Mono-anionic phosphopeptides produced by unexpected histidine alkylation exhibit high plk1 polo-box domain-binding affinities and enhanced antiproliferative effects in hela cells

Article abstract of DOI:10.1002/bip.22569

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on-resin N(τ)-alkylation of histidine residues already bearing a N(π)- alkyl group. This resulted in cationic imidazolium-containing pThr peptides, several of which exhibit single-digit nanomolar PBD-binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio-reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD-binding peptides and peptide mimetics.

Full text of DOI:10.1002/bip.22569

Mono-Anionic Phosphopeptides Produced by Unexpected Histidine
Alkylation Exhibit High Plk1 Polo-Box Domain-Binding Affinities and
Enhanced Antiproliferative Effects in HeLa Cells
Wen-Jian Qian,1 Jung-Eun Park,2 Dan Lim,3 Christopher C. Lai,1 James A. Kelley,1 Suk-Youl Park,4
Ki Won Lee,4,5 Michael B. Yaffe,3 Kyung S. Lee,2 Terrence R. Burke Jr.1
1Chemical Biology Laboratory, Center for Cancer Research, National Institutes of Health, NCI at Frederick, Frederick, MD
21702
2Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda,
MD 20892
3Department of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cam-
bridge, MA 02139
4Advanced Institutes of Convergence Technology, Seoul National University, Suwon 443-270, Republic of Korea
5World Class University Biomodulation Major and Department of Agricultural Biotechnology, Seoul National University,
Seoul, 151-742, Republic of Korea
Received 25 August 2014; revised 12 September 2014; accepted 27 September 2014
Published online 4 October 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22569
or its mimetics. In our current article we report the unan-
ABSTRACT:
ticipated on-resin N(s)-alkylation of histidine residues
Binding of polo-like kinase 1 (Plk1) polo-box domains
already bearing a N(p)- alkyl group. This resulted in cat-
(PBDs) to phosphothreonine (pThr)/phosphoserine
ionic imidazolium-containing pThr peptides, several of
(pSer)-containing sequences is critical for the proper
which exhibit single-digit nanomolar PBD-binding affin-
function of Plk1. Although high-affinity synthetic pThr-
ities in extracellular assays and improved antimitotic effi-
containing peptides provide starting points for developing
cacies in intact cells. We enhanced the cellular efficacies of
PBD-directed inhibitors, to date the efficacy of such pep-
these peptides further by applying bio-reversible pivaloy-
tides in whole cell assays has been poor. This potentially
loxymethyl (POM) phosphoryl protection. New structural
reflects limited cell membrane permeability arising, in
insights presented in our current study, including the
part, from the di-anionic nature of the phosphoryl group
potential utility of intramolecular charge masking, may
be useful for the further development of PBD-binding
Additional Supporting Information may be found in the online version of this
C
article.
V
peptides and peptide mimetics. 2014 Wiley Periodicals,
Correspondence to: Terrence R. Burke, Jr., National Cancer Institute, National Insti-
tutes of Health, Building 376 Boyles St., NCI-Frederick, Frederick, MD 21702; e-
mail: tburke@helix.nih.gov and Kyung S. Lee, National Cancer Institute, National
Institutes of Health, 9000 Rockville Pike, Building 37, Room 3118, Bethesda, MD
20892; e-mail: kyunglee@mail.nih.gov
Inc. Biopolymers (Pept Sci) 102: 444–455, 2014.
Keywords: Plk1; polo kinase; polo-box domain; crystal
structure; cationic dialkyl histidine; intramolecular
charge masking; phosphopeptides
Wen-Jian Qian and Jung-Eun Park contributed equally to this work.
Contract grant sponsor: National Institutes of Health
Contract grant numbers: ES015339, GM104047
Contract grant sponsors: Intramural Research Program of the NIH; Center for
Cancer Research; NCI-Frederick and the National Cancer Institute; NCI Director’s
Innovation Career Development Award
This article was originally published online as an accepted
preprint. The “Published Online” date corresponds to the
C
V
2014 Wiley Periodicals, Inc.
444
PeptideScience Volume 102 / Number 6

Antiproliferative Effects in HeLa Cells
445
could potentially result from poor cell membrane permeability,
which may be attributable in part to the peptide phosphoryl
di-anionic charge.
preprint version. You can request a copy of any preprints from the
past two calendar years by emailing the Biopolymers editorial
office at biopolymers@wiley.com.
Overcoming limitations imposed by poor cell membrane
permeability of phosphoryl functionality is a general challenge
to the development of inhibitors of phospho-dependent PPIs.
Traditionally, enhancing cellular uptake by minimizing anionic
INTRODUCTION
he polo-like family of serine/threonine protein charge has been approached either by using less charged phos-
kinases (Plk1–Plk3 collectively called Plks) plays piv- phoryl mimetics or by blocking the acidic phosphoryl hydrox-
otal roles in cell cycle regulation and cell prolifera- yls with bio-reversible prodrug moieties. In our current article
tion.1–5 Plk1–Plk3 contain N-terminal catalytic we report the unexpected on-resin Mitsunobu-catalyzed His
domains, as well as C-terminal polo-box domains N(s)-alkylation of 2a to yield imidazolium-containing peptides
T
(PBDs), which bind to “Ser- pSer/pThr”-containing motifs. (3, Figure 1) that exhibit enhanced cellular efficacies, poten-
These latter phospho-dependent protein-protein interactions tially through intramolecular “charge masking.”36 As reported
(PPIs) mediate the sub-cellular localization required for herein, we obtained further optimization of cellular efficacy by
proper Plk function.6–10 Plk1 has been recognized among Plk combining N(s)-alkylation with bioreversible phosphoryl pro-
family members as a potential anticancer therapeutic target drug protection.
and significant effort has been directed at developing Plk1
kinase domain inhibitors.11–20 However, a potential draw-
back of kinase domain inhibitors is low specificity resulting RESULTS AND DISCUSSION
from similarities among the ATP-binding clefts of Plks.
Down regulation of Plk1 along with concomitant inhibition Unanticipated On-Resin Mitsunobu-Catalyzed His
of the closely-related Plk2 or Plk3 is undesirable, due to the N(s) Alkylation
positive roles these latter kinases play in maintaining Using 2a as a platform, we had previously examined replacing
genomic stability.21,22 The uniqueness of PBDs to the Plk the pThr residue with mono-anionic non-phosphate-contain-
family makes disruption of PBD-dependent protein–protein ing pThr mimetics. However, the resulting peptides exhibited
interactions (PPIs) an attractive alternative to kinase domain significantly reduced Plk1 PBD-binding affinities.37 Therefore,
inhibitors.10,23–29
our current work began as an attempt to revisit the issue of
In our efforts to develop highly potent Plk1 PBD-binding mono-anionic pThr esters within the context of the 2a plat-
inhibitors, we recently reported the introduction of long chain form. The objective was to prepare mono-phosphoryl esters of
alkyl phenyl groups onto the N(p) nitrogen (N3) of the His type 2b–2n (Figure 1). In our earlier studies, when we sub-
imidazole ring in the polo-box interacting protein 1 (PBIP1)- jected the resin-bound sequence, “Ac-Pro-Leu-His[N(s)-Trt]-
derived peptide “PLHSpT” (1) to form peptides such as 2a Ser(OtBu)-pThr[(OBn)(OH)]-amide resin” to Mitsunobu
(Figure 1). This modification can impart up to 1000-fold coupling using a series of alcohols, the major product resulted
PBD-binding affinity enhancement through interaction of the from esterification of a single pThr phosphoryl hydroxyl, as
N(p) His-adduct moiety within a cryptic hydrophobic binding expected. However in general, these mono-esters displayed
pocket on the surface of the PBD (PDB accession code reduced PBD-affinities relative to 1.30 As we indicated in our
3RQ7).30 Our subsequent work31–33 as well as the work of prior report, along with the major pThr esterified peptides, we
others,34,35 has demonstrated that aryl functionality tethered also identified minor byproducts arising from His N(p)-alkyla-
from a variety of locations along the peptide chain can bind in tion and some of these exhibited extremely high PBD-binding
the newly discovered hydrophobic channel. Among the pep- affinities. We subsequently confirmed that under Mitsunobu
tides reported to date, the N(p)-alkyl-His residue in peptide 2a conditions, N(p)-alkylation of N(s)-Trt-protected His residues
may be viewed as being “privileged” in its ability to access this can occur, but in very limited fashion.38
new binding region, because of its close proximity to the key
In our current work, we repeated the on-resin Mistunobu
signature affinity residues, “Ser-pThr.” The compact nature of reactions as outlined above, except that instead of a His[N(s)-
the “(alkyl)His-Ser-pThr” motif makes peptides such as 2a Trt] residue we employed a His[N(p)-(CH2)8Ph] residue (des-
attractive starting points for inhibitor design. However, in spite ignated as “His*”).38 Following acid-catalyzed resin cleavage
of low-nanomolar PBD-binding affinities in in vitro assays, and HPLC purification we obtained as the major products,
peptides related to 2a achieve effects in cell culture assays only peptides having molecular weights corresponding to the antici-
at much higher concentrations.30 This low cellular efficacy pated phosphoryl esters 2b–2n. To unambiguously confirm
Biopolymers (Peptide Science)

446
Qian et al.
FIGURE 1 Structures of peptides discussed in the text.
the product structures, we subjected all members of the series
to high-resolution electrospray ionization (ESI) LC-MS/MS
analysis. As described below, to our surprise these analyses
showed that rather than derivatizing a phosphoryl oxygen as
had been anticipated, in most cases alkylation had occurred
predominately at the His N(s)-position. This yielded a series
of isomeric products 3b–3n (Figure 1).
Mass Spectral Characterization of His N(p)N(s)
Bis-alkylated Products
The mass spectral characterization of peptide 3m provides a
model of the analytical approach that was applied to all mem-
bers of the series. Electrospray ionization (ESI) of 3m gener-
ated both the protonated peptide (MH1) and the double-
protonated peptide (M12H)21, which are the two ions that
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Antiproliferative Effects in HeLa Cells
447
would also be expected for the isomeric phosphopeptide ester resolution (20 mDa extraction window encompassing the tar-
2m. Structural differentiation of these two isomers required geted ion) that is matched to the resolution and mass accuracy
MS/MS analysis of these ions derived from the intact peptide of the MS/MS analysis (Figure 2C, middle and bottom traces).
(as illustrated schematically in Figure 2A). The primary MS/ Here it can be seen that any 2m byproduct present in 3m is
MS fragmentation observed for pSer- and pThr-containing minimal since the diagnostic ion abundance is at the level of
peptides is loss of neutral phosphoric acid (H3PO4) or HPO3, instrument noise. The peptides prepared by the on-resin Mit-
with a much reduced fragmentation of the amide bond peptide sunobu chemistry were primarily the N(p), N(s)-bis-alky-His
backbone to generate “b” and “y” sequence-indicating ions.39 peptides (3). Only in the cases of 3d and 3f were more than
The abundance of these ions depends on the charge-state of 2% of the mono-esterified pThr isomers (2d and 2f) produced
the precursor ion, the structure of the peptide and the nature as byproducts and these were estimated at 15 and 17%,
of the collision-induced dissociation (CID).40 For the phos- respectively.
phopeptide 3m, which contains a cationic, N(p),N(s)- bis-
alky-His residue and a free phosphate, the predominant CID-
Evaluation in Plk1 PBD ELISA Inhibition Assays
mediated neutral loss should be that of H3PO4 (98 da), result-
When we examined the peptide products (3b–3n) in ELISA-
ing in a product ion at m/z 853.5182. For the corresponding
based Plk1 PBD inhibition assays, we found that, in contrast to
phosphopeptide isomer 2m, which contains a N(p)-mono-
the loss of potent binding affinity observed with mono-anionic
alkylated His residue and an esterified pThr moiety, the
esters of 1,30 members of the currents series retained good
expected neutral loss would be H2PO3(OR), where
PBD-binding affinity, which in some cases equaled or exceeded
R 5CH2CH2CH(OH)CH2OH, to result in an ion at m/z
that of the parent peptide 2a (Table I and Supporting Informa-
765.4658 (Figure 2A). The “b3”and “b4” sequence-indicating
tion Figure S3).
ions would also be distinct, due to the difference in the nature
Given the similarities in inhibitory potencies among the
of His alkylation.
series 3 in ELISA competition assays, we considered peptides
A loss of both HPO3 and H3PO4, indicative of the free
showing marginally better IC50 values as potential candidates
phosphate of compound 3m, was observed in both the CID
for cell-based studies (3c, 3d, 3e, 3l, and 3m; IC50 5 0.001 mM,
(Figure 2B) and HCD MS/MS spectra derived from MH1. The
Table I). Of these, 3e was omitted due to the presence of an
expected fragment ion resulting from loss of H2PO3(OR), in
anionic carboxylic acid group. Preliminary cellular evaluation
the case of a mono-esterified pThr (e.g. 2m) was not detected.
of the remaining members of the series showed that the
Furthermore, b3, b4 and y3 ions, which result from amide-
hydroxyl-containing analogs (3c, 3l, and 3m) exhibited signifi-
bond cleavage and contain the bis-alkyl-His residue, were
cantly higher potencies than the non-hydroxyl-containing ana-
clearly observed in all MS/MS spectra of 3m. MSn (n 5 2 and
log (3d). This may potentially indicate a beneficial role for
3) analysis of the remaining members of the series 3b–3n,
hydroxyl functionality in these assays. Interestingly, 3l and 3m
including mono-esterified pThr peptides prepared by alternate
showed identical potencies in spite of the diastereomeric
synthetic procedures, confirmed the validity of the above
hydroxyls in their N(s)-alkyl functionalities. On the basis of
observations. Because the isomeric N-alkylated and esterified
the above, we selected peptides 3c and 3m for more in-depth
peptides could not be completely separated by HPLC, a high-
evaluation.
resolution MS/MS extracted ion chromatogram method was
To rule out promiscuous modes of binding,42,43 the Ser to
developed to define the ratio of isomers (Figure 2C). As dis-
Ala mutants (S4A) of 3c and 3m were prepared, since the Ser
cussed above, for each phosphopeptide isomer pair, a set of
residue provides key recognition features for canonical Plk1
unique MS/MS product ions resulting from loss of a neutral
PBD binding and replacement with an Ala residue is known to
phosphate-containing moiety is preferentially generated from
significantly reduce binding affinity.6 Consistent with non-
MH1. For the bis-alkyl-His isomer, this is the product ion
promiscuous binding, our current study showed that the Ser
resulting from loss of H3PO4, e.g. m/z 853.5182 for 3m. For the
to Ala substituted peptides exhibited more than two orders-of-
phosphate ester isomer, the comparable product ion results
magnitude reduced inhibitory potencies [3c(S4A) IC50 5 0.8
from loss of the corresponding phosphoric acid ester or, in the
mM and 3m(S4A) IC50 5 0.15 mM] (Table I and Supporting
case of 2m, m/z 765.4658. Because these are major product
Information Figure S3).
ions with expected similar abundances, the relative amounts of
each can be estimated by extracting and comparing the abso-
lute ion abundances when they are analyzed under similar con- Plk1 PBD Co-Crystal Studies
ditions, i.e., LC/MS/MS. Certainty that these are the only ions To unambiguously establish its mode of binding, we solved
extracted is increased by carrying out this procedure at a high the X-ray cocrystal structure of 3m bound to the Plk1 PBD
Biopolymers (Peptide Science)

FIGURE 2 High resolution LC-MS/MS analysis of pThr-pentapeptide 3m. (A) Structures and
expected CID fragmentation of MH1 (m/z 951.4951) for the isomers 2m and 3m, respectively; (B)
Partial, high resolution CID MS/MS mass spectrum of the MH1 of 3m. The unequivocal presence
of a free phosphate moiety is indicated by the product ions b3, a4, b4, y3, MH1-H3PO4, and MH1-
HPO3. A more complete assignment of product ions and masses is available in the Supporting
Information. (C) LC-MS/MS estimation of the isomeric purity of 3m. Top trace—full total ion
chromatogram (TIC) for CID MS/MS of MH1, middle trace—high-resolution (30 mDa window)
MS/MS extracted ion chromatogram (HR-MS2-XIC) for MH1-H3PO4 of 3m (absolute peak
height 5 4.79 X 106 counts), bottom trace—HR-MS2-XIC for MH1-H2PO3(OR) of 2m where
R 5CH2CH2CH(OH)CH2OH (absolute peak height 5 5.91 3 103 counts). Based on this analysis,
pThr-pentapeptide 3m is predominantly the free phosphate (>99%) and contains very little phos-
phopeptide ester 2m byproduct (ꢀ0.1%).

Antiproliferative Effects in HeLa Cells
449
Table I Results of ELISA Plk1 PBD Competition Assaysa
Entry
Compd.
IC50 (mM)
1
PLHST
1
NA
9
2
3
2a
0.003
0.003
0.005
0.001
0.8
4
2a†b
3b
5
6
3c
7
3c(S/A)
3d
3e
8
0.001
0.001
0.004
0.005
0.006
0.04
9
10
11
12
13
14
15
16
17
18
19
3f
3g
3h
3i
FIGURE 3 Plk1 PBD co-crystal structure of 3m superimposed on
the co-crystal structure of PBD-bound 2a (PDB 3RQ7), highlight-
ing the high degree of correspondence. The 3m structure is ren-
dered with the protein backbone shown as grey ribbon and the
ligand colored according to element (carbon 5 grey, nitro-
gen 5 blue and oxygen 5 red). The 2a structure is rendered with the
protein backbone shown as blue ribbon and the ligand colored yel-
low. The red arrow indicates the location of the resolved portion of
the N(s) substituent. Most of the alkyldiol substituent is
unresolved.
3j
3k
0.003
0.002
0.001
0.001
0.15
3l
3m
3m(S/A)
3n
0.015
a Determined by ELISA competition assays as described. Binding curves
are shown in Supporting Information Figures S3.
b As defined in the text, 2a† is a variant of 2a, in which the pThr residue
is replaced by a (2S,3R)22-amino-3-methyl-4-phosphonobutyric amide res-
idue41 and an N-terminal PEG group has been appended to increase water
solubility.30
for 2a, that such modification had little effect on binding affin-
ity.30 In the current work, we found that 2a*, 3c*, and 3m*
(Figure 3 and Supporting Information Figure S4). The Plk1 bound to PBD1 with affinity constants (Kd values) of 12, 2,
PBD interactions of the parent peptide 2a have been reported and 3 nM, respectively (Table II). Selectivities of 2a*, 3c*, and
(PDB: 3RQ7).30 In our current work, we observed that both 3m* for PBD1 relative to PBD2 were 33-fold (400 nM), 21-
the protein backbone and the peptide ligand in the PBD•3m fold (43 nM) and 8-fold (24 nM), respectively, while the corre-
complex were essentially superimposable with the PBD•2a sponding selectivities for PBD1 relative to PBD3 were 25-fold
structure (Figure 3). Importantly, interactions of the His N(p)- (306 nM), 120-fold (240 nM) and 20-fold (62 nM), respec-
(CH2)8Phe groups within a hydrophobic region identified in tively. In the absence of crystal structures for PBD2 and PBD3,
our earlier report, were essentially superimposable. Extra elec- the basis for selective binding to PBD1 relative to PBD2 and
tron density in the region of the His N(s)- position was con- PBD3 are not obvious. However, alignment of the PBD
sistent with a substituent at this position (Supporting sequences6,9 provides insights into potential reasons for ligand
Information Figure S4). The absence of more extended
resolved density could indicate a degree of conformational
Table II Determination of PBD Selectivity
flexibility for the substituent.
Kd (nM)a
Entry
Compd.b
PBD1
PBD2c
PBD3c
Determination of Plk1 PBD-Binding Specificities
using Fluorescence Polarization
1
2
3
2a*
3c*
12
2
400 [33x]
43 [21]
24 [8]
306 [25x]
240 [120]
62 [20]
To evaluate Plk1 specificity, we introduced fluorescein isothio-
cyanate (FITC) groups into 2a, 3c, and 3m via N-terminal
PEG tethers (designated as 2a*, 3c*, and 3m*) (Supporting
Information Figure S5) and determined binding constants to
PBDs from Plk1, 2 and 3 (designated as PBD1, PBD2 and
PBD3, respectively) using fluorescence polarization techniques
3m*
3
a Determined by fluorscence polarization assays using FITC-labeled
ligands.
b Designation with an “*” indicates the presence of an N-terminal PEG-
FITC group as described in Supporting Information Figure S5.
c Values in brackets refer to the fold-change in binding affinity relative to
(Supporting Information Figure S6). We had previously shown PBD1. Binding curves are shown in Supporting Information Figure S6.
Biopolymers (Peptide Science)

450
Qian et al.
selectivity. These include interactions of the N-terminal Pro-
Leu residues with a region defined by Arg516 and Phe535 in
PBD1 but which corresponds to Lys and Tyr residues, respec-
tively, in PBD2 and PBD3.44
Evaluation of Bis-Alkyl-His Analogs in Cell-Based
Studies
Next, we examined the effects of selected phosphopeptides in
cultured HeLa cells. In these experiments, we employed 2a† as
a reference. This is a previously reported variant of 2a, in
which the pThr residue is replaced by a (2S,3R)22-amino-3-
methyl-4-phosphonobutyric amide residue41 and an N-termi-
nal PEG group has been appended to increase water solubil-
ity.30 This analog represents the most potent peptide we have
yet reported in cell-based assays.30 In our current work 2a† was
able to induce partial mitotic block in asynchronously growing
HeLa cells in a dose-dependent fashion, with the percentage of
mitotically arrested cells at 24 h after treatment being more
than doubled the percentage at 12 h after treatment. A maxi-
mal mitotic index of ꢀ35% was observed after exposure for 24
h at the highest concentration used (600 mM) (Figure 4).
Under these conditions, treatment with 3c achieved significant
levels of mitotic block, with 200 and 400 mM concentrations
resulting in mitotic indices of ꢀ40 and 60%, respectively, after
24 h. Treatment with 3m resulted in similar, although slightly
reduced, effects. In both cases, the corresponding S/A mutants,
3c(S4A) and 3m(S4A), respectively, gave significantly attenu-
ated effects, supporting a Plk PBD-dependent mechanism of
action (Figure 4).
FIGURE 4 Percent of cultured HeLa cells in mitosis after treat-
ment with the indicated concentrations of peptides for 12 h (upper)
and 24 h (lower).
phosphoryl esters and contain N-terminal acetyl groups, 2a†
employs the phosphonic acid-based pThr mimetic, Pmab, and
has an N-terminal PEG group. A component of the improved
cellular efficacy of 3c and 3m may arise from improved cell
membrane transit, potentially achieved as a consequence of
neutralizing one phosphoryl anionic charge through intramo-
lecular charge masking by the His imidazolium cation.36 We
reasoned that neutralization of a second anionic charge could
further enhance cellular activity.
We have previously shown that treatment of cells with 2a†
decreases cellular proliferation rate due to PBD inhibition-
induced apoptotic cell death.30 In a similar fashion, the mitotic
block observed in our current work is reflected in even stronger
antiproliferative effects, which were measured by plotting rela-
tive cell numbers for each dose at 12, 24, and 48 h (Figure 5).
An IC50 value of 320 mM for 2a† is consistent with the previ-
ously reported IC50 value of 380 mM.30 Approximate IC50 val- Phosphoryl Prodrug Derivatization
ues of 85 and 90 mM obtained for 3c and 3m, respectively, A general strategy for increasing the bioavailability of phos-
represent marked improvements relative to 2a†, while the cor- phates is to block their acidic functionality with “prodrug”
responding S/A mutants, 3c(S4A) and 3m(S4A), showed sig- groups that can be removed enzymatically once the agent is
nificantly reduced effects.
within the cell.45,46 The pivaloyloxymethyl (POM) moiety is a
In ELISA PBD inhibition assays of the current study, 2a, form of phosphoryl prodrug protection that has been widely
2a†, 3c, and 3m all displayed similar IC50 values (Table I). The employed in nucleotides.46 The POM group is removed enzy-
lower IC50 value of 2a† relative to our previously reported matically by esterase-mediated cleavage of the pivaloyloxy-
value for this peptide (3 nM versus 30 nM)30 stems from vari- methyl ester linkage. This yields pivalic acid and an unstable
ability in the ELISA assay (the ELISA O.D. 450 values vary hydroxymethyl ester group, which decomposes to formalde-
depending on the length of developing time). The enhanced hyde with liberation of the free phosphoryl hydroxyl group. To
cellular activities shown by 3c and 3m relative to 2a† could be our knowledge, with the exception of our recent synthetic
attributable to a number of factors. While 3c and 3m utilize methods paper,47 there have been no prior applications of
Biopolymers (Peptide Science)

Antiproliferative Effects in HeLa Cells
451
FIGURE 5 Dose-dependent inhibition for cell proliferation by the indicated peptides. Asynchro-
nously growing HeLa cells were treated with various concentrations of the indicated compounds.
The resulting cells were harvested at the 0, 12, 24, and 48 h time points and then quantified. The
relative cell numbers for the indicated time points were determined in comparison to those of the
respective 0 h time point sample. The IC50 values of cell proliferation inhibition for indicated com-
pounds were calculated using the data points from the 48 h samples.
POM phosphoryl protection of pThr or pSer-containing poly- site of action, yet once there, that enzymatic activation occurs
peptides within a biological context. Accordingly, we utilized within a biologically relevant time frame. Stability of esterase-
our recently reported reagent, N-Fmoc-Thr[PO(OPOM)OH]- labile prodrugs is often approximated using in vitro assays that
OH,47 along with our previously reported N-Fmoc-His*-OH38 employ readily available pig liver esterase (PLE). Because it was
to prepare resin-bound “Ac-Pro-Leu-His*-Ser-Thr[PO(OPO- also important to examine the stability of the POM group
M)OH)]-amide resin. Mitsunobu alkylation followed by acid- within the more relevant contexts of cell culture media and
catalyzed resin cleavage provided 4c as a variant of 3c bearing intracellular milieu, we performed these experiments as well.
mono-POM protection of the phosphoryl group (see Support- We found that conversion of 4c to 3c occurred with a half-life
ing Information Figure S7).
of ꢀ240 min in control PLE (Supporting Information Figure
S9A). In culture media the half-life of 3c at a concentration of
1 mM was ꢀ400 min (Supporting Information Figure S9B). In
addition, at a more relevant concentration of 200 mM, conver-
Esterase Stability Assays
A successful prodrug strategy requires that the chosen deriva- sion of 4c to 3c in culture media did not occur to any appreci-
tive exhibit sufficient stability to allow delivery to the desired able extent. In contrast, incubating 1 mM concentration of 4c
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452
Qian et al.
Table III Pre-Incubation Dependent Plk1 PBD Binding
OH and Fmoc-His[N(p)-(CH2)8Ph)]-OH was prepared according to
literature procedures.38 Peptides were synthesized on NovaSyn TGR
R
V
R
V
resin (Novabiochem, cat. no. 01–64-0060) or NovaSyn TG Siber
IC50 (mM)
resin (Novabiochem, cat. no. 01–64-0092) using standard Fmoc-
based solid-phase protocols in N-methyl-2-pyrrolidone (NMP). The
1-O-Benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexafluoro-phos-
phate (HBTU) (5.0 eq.), hydroxybenzotriazole (HOBT) (5.0 eq.) and
N,N-diisopropylethylamine (DIPEA) (10.0 eq.) were used as coupling
reagents. Amino terminal acetylation was achieved using 1-
acetylimidazole (10 eq.) in DMF. The finished resins SI-1 or SI-2 (0.1
mmol) (Supporting Information Figure S1) were swelled in CH2Cl2
(15 min) and then treated with triphenylphosphine (262 mg, 1.0
mmol), diethyl azidodicarboxylate (DEAD) (0.46 mL, 40% solution
in toluene, 1.0 mol) and alcohols b–n (Supporting Information Figure
S1) (1.0 mmol) in dry CH2Cl2 at room temperature (4 h), then
washed (CH2Cl2) and dried under vacuum (over night). Resins were
then cleaved by treatment with trifluoroacetic acid (TFA): triispropyl-
silane (TIS) : H2O (95 : 2.5 : 2.5) (5 mL, 4 h). The resin was removed
by filtrations and the filtrate was concentrated under vacuum, the resi-
due was dissolved in 50% aqueous acetonitrile (CH3CN) (5 mL) and
purified by reverse phase preparative HPLC using a Phenomenex C18
column (21 mm dia 3 250 mm, cat. no: 00G-4436-P0) with a linear
gradient from 20% aqueous CH3CN (0.1% TFA) to 90% CH3CN
(0.1% TFA acid) over 30 min at a flow rate of 10.0 mL min21. Lyoph-
ilization gave the products 3b–3n as white powders (Supporting
Information Figure S1). Peptide 2a was prepared as previously
reported.38 Analytical data are provided in Supporting Information
Table SI. Structure confirmation by MS/MS is described in the Sup-
porting Information.
Entry
Compd.
Withouta
Withb
1
2
3
6
4
7
5
8
PLHSpT (1)
2a
3c
16
0.002
0.002
1.3
2
0.001
0.001
0.2
3c(S4A)
3m
0.002
0.3
0.001
0.06
0.002
1
3m(S4A)
4c
4c(S4A)
0.02
28
a Cell lystates were incubated (1.5 h) without peptide prior to perform-
ing binding studies (1-h incubations).
b Cell lysates were preincubated with peptide (1.5 h) prior to performing
binding studies (1-h incubations). Binding curves are shown in Supporting
Information Figure S8.
with cell lysates showed that 50% conversion to 2c occurred in
ꢀ90 min (Supporting Information Figure S9C). These data
indicate that in cell culture studies, 4c should persist in rela-
tively unchanged form in the extracellular media, yet be rapidly
converted to the active form 3c once inside the cell. Interest-
ingly, since the in vitro ELISA-based PBD-inhibition assay uti-
lizes cell lysates, conversion of 4c to 3c could occur during the
course of a typical assay. Indeed, the inhibitory potency of 4c
was found to increase from 0.02 to 0.002 mM by a 1.5 h prein-
cubation prior to conducting the standard assay (Table III and
Supporting Information Figure S8).
Synthesis of Fluorescein Isothiocyante (FITC)-
Labeled Peptides (3*c and 3*m) for Plk Specificity
Determination
The resin-bound peptide SI-3 (Supporting Information Figure S5) was
synthesized by standard Fmoc solid-phase peptide-protocols as out-
Evaluation of the Effects of Prodrug Protection in
Cell-Based Studies
lined above, and the amino-terminus was acylated with Fmoc-N-
R
V
amido-dPEG 8-NHS ester (3.0 eq.) (Quanta Biodesign, Cat# 10995)
The effect of POM-protection in 4c was examined in asynchro-
nously growing HeLa cells, as described above. These studies
demonstrated that relative to parent 3c, peptide 4c showed an
improved ability to induce mitotic block, reaching a maximum
mitotic index of ꢀ80% at 24 h at a concentration of 400 mM,
as compared to ꢀ60% for 3c under the same conditions and
roughly 18% for 2a† (Figure 5). The potency of the S/A mutant
4c(S4A) was very similar to vehicle control, strongly support-
ing the PBD-dependence of mitotic block by 4c (Figure 5).
The anti-proliferative potency of 4c (IC50 5 55 mM) was also
improved relative to 3c (IC50 5 85 mM) (Figure 5).
by reacting with HBTU (5.0 eq.), HOBt (5.0 eq.) and DIPEA (10.0 eq.)
at room temperature (4 h). On-resin Mitsunobu His-N(s) alkylation
was then performed as indicated above for the synthesis of peptides 3
(Supporting Information Figure S1) to form the resin-bound peptides
SI-4 and SI-5 (Supporting Information Figure S5). Following Fmoc
deprotection, the resins were treated with the fluorescein isothiocyanate
(FITC) (open-form isomer I) (5.0 eq.) and DIPEA (10.0 eq.) in NMP
(12 h). [Note: Performing installation of the FITC group prior to Mit-
sunobu esterification yielded complex products following resin cleav-
age.] Finished resins were washed sequentially with DMF, MeOH,
CH2Cl2 and ether, dried under vacuum (1 h) and subjected to TFA-
mediated cleavage as described above for the synthesis of peptides 3
and purified by HPLC to provide the desired peptides 3c* and 3m* as
yellow powders. Analytical data are provided in Supporting Informa-
tion Figure S5. Peptide 2a* was synthesized as previously described.30
MATERIALS AND METHODS
On-resin Synthesis of Peptides Containing Synthesis of the POM Prodrug-Protected Peptides 4c
N(p),N(s)-alkyl His Residues (3b–3m) and 4c(S4A)
Fmoc-protected amino acids were purchased from Novabiochem. Synthesis of peptides 4c and 4c(S4A), which represent peptides 3c
Phosphothreonine was incorporated using Fmoc-Thr[PO(OBzl)OH]- and 3c(S4A) having their one phosphoryl hydroxyl derivatized as a
Biopolymers (Peptide Science)

Antiproliferative Effects in HeLa Cells
453
pivaloyloxymethyl (POM) ester, was achieved as outlined in Support- esterified histidyl phosphopeptide 2 present in each sample. A sum-
ing Information Figure S7. Solid-phase peptide synthesis was con- mary of the high resolution MS1 and MS2 data for each compound
ducted as indicated above for the synthesis of resin-bound peptides can be found beginning in the Supporting Information on p S-6.
SI-1 and SI-2 (Supporting Information Figure S1) except that Fmoc-
pThr[PO(OPOM)(OH)]-OH (SI-6)47 was used in place of Fmoc-
ELISA-based PBD-Binding Inhibition Assays
Thr(PO(OBzl)OH)-OH to yield the resin-bound peptides SI-7 and
SI-8 (Supporting Information Figure S7). On-resin Mitsunobu His-
N(s) alkylation was then performed as indicated above for the synthe-
sis of peptides 3. The resulting resins were cleaved using TFA as indi-
cated above and purified by HPLC to provide peptides 4c and
4c(S4A) as white powders. Analytical data are provided in Supporting
Information Table SI.
A biotinylated p-T78 peptide was first diluted with 13 coating solu-
tion (KPL, Gaithersburg, MD) to the final concentration of 0.3 mM,
and then 100 mL of the resulting solution was immobilized onto a 96-
well streptavidin-coated plate (Nalgene Nunc, Rochester, NY). The
wells were washed once with PBS plus 0.05% Tween20 (PBST), and
incubated with 200 mL of PBS plus 1% BSA (blocking buffer) for 1 h
to prevent non-specific binding. Mitotic 293A lysates expressing HA-
EGFP-Plk1 were prepared in TBSN buffer (ꢀ60 mg total lysates in 100
mL buffer), mixed with the indicated amount of the competitors (p-
T78 peptide and its derivative compounds), provided immediately
onto the biotinylated peptide-coated ELISA wells, and then incubated
with constant rocking for 1 h at 25ꢁC. Following the incubation,
ELISA plates were washed four times with PBST. To detect bound
HA-EGFP-Plk1, the plates were probed for 2 h with 100 mL well21 of
anti-HA antibody at a concentration of 0.5 mg mL21 in blocking
buffer and then washed 5 times. The plates were further probed for 1
h with 100 mL well21 of HRP-conjugated secondary antibody (GE
Healthcare, Piscataway, NJ) at a 1:1000 dilution in blocking buffer.
The plates were washed five times with PBST and incubated with 100
mL well21 of 3,3’,5,5’-tetramethylbenzidine (TMB) solution (Sigma,
St. Louis, MO) as a substrate until a desired absorbance was reached.
The reactions were stopped by the addition of 100 mL well21 of stop
solution (Cell Signaling Technology, Danvers, MA). The optical den-
sity (O.D.) was measured at 450 nm by using an ELISA plate reader
(Molecular Devices, Sunnyvale, CA). Data are shown in Table I and
Supporting Information Figure S3.
Mass Spectrometry Analysis
Low resolution positive ion, electrospray ionization (ESI) mass spectra
were obtained by LC/MS analysis on an Agilent LC/MSD single quad-
rupole system that was also equipped with an in-line diode-array UV
detector. A narrow-bore (100 3 2.1 mm2), small-particle (3.5 mm),
Zorbax Rapid-Resolution reversed-phase C18 column coupled with a
C
18 guard column (12.5 3 2.1 mm2) was eluted with a 5–90% gradi-
ent of CH3OH/H2O containing 0.1% CH3COOH at a flow rate of
300 mL min21 to separate the components of crude reaction isolates,
enzymatic and cellular incubation mixtures, and purified final prod-
ucts. In the case of the latter, both the total-ion chromatogram (TIC)
and the UV-chromatogram at 220 nm were used to assess compound
purity. Enzymatic and hydrolytic stability was monitored and quanti-
fied by generating the appropriate compound-indicating extracted ion
chromatograms using the LC/MSD ChemStation software (version
B.04.02 SP1).
High resolution LC/MS and LC/MS/MS analyses were conducted
on a Thermo-Fisher LTQ-XL Orbitrap hybrid mass spectrometer sys-
tem operated under Xcalibur (version 2.1.0 SP1) control for data
acquisition and qualitative analysis. A narrow-bore (150 3 2 mm2),
small particle (3.0 mm) Imtakt, Cadenza CD-C18 reversed-phase col-
umn coupled with a guard column (5 3 2 mm2) of the same station-
ary phase was eluted with a two-stage linear gradient of CH3CN/H2O
containing 0.1% HCOOH at 250 mL min21. An initial shallow gradi-
ent of 2–50% CH3CN/H2O over 20 min was followed by a steeper
gradient of 50–90% CH3CN/H2O in 10 min with a 3-min hold at the
final conditions before LC reset and equilibration. Primary mass spec-
tra (MS1) for accurate mass measurement of a molecular species
([M1H]1, [M1Na]1 or [M12H]12) were obtained at a resolution
of 30,0000 (FWHM), while MS/MS (MS2) studies employing
collision-induced dissociation (CID) or higher energy CID (HCD)
were conducted at a resolution of 15,000. For these MS2 studies,
[M1H]1 or [M12H]12 precursor ions were selected using a mass
window that encompassed the isotopic profile of the ion of interest.
For the compounds in question, CID and HCD were carried out at an
optimized energy setting of 30 and 27.5, respectively, in order to give
the highest intensity product ion mass spectrum. Product ion spectra
PBD Fluorescence Polarization Binding Assays for
Plk1, Plk2, and Plk3
The 5-carboxyfluorescein-labeled peptides 2a*, 3c*, and 3m* were
incubated, at a final concentration of 2 nM, with various concentra-
tions of bacterially-expressed purified PBDs of Plk1, Plk2 and Plk3 in
a binding buffer containing 10 mM Tris (pH 8.0), 1 mM EDTA,
50 mM NaCl, and 0.01% Nonidet P-40. Fluorescence polarization was
analyzed 10 min after mixing of all components in a 384-well format
using a Molecular Devices SpectraMax Paradigm Multi-Mode Micro-
plate Detection Platform. All experiments were performed in tripli-
cate. Obtained data were plotted using GraphPad Prism software
version 6. IC50 values are provided in Table II and binding curves are
shown in Supporting Information Figure S6.
Stability of Prodrug Protected Peptide 4c to
Enzymatic Deprotection
containing multiply charged ions (i.e., those from [M12H]12 precur- Pig Liver Esterase Hydrolysis of Peptide 4c. Following litera-
sors) were deconvoluted to a single charge state using the Xtract soft- ture procedures,48 0.05M potassium phosphate buffer (pH 7.4) was
ware module of Xcalibur in order to facilitate structural assignment placed in centrifuge tube and a solution of peptide 4c in MeOH was
and analysis. MS2 high-resolution extracted ion chromatograms (HR- added to the buffer to achieve a concentration of 200 lM of 4c with
XICs) were generated post-analysis using a 20 or 30 mDa mass win- MeOH being less than 1%. To a 1-mL aliquot of the above solution
dow centered on the observed accurate mass of the appropriate diag- was added pig liver esterase (57.6 U) and the reaction mixture was
nostic ions. These HR-XICs were then used to locate and measure the incubated at 37ꢁC with gentle agitation. At various time points, ali-
relative amounts of isomeric dialkylhistidyl phosphopeptide 3 and quots of the reaction mixture (50 lL) were transferred to an
Biopolymers (Peptide Science)

454
Qian et al.
Eppendorf tube containing MeCN (50 lL). Following filtration, the equal volume of reservoir solution consisting of 13% (w/v) PEG 3350,
hydrolysis product was monitored by LC-MS. Data are shown in Sup- 0.1M HEPES pH 7.5 and 100 mM NaCl. Crystals began appearing
porting Information Figure S9.
overnight and reached maximum size over several days. Crystals grew
in clusters that were manually broken up to obtain sufficiently single
crystals suitable for data collection.
Hydrolysis of Peptide 4c in Cell Culture Media. To an aliquot
of culture medium consisting of Dulbecco’s modified eagle medium
(DMEM) [high glucose, no glutamine, (cat. 11960, Life Technolo-
gies)]49 (500 mL); fetal bovine serum (FBS) (cat. SH30396, Thermo
Scientific) (50 mL); Antimycotic (cat. 15240-112, Life Technologies),
(5 mL), Glutamax (cat. 35050-079, Life Techonologies) (5 mL) and
Hepes buffer (cat. 15630-130, Life Technologies), (12.5 mL) was
added a solution of peptide 4c in MeOH to achieve a concentration of
1 lM of 4c with MeOH being <1%. The reaction mixture was then
incubated at 37ꢁC with gentle shaking. At various time points, ali-
quots of the reaction mixture (50 lL) were transferred to an Eppen-
dorf tube containing MeCN (50 lL). Following filtration, the
hydrolysis product was monitored by LC-MS. Data are shown in Sup-
porting Information Figure S9.
Data Collection and Structure Determination and Refine-
ment. Crystals were cryo-protected in 26% (w/v) PEG 3350, 0.1M
HEPES pH 7.5, 150 mM NaCl, 6% (v/v) glycerol, 1 mM 3m, 1% (v/v)
DMSO and 10 mM DTT, and data were collected at 100 K on a
Rigaku Raxis-IV image plate detector with a Rigaku RU-300 home X-
ray source. The data were processed with the HKL50 and CCP451 soft-
ware suites. The structure was solved by molecular replacement using
AMoRe52 using chain A of structure 3FVH28 (RCSB accession code)
as a search model, and refined using PHENIX53 with manual fitting
in XtalView.54 Refinement statistics, a sigmaA weighted 2Fo – Fc elec-
tron density map and a SA omit Fo – Fc electron map contoured at 3r
˚
and 1.45 A are provided in Supporting Information Figure S4.
Hydrolysis of Peptide 4c by Total Cell Lysates. Mitotic 293A
cells expressing HA-EGFP-Plk1 were lysed at 4ꢁC using lysis buffer
consisting of 50 mM Tris--HCl, pH 8.0; 120 mM NaCl; 5% nonyl phe-
noxypolyethoxylethanol (Tergitol-type NP-40); 5 mM ethylene glycol
tetraacetic acid (EGTA); 1.5 mM ethylenediaminetetraacetic acid
(EDTA); 4-nitrophenyl phosphate di(tris) salt [PNPP (cat. N3254,
Aldrich)] (0.1 g/10 mL) and protease inhibitor [complete EDTA-free
(cat. 11873580001, Roche]. The lysate was centrifuged (13,000 rpm, 15
min, 4ꢁC) and the resulting cell lysate supernatant was placed in 96-
well plates. To the wells was added a solution of peptide 4c in DMSO
so that the concentration of the peptide was 1 lM with DMSO being
at a concentration of <1% and a total volume of 125 mL. The reaction
mixtures were incubated at 37ꢁC with gentle shaking. At different time
points, MeCN (125 lL) was added to each well to quench the reac-
tions. Following filtration, the hydrolysis products were monitored by
LC-MS. Data are shown in Supporting Information Figure S9.
CONCLUSIONS
In spite of their potential therapeutic applications, inhibitors
of phospho-dependent protein–protein interactions present
particular challenges for the development of cell permeable
binding antagonists, due to the critical roles traditionally
played by the di-anionic phosphoryl moiety in ligand affinity
and the impediments that these charged groups present to cell
membrane transit. Our current work is significant in that it
has resulted in the discovery of unanticipated N(p),N(s)- bis-
alky-His-containing peptides having low-nanomolar inhibitory
potencies in in vitro PBD-binding assays. Importantly, the effi-
cacies in cellular assays of some of these peptides are improved
relative to the dianionic peptide 2a†. A key structural aspect of
the improved cellular potencies may be related to reduction of
overall net peptide charge through intramolecular charge
masking of one phosphoryl anionic charge by the His imidazo-
lium cations. We have also demonstrated that further improve-
ment in cellular profiles can be achieved by utilizing POM
phosphoryl protection. The strategy described herein repre-
Cell Culture, Analysis of Cellular Proliferation,
Aberrant Mitotic Population and Indirect
Immunofluorescence Microscopy
HeLa cervical carcinoma CCL2 and 293A cells were cultured as rec-
ommended by the American Type Culture Collection (Manassas, VA).
To prepare mitotic 293A cells expressing HA-EGFP-Plk1, cells were
infected with adenovirus expressing HA-EGFP-Plk1 and arrested with sents a notable advance in the development of PBD-binding
200 ng mL21 of nocodazole for 16 h. To analyze the effect of the indi-
inhibitors that may also find use in the design and develop-
cated compounds in cultured cells, logarithmically growing HeLa cells
ment of phosphate-containing inhibitors in other contexts.
were treated with various concentrations of the indicated compounds
for 12, 24, and 48 h. Images of HeLa cells were acquired using a Zeiss
Appreciation is expressed to Wei Dai, New York University
Axiovert 100M microscope (Carl Zeiss MicroImaging, Thornwood,
School of Medicine, NY for reagents. The authors have no com-
NY). Effects of treatment on mitotic block and cellular proliferation
peting financial interests.
are shown graphically in Figures 4 and 5.
X-ray Crystallography
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