Non-proteinogenic amino acids in the pThr-2 position of a pentamer peptide that confer high binding affinity for the polo box domain (PBD) of polo-like kinase 1 (Plk1)

Article abstract of DOI:10.1016/j.bmcl.2012.10.093

We report herein that incorporating long-chain alkylphenyl-containing non-proteinogenic amino acids in place of His at the pT-2 position of the parent polo-like kinase 1 (Plk1) polo box domain (PBD)-binding pentapeptide, PLHSpT (1a) increases affinity. Fo

Full text of DOI:10.1016/j.bmcl.2012.10.093

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7306–7308
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Non-proteinogenic amino acids in the pThr-2 position of a pentamer
peptide that confer high binding affinity for the polo box domain (PBD)
of polo-like kinase 1 (Plk1)
Wen-Jian Qian a,1, Jung-Eun Park b,1, Kyung S. Lee b, , Terrence R. Burke Jr. a,
⇑
⇑
a Chemical Biology Laboratory, Molecular Discovery Program, Frederick National Laboratory for Cancer Research, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Frederick, MD 21702, USA
b Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein that incorporating long-chain alkylphenyl-containing non-proteinogenic amino acids in
place of His at the pT-2 position of the parent polo-like kinase 1 (Plk1) polo box domain (PBD)-binding
pentapeptide, PLHSpT (1a) increases affinity. For certain analogs, approximately two orders-of-magnitude
improvement in affinity was observed. Although, none of the new analogs was as potent as our previously
Received 4 September 2012
Revised 16 October 2012
Accepted 19 October 2012
Available online 27 October 2012
described peptide 1b, in which the pT-2 histidine imidazole ring is alkylated at its
p nitrogen (N3), our
current finding that the isomeric His(N1)-analog (1c) binds with approximately 50-fold less affinity than
1b, indicates the positional importance of attachment to the His imidazole ring. Our demonstration that a
range of modified residues at the pT-2 position can enhance binding affinity, should facilitate the devel-
opment of minimally-sized Plk1 PBD-binding antagonists.
Keywords:
Polo-like kinase
Polo box domain
Non-proteogenic amino acids
Peptide ligands
Published by Elsevier Ltd.
Signal transduction
Mitosis represents a highly complex and orchestrated process
that involves proper functioning of the serine/threonine polo-like
kinase 1 (Plk1).1 Plk1 contains a catalytic domain and C-terminal
polo box domain (PBD) composed of two linked polo boxes (PB1
and PB2). The PBD serves an important role by directing the Plk1
to specific phosphoserine (pS) and phosphothreonine (pT)-containing
sequences.2,3 The ability of Plk1 to promote oncogenic transforma-
tion4–6 has made Plk1 a potential anticancer drug target for both
catalytic domain and PBD-directed agents.7,8
Peptide-based ligands offer an approach for developing PBD-
binding antagonists. One objective in the design of such agents is
to maximize binding interactions in the most efficient manner.
The Plk1 PBD has been shown to recognize sequences of the gen-
These show that the ‘‘SpT’’ motif forms an anchor that is held in
place by means of an electrostatic ‘‘pincer’’ arising from the inter-
action of the negatively-charged phosphoryl group with H538 and
K540 present the PB2 unit. The importance of the H538 and K540
residues for phospho-dependent binding has been confirmed by
mutational studies.10 Interactions arising from within this SpT-
binding region will likely be critical for high affinity binding of
any peptide or peptide mimetic.
The polo box-interacting protein 1 (PBIP1) is a Plk1 substrate
that undergoes phosphorylation at T78 to form a Plk1 PBD-binding
ligand.14 We have shown that the PBIP1-derived 5-mer peptide,
74-PLHSpT-78 (1a, Figure 1) can serve as a minimal sequence that
retains high Plk1 PBD-binding affinity.13 Peptide 1a affords a
potentially attractive starting point for developing compact PBD-
binding ligands. Abell has demonstrated by using a more extended
9-mer PBIP1 sequence, 71-FDPPLHSpTA-79, that the amino-terminal
F71 residue can enter into binding interactions with a newly
formed pocket defined by PBD residues V415, Y417, Y421, L478,
Y481, F482 and Y485.15 This pocket is not present or accessible
in the crystal structure of the shorter PBD-bound 5-mer peptide
(1a) (PDB: 3HIK).13 However, we recently discovered that struc-
tural variants of the parent peptide 1a, in which the pT-2 histidine
eral form, [P/F]-[U/P]-[U]-[T/Q/H/M]-S-[pT/pS]-[P/U], where U
designates a hydrophobic residue.9,10 The ‘‘S-pT’’ dipeptide seg-
ment represents a critical element that is necessary for high bind-
ing affinity. Several crystal structures of the Plk1 PBD in complex
with different phosphopeptides have been reported, including
MQSpTPL (PDB: 1Q4K),11 PMQSpTPL (PDB: 1UMW),10 LLCSpTPN
(PDB: 3BZI),12 LHSpTA (PDB: 3FVH),13 and PPHSpT (PDB: 3C5L).13
⇑
Corresponding authors. Tel.: +1 301 496 9635; fax: +1 301 496 8419 (K.S.L.);
imidazole ring is alkylated at its
p nitrogen (N3), can afford excep-
tionally high affinity.16 This is exemplified by peptide 1b, which
contains a C6H5(CH2)8– group at this position and which shows
an approximate 1000-fold affinity enhancement relative to 1a.16
tel.: +1 301 846 5906; fax: +1 301 846 6033 (T.R.B.).
E-mail addresses: kyunglee@mail.nih.gov (K.S. Lee), burkete@helix.nih.gov (T.R.
Burke).
1
These authors contributed equally to this work.
0960-894X/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.10.093

W.-J. Qian et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7306–7308
7307
Figure 2. ELISA-based Plk1 PBD-binding results (OD: optical density) generated as
reported in Refs. 13,16. A representative graph from three independent experiments
is shown.
even at the highest concentration tested (300 lM), confirming
the phospho-dependent nature of binding. The low micromolar
affinity observed for the parent pT78-derived pentapeptide 1a
(IC50 = 16 l
M, Figure 2) is consistent with previous reports.16–18
For the all newly prepared peptides, introduction of non-canonical
residues bearing long-chain alkylphenyl groups at the pT-2 posi-
tion conferred additional affinity to the parent peptide 1a. Model-
ing studies comparing the crystal structure of PBD-bound 1b (PDB
Accession Code: 3RQ7)16 with docked and energy minimized struc-
tures of the peptides 1c–1h, showed that with the exception of
their pT-2 side chains, all other portions of the peptides were
nearly unchanged relative to 1b (Figure 3). In comparison to 1b
(IC50 = 5.6 nM),16 the isomeric His(N1)-analog (1c) bound with
Figure 1. Structures of peptides and modified N-Fmoc-protected amino acids.
A crystal structure of 1b bound to the PBD (PDB Accession Code:
i3RQ7)16 demonstrates that binding of the C6H5(CH2)8-moiety oc-
cur in a well-formed hydrophobic channel, which is created by dra-
matic re-orientation of the Y481 aryl ring. Elements of binding
within this channel are reminiscent of what had been reported
for the F71 residue of the 9-mer-peptide.15
approximately 50-fold less affinity (IC50 = 0.32 lM). This indicates
the positional importance of attachment to the His imidazole ring.
Recently, this PBD binding region has been accessed from differ-
ent locations on peptides.17–19 It is noteworthy that for these ana-
logs, the residues that interact with the binding channel originate
from positions in the peptide chain further removed from the crit-
ical SpT motif than does the alkyl-His residue in peptide 1b. There-
fore, the pT-2 position may be viewed as ‘‘privileged’’ in its ability
to access this new binding region, while maintaining close proxim-
ity to the signature ‘‘S-pT’’ residues. The current work was under-
taken to examine whether modified residues other than His at the
pT-2 position of 1a can also impart high affinity.
The reasons for the difference in affinities shown by 1b and 1c are
not clear. Modeling studies show that relative to 1b, the v1 and v2
angles of 1c exhibit slight rotations that result in misalignment of
the respective imidazole rings. While the His(N3)-alkyl side chain
of 1b is directed toward the protein surface, the orientation of
the His imidazole ring in 1c directs the (N1)-alkyl side chain
slightly away from the protein surface, resulting in a 1.7 Å upward
displacement of the first methylene group and displacement of the
succeeding three methylene units (Figure 3A).
The Trp adduct (1d, IC50 = 9.3 lM) bound with approximately
Aryl functionality has been shown to bind preferentially in the
hydrophobic pocket being targeted by the present work.15–19
Accordingly, long chain phenylalkyl groups were introduced by a
variety of approaches at the pT-2 position of peptide 1. These in-
clude modifying the side chain nitrogens of Trp (1d), Asn (1e),
Gln (1f), ornithine (Orn) (1g) and Lys (1h) (Figure 1). In addition,
the His(N1) alkyl derivative was prepared (1c), since only isomeric
His(N3) alkyl adducts had been examined previously.16,20 The
C6H5(CH2)8– group was employed for His, Trp and Asn residues,
while shorter chains were utilized for Gln and Orn [C6H5(CH2)7–]
and Lys [C6H5(CH2)5–] (Figure 1). The synthesis of peptides 1a
and 1b has been reported.16,20 Synthesis of the remaining peptides
was by solid-phase Fmoc-based protocols using acid labile resin
(Supporting Information). Introduction of the pT-2-modified resi-
dues bearing long chain phenylalkyl groups was accomplished
using appropriate non-coded N-Fmoc-protected amino acids 2c–
2h (Figure 1), which were prepared as described in the Supporting
Information. Completed peptides were cleaved from the resin and
purified by HPLC to provide the desired products 1c–1h as white
amorphous powders.
30-fold less affinity than 1c. This is in spite of the fact that the pyr-
role portion of the indole ring of 1d aligns closely with the imidaz-
ole ring of 1c and that their respective C6H5(CH2)8– groups are
essentially superimposable (Figure 3B). The loss of affinity by 1d
may result from entropy penalties incurred by the ordering of sol-
vent molecules about the Trp indole phenyl ring, which extends
into the solvent and away from the protein (Figure 3B). The Asn-
based adduct (1e) was shown to bind in nearly superimposable
fashion with 1b (Figure 3C). However, in order to achieve this ori-
entation, its side chain amide bond must assume a highly unfavor-
able cis-conformation. The corresponding energy penalties paid to
achieve this conformation could contribute to its approximately
200-fold loss of affinity relative to 1b (IC50 = 1.1
duct (1f) exhibited nearly 10-fold higher affinity (IC50 = 0.14
l
M). The Gln ad-
M)
l
when compared to the Asn adduct (1e). This higher affinity may
be due in part to the fact that the side chain amide bond of 1f exists
in an energetically-favorable trans-orientation (Figure 3D). The
higher affinity of 1f may also be due in part to the formation of a
hydrogen bond between its amide carbonyl and the phenolic hy-
droxyl of Tyr485 (distance 2.3 Å), which is not possible with 1e.
All peptides were evaluated for their ability to compete with
immobilized PBIP1-derived 13-mer p-T78 peptide for binding to
Plk1 PBD in an ELISA-based inhibition assay that employed HEK
293A cell lysates expressing GFP-HA-fused Plk1 (Figure 2).13,16 In
this assay the control peptide, ‘‘PLHST’’ exhibited negligible affinity,
The ornithine adduct (1g, IC50 = 0.17 lM) was found to bind
with affinity equal to the Gln adduct (1f). This may reflect the fact
that similar to 1g, the side chain amide group of 1f is in an energet-
ically-favorable trans-orientations and that it can also form a
hydrogen bond between its amide carbonyl and the phenolic

7308
W.-J. Qian et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7306–7308
functionality in an energetically-favorable trans-orientation and
to make strong hydrogen bonds with the phenolic hydroxyl of
Tyr485. Although, none of the new analogs was as potent as our
previously described peptide 1b, our finding that the isomeric
His(N1)-analog (1c) binds with approximately 50-fold less affinity
than 1b, indicates the positional importance of attachment to the
His imidazole ring. Our current work demonstrates that a range
of modified residues at the pT-2 position can enhance binding. This
should facilitate the development of minimally-sized Plk1 PBD-
binding antagonists.
Acknowledgments
This work was supported by the Intramural Research Program
of the NIH, Center for Cancer Research, Frederick National Labora-
tory for Cancer Research and the National Cancer Institute,
National Institutes of Health.
A. Supplementary data
Supplementary data (synthetic experimental procedures and
analytical data for synthetic products, Plk1 PBD-binding assay pro-
tocols and modeling procedures) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2012.10.093.
References and notes
1. Barr, F. A.; Sillje, H. H. W.; Nigg, E. A. Nat. Rev. Mol. Cell Biol. 2004, 5, 429.
2. Lowery, D. M.; Lim, D.; Yaffe, M. B. Oncogene 2005, 24, 248.
3. Park, J.-E.; Soung, N.-K.; Johmura, Y.; Kang, Y. H.; Liao, C.; Lee, K. H.; Park, C. H.;
Nicklaus, M. C.; Lee, K. S. Cell. Mol. Life Sci. 2010, 67, 1957.
4. Eckerdt, F.; Yuan, J.; Strebhardt, K. Oncogene 2005, 24, 267.
5. Taka, N.; Hamanaka, R.; Yoshimatsu, J.; Miyakawa, I. Oncogene 2005, 24, 287.
6. Lu, L.-Y.; Yu, X. Cell Div. 2009, 4. http://dx.doi.org/10.1186/1747-1028-1184-
1184.
7. McInnes, C.; Mezna, M.; Fischer, P. M. Curr. Top. Med. Chem. 2005, 5, 181.
8. Strebhardt, K. Nat. Rev. Drug Disc. 2010, 9, 643.
9. Elia, A. E. H.; Cantley, L. C.; Yaffe, M. B. Science 2003, 299, 1228.
10. Elia, A. E.; Rellos, P.; Haire, L. F.; Chao, J. W.; Ivins, F. J.; Hoepker, K.;
Mohammad, D.; Cantley, L. C.; Smerdon, S. J.; Yaffe, M. B. Cell 2003, 115, 83.
11. Cheng, K.-Y.; Lowe, E. D.; Sinclair, J.; Nigg, E. A.; Johnson, L. N. EMBO J. 2003, 22,
5757.
Figure 3. In silico docking of peptides 1c–1h (carbons shown in various colors)
onto the Plk1 PBD overlaid with the parent peptide 1b (carbons shown in gray: PDB
accession code: 3RQ7). Surface is shown as semitransparent electrostatic potential
with Tyr485 and key hydrogen bonds indicated.
12. Garcia-Alvarez, B.; de Carcer, G.; Ibanez, S.; Bragado-Nilsson, E.; Montoya, G.
Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 3107.
13. Yun, S.-M.; Moulaei, T.; Lim, D.; Bang, J. K.; Park, J.-E.; Shenoy, S. R.; Liu, F.;
Kang, Y. H.; Liao, C.; Soung, N.-K.; Lee, S.; Yoon, D.-Y.; Lim, Y.; Lee, D.-H.; Otaka,
A.; Appella, E.; McMahon, J. B.; Nicklaus, M. C.; Burke, T. R., Jr.; Yaffe, M. B.;
Wlodawer, A.; Lee, K. S. Nat. Struct. Mol. Biol. 2009, 16, 876.
14. Kang, Y. H.; Park, J.-E.; Yu, L.-R.; Soung, N.-K.; Yun, S.-M.; Bang, J. K.; Seong, Y.-
S.; Yu, H.; Garfield, S.; Veenstra, T. D.; Lee, K. S. Mol. Cell 2006, 24, 409.
15. Sledz, P.; Stubbs, C. J.; Lang, S.; Yang, Y.-Q.; McKenzie, G. J.; Venkitaraman, A. R.;
Hyvoenen, M.; Abell, C. Angew. Chem. Int. Ed. 2011, 50, 4003.
16. Liu, F.; Park, J.-E.; Qian, W.-J.; Lim, D.; Graber, M.; Berg, T.; Yaffe, M. B.; Lee, K.
S., ; Burke, T. R., Jr. Nat. Chem. Biol. 2011, 7, 595.
17. Liu, F.; Park, J.-E.; Qian, W.-J.; Lim, D.; Scharow, A.; Berg, T.; Yaffe, M. B.; Lee, K.
S.; Burke, T. R. ACS Chem. Biol. 2012, 7, 805.
18. Liu, F.; Park, J.-E.; Qian, W.-J.; Lim, D.; Scharow, A.; Berg, T.; Yaffe, M. B.; Lee, K.
S.; Burke, T. R. ChemBioChem 2012, 13, 1291.
19. Sledz, P.; Lang, S.; Stubbs, C. J.; Abell, C. Angew. Chem., Int. Ed. Engl. 2012, 124,
7800.
20. Qian, W.-J.; Park, J.-E.; Liu, F.; Lee, K. S.; Burke, T. R., Jr. Bioorg. Med. Chem. 2012.
http://dx.doi.org/10.1016/j.bmc.2012.05.036.
hydroxyl of Tyr485 (distance 2.3 Å) (Figure 3E). Compared with 1f
and 1g, the Lys adduct (1h) binds with approximately fourfold less
affinity (IC50 = 0.65 lM). Although like 1f and 1g, the amide bond
of 1h exists in a trans-orientation, its ability to hydrogen bond with
the phenolic hydroxyl of Tyr485 is significantly weaker (2.99 Å dis-
tance between the amide proton of 1h and the phenolic oxygen)
and this may contribute to its reduced affinity (Figure 3F).
The purpose of the current work was to examine the effects of
incorporating long-chain alkylphenyl-containing non-proteino-
genic amino acids at the pT-2 position of 1a. We found that in all
cases, peptides modified in this fashion bound with higher affinity
than 1a. For certain analogs, affinity enhancements of approxi-
mately two orders-of-magnitude were observed (1f and 1g). High-
er affinity was associated with an ability to bind carboxamide