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

Andrew K. Urick, Laura M.L. Hawk, Melissa K. Cassel, Neeraj K. Mishra, Shuai Liu, Neeta Adhikari, Wei Zhang, Camila O. Dos Santos, Jennifer L. Hall, William C.K. Pomerantz

Research output: Contribution to journalArticle

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Abstract

Bromodomain-containing protein dysregulation is linked to cancer, diabetes, and inflammation. Selective inhibition of bromodomain function is a newly proposed therapeutic strategy. We describe a 19F NMR dual screening method for small molecule discovery using fluorinated tryptophan resonances on two bromodomain-containing proteins. The chemical shift dispersion of 19F resonances within fluorine-labeled proteins enables the simultaneous analysis of two fluorinated bromodomains by NMR. A library of 229 small molecules was screened against the first bromodomain of Brd4 and the BPTF bromodomain. We report the first small molecule selective for BPTF over Brd4, termed AU1. The Kd = 2.8 for AU1, which is active in a cell-based reporter assay. No binding is detected with Brd4. Three new Brd4 inhibitors with submicromolar affinity were also discovered. Brd4 hits were validated in a thermal stability assay and potency determined via fluorescence anisotropy. The speed, ease of interpretation, and low protein concentration needed for protein-observed 19F NMR experiments in a multiprotein format offers a new method to discover and characterize selective ligands for bromodomain-containing proteins.

Original languageEnglish (US)
Pages (from-to)2246-2256
Number of pages11
JournalACS Chemical Biology
Volume10
Issue number10
DOIs
StatePublished - Oct 16 2015

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Fluorine
Screening
Nuclear magnetic resonance
Molecules
Proteins
Assays
Small Molecule Libraries
Fluorescence Polarization
Chemical shift
Medical problems
Tryptophan
Anisotropy
Thermodynamic stability
Hot Temperature
Fluorescence
Ligands
Inflammation
Neoplasms
Experiments

Cite this

Dual Screening of BPTF and Brd4 Using Protein-Observed Fluorine NMR Uncovers New Bromodomain Probe Molecules. / Urick, Andrew K.; Hawk, Laura M.L.; Cassel, Melissa K.; Mishra, Neeraj K.; Liu, Shuai; Adhikari, Neeta; Zhang, Wei; Dos Santos, Camila O.; Hall, Jennifer L.; Pomerantz, William C.K.

In: ACS Chemical Biology, Vol. 10, No. 10, 16.10.2015, p. 2246-2256.

Research output: Contribution to journalArticle

Urick, AK, Hawk, LML, Cassel, MK, Mishra, NK, Liu, S, Adhikari, N, Zhang, W, Dos Santos, CO, Hall, JL & Pomerantz, WCK 2015, 'Dual Screening of BPTF and Brd4 Using Protein-Observed Fluorine NMR Uncovers New Bromodomain Probe Molecules', ACS Chemical Biology, vol. 10, no. 10, pp. 2246-2256. https://doi.org/10.1021/acschembio.5b00483
Urick, Andrew K. ; Hawk, Laura M.L. ; Cassel, Melissa K. ; Mishra, Neeraj K. ; Liu, Shuai ; Adhikari, Neeta ; Zhang, Wei ; Dos Santos, Camila O. ; Hall, Jennifer L. ; Pomerantz, William C.K. / Dual Screening of BPTF and Brd4 Using Protein-Observed Fluorine NMR Uncovers New Bromodomain Probe Molecules. In: ACS Chemical Biology. 2015 ; Vol. 10, No. 10. pp. 2246-2256.
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