Exploiting the P-1 pocket of BRCT domains toward a structure guided inhibitor design

Article abstract of DOI:10.1021/ml200147a

Breast cancer gene 1 carboxy terminus (BRCT) domains are found in a number of proteins that are important for DNA damage response (DDR). The BRCT domains bind phosphorylated proteins, and these protein-protein interactions are essential for DDR and DNA repair. High affinity domain specific inhibitors are needed to facilitate the dissection of the protein-protein interactions in the DDR signaling. The BRCT domains of BRCA1 bind phosphorylated protein through a pSXXF consensus recognition motif. We identified a hydrophobic pocket at the P-1 position of the pSXXF binding site. Here we conducted a structure-guided synthesis of peptide analogues with hydrophobic functional groups at the P-1 position. Evaluation of these led to the identification of a peptide mimic 15 with a inhibitory constant (K_i) of 40 nM for BRCT(BRCA1). Analysis of the TopBP1 and MDC1 BRCT domains suggests a similar approach is viable to design high affinity inhibitors.

Full text of DOI:10.1021/ml200147a

LETTER
pubs.acs.org/acsmedchemlett
Exploiting the P-1 Pocket of BRCT Domains Toward a Structure
Guided Inhibitor Design
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Ziyan Yuan,† Eric A. Kumar,†, Stephen J. Campbell,§, Nicholas Y. Palermo,†, Smitha Kizhake,†
J. N. Mark Glover,§ and Amarnath Natarajan*,†,‡
†Eppley Institute for Cancer Research, ‡Departments of Pharmaceutical Sciences, Genetics Cell Biology and Anatomy,
University of Nebraska Medical Center, Omaha, Nebraska 68022, United States
§Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
S Supporting Information
b
ABSTRACT: Breast cancer gene 1 carboxy terminus (BRCT)
domains are found in a number of proteins that are important
for DNA damage response (DDR). The BRCT domains bind
phosphorylated proteins, and these proteinÀprotein interactions
are essential for DDR and DNA repair. High affinity domain
specific inhibitors are needed to facilitate the dissection of the
proteinÀprotein interactions in the DDR signaling. The BRCT
domains of BRCA1 bind phosphorylated protein through a
pSXXF consensus recognition motif. We identified a hydropho-
bic pocket at the P-1 position of the pSXXF binding site. Here we
conducted a structure-guided synthesis of peptide analogues with hydrophobic functional groups at the P-1 position. Evaluation of
these led to the identification of a peptide mimic 15 with a inhibitory constant (Ki) of 40 nM for BRCT(BRCA1). Analysis of the
TopBP1 and MDC1 BRCT domains suggests a similar approach is viable to design high affinity inhibitors.
KEYWORDS: ProteinÀprotein interface, peptide mimics, BRCT inhibitors and breast cancer
igh affinity small molecule inhibitors of proteinÀprotein
interactions (PPIs) are emerging as effective tools to dissect
presence of a hydrophobic patch formed by Val1654, Leu1657,
H
Pro1659, and Phe1662 (VLPF) at the N-terminus of the phos-
phorylated serine residue (Figure 1).13,14,26 Here we report a
structure-guided synthesis of peptide analogues that occupy the
VLPF hydrophobic cluster. Conjugating a phenyl ring with a
constrained three-carbon chain through an amide bond to the
N-terminus of the phosphoserine residue of the pSPVF peptide
resulted in peptide mimics with low nanomolar inhibitory
constants (Ki). We conducted a computational study to explore
if this strategy will lead to peptide mimics with increased affinity
for other BRCT containing proteins (TopBP1 and MDC1). The
study suggests that conjugating the hydrophobic fragments to the
N-terminus of phosphorylated peptides that bind other BRCT
domain containing proteins (TopBP1 and MDC1) may lead to
peptide mimics with increased binding affinity.
We used four tetrapeptides (1À4) and three fluorescent
probes (Flu-short, Flu-long, and TMR-long) to determine the
optimal probe and an equation to determine Ki values for this,
and the Ki values are shown in Table 1. The Kd for the TMR-long
probe was ∼3-fold better than that for Flu-long and ∼30-fold
better than that for Flu-short. The dissociation constants (Kd) of
the fluorescent probes are comparable to those previously
reported (Figure S1)14,24,28,29 We next subjected four unlabeled
signaling cascades and as potential therapeutics.1 Tandem car-
boxy terminus domains of breast cancer gene 1 (BRCT) are
considered phosphoprotein binding modules.2,3 Of particular
interest to us are the BRCT domains of the early onset of breast
cancer gene 1 (BRCA1). The BRCT-BRCA1 domains recognize
and bind phosphorylated proteins such as Abraxas, BACH1, and
CtIP.4À12 PPIs mediated by BRCT-BRCA1 are involved in the
regulation of cellular functions such as DNA damage response
and repair.2À14 Mutations in the BRCT-BRCA1 domains are
known to alter their binding affinity for peptides derived from
their binding partners.9,15,16 This has been suggested as the
molecular basis for predisposing women to breast and ovarian
cancers.2 It is well-known that cancer cells with truncation muta-
tions in the C-terminus of BRCA1 are sensitive to DNA damage
based therapeutics, while expression of the wild type BRCA1 in
these cells reverses this phenotype.17À19 Therefore, high affinity
BRCT-BRCA1 inhibitors will not only be useful as chemical
probes to dissect BRCT-BRCA1 specific signaling but also have
the potential to be developed as adjuvants for DNA damage
based therapeutics.
Patches and crevices surrounding the hot spot of a PPI can be
exploited to enhance the binding of peptide analogues with
orthogonal functional groups.13,20À23 The BRCT-BRCA1 binds
tetrapeptides (pSPXF) with micro- to nanomolar affinities.13,14,24,25
Analysis of BRCT-BRCA1Àtetrapeptide structures indicates the
Received: June 20, 2011
Accepted: August 17, 2011
Published: August 17, 2011
r
2011 American Chemical Society
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Table 1. BRCT-BRCA1 Inhibition (Ki Values)
Figure 1. Packing interactions of pSXXF peptide with BRCT-BRCA1
(pdb id: 3K0K). The tetrapeptide and BRCT-BRCA1 are rendered as
sticks and cartoon, respectively. The hydrophobic cluster (Val1654
,
Leu1657, Pro1659, and Phe1662) at the P-1 position is shown as a blue
surface. The graphic was generated by Pymol.27.
peptides (1À4) to a competition assay with each probe-protein.
To determine Ki values, we used six available equations, ChengÀ
Prussof, ColeskaÀWang, Huang, Kenakin, MunsonÀRodbard,
and RoehrlÀWagner (Table S1).30À35 The results from these
studies suggest that the TMR-long probe and the ColeskaÀWang
equation were optimal to identify high affinity inhibitors for the
BRCT-BRCA1 system (Table S2).
Peptides 5À8 with an unnatural amino acid (Nap = napthyl
side chain) at the P-1 position were generated to explore the
VLPF patch. Peptides 5 and 6 would examine the effect of the
stereocenter (R and S) at the P-1 position while peptide 7 and 8
would determine if the C-terminus of the peptide has an effect on
VLPF occupancy. Conjugating the Nap amino acid to the
N-terminus of pSPXF peptides results in an increase in the Ki
values (μM) when compared to the parent peptides (5 vs 1 =
1.24 vs 1.85, 7 vs 3 = 0.54 vs 1.96, and 8 vs 4 = 0.13 vs 0.20).
Reversal of the stereochemistry from S to R by incorporating
the unnatural Nap amino acid results in ∼15-fold loss of activity
(6 vs 5). These results suggest that incorporating a hydrophobic
group at the P-1 position to occupy the VLPF cluster is an
approach that would lead to BRCT-BRCA1 inhibitors with
increased affinity.
Next we generated a set of seven peptide mimics (9À15) with
varying functionalities at the P-1 position to determine the
optimal linker length, the constraint on the linker, and the size
of the hydrophobic group. We observed a decrease in activity
with increase in the chain lengths through peptide mimics 9À11,
with mimic 9 showing a ∼2-fold higher activity than the parent
peptide 4. Analysis of the Ki values for mimics 9, 10, and 11
suggests that the pocket can tolerate no more than a 3 carbon
linker. We next explored the size of the VLPF hydrophobic
cluster using a napthyl ring and a 3,4-dimethoxy substituted
phenyl ring instead of the phenyl ring in mimics 12 and 13. With
these mimics we observed ∼40- and ∼150-fold loss of activity
compared to the case of 9, suggesting that the phenyl ring has the
optimal size to occupy the VLPF patch. We next explored if linker
flexibility as opposed to linker length is responsible for this
change in activity. Mimics 14 and 15 were generated with
different levels of linker flexibility. Indeed, we observed increased
activity (6À12 fold) in 14 and 15 with restricted linkers
compared to 10 (Figure 2). To summarize, this systematic
approach resulted in the identification of mimic 15 with a Ki
value of 40 nM for BRCT-BRCA1.
We next conducted a hybrid docking molecular dynamics
simulation to determine the probable structure and computa-
tionally determine the ΔΔG (14 À 4 and 15 À 4). The
coordinates for peptide 2 bound to BRCT-BRCA1 were ob-
tained from the structure (pdb id: 3K0K) and the hydroxyl on
P+2 threonine was replaced with the methyl to generate peptide 4.
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The beads were then isolated, and the bound protein was
subjected to polyacrylamide gel electrophoresis and exposed to
phosphor screen. The relative band intensity was quantified by
Image-J software, and the pulldown IC50 values were determined
through curve fitting using SigmaPlot. In summary, the most
potent peptide mimics 14 and 15 identified from the FP assay
were also the most potent in the in vitro transcription transla-
tion assay.
In conclusion, analysis of the BRCTÀBRCA1ÀpSXXF com-
plex structures suggested the presence of a hydrophobic cluster
(VLPF) at the P-1 position of the pSXXF binding site. A systematic
structure guided iterative synthesis and screening of peptide
mimics resulted in the identification of BRCTÀBRCA1 binders
with higher affinity. A constrained 3-carbon linker with a phenyl
ring conjugated to the N-terminus of pSPVF peptide results in
mimics with low nanomolar Ki values. A systematic analysis of the
available equations led to the identification of the ColeskaÀWang
equation as the optimal model for determining the Ki values for
this system. A computational study suggests such an approach
can be extended to other BRCT containing proteins (MDC1 and
TopBP1). Finally, the potencies of peptide mimics identified in
the FP assay correlate with the in vitro biochemical data.
Figure 2. Packing interactions of peptide mimics 14 and 15 with
BRCT-BRCA1 (pdb id: 3K0K). The coordinates were derived from a
hybrid docking molecular dynamics simulation, and the graphic was
generated by Pymol.27
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental methods, Tables
b
S1ÀS3 (models to determine Ki values, Ki values of 1À4, and
computational study to explore the P-1 site on BRCT domains),
and Figures S1ÀS5 (determination of Ki values, LC traces, and
mass spectra). This material is available free of charge via the
Internet at http://pubs.acs.org.
Figure 3. Competitive streptavidin pulldown of the BRCT-BRCA1/
BACH1 peptide interaction with peptide mimics (7, 10, 11, 14, and 15).
Control indicates binding of the BRCT-BRCA1 to an immobilized
phospho BACH1 peptide on streptavidin agarose beads in the presence
of vehicle. Titrations are shown with increasing concentration of peptide
mimics. The band intensities were quantified using Image-J software,
and pulldown IC50 values were determined through curve fitting using
SigmaPlot.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (402) 559 3793. Fax: (402) 559 8270. E-mail: anatarajan@
unmc.edu.
Author Contributions
Equal contribution.
R groups of peptides (14 and 15) were substituted using
YASARA.36 The positions of all atoms of the receptor and the
parent peptide residues (pSPVF) were fixed. Water and the simula-
tion box were added using the YASARA “Cell neutralization and
pKa prediction” experiment. Each ligand was then refined for
50 ps using the YAMBER force field. The energies of binding were
calculated using AutoDock Vina in single calculation mode.37
The ΔΔG values obtained computationally were 0.4 and 0.9 kcal
for 14 and 15, respectively, and are consistent with the experi-
mental results. We conducted a similar study with pSQEY-
BRCT-MDC1 (pdb id: 3K05)25 and pTPELY-BRCT-TopBP1
(pdb id: 3AL3).38,39 The ΔΔG values with and without the
R groups in 14 and 15 ranged from 0.15 to 0.53 kcal (Table S3).
In summary, these studies suggest that exploring crevices and
patches adjacent to the hot spots of PPIs is a viable strategy to
improve the binding affinity of ligands.
To determine the relative affinities of the peptide mimics for
BRCT-BRCA1 in an orthogonal biochemical study, we con-
ducted a competitive streptavidin pulldown study using an in
vitro transcription translation matrix (Figure 3). BRCT-BRCA1
(1646À1859) was expressed by in vitro transcription translation
in the presence of 35-S labeled methionine. A mixture of
phospho-BACH1 peptide immobilized on streptavidinÀagarose
beads and peptide mimics was incubated with the BRCT-BRCA1.
Funding Sources
Funding SourcesThis project was supported in part by NIH
T32CA009476 (E.A.K. and N.Y.P.), Alberta Cancer Board—
G218000116 (J.N.M.G.), and NIH R01CA127239 (A.N.).
’ ACKNOWLEDGMENT
We would like to thank the UNMC mass spectrometry core
facility for MS analysis.
’ ABBREVIATIONS
DDR, DNA damage response; PPI, proteinÀprotein interaction;
BRCT, carboxy terminus domains of breast cancer gene 1; BRCA1,
breast cancer gene 1
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