Dual Screening of BPTF and Brd4 Using Protein-Observed Fluorine NMR Uncovers New Bromodomain Probe Molecules

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Bromodomain-containing proteins are implicated in diseases such as cancer, diabetes, and inflammation.
• Selective inhibition of bromodomain function presents a promising therapeutic strategy.
• Targeting bromodomains requires efficient methods for small molecule discovery and characterization.

Purpose of the Study:

• To develop and validate a novel (19)F NMR dual screening method for identifying selective small molecule inhibitors of bromodomain-containing proteins.
• To discover selective inhibitors for the BPTF bromodomain and Brd4 bromodomain.
• To demonstrate the utility of protein-observed (19)F NMR in multiprotein screening formats.

Main Methods:

• Utilized a (19)F NMR dual screening approach employing fluorinated tryptophan resonances on two distinct bromodomain proteins.
• Screened a library of 229 small molecules against the first bromodomain of Brd4 and the BPTF bromodomain.
• Validated identified inhibitors using thermal stability assays and fluorescence anisotropy.

Main Results:

• Discovered AU1, the first reported small molecule selective for the BPTF bromodomain over Brd4, with a Kd of 2.8 μM and cellular activity.
• Identified three novel Brd4 inhibitors with submicromolar affinity.
• Demonstrated the simultaneous analysis of two fluorinated bromodomains by NMR, enabling efficient screening.

Conclusions:

• The developed (19)F NMR dual screening method is a rapid, sensitive, and interpretable approach for discovering selective ligands for bromodomain proteins.
• This method facilitates the characterization of small molecules targeting disease-associated bromodomains.
• The findings provide new chemical tools for studying bromodomain function and developing targeted therapies.