commercially available and in-house 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 bromodomain inhibitor engagement. This tool effectively targets BPTF and other class I bromodomains, aiding drug discovery for diseases like cancer.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Bromodomain proteins play roles in diseases like cancer and inflammation.
- Non-BET bromodomains are understudied, and high-throughput assays for inhibitors are limited.
- Existing inhibitors like BZ1 show non-selectivity across class I bromodomains.
Purpose of the Study:
- To design a NanoBRET tracer for assessing live-cell target engagement of bromodomain inhibitors.
- To evaluate the tracer's broad applicability across different class I bromodomains.
- To demonstrate the tracer's utility in inhibitor development and target engagement studies.
Main Methods:
- Development of a NanoBRET tracer (MS-1) based on a BZ1 analog.
- Utilizing live-cell assays to measure target engagement.
- Testing MS-1 against BPTF, PCAF, GCN5, and CECR2 bromodomains.
- Determining IC50 values for various inhibitors.
Main Results:
- MS-1 effectively binds BPTF, PCAF, GCN5, and CECR2 bromodomains in HEK293T cells.
- Binding occurs at low to sub-micromolar concentrations.
- The tracer demonstrated versatility for assessing inhibitor activity against multiple class I bromodomains.
Conclusions:
- MS-1 is a broadly applicable NanoBRET tracer for class I bromodomains.
- This tracer facilitates functional assessment of inhibitor activity in live cells.
- MS-1 aids future target engagement studies and inhibitor development for epigenetic targets.
