Protein-observed 19F-NMR for fragment screening, affinity quantification and druggability assessment

Clifford T. Gee, Keith E. Arntson, Andrew K. Urick, Neeraj K Mishra, Laura Hawk, Andrea J. Wisniewski, William C Pomerantz

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

NMR spectroscopy can be used to quantify the binding affinity between proteins and low-complexity molecules, termed 'fragments'; this versatile screening approach allows researchers to assess the druggability of new protein targets. Protein-observed 19F-NMR (PrOF NMR) using 19F-labeled amino acids generates relatively simple spectra that are able to provide dynamic structural information toward understanding protein folding and function. Changes in these spectra upon the addition of fragment molecules can be observed and quantified. This protocol describes the sequence-selective labeling of three proteins (the first bromodomains of Brd4 and BrdT, and the KIX domain of the CREB-binding protein) using commercially available fluorinated aromatic amino acids and fluorinated precursors as example applications of the method developed by our research group. Fragment-screening approaches are discussed, as well as K d determination, ligand-efficiency calculations and druggability assessment, i.e., the ability to target these proteins using small-molecule ligands. Experiment times on the order of a few minutes and the simplicity of the NMR spectra obtained make this approach well-suited to the investigation of small- to medium-sized proteins, as well as the screening of multiple proteins in the same experiment.

Original languageEnglish (US)
Pages (from-to)1414-1427
Number of pages14
JournalNature Protocols
Volume11
Issue number8
DOIs
StatePublished - Aug 1 2016

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Screening
Nuclear magnetic resonance
Proteins
Molecules
CREB-Binding Protein
Ligands
Protein folding
Aromatic Amino Acids
Protein Folding
Structural dynamics
Labeling
Nuclear magnetic resonance spectroscopy
Magnetic Resonance Spectroscopy
Experiments
Research Personnel
Amino Acids
Research

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Protein-observed 19F-NMR for fragment screening, affinity quantification and druggability assessment. / Gee, Clifford T.; Arntson, Keith E.; Urick, Andrew K.; Mishra, Neeraj K; Hawk, Laura; Wisniewski, Andrea J.; Pomerantz, William C.

In: Nature Protocols, Vol. 11, No. 8, 01.08.2016, p. 1414-1427.

Research output: Contribution to journalArticle

Gee, Clifford T. ; Arntson, Keith E. ; Urick, Andrew K. ; Mishra, Neeraj K ; Hawk, Laura ; Wisniewski, Andrea J. ; Pomerantz, William C. / Protein-observed 19F-NMR for fragment screening, affinity quantification and druggability assessment. In: Nature Protocols. 2016 ; Vol. 11, No. 8. pp. 1414-1427.
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