to characterize binding of known hits or aid in hit development [83]. This method is
especially useful to identify the binding site and key amino acids when a co-crystal
structure cannot be obtained. Additionally, 2D-NMR methods have been used to
study the interaction between bromodomains and acetylated histone peptide mimics
[25, 84–87]. As an example, Ferguson et al. used
1 H15 N-HSQC chemical shift and
CLEAN-chemical exchange experiments to determine hot spot amino acids involved
in the native interaction between H3K14ac and BAZ2B [88]. Multidomain
proteins have also been studied by
1 H15 N-HSQC NMR with chemically defined
nucleosomes [35].
4.2.3 PrOF NMR Is a Sensitive Technique That Gives Simple Spectra
to Analyze for Binding Interactions
Protein-observed fluorine (PrOF) NMR is an alternate protein-observed method that
is fast and gives simple spectra to interpret. For PrOF NMR, bromodomains are
fluorine labeled by the incorporation of fluorinated amino acids. Fluorine is an ideal
NMR reporting atom, because it is the second most sensitive stable NMR-active
nuclei (83% compared to
1 H), its hyperresponsiveness to change in its environment
evident by its ~400 ppm chemical shift range,
19
F is 100% isotopically naturally
abundant, and fluorine is not present in biomolecules, resulting in no background
signal [89, 90]. Aromatic amino acids are enriched at the bromodomain binding
interface but not prevalent throughout the rest of the protein [91, 92]. Thus,
nonnatural amino acids such as 5-fluorotryptophan (5FW), 3-fluorotyrosine (3FY),
or 4-fluorophenylalanine (4FF) may be incorporated as sensitive NMR reporter side
chains. A detailed protocol for fluorinated amino acid incorporation and strategies
for fluorine labeling and screening have been described [93]. PrOF NMR experiments with bromodomains are fast (2–5 min with 50 μM protein on a 500 MHz
instrument) when cryoprobes are used and the chemical shift perturbations are
reproducible making it a good method for screening [94]. Additionally, having
multiple fluorine labels allows for monitoring of protein behavior and assessment
of where small molecules are binding to the protein (Fig. 11).
A 2015 paper by Urick et al. exemplifies a screening platform that takes advantage of the strengths of PrOF NMR, DSF, FP, and ITC to identify hits, rank order
hits, and characterize binding affinity for compounds targeting BRD4 BD1 and
BPTF [71]. An advantage of PrOF NMR over ligand-observed NMR methods is
the ability to screen two proteins in the same experiment (single NMR tube) because
the PrOF NMR spectra are simple, essentially completing two screens at once. Dualprotein PrOF NMR screens give bromodomain selectivity information at the onset of
the inhibitor discovery process. The HSQC analog of this experiment, RAMPED-UP
NMR, was previously described by Zartler et al. [95].
Urick et al. completed a dual-protein PrOF NMR screen to identify selective hits
for both BRD4 and BPTF. BRD4 BD1 and BPTF were sequence selectively labeled
with 5-fluorotryptophan (5FW). BRD4 BD1 has three tryptophan residues. W81 is
located on the WPF near the binding site and acts as the primary reporting residue.
Applied Biophysics for Bromodomain Drug Discovery
311
especially useful to identify the binding site and key amino acids when a co-crystal
structure cannot be obtained. Additionally, 2D-NMR methods have been used to
study the interaction between bromodomains and acetylated histone peptide mimics
[25, 84–87]. As an example, Ferguson et al. used
1 H15 N-HSQC chemical shift and
CLEAN-chemical exchange experiments to determine hot spot amino acids involved
in the native interaction between H3K14ac and BAZ2B [88]. Multidomain
proteins have also been studied by
1 H15 N-HSQC NMR with chemically defined
nucleosomes [35].
4.2.3 PrOF NMR Is a Sensitive Technique That Gives Simple Spectra
to Analyze for Binding Interactions
Protein-observed fluorine (PrOF) NMR is an alternate protein-observed method that
is fast and gives simple spectra to interpret. For PrOF NMR, bromodomains are
fluorine labeled by the incorporation of fluorinated amino acids. Fluorine is an ideal
NMR reporting atom, because it is the second most sensitive stable NMR-active
nuclei (83% compared to
1 H), its hyperresponsiveness to change in its environment
evident by its ~400 ppm chemical shift range,
19
F is 100% isotopically naturally
abundant, and fluorine is not present in biomolecules, resulting in no background
signal [89, 90]. Aromatic amino acids are enriched at the bromodomain binding
interface but not prevalent throughout the rest of the protein [91, 92]. Thus,
nonnatural amino acids such as 5-fluorotryptophan (5FW), 3-fluorotyrosine (3FY),
or 4-fluorophenylalanine (4FF) may be incorporated as sensitive NMR reporter side
chains. A detailed protocol for fluorinated amino acid incorporation and strategies
for fluorine labeling and screening have been described [93]. PrOF NMR experiments with bromodomains are fast (2–5 min with 50 μM protein on a 500 MHz
instrument) when cryoprobes are used and the chemical shift perturbations are
reproducible making it a good method for screening [94]. Additionally, having
multiple fluorine labels allows for monitoring of protein behavior and assessment
of where small molecules are binding to the protein (Fig. 11).
A 2015 paper by Urick et al. exemplifies a screening platform that takes advantage of the strengths of PrOF NMR, DSF, FP, and ITC to identify hits, rank order
hits, and characterize binding affinity for compounds targeting BRD4 BD1 and
BPTF [71]. An advantage of PrOF NMR over ligand-observed NMR methods is
the ability to screen two proteins in the same experiment (single NMR tube) because
the PrOF NMR spectra are simple, essentially completing two screens at once. Dualprotein PrOF NMR screens give bromodomain selectivity information at the onset of
the inhibitor discovery process. The HSQC analog of this experiment, RAMPED-UP
NMR, was previously described by Zartler et al. [95].
Urick et al. completed a dual-protein PrOF NMR screen to identify selective hits
for both BRD4 and BPTF. BRD4 BD1 and BPTF were sequence selectively labeled
with 5-fluorotryptophan (5FW). BRD4 BD1 has three tryptophan residues. W81 is
located on the WPF near the binding site and acts as the primary reporting residue.
Applied Biophysics for Bromodomain Drug Discovery
311