Isoxazole-Derived Amino Acids are Bromodomain-Binding Acetyl-Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3

Article abstract of DOI:10.1002/anie.201602908

A range of isoxazole-containing amino acids was synthesized that displaced acetyl-lysine-containing peptides from the BAZ2A, BRD4(1), and BRD9 bromodomains. Three of these amino acids were incorporated into a histone H4-mimicking peptide and their affinity for BRD4(1) was assessed. Affinities of the isoxazole-containing peptides are comparable to those of a hyperacetylated histone H4-mimicking cognate peptide, and demonstrated a dependence on the position at which the unnatural residue was incorporated. An isoxazole-based alkylating agent was developed to selectively alkylate cysteine residues in situ. Selective monoalkylation of a histone H4-mimicking peptide, containing a lysine to cysteine residue substitution (K12C), resulted in acetyl-lysine mimic incorporation, with high affinity for the BRD4 bromodomain. The same technology was used to alkylate a K18C mutant of histone H3.

Full text of DOI:10.1002/anie.201602908

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International Edition: DOI: 10.1002/anie.201602908
German Edition: DOI: 10.1002/ange.201602908
Protein Modifications
Isoxazole-Derived Amino Acids are Bromodomain-Binding Acetyl-
Lysine Mimics: Incorporation into Histone H4 Peptides and Histone
H3
Angelina R. Sekirnik (nꢀe Measures)+, David S. Hewings+, Natalie H. Theodoulou,
Lukass Jursins, Katie R. Lewendon, Laura E. Jennings, Timothy P. C. Rooney,
Tom D. Heightman, and Stuart J. Conway*
Abstract: A range of isoxazole-containing amino acids was
synthesized that displaced acetyl-lysine-containing peptides
from the BAZ2A, BRD4(1), and BRD9 bromodomains. Three
of these amino acids were incorporated into a histone H4-
mimicking peptide and their affinity for BRD4(1) was
assessed. Affinities of the isoxazole-containing peptides are
comparable to those of a hyperacetylated histone H4-mimick-
ing cognate peptide, and demonstrated a dependence on the
position at which the unnatural residue was incorporated. An
isoxazole-based alkylating agent was developed to selectively
alkylate cysteine residues in situ. Selective monoalkylation of
a histone H4-mimicking peptide, containing a lysine to cysteine
residue substitution (K12C), resulted in acetyl-lysine mimic
incorporation, with high affinity for the BRD4 bromodomain.
The same technology was used to alkylate a K18C mutant of
histone H3.
ized.[9] Neutralization of lysineꢀs positive charge by acetyla-
tion weakens the interaction of histone with DNA, thus
resulting in the relaxed, transcriptionally active form of
DNA—euchromatin. In addition, transcriptional machinery
is recruited to chromatin through the interaction of KAc with
reader protein modules: bromodomains.[10–12] The study of
lysine acetylation is therefore of high interest and the
generation of homogeneously modified proteins and peptides
is essential to achieve this. To this end, elegant chemical
strategies have been developed to synthesize single homoge-
neous forms of proteins, containing either KAc or an
analogue which functions similarly.[13–18] The value of these
modifications in uncovering the mechanistic roles of histone
modifications has been demonstrated,[19,20] particularly in the
study of sirtuins (see Figure S1 in the Supporting Informa-
tion).[21–27] However, only two of these KAc mimics have been
employed in the study of bromodomains: N-methanesulfonyl-
lysine (6; see Figure 2) has been reported as a stable KAc
mimic which binds to bromodomains when incorporated into
a p53-mimicking peptide;[28] KAc mimics containing methyl-
thiocarbonylthialysine (MTCTK; 7; Figure 2) interact two- to
fourfold less efficiently with the bromodomain of BRDT
compared to KAc.[18]
During the development of ligands for the bromodomain
and extra-terminal domain (BET) family of bromodomain-
containing proteins (BCPs), we identified the 3,5-dimethyl-
isoxazole (DMI) moiety as an effective KAc mimic.[29–31]
Sharp et al. subsequently showed that the DMI moiety is
most potent based on a comparison of KAc mimics.[32] Our
analysis of the ChEMBL data base (https://www.ebi.ac.uk/
chembl/) revealed that 4-phenyl-3,5-dimethylisoxazole (1;
Figure 1A) possesses the highest binding efficiency index and
surface efficiency index[33] for the BET BCPs, BRD2 and
BRD4. We therefore decided to investigate whether the DMI
motif is a general KAc mimic which can be incorporated into
unnatural amino acids, and whether peptides in which KAc
has been substituted for these amino acids would retain
affinity for bromodomains. If successful, these amino acids
would be of high utility in generating homogenous peptides
and proteins in which the DMI functions as a stable replace-
ment for a KAc mark.
L
ysine acetylation is a prevalent post-translational modifi-
cation (PTM) which plays a fundamental role in regulating
protein function.[1,2] Although acetylated lysine (KAc) resi-
dues are found throughout the cellular environment,[3–5] it is
the role of this PTM in chromatin function which has
garnered most recent interest.[6] KAc is viewed as one of
the “marks” that comprises the epigenetic code,[7,8] and
enzymes that write (histone acetyl transferases) and erase
(histone deacetylases) this modification are well character-
[*] Dr. A. R. Sekirnik (nꢀe Measures),[+] Dr. D. S. Hewings,[+]
Dr. N. H. Theodoulou, L. Jursins, K. R. Lewendon, Dr. L. E. Jennings,
Dr. T. P. C. Rooney, Prof. Dr. S. J. Conway
Department of Chemistry, Chemistry Research Laboratory, University
of Oxford, Mansfield Road, Oxford, OX1 3TA (UK)
E-mail: stuart.conway@chem.ox.ac.uk
Homepage: http://conway.chem.ox.ac.uk/
Dr. T. D. Heightman
Nuffield Department of Clinical Medicine, Structural Genomics
Consortium, University of Oxford, Old Road Campus Research
Building, Roosevelt Drive, Oxford, OX3 7DQ (UK)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
1002/anie.201602908.
Analysis of the X-ray crystal structure of OXFBD02 (2;
Figure 1A)[31] bound to BRD4(1) (PDB code: 4J0S, carbon:
yellow) and the X-ray crystal structure of the histone H4-
mimicking peptide H41-12KAc5KAc8 (PDB code: 3UVW,
carbon: purple) in complex with BRD4(1) (Figure 1B)
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KGaA. This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited.
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allow formation of further hydrogen bonds with the bromo-
domain (5 and 10–14). The phenyl ring of the phenylalanine
derivatives 8 and 9 was predicted to interact with the
hydrophobic residues in the ZA channel region of the binding
pocket. As the aim was to identify general KAc mimics, the
structures of these compounds were kept flexible to facilitate
binding to the maximum number of bromodomains. Although
some isoxazole-containing amino acids have been reported
previously,[34,35] in these compounds the isoxazole is attached
directly to the a-carbon atom, and so is unlikely to penetrate
the KAc binding pocket effectively.
KAc has a low affinity for bromodomains.[36] In histone
peptides the surrounding residues are thought to provide
much of the bromodomain affinity, which complicates the
assessment of the bromodomain-binding abilities of individ-
ual amino acids. Nonetheless, the percentage inhibition
AlphaScreen data, obtained at amino-acid concentrations of
50 mm or 250 mm, were used to assess the binding of the
individual amino acids to the phylogenetically diverse
BAZ2A, BRD4(1), and BRD9 bromodomains (Table 1).
All of the amino acids showed some concentration-dependent
binding to at least one bromodomain. Interestingly, some of
the amino acids showed selectivity for one or two of the
bromodomains, with the phenyl-containing amino-acid 8
showing the highest affinity for BRD4(1), but no binding to
BRD9. The compound 5 showed the highest affinity for both
BAZ2A and BRD9. These data indicate that isoxazole-
containing amino acids can act as KAc mimics in the context
of bromodomain recognition. The data also suggest that
ligand selectivity between bromodomains can be achieved by
altering interactions in the KAc binding pocket, in addition to
the peptide-binding region (see the Supporting Information).
Figure 1. A) Structures of 4-phenyl-3,5-dimethylisoxazole (1), which has
a high binding efficiency index (BEI) and surface efficiency index (SEI)
for the BRD4 bromodomains, and OXFBD02 (2). B) An overlay of the
X-ray crystal structure of the BET bromodomain ligand 2 bound to
BRD4(1) (PDB code: 4J0S, carbon: yellow) with the X-ray crystal
structure of the histone H4-mimicking peptide H41–12KAc5KAc8 (PDB
code: 3UVW, carbon: purple) bound to BRD4(1). Some residues are
omitted for clarity. C) The structures of KAc and the isoxazole-contain-
ing amino-acids 3–5 incorporated into peptides in this study.
Table 1: Percentage inhibition of bromodomain-KAc recognition by
isoxazole-containing amino acids.[a]
Figure 2. The structures of previously reported acetyl-lysine mimicking
amino acids 6 and 7, and the isoxazole-containing amino-acids 3–5
and 10–14.
informed the design of ten isoxazole-containing amino acids
(3–5 and 8–14; Figure 2). Varying chain lengths and locations
of hydrogen-bond donors and acceptors were incorporated
(see the Supporting Information for syntheses). Substitution
of the isoxazole core at either the 3-, 4-, or 5-position was
intended to probe the optimum orientation for KAc pocket
binding (12–14). Inclusion of an amide on the linkage might
[a] n.d=not determined.
2
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Having established that isoxazole-containing amino acids
Affinities of the modified peptides for BRD4(1) were
can bind to diverse bromodomains, we next sought to dependent on the position at which the unnatural residue was
determine how effectively these amino acids could emulate incorporated. Substitution at either K5 or K8 reduced the
KAc when incorporated into a histone-mimicking peptide. affinity compared to H4(KAc)4, with these peptides showing
For these studies we selected amino-acids 3–5, based on IC50 values in the 12.6–37.8 mm range. The alkyl-based amino-
a combination of their bromodomain affinity and structural acid 3 had the least detrimental effect on affinity when
flexibility. We decided to focus on BRD4(1) and an H4- incorporated at these positions. However, substitution at
mimicking peptide as a model system, as the binding of either K12 or K16 resulted in peptides with similar affinities
variously acetylated histone H4-mimicking peptides, and compared to that of the reference peptide (IC50 = 6.4–
a large number of small-molecule ligands, to BRD4(1) have 10.1 mm). When incorporated at these positions, the amide-
been well characterized.[37]
linked isoxazole 5 bound more strongly than the control
The histone H4 tail possesses four lysine residues which peptide, with IC50 values of (6.8 Æ 0.9) and (8.4 Æ 0.8) mm for
can be acetylated, and are located at positions 5, 8, 12, and 16. the K12 and K16 position, respectively. Positional sensitivity
The tetra-acetylated histone H4-mimicking peptide H41–20
-
was consistent with studies on a histone H4-mimicking
KAc5KAc8KAc12KAc16 [H4(KAc)4] shows the highest peptide, which demonstrated that dual acetylation at K5
affinity for BRD4(1), with reported KD values of 3.1 mm and K8 was almost as potent as H4(KAc)4.[39] Given the
(ITC)[36] and 4.8 mm (ITC).[38] BRD4(1) can recognize two apparent high contribution of KAc at these positions to the
adjacent KAc residues on the same peptide at any one time.[39] overall peptide affinity, it seems reasonable that their
X-ray crystal structures reveal the N-terminal KAc forms substitution is less well tolerated. Overall, these data indicate
a hydrogen-bonding interaction with the conserved aspara- that amino-acids 3–5 are able to mimic KAc when incorpo-
gine residue, while flexible glycine residues allow a peptide rated into a peptide.
conformation where the C-terminal KAc fits into the grooves
We next sought to develop a complementary, tag-and-
created by the ZA and BC loops (PDB: 3UVW, 3UVX, modify, approach in which a cysteine residue could be
3UVY). It was not clear whether an isoxazole-containing alkylated by an isoxazole-containing electrophile. Based on
amino acid could effectively mimic a KAc residue in either of the alkylation procedure used in the synthesis of the serine
these binding modes, or whether two adjacent isoxazole- derivative 4 (see Scheme S8), we investigated whether 5-
containing amino acids would be accommodated by BRD4- (chloromethyl)-3-methylisoxazole (Cl-DMI) could be used to
(1). Therefore, the amino-acids 3–5 were sequentially intro- modify a cysteine within a peptide in situ. Trials were first
duced into H4(KAc)4 in place of KAc at either position 5, 8, performed with this alkylating agent on a model tripeptide
12, or 16. IC50 values for the resulting peptides were (YCK), thus indicating that selective alkylation on a cysteine
determined using an AlphaScreen assay (Figure 3) [inhibiting residue could be achieved (see Figure S2). The reaction was
the interaction of BRD4(1) with biotinylated H4(KAc)4], then carried out using an H4-mimicking peptide with
with inclusion of unbiotinylated H4(KAc)4 to determine a cysteine residue replacing lysine 12, H41-20KAc5K-
whether the KAc mimics could interact more strongly than Ac8K12CKAc16 [H4(KAc)3K12C; see Figure S3], as our
KAc with bromodomains.
data suggested that an isoxazole-containing amino acid would
be well tolerated at this position.
The degree of alkylation was determined using analytical
HPLC, which allowed separation of the reactant and product,
and identification of by-products. In addition, the reaction
could be followed using MALDI-TOF-MS (Figure 4). HPLC
integral ratios from reaction mixtures correlate well with peak
ratios in MALDI-TOF-MS spectra (see Figure S7). The site
of alkylation was confirmed as the cysteine residue by
targeted nano-LC tandem MS/MS (see Figure S8) and
MALDI-TOF-MS/MS, with laser-induced fragmentation
(see Figure S9). Importantly, the characteristic peak resulting
from amide-bond cleavage adjacent to the alkylated cysteine
was observed at m/z 1148.75 (expected m/z 1148.58), but was
not present in the starting peptide.
Only one equivalent of Cl-DMI was required for almost
complete alkylation after 1 hour, whereas excess (ꢀ 5 equiv)
reagent gave rise to unidentified by-products (see Fig-
ure S10). Once the alkylation had been optimized (see
Table S2) the desired product was purified using HPLC.
Isolated H4(KAc)3K12C(DMI) was lyophilized and its affin-
ity to BRD4(1) assessed in an AlphaScreen assay, and
compared to the affinities of H4(KAc)3 peptides containing
the amino-acids 3–5 at position 12 (Figure 5). The thioether-
linked DMI showed an IC50 value of (2.87 Æ 0.3) mm, which is
Figure 3. IC50 values for H4(KAc)4-mimicking peptides bearing isoxa-
zole-containing amino acids (substitution position indicated numeri-
cally), obtained using an AlphaScreen assay inhibiting the interaction
of BRD4(1) with biotinylated H4(KAc)4. The guideline shows the
IC50 value of control H4(KAc)4 peptide for comparison. IC50 values (in
mm above bars) are geometric means, from 4-parameter fittings[40] to
data obtained in triplicate. Error bars represent the error of the fit.
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Figure 4. A) A cartoon representing H4(KAc)3K12C alkylation.
B) Regions of MALDI-TOF-MS spectra of samples taken at the time
indicated during the alkylation of H4(KAc)3K12C under optimized
conditions: 1 mm H4(KAc)3K12C, 4m guanidine hydrochloride, 10 mm
methionine, 5 mm TCEP, 1m CHES (pH 9), 5 mm Cl-DMI, showing
conversion from starting peptide (observed m/z 2255.09; expected
m/z 2255.21) to alkylated H4(KAc)3K12C(DMI) peptide (observed m/z
2350.07; expected m/z 2350.24).
Figure 6. A) A cartoon representing histone H3 alkylation. B) LCMS
monitoring H3K18C protein alkylation with Cl-DMI. Deconvoluted
masses (range 10.0 kDa–20.0 kDa, resolution 1.0 Da, zoomed region
15.0 kDa-15.5 kDa) observed at time indicated bottom right. Spectra
show progression from starting protein [H3K18C expected mass
15214] to monoalkylated product [H3K18C(DMI) expected mass
15309]. See the Supporting Information for full spectra (see Fig-
ure S12).
Figure 5. IC50 values for H4(KAc)4 analogue peptides bearing isoxazole-
containing amino-acid substitutions at K12 (identity of substitution is
shown below bars). Data were obtained from serial dilutions in an
AlphaScreen assay inhibiting the interaction of BRD4(1) with biotiny-
lated H4(KAc)4. Guideline shows IC50 value of the control H4(KAc)4
peptide for comparison. IC50 values (in mm above bars) are geometric
means, from 4-parameter fittings[40] to data obtained in triplicate. Error
bars represent the error of the fit. See Figure S11.
human histone H3, a position that is endogenously acetylated,
was treated with Cl-DMI under the optimized alkylation
conditions (Figure 6). After 3 hours, a mass corresponding to
the monoalkylated protein was the sole species observable by
LCMS (see Figure S13: observed mass 15309 Da). These data
indicate selective incorporation of the DMI into the histone
H3 protein.
In conclusion, ten structurally diverse isoxazole-contain-
ing amino acids were synthesized. They are the first unpro-
tected monomeric amino acids able to prevent a histone-
mimicking peptide binding to the bromodomains of BAZ2A,
BRD4(1), and BRD9. Incorporation into histone H4-mim-
icking peptides confirmed that these isoxazole-containing
the lowest observed for any of the reported peptides in this
assay. The decreased affinity of the H4(KAc)3K12C starting
peptide, relative to H4(KAc)4, was confirmed by ITC [KD =
(32.4 Æ 6.7) mm; see Figure S12]. This result corroborates the
ability of this isoxazole-containing amino acid to mimic the
interactions of KAc with bromodomains.
To determine whether an isoxazole-derived KAc mimic
could be incorporated into a protein, a K18C mutant of
4
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Revised: May 8, 2016
Published online: && &&, &&&&
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Protein Modifications
Hang ten: Ten isoxazole-containing
amino acids were synthesized, three of
which were incorporated into a histone
H4-mimicking peptide, and shown to
bind to the first bromodomain of BRD4. A
complementary tag and modify strategy
allows addition of either a 3-, 4-, or 5-
dimethylisoxazole onto the cysteine resi-
dues of a histone H4-mimicking peptide,
and the full histone H3 protein.
A. R. Sekirnik (nꢁe Measures),
D. S. Hewings, N. H. Theodoulou,
L. Jursins, K. R. Lewendon, L. E. Jennings,
T. P. C. Rooney, T. D. Heightman,
S. J. Conway*
&&&& — &&&&
Isoxazole-Derived Amino Acids are
Bromodomain-Binding Acetyl-Lysine
Mimics: Incorporation into Histone H4
Peptides and Histone H3
6
www.angewandte.org
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!