Peptoid-Peptide Hybrid Ligands Targeting the Polo Box Domain of Polo-Like Kinase 1

Article abstract of DOI:10.1002/cbic.201200206

We replaced the amino terminal Pro residue of the Plk1 polo-box-domain-binding pentapeptide (PLHSpT) with a library of N-alkyl-Gly peptoids , and identified long-chain tethered phenyl moieties giving greater than two-orders-of-magnitude affinity enhancement. Further simplification by replacing the peptoid residue with appropriate amides gave low-nanomolar affinity N-acylated tetrapeptides. Binding of the N-terminal long-chain phenyl extension was demonstrated by X-ray co-crystal data.

Full text of DOI:10.1002/cbic.201200206

DOI: 10.1002/cbic.201200206
Peptoid–Peptide Hybrid Ligands Targeting the Polo Box
Domain of Polo-Like Kinase 1
Fa Liu,[a] Jung-Eun Park,[b] Wen-Jian Qian,[a] Dan Lim,[c] Andrej Scharow,[d] Thorsten Berg,[d]
Michael B. Yaffe,[c] Kyung S. Lee,*[b] and Terrence R. Burke, Jr.*[a]
We replaced the amino terminal Pro residue of the Plk1 polo-
box-domain-binding pentapeptide (PLHSpT) with a library of
N-alkyl-Gly “peptoids”, and identified long-chain tethered
phenyl moieties giving greater than two-orders-of-magnitude
affinity enhancement. Further simplification by replacing the
peptoid residue with appropriate amides gave low-nanomolar
affinity N-acylated tetrapeptides. Binding of the N-terminal
long-chain phenyl extension was demonstrated by X-ray co-
crystal data.
Introduction
The polo-like serine/threonine protein kinases (Plks) are impor-
tant mediators of cell-cycle regulation that contain C-terminal
polo-box domains (PBDs) whose function is to bind to phos-
phoserine (pS)/phosphothreonine (pT)-containing sequences.
Because Plk1 can promote oncogenic transformation, efforts
are underway to develop PBD-binding inhibitors as potential
downregulators of Plk1 function.[1] We have taken the crystal
structure of the pentapeptide PLHSpT (1) bound to the Plk1
PBD (PDB ID: 3HIK) as a starting point for antagonist develop-
ment.[2] The structure shows that the N-terminal Pro residue in-
teracts with the PBD surface formed by the two aromatic resi-
dues Trp414 and Phe535 and by hydrogen bonding of its car-
bonyl group with the guanidinium moiety of Arg516. This ori-
entation differs greatly from that of the N-terminal Pro residue
of PBD-bound PPHSpT (PDB ID: 3C5L), such that the combined
protein-binding surface encompassed by these two orienta-
tions covers a broad region. Therefore, we speculated that the
N-terminal Pro residue of 1 could potentially provide attractive
opportunities for structural elaboration. Proline is distinguished
among the encoded amino acids by having a secondary a-
amino group that is derived from intramolecular alkylation to
form a pyrrolidine ring. N-substituted glycines (NSGs) are a
class of secondary amine-containing constructs that present
features reminiscent of Pro residues.[3] Pseudopeptides com-
posed of NSGs (termed “peptoids”)[4] have shown utility in
drug discovery,[5] as have peptide–peptoid hybrids, which con-
tain both peptide and NSG residues.[6] Our current study was
undertaken to optimize the N-terminal PBD binding interac-
tions of peptide 1 by using a peptide–peptoid hybrid ap-
proach that employed NSG residues in place of the Pro residue
at the pT-4 position.
ly deprotected to provide a free Leu residue (representing the
À3 position of parent peptide 1), which was bromoacetylated
to yield the corresponding a-bromoacetamide 3 (Scheme 1).
This peptide served as the common intermediate for the
synthesis of subsequent analogs. The resin was divided into
portions and each portion was treated with an appropriate
amine (4a–r, Scheme 1) to yield the corresponding NSG-con-
taining derivative (5a–r). Acetylation of the terminal secondary
amines gave the fully protected resin-bound congeners, which
were cleaved under acidic conditions to provide the final pep-
toid–peptide hybrids as their C-terminal carboxamides (6a–r,
Scheme 1).
The Plk1 PBD binding affinities of the synthetic products
were evaluated using an ELISA-based 96-well assay that meas-
ures their ability to compete with a surface-bound high-affinity
pT-containing peptide for binding to Plk1 in mitotic 293A cell
lysates.[2] Hybrids 6d and 6e had dramatically diminished bind-
ing affinity relative to the parent peptide. This indicated that
both positive and negative charges are not well tolerated in
this region of the molecule. The hybrids 6 f, 6j and 6k showed
affinities similar to the parent peptide 1, while 6a, 6c, 6g and
[a] Dr. F. Liu,+ Dr. W.-J. Qian, Dr. T. R. Burke, Jr.
Chemical Biology Laboratory, Frederick National Lab, NCI, NIH
Frederick, MD 21702 (USA)
E-mail: tburke@helix.nih.gov
[b] Dr. J.-E. Park,+ Dr. K. S. Lee
Laboratory of Metabolism, National Cancer Institute, NIH
Bethesda, MD 20892 (USA)
E-mail: kyungle@mail.nih.gov
[c] Dr. D. Lim, Prof. M. B. Yaffe
Center for Cancer Research, Massachusetts Institute of Technology
Cambridge, MA 02139 (USA)
[d] A. Scharow, Prof. T. Berg
Institute of Organic Chemistry, University of Leipzig
04103 Leipzig (Germany)
Results and Discussion
[+] These authors contributed equally to this project.
We synthesized a library of peptide–peptoid hybrids using acid
sensitive NovaSyn TGR resin using Fmoc protocols and a “sub-
monomer approach”.[7] Resin-bound peptide 2 was N-terminal-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200206.
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K. S. Lee, T. R. Burke, Jr., et al.
may reflect “promiscuous” bind-
ing mechanisms that do not rely
on specific interactions with the
PBD.[8] To examine this possibili-
ty, we made use of the fact that
the “SpT” dipeptide motif is cru-
cial for specific high-affinity Plk1
PBD binding and that substitu-
tion of the Ser residue by Ala
markedly reduces affinity.[9] We
replaced the Ser residue in 6q
with an Ala residue [6q(S4A)]
and found that this resulted in
a significant reduction in binding
affinity (Figure 1A). We conclud-
ed from this that binding of 6q
was specific.
Our previous work has shown
that the tetrapeptide LHSpT, ob-
tained by deletion of the N-ter-
minal Pro (À4 residue), exhibits
significantly less Plk1 PBD-bind-
ing affinity than the parent pep-
tide 1.[10] However, the binding
affinity of 6q appeared to derive
significantly from the tethered
phenyl functionality. Therefore,
we prepared the tetrapeptide 7
by acylating the resin-bound 2
with an N-terminal Phe-(CH2)8-
CO- group prior to cleavage
from the resin (Scheme 2). The
total extension of the tethered
phenyl moiety in 7 is approxi-
mately the same as the alkylated
NSG residue in 6q. The Plk1
PBD-binding
affinity of
7
equaled that of the pentapep-
tide 6q. This indicated that the
N-terminal portions of both pep-
tides may bind in a similar fash-
ion (Figure 1B).
Scheme 1. Submonomer solid-phase synthesis of 6a–r. a) Bromoacetic acid, DIC, DIPEA, NMP, 1 h; b) amine 4a–r
(2.0 m in DMSO), RT, 4 h; c) N-acetylimidazole, NMP, overnight; d) acetic anhydride, DIPEA, 1,2-dichloroethane, 4 h;
e) TFA/TIS/H2O (90:5:5), 4 h.
Because the tether chains in
both 6q and 7 are highly flexi-
6i showed slightly higher binding affinities than 1 and the an-
alogues 6b, 6h and 6l were weaker binders than 1 (see Fig-
ure S1 in the Supporting Information). The binding affinities of
6a–l indicated that uncharged, hydrophobic groups of limited
bulk were preferred.
ble, it was unclear what the effects would be of introducing
conformational restriction in this region of the peptide. Cop-
per(I)-catalyzed [3+2] Huisgen cycloaddition “click chemistry”
of azides and alkynes is a widely used means of functional
group ligation that yields substituted triazole rings having
fixed geometries.[11] Accordingly, we converted resin-bound 2
to its N-terminal pent-4-ynamide (8) and subjected this to cop-
per(I)-catalyzed Huisgen cycloaddition using azides 9 (three-
carbon chain) and 10 (four-carbon chain, Scheme 2). When the
reactions were performed in DMSO at room temperature over-
night the 1,4-substituted triazole products (11 a and 12a) were
obtained. However, when the reactions were run in DMF at
1008C for 48 h without copper, the sterically more crowded
Focusing on analogues 6 f–j, we explored phenyl functionali-
ty tethered by a series of (CH2)n-based linkers having sequen-
tially increasing chain lengths from n=2 to n=7 (6m–r,
Scheme 1). The binding affinities of these peptoid–peptide hy-
brids increased with increasing linker length, reaching a maxi-
mum at n=6 (6q), with n=7 (6r) being of equal potency (Fig-
ure 1A). Because n-alkylphenyl groups impart considerable
hydrophobic character, we were concerned that these results
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Targeting the Polo Box Domain of Plk1
1,5-substituted products (11 b and 12b) were also obtained
along with the 1,4-substituted products (Scheme 2). The bind-
ing affinities of all four triazole-containing peptides were re-
duced relative to 6q and 7 (Figure 1B). This potentially indi-
cates unfavorable conformationally induced interactions. The
reduced affinities of 11 a and 12a are noteworthy, since their
1,4-substituted triazoles closely mimic the NSG residues in pep-
toid–peptide hybrids 6p and 6q, respectively.
In order to compliment ELISA binding data obtained on
solid supports, we performed Plk1 PBD competition assays
using fluorescence polarization techniques as previously de-
scribed.[12] This assay measures the ability of unlabeled pep-
tides to compete with 5-carboxyfluorescein-GPMQSpTPLNG-
OH for binding to PBD protein in solution. It was found that
the parent peptide 1 showed 40Æ0.2% inhibition at the high-
est concentration tested (2.56 mm).[2b] In contrast, the peptoid–
peptide hybrid 6q, which along with 6r, was identified by
ELISA assays as being the most potent analogue tested, exhib-
ited greater than 170-fold higher affinity (IC50 =15Æ3 nm;
Table 1). Also consistent with the ELISA data in Figure 1B was
the finding that the tetrapeptide 7 (IC50 =19Æ2 nm) was only
slightly less potent than 6q. We determined the effects of
replacing the pThr residue with the phosphatase-stable pThr
mimetic (2S,3R)-2-amino-3-methyl-4-phosphonobutyric acid
(Pmab).[13] Peptides containing Pmab could be potentially ad-
vantageous over their pThr-con-
Figure 1. ELISA-based Plk1 PBD-binding results. Representative graphs from
three independent experiments are shown (OD: optical density).
taining counterparts in cellular
studies, where cytoplasmic phos-
phatases can cleave phosphoryl
ester bonds.[2] When the pThr
residue in 6q was replaced with
Pmab to give the corresponding
peptide 13 (Scheme 3) a moder-
ate reduction in affinity was ob-
served (13, IC50 =71Æ17 nm).
However, binding was still spe-
cific, since the mutant 13(S4A)
variant resulted in a nearly 500-
fold loss of affinity (Table 1).
A
fluorescein-isothiocyanate-
labeled PEGylated version of 6q
was prepared (14, Scheme 3) for
use in fluorescence polarization-
based determination of binding
affinities to the PBDs of Plk1, 2
and 3.[12] For each domain, Kd
values were extrapolated from
the fluorescence polarization
binding curves as the concentra-
tion at which 50% of the fluores-
cein-labeled peptide 14 is pro-
tein-bound (Figure S3). Against
the Plk1 PBD, a Kd value of
26.7Æ2.8 nm was obtained,
which is consistent with the IC50
value of 15 nm found in fluores-
cence polarization competition
Scheme 2. Synthesis of N-terminally modified tetrapeptides.
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K. S. Lee, T. R. Burke, Jr., et al.
Table 1. Plk1 PBF-binding IC50 values.[a]
No
IC50 [mm]
No
IC50 [mm]
1
6q
7
[b, c]
13
13(S4A)
0.071Æ0.017
34.02Æ2.88
0.015Æ0.003
0.019Æ0.002
[a] Determined by competition against binding of 5-carboxyfluorescein-
GPMQSpTPLNG-OH (5-CF-15) and the Plk1 PBD as determined by fluores-
cence polarization assays. [b] 40Æ2% inhibition at 2.56 mm.[2b] [c] Auto-
fluorescence-limited.
Figure 2. Comparison of Plk1 PBD in complex with peptide 1 (carbons in
magenta) superimposed with bound 6q (carbons in gray). For 6q, protein
side chains lining the Phe(CH2)6- binding channel are shown in yellow with
the Y481 side chain associated with bound 1 shown in magenta.
ing from the histidine imidazole ring of 1 (PDB ID: 3RQ7; Fig-
ure 3B).[2a] Additionally, the binding of the phenyl ring of 6q is
also related to what has been reported for the phenyl ring in
the N-terminal Phe residue of FDPPLHSpTA (PDB ID: 3P37;
Figure 3C).[14] It can also be surmised for tetrapeptide 7 that
the N-terminal Phe(CH2)8-CO- group binds in a fashion similar
to 6q. Taken together, these findings indicate a high degree of
plasticity in the protein that permits diverse structures to
access the auxiliary pocket.
Scheme 3. Variants of peptide 6q.
assays (see above). In contrast, Kd values against the PBDs of
Plk2 and Plk3 were more than two-orders of magnitude higher
(>2560 nm; Figure S3).
Conclusions
In order to determine the molecular basis for the enhanced
binding affinity of 6q relative to the parent peptide 1, we
solved the X-ray co-crystal structure of 6q in complex with
Plk1 PBD (Table S4 and Figure S4). The residues HSpT of 6q
were nearly superimposable with those of the PBD-bound 1 in
the 3HIK structure (Figure 3). However, significant structural
differences were observed with the pT-3 Leu residue, where
the psi angles (y=À1.98 and À141.58 for 1 and 6q, respec-
tively) place the N-terminal functionality of these peptides in
nearly opposing directions. In this new orientation, the peptoid
Phe(CH2)6- group of 6q is directed across the b2 and b3 sheets
of the PBD protein, where it terminates with its phenyl ring
nestled against the aB helix. The net effect is to reveal a chan-
nel that is absent in the binding of the parent peptide 1. Reve-
lation of this binding channel is made possible by extensive
downward rotation of the Y481 side chain, which occludes
access in the 3HIK structure. Other residues lining the new
channel (V415, Y421, F482 and Y485) have orientations only
modestly different from those observed in the 3HIK structure
(Figure 2).
The original intent of this study was to optimize N-terminal
PBD-binding interactions of the wild-type peptide 1 by replac-
ing its Pro residue with NSG constructs. These efforts were
highly successful. They yielded greater than two orders-of-
magnitude improved affinity over the parent peptide while
maintaining good Plk1 PBD selectivity. We achieved further
simplification by replacing the peptoid altogether. Our findings
expand the design parameters available for development of
Plk1 PBD-binding antagonists by showing that low-nanomolar
affinity tetrapeptide analogues can be derived by appropriate
N-terminal acylation. These results should be useful for further
development of high affinity PBD-binding peptides and pep-
tide mimetics.
Experimental Section
General synthetic procedures: All experiments involving mois-
ture-sensitive compounds were conducted under dry conditions
(positive argon pressure) using standard syringe, cannula, and
septa apparatus. Solvents: All solvents were purchased anhydrous
(Aldrich) and used directly. HPLC-grade hexanes, EtOAc, CH2Cl2,
and MeOH were used in chromatography. TLC: Analytical TLC was
performed on Analtech pre-coated plates (Uniplate, silica gel GHLF,
250 microns) containing a fluorescence indicator; NMR spectra
Interactions of the peptoid Phe(CH2)6- group of 6q are
nearly superimposable with what is seen when 4R-Phe(CH2)4-
O- functionality is introduced at the amino-terminal Pro resi-
due of the parent peptide 1 (PBD ID: 4DFW; Figure 3A).[2b] We
observed similar interactions with a Phe(CH2)8 adduct originat-
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Targeting the Polo Box Domain of Plk1
triazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate
(HBTU; 5.0 equiv), hydroxybenzotriazole (HOBT; 5.0 equiv) and N,N-
diisopropylethylamine (DIPEA; 10.0 equiv) were used as coupling
reagents. Coupling of the Leu residue and Fmoc-deprotection pro-
vided the resin-bound peptide 2 having a free terminal amine
group. Treatment with bromoacetic acid (10.0 equiv), diisopropyl-
carbodiimide (DIC, 5.0 equiv) and DIPEA (20.0 equivalents) in NMP
(30 min), followed by washing, provided the corresponding resin-
bound bromoacetylated peptide (3). Aliquots of the resin were
treated with primary amines 4a–r in DMSO (ꢀ2.5m, overnight) to
provide the corresponding resin-bound peptides 5a–r (Scheme 2).
Amines 4a–o were purchased from Sigma–Aldrich, while amines
4p–r were prepared according to the literature procedures.[15] Ter-
minal alkylpeptide amines were acetylated using either N-acetyl-
imidazole (5.0 equiv in DMF, overnight for peptides 5a–l), or acetic
anhydride (10.0 equiv) in the presence of DIPEA (20.0 equiv) in
1,2-dichloroethane (RT, 4 h for peptides 5m–r). Incorporation of
(2S,3R)-2-amino-3-methyl-4-phosphonobutyric acid (Pmab) in place
of the pThr residue was accomplished as previously reported.[13]
For peptide pull-down assays, a cysteine residue was attached to
the amino-terminus by means of a 6-aminohexanoylamide (Ahex)
spacer. Completed resins were sequentially washed with DMF,
MeOH, CH2Cl2 and ether and then dried under vacuum (overnight).
Resins (200 mg) were cleaved by treatment with a mixture of TFA/
triisopropylsilane (TIS)/H2O (90:5:5, 5 mL, 4 h) and concentrated
under vacuum and then diluted with cold ether to give precipitat-
ed products, which were washed with ether. The resulting solids
were dissolved in 50% aqueous acetonitrile (5 mL) and purified by
reversed-phase preparative HPLC using a Phenomenex C18 column
(21 mm ø ꢁ250 mm, cat. no: 00G-4436-P0) with a linear gradient
from 0% aqueous acetonitrile (0.1% TFA) to 100% acetonitrile
(0.1% TFA) over 30 min at a flow rate of 10.0 mLminÀ1. Lyophiliza-
tion gave product peptides 6a–r as white powders. Mass spectra
and HPLC data are provided in Tables S1–S3.
On-resin modification of peptides by click chemistry: Synthesis
of peptides 12 and 13: Resin-bound peptide 2 (0.20 mmol) was
converted to the corresponding amino-terminal pent-4-ynylamide
(8) by reaction with 4-pentynoic acid (5.0 equiv), DIC (5.0 equiv),
HOBT (5.0 equiv) and DIPEA (20.0 equiv) in NMP (3.0 mL) at room
temperature (2 h). The resin was washed with DMF, CH2Cl2, MeOH
and ether and dried under high vacuum (overnight). Conversion of
3-phenylpropanol and 4-phenylbutanol to the corresponding 3-
and 4-azido derivatives 9 and 10 respectively, was accomplished
by treatment with methanesulfonyl chloride followed by sodium
azide. Copper-catalyzed azide–alkyne cycloaddition reactions were
conducted both with and without warming to provide the corre-
sponding 1,4-triazole adducts (11 a and 12a) and 1,5-triazole ad-
ducts (11 b and 12b), respectively. For reactions performed without
heat, dried resin (8, 100 mg) was suspended in acetonitrile (4.0 mL)
with DMSO (1.0 mL) in a plastic tube and degassed under argon
(5 min). To this was added DIPEA (10 mL), CuI (19 mg) and phenyl-
alkyl azide (9 or 10, 10.0 equivalents) and the tube was sealed and
agitated at RT (overnight). The resin was washed sequentially with
DMF, H2O, MeOH and ether and dried under high vacuum (4 h).
Cleavage as described above provided 1,4-triazole adducts (11 a
and 12a) following HPLC purification. For reactions performed
with heating, dried resin (8, 100 mg) and phenylalkyl azide (9 or
10, 10.0 equivalents) were mixed in DMF (2.0 mL) in a flask and
heated (1008C, 2 days). The resins were then washed sequentially
with DMF, H2O, MeOH and ether and then dried under high
vacuum (4 h). Cleavage as described above provided 1,5-triazole
adducts (11 b and 12b), respectively following HPLC purification.
Mass spectral and HPLC data are provided in Table S2.
Figure 3. Superposition of Plk1 PBD-bound 6q (carbons in gray) over previ-
ously reported PBD-bound peptides. PDB IDs for bound peptides: A) 4DFW;
B) 3RQ7; C) 3P37.
were recorded using a Varian Inova 400 MHz spectrometer. Cou-
pling constants are reported in Hertz, and peak shifts are reported
in d (ppm) relative to TMS. Low-resolution mass spectra (ESI) was
measured on an Agilent 1200 LC/MSD-SL system, and high-resolu-
tion mass spectra (ESI or APCI) were measured at the UCR Mass
Spectrometry Facility, Department of Chemistry, University of Cali-
fornia, 3401 Watkins Dr., Riverside CA, 92521.
Analytical HPLC analysis: The purities were determined by using
Phenomenex C18 column (4.60 mm ø ꢁ 250 mm, cat. no: 00G-4435-
E0) with a linear gradient from 5% aqueous acetonitrile (0.1% tri-
fluoroacetic acid; TFA) to 100% acetonitrile (0.1% TFA) over 25 min
at a flow rate of 1.0 mLminÀ1, followed by 100% acetonitrile for
another 5 min.
Synthesis of peptoid–peptide hybrids (6a–r): Fmoc-Thr(PO-
(OBzl)OH)-OH and other Fmoc-protected amino acids were pur-
chased from Novabiochem. Peptides were synthesized on NovaSyn
TGR resin (Novabiochem, cat. no. 01-64-0060) using standard Fmoc
solid-phase protocols in N-methyl-2-pyrrolidone (NMP). 1-O-Benzo-
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ELISA-based PBD-binding inhibition assays: Peptide pull-down
assays were carried out essentially as described previously.[2,10]
Keywords: crystallography · kinases · molecular recognition ·
proteins
A
biotinylated p-T78 peptide was first diluted with 1ꢁ coating solu-
tion (KPL Inc., Gaithersburg, MD) to a final concentration of 0.3 mm,
and then an aliquote of the resulting solution (100 mL) was immo-
bilized onto a 96-well streptavidin-coated plate (Nalgene Nunc,
Rochester, NY). The wells were washed once with PBS plus 0.05%
Tween 20 (PBST), and incubated with PBS (200 mL) plus 1% BSA
(blocking buffer; 1 h) to prevent non-specific binding. Mitotic 293A
lysates expressing HA-EGFP-Plk1 were prepared in TBSN buffer (ca.
60 mg total lysates in 100 mL buffer), mixed with the indicated
amount of peptide ligands and applied immediately onto the bio-
tinylated p-T78 peptide-coated ELISA wells, and then incubated
with constant rocking (1 h at 258C). Following incubation, the
ELISA plates were washed with PBST (4ꢁ). To detect bound HA-
EGFP-Plk1, the plates were probed (2 h) with anti-HA antibody
(100 mL per well, 0.5 mgmLÀ1 in blocking buffer) and then washed
(5ꢁ). The plates were further probed (1 h) with HRP-conjugated
secondary antibody (100 mL per well, a 1:1000 dilution in blocking
buffer; GE Healthcare). The plates were washed with PBST (5ꢁ) and
incubated with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate solu-
tion (100 mL per well; Sigma) until a desired absorbance was ach-
ieved. The reactions were stopped by the addition of stop solution
(100 mL per well; Cell Signalling Technology, Danvers, MA) and the
optical densities were measured at 450 nm using an ELISA plate
reader (Molecular Devices, Sunnyvale, CA). Data are shown in Fig-
ures 1 and S1.
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Plk1 PBD fluorescence polarization competition binding assays:
Competition assays for the Plk1 PBD were performed essentially as
previously described.[12] In brief, 5-carboxyfluorescein-GPMQSpTP-
LNG-OH (final concentration: 2 nm) was incubated with the Plk1
PBD (final concentration: 20 nm) in the presence of the test pep-
tides (final concentrations of buffer components: NaCl (50 mm),
Tris (10 mm, pH 8.0), EDTA (1 mm), 0.1% Nonidet P-40 substitute,
dithiothreitol (1 mm), and 2% DMSO). Fluorescence polarization
was analyzed after 60 min. Inhibition curves were fitted using Sig-
maPlot (SPSS) and shown in Figure S2. All experiments were per-
formed in triplicate. IC50 values are shown in Table 1.
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X-ray crystallography: Procedures, refinement statistics (Table S4)
and sigma-weighted 2FoÀFc electron density map contoured at 1s
and 1.55 ꢂ resolution around the ligand 6q are provided in Fig-
ure S4.
[13] F. Liu, J.-E. Park, K. S. Lee, T. R. Burke, Tetrahedron 2009, 65, 9673–9679.
´
´
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man, M. Hyvçnen, C. Abell, Angew. Chem. 2011, 123, 4089–4092;
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Acknowledgements
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This work was supported in part by the Intramural Research Pro-
gram of the NIH, Center for Cancer Research, NCI-Frederick and
the National Cancer Institute, National Institutes of Health (F.L.,
J.-E.P., W.-J.Q., K.S.L. and T.R.B.) and National Institutes of Health
grant R01 GM60594 and ES015339 (M.B.Y.) and the Deutsche For-
schungsgemeinschaft grant BE 4572/1-1 (T.B).
Received: March 26, 2012
Published online on && &&, 0000
&6
&
www.chembiochem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 7
ÝÝ
These are not the final page numbers!

FULL PAPERS
Putting the hole in the polo: Replacing
the amino terminal Pro residue of the
Plk1 polo box domain (PBD)-binding
pentamer peptide, PLHSpT, with a library
of N-alkyl-Gly “peptoids” gave low-nano-
molar affinity peptide–peptoid hybrids.
An X-ray crystal structure of one PBD-
bound hybrid showed that a long-chain
alkylphenyl group created a hydropho-
bic channel (right) that was not present
in the PLHSpT-bound complex (yellow
circle, left).
F. Liu, J.-E. Park, W.-J. Qian, D. Lim,
A. Scharow, T. Berg, M. B. Yaffe, K. S. Lee,*
T. R. Burke, Jr.*
&& – &&
Peptoid–Peptide Hybrid Ligands
Targeting the Polo Box Domain of
Polo-Like Kinase 1
ChemBioChem 0000, 00, 1 – 7
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
&
7
&
ÞÞ
These are not the final page numbers!