Functional examination of novel kisspeptin phosphinic peptides

Article abstract of DOI:10.1371/journal.pone.0195089

Kisspeptins acting on their cognate G protein-coupled receptor, kisspeptin receptor, play important roles in the suppression of cancer cell metastasis and regulation of the reproductive system, and therefore are important for therapeutic intervention. All native functional human kisspeptins (kisspeptin-54, kisspsptin-14 and kisspeptin-13) share the 10 amino acids of kisspeptin-10 at their C-terminus (45-54). However, they are inactivated rapidly by matrix metalloproteinases (MMPs) through the cleavage of the peptide bond between glycine 51 and leucine52, which limits their clinical applications. Development of MMP-resistant analogues of kisspeptins may provide better therapeutic outputs. In the present study, two kisspeptin phosphinic peptides were designed and synthesized, and their ability to induce phosphorylation of ERK1/2 through kisspeptin receptor and their inhibition on MMP-2 and MMP-9 whose activity correlates with cancer metastasis were assessed. The results showed that one analogue, phosphinic kisspeptin R isomer (PKPR), exhibited kisspeptin receptor-agonistic activity and also inhibitory activity on MMP-2, indicating that PKPR may serve as a lead for the further development of kisspeptin analogues for therapeutic purpose.

Full text of DOI:10.1371/journal.pone.0195089

RESEARCH ARTICLE
Functional examination of novel kisspeptin
phosphinic peptides
Xiaoyang Zhang1, Magdalini Matziari2, Yixin Xie2, David Fernig3, Rong Rong1, Jia Meng1,
Zhi-Liang Lu1*
1 Department of Biological Sciences, Xi’an Jiaotong-Liverpool University, Suzhou, Jiangsu, China,
2 Department of Chemistry, Xi’an Jiaotong-Liverpool University, Suzhou, Jiangsu, China, 3 Department of
Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom
* zhiliang.lu@xjtlu.edu.cn
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Abstract
Kisspeptins acting on their cognate G protein-coupled receptor, kisspeptin receptor, play
important roles in the suppression of cancer cell metastasis and regulation of the reproduc-
tive system, and therefore are important for therapeutic intervention. All native functional
human kisspeptins (kisspeptin-54, kisspsptin-14 and kisspeptin-13) share the 10 amino
acids of kisspeptin-10 at their C-terminus (45–54). However, they are inactivated rapidly by
matrix metalloproteinases (MMPs) through the cleavage of the peptide bond between gly-
cine51 and leucine52, which limits their clinical applications. Development of MMP-resistant
analogues of kisspeptins may provide better therapeutic outputs. In the present study, two
kisspeptin phosphinic peptides were designed and synthesized, and their ability to induce
phosphorylation of ERK1/2 through kisspeptin receptor and their inhibition on MMP-2 and
MMP-9 whose activity correlates with cancer metastasis were assessed. The results
showed that one analogue, phosphinic kisspeptin R isomer (PKPR), exhibited kisspeptin
receptor-agonistic activity and also inhibitory activity on MMP-2, indicating that PKPR may
serve as a lead for the further development of kisspeptin analogues for therapeutic purpose.
OPEN ACCESS
Citation: Zhang X, Matziari M, Xie Y, Fernig D,
Rong R, Meng J, et al. (2018) Functional
examination of novel kisspeptin phosphinic
peptides. PLoS ONE 13(4): e0195089. https://doi.
org/10.1371/journal.pone.0195089
Editor: Aamir Ahmad, University of South Alabama
Mitchell Cancer Institute, UNITED STATES
Received: November 14, 2017
Accepted: March 18, 2018
Published: April 3, 2018
Copyright: © 2018 Zhang et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Introduction
Kisspeptin receptor, that is previously known as GPR54 [1], AXOR12 [2] and hOT7T175 [3],
is a member of G protein-coupled receptor (GPCR) superfamily and is coupled to Gq/11 pro-
teins [4]. Its endogenous ligands are firstly isolated from human placental extracts and termed
either metastin (54-amino acid peptide) due to its capability to inhibit cancer metastasis [3] or
kisspeptins (KPs) with different length in amino acids (54-, 14- and 13-amino acid peptides)
[5]. In humans, KP-54 is cleaved from a 145-amino acid polypeptide precursor encoded by
KiSS1 gene [3]. KP-54 is further hydrolysed into several shorter and biologically functional
variants, i.e. KP-14, KP-13 and KP-10. The different forms of KPs possess similar receptor
binding affinity and potency [3] and share the 10 amino acids of KP-10 at their C-terminus,
which are highly conserved among all vertebrate species [6].
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This work was supported by the National
Natural Science Foundation of China (81373469)
(http://www.nsfc.gov.cn/) and the Research
Development Fund of Xi’an Jiaotong-Liverpool
University (http://www.xjtlu.edu.cn/en/) to ZL. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
KPs and their cognate receptor are key regulators of the mammalian reproductive system.
Natural mutations of the human kisspeptin receptor lead to idiopathic hypogonadotropic
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Novel kisspeptin phosphinic peptides
Competing interests: The authors have declared
hypogonadism [7–10]. Administration of KPs stimulates the release of both luteinizing hor-
mone (LH) and follicle-stimulating hormone in mammalian species, including humans [11,
12], mice [13, 14], rats [15] and also many domestic species such as cattle and sheep [16].
Detailed studies in the past reveal that KP-induced release of LH is inhibited by GnRH recep-
tor antagonists, indicating that GnRH is the mediator of KP-induced release of gonadotropins
[15]. The expression of kisspeptin receptor at mRNA [14, 15] and protein levels [13] is also
observed in the GnRH neurons. These results find that KPs regulate the reproductive system
by activating their receptor presented on the GnRH neurons, which leads to the secretion of
GnRH and, consequently, the release of gonadotropins.
that no competing interests exist.
Originally, the KiSS1 gene is identified as a metastasis suppresser gene due to the ability of
KP-54 to inhibit cancer cell metastasis [17]. KPs inhibit metastasis in a variety of cancers,
including melanoma [3], breast [18], endometrial [19], gastric [20] ovarian cancers [21], and
others. Therefore, KP analogues may act as inhibitors of cancer metastasis which is the major
cause of cancer death.
However, native KPs are metabolically unstable in blood and are hydrolysed by various
serum-containing proteases and MMPs such as MMP-2 and MMP-9 [22, 23]. MMPs cleave the
peptide bond between Gly51-Leu52 of KPs [24]. The expression level of MMP-2 and MMP-9
increases during the progression of many cancers, such as breast [25, 26], endometrial [27, 28]
and ovarian [29, 30] cancers, which closely correlates with cancer migration and poor prognosis.
Therefore, development of KP analogues which activate kisspeptin receptor and resist or inhibit
MMPs may provide a novel treatment to inhibit cancer cell metastasis or for hormone replace-
ment therapies with enhanced potency. Two main approaches are used to improve metabolic sta-
bility of KPs, including reduction of peptide length of KPs [31] and/or substitutions of the
cleavage sites with nonhydrolysable isosteres [23, 32]. In the present study, two penta-peptides are
designed and synthesized based on the primary structure of KP-10 among which the last C-termi-
nal 6 amino acids are crucial for receptor binding [32]. To develop KP agonistic analogues which
bind and inhibit MMPs, the peptide bond between Gly-Leu of the C-terminal 6 amino acid pep-
tide is replaced by a phosphinic acid moiety, -PO2-CH2-. The synthetic peptides containing sub-
stitution of a peptide bond with the phosphinic acid moiety are termed KP phosphinic peptides.
Phosphinic peptides are potent and selective inhibitors of various proteases, such as MMPs [33,
34], as their chemical structure mimics the intermediate transition state formed during the hydro-
lysis of peptides by proteases. In the present study, the kisspeptin receptor-agonistic activity and
the inhibitory activity on MMP-2 and MMP-9 of the synthetic peptides are examined, finding
that one synthetic peptide analogue activates kisspeptin receptor and inhibits MMP-2 activity.
Materials and methods
Materials
The cDNA of flag-tagged kisspeptin receptor was kindly provided by Dr. Andy Babwah, the
University of Western Ontario, Canada. KP-10 and Sulfo-Cy5-KP-18 were commercially syn-
thesized by Chinese Peptides (Hangzhou, China) to a purity >95%. A water soluble Sulfo-Cy5-
NHS was used to conjugate with the N-terminal amine of KP-18. Rabbit monoclonal anti-
ERK1/2 and mouse monoclonal anti-β-actin antibodies were purchased from Cell Signalling
Technology (Boston, USA). IRDye1 goat anti-mouse or anti-rabbit IgG (H + L) secondary
antibody was from LI-COR (Lincoln, USA). Fetal bovine serum (FBS) was purchased from
Bovogen (East Keilor, Australia) and all other reagents used in cell culture were purchased
from Gibco (Waltham, USA). The MMP colorimetric drug discovery kits were purchased
from Enzo Life Sciences (New York, USA). All other reagents were purchased from Sigma-
Aldrich (St. Louis, USA) and Thermofisher Scientific (Waltham, USA).
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Novel kisspeptin phosphinic peptides
Synthesis and purification of KP phosphinic peptides
The applied synthetic procedure is depicted in Fig 1. The phosphinic pseudodipeptide block 10
was synthesized by a 3-component condensation [35] in order to avoid the cumbersome and
low-yielding synthesis of the aminophosphinic Gly analogue. In detail, the acrylate precursor of
Leu 5 was synthesized by alkylation of the malonate ester 1 that led to 3, selective monosaponifi-
cation produced 4 which upon Knoevenagel condensation with formaldehyde led to 5 [36]. The
synthesis of 7 was accomplished by a Michael-type addition of hypophosphorus ammonium
salt 6 under inert atmosphere using HMDS as the silylation agent. The phosphinic acid 7 was
subsequently condensed with formaldehyde and Cbz-carbamate in AcCl that acts as both sol-
vent and acetylation reagent which facilitates the intermediate imine formation. The phosphi-
nate pseudodipeptidic block 8 was afterwards protected at the phosphinate with the adamantyl
group towards 9 [37], which was then saponified to lead to compound 10. This block was used
in solid-phase peptide synthesis by using Rink-amide lanterns and standard Fmoc-protocol.
The 2 isomers of 11 were further separated by RP-HPLC. All intermediates and the final prod-
ucts were identified and characterized by HRMS and NMR. The assignment of the absolute
configuration was based on previous studies of similar phosphinic peptide structures [38].
Cell culture and receptor transfection
HEK293 (ECACC 85120602) and MCF-7 (ECACC 86012803) cells were cultured in Dulbec-
co’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) FBS, 2 nM L-gluta-
mine, 0.2 unit/ml penicillin, 0.2 μg/ml streptomycin and were maintained at 37˚C in a
humidified atmosphere containing 5% (v/v) CO2. Both cell lines were reported to express
kisspeptin receptor naturally [39, 40], but no receptor binding and activity were detected in
our cells. HEK293 and MCF-7 cells were transiently transfected with 10 μg cDNA of human
kisspeptin receptor by electroporation using the Bio-Rad Gene Pulser Xcell™ system and
Bio-Rad 0.4 cm electroporation cuvettes. HEK293 cells were electroporated at 300 volts
with a capacitance of 950 μF, while MCF-7 cells were electroporated at 320 volts with a
capacitance of 950 μF.
Western blot analysis of the phosphorylation of ERK1/2
HEK293 and MCF-7 cells transiently transfected with kisspeptin receptor were used for phos-
phorylation assay of the extracellular signal-regulated kinase 1/2 (ERK1/2) After 24 hours follow-
ing transfection, the cells were serum-starved in a serum-free medium (DMEM supplemented
with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.1% (w/v) bovine
serum albumin (BSA), 2 nM L-glutamine, 0.2 unit/ml penicillin and 0.2 μg/ml streptomycin)
overnight prior to any treatment. Assays were then conducted by stimulating the cells with KP-
10 or KP phosphinic peptides. Following stimulation, the cells were placed on ice and solubilized
with SDS-PAGE sample loading buffer supplemented with 5 mM sodium orthovanadate and
completeTM protease inhibitor cocktail. Solubilized samples were boiled and then subjected to
SDS-PAGE. Subsequently, proteins were electrotransferred onto polyvinylidene difluoride
(PVDF) membranes for immunoblotting. Immunoblots were then assayed for phosphorylated
ERK1/2 using a phospho-specific rabbit monoclonal antibody (1:1000) (phospho-p42/44 ERKs,
Thr202/Tyr204) and for β-actin using a mouse monoclonal antibody (1:1000). Following incuba-
tion with IRDye1 goat anti-mouse (1:5000) or anti-rabbit (1:10000) IgG (H + L) secondary
antibody, the PVDF membranes were visualised using Odyssey1 infrared imaging system
(LI-COR, Lincoln, USA). Immunoblots were quantified by densitomery using the Odyssey1
application software (version 3.0).
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Novel kisspeptin phosphinic peptides
Fig 1. Synthesis of the KP phosphinic peptides.
https://doi.org/10.1371/journal.pone.0195089.g001
Evaluation of receptor binding with Sulfo-Cy5-KP-18
HEK293 cells transiently transfected with kisspeptin receptor were seeded onto 10-cm cell cul-
ture dishes. After 24 hour-incubation in complete medium, the cells were washed twice with
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Novel kisspeptin phosphinic peptides
phosphate-buffered saline (PBS), trypsinised and seeded onto 96-well cell culture plates (8 x
104 cells/well). On the following day, the cells were washed once with PBS and then incubated
with either vehicle (0.02% (v/v) propylene glycol) or various concentrations of Sulfo-Cy5-KP-
18 in the serum-free DMEM containing 10 mM HEPES and 0.1% BSA for 4 hours at 4˚C. The
non-specific binding was determined in the presence of 10 μM KP-10. After incubation, free
ligands were removed by three rapid washes with ice-cold PBS. The fluorescence intensity was
measured using PHERAStar FS microplate reader (BMG LABTECH, Germany).
Sulfo-Cy5-KP-18-induced internalisation of kisspeptin receptor
MCF-7 cells transiently transfected with kisspeptin receptor were seeded onto 96-well plates.
After 48 hours, the cells were incubated with a mixture of 50 nM Sulfo-Cy5-KP-18 and various
concentrations of tested peptides or their corresponding vehicles in the complete medium
respectively for 30 minutes at 37˚C. After incubation, free ligands were removed by rapid
washes with ice-cold PBS and the non-internalized ligands were removed by a wash with ice-
cold 0.1 M glycine-HCl buffer (pH 3.0) at 4˚C for 5 minutes. The fluorescence intensity was
measured using PHERAStar FS microplate reader.
Measurement of the inhibition of KP phosphinic peptides on the activity of
MMPs
The activity of MMPs was measured using MMP colorimetric drug discovery kits in a 96-well
microtiter plate according to the manufacturer’s instructions. Different concentrations of the
phosphinic peptides or 1.3 μM N-Isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic
acid (NNGH), a positive inhibitor for MMPs, were, respectively, added to the assay mixtures
containing MMP enzyme (MMP-2 or MMP-9; 9 mU/μL). The plates were then incubated at
37˚C for 1 hour for enzyme reaction. After that, a final concentration of 100 μM colorimetric
substrate (Ac-PLG-[2-mercapto-4-methyl-pentanoyl]-LGOC2H5) was added to each well and
the plates were then read at 412 nm every minute for at least 20 minutes using PHERAStar FS
microplate reader. The full enzyme activity (control) was measured without addition of any
inhibitor or tested peptides. The reaction velocity (V) was determined by calculating the slope
of the fitted line to the linear portion of the data collected. The slope of blank (without addition
of MMP enzyme) was subtracted from all samples and the remaining activity of the enzyme
was calculated using following formula.
VðinhibitorÞ
% Enzyme activity ¼
à 100
VðcontrolÞ
Data analysis
The means SD are shown for values obtained for the numbers of independent experiments
indicated in the figure legends. GraphPad Prism software 6.0 (Graph Pad, San Diego, USA)
was used to analyse the data for statistical significance. The statistical significance was calcu-
lated by one-way or two-way ANOVA.
Results
Design and synthesis of KP phosphinic peptides
The KP phosphinic peptides were designed from the C-terminal 5 amino acids of KP-10 by
replacement of the amide bond between Gly51 and Leu52 with the phosphinate (-P(O)(OH)
CH2) bond which is resistant to hydrolysis of MMPs (Fig 2). The N-terminus of the KP phos-
phinic peptides was protected by the Cbz group which resembles the Phe50 side-chain of KP-
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Novel kisspeptin phosphinic peptides
Fig 2. Structures of KP-10 and its derived phosphinic peptides. The amino acid sequence of KP-10 is shown in A). The predicted structures of
phosphinic kisspeptin S isomer (PKPS) and phosphinic kisspeptin R isomer (PKPR), which are diastereoisomers, are shown in B) and C).
https://doi.org/10.1371/journal.pone.0195089.g002
10. The synthesis is described in section 2.2. The KP phosphinic peptides have S isomer
(PKPS) and R isomer (PKPR) which are diastereoisomers (Fig 2).
Effect of KP phosphinic peptides on kisspeptin receptor-elicited ERK1/2
phosphorylation
The agonistic activity of the phosphinic peptides on the kisspeptin receptor was evaluated by
measuring their stimulation of the dual phosphorylation of ERK1/2 in HEK293 cells tran-
siently expressing kisspeptin receptor. KP-10 at a final concentration of 100 nM was used as a
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Novel kisspeptin phosphinic peptides
positive control. Three different concentrations of each KP phosphonic peptide were used to
stimulate the cells for 3 minutes at which the maximum response was achieved for KP-10 (S1
Fig). Stimulation of HEK293 cells transiently expressing kisspeptin receptor with KP-10 (100
nM) led to a 1.60-fold increase (P < 0.005) in the phosphorylation of ERK1/2 (Fig 3), while
neither lower concentrations of PKPR (< 1 μM, Fig 3B) nor PKPS up to 10 μM (Fig 3A)
induced phosphorylation of ERK1/2. However, PKPR at a concentration of 10 μM increased
the level of phosphorylation of ERK1/2 to a 1.53 0.175-fold over basal (P < 0.01) (Fig 3B),
indicating that PKPR is biologically active.
Examination of the antagonistic activity of KP phosphinic peptides on
kisspeptin receptor
To examine whether the KP phosphinic peptides exhibit antagonistic activity on the kisspeptin
receptor, their ability to inhibit KP-10-induced phosphorylation of ERK1/2 was tested. MCF-7
cells transiently expressing kisspeptin receptor were used as model cells. The cells were first treated
with 10 μM of PKPS or PKPR respectively for 30 minutes. After that, KP-10 (100 nM, final con-
centration) was added for a further 10-minute stimulation which itself elicited a significant
(P < 0.001) phosphorylation of ERK1/2 (S1 Fig). However, PKPS (Fig 4; white bars) or PKPR
(Fig 4; grey bars) at 10 μM did not inhibit KP-10-induced phosphorylation of ERK1/2, while treat-
ment of the cells with PKPR alone, i.e., in the absence of KP-10, gave a 1.46 0.176-fold increase
(P < 0.05) in the phosphorylation of ERK1/2, similar to the result obtained above.
Evaluation of the binding of KP phosphinic peptides to kisspeptin receptor
A fluorophore-labelled KP-18 was designed and synthesized in order to determine the binding
of PKPS and PKPR to kisspeptin receptor. To minimize the effect of the conjugate of the
water-soluble fluorophore Sulfo-Cy5 on the function of the C-terminal 10 amino acids of KPs,
Sulfo-Cy5 was introduced to the N-terminal amine of KP-18, as the introduction of Cy5.5 to
the N-terminal amine of KP-10 led to inactivity of the Cy5.5-KP-10 in binding with kisspeptin
receptor [41]. A saturation binding assay was carried out in HEK293 cells transiently express-
ing kisspeptin receptor to detect the binding of Sulfo-Cy5-KP-18 to kisspeptin receptor. A sig-
nificant specific binding was detected at 50 nM or higher concentrations of Sulfo-Cy5-KP-18
(Fig 5).
Since the saturation of Sulfo-Cy5-KP-18 binding was not observed even though high con-
centrations of Sulfo-Cy5-KP-18 were used, the dissociation constant, Kd value of Sulfo-Cy5-
KP-18, could not be determined. The lower sensitivity of the Sulfo-Cy5-KP-18 binding assay
made it impossible to be used as a labelled ligand for competition binding assay to measure the
binding affinity of the KP phosphinic peptides. As an alternative approach, the binding of the
KP phosphinic peptides to kisspeptin receptor was examined by measuring their inhibition on
Sulfo-Cy5-KP-18-induced internalisation of the kisspeptin receptor. MCF-7 cells transiently
expressing with kisspeptin receptor were co-incubated with 50 nM Sulfo-Cy5-KP-18 and vehi-
cle, different concentrations of KP-10, PKPS or PKPR respectively. Addition of KP-10 at 5 μM
led to a maximum (22.3% 7.96) inhibition (P < 0.005) on Sulfo-Cy5-KP-18-induced interna-
lisation of the kisspeptin receptor (Fig 6; black bars). However, neither PKPS nor PKPR inhib-
ited the internalisation of Sulfo-Cy5-KP-18 at any concentrations tested (Fig 6).
Inhibition of KP phosphinic peptides on MMPs
The effect of the KP phosphinic peptides on MMP-2 and MMP-9 enzyme activity was detected
using MMP colorimetric drug discovery kits. NNGH, a potent inhibitor of MMPs, was pro-
vided by the kits and used as a positive control. NNGH at 1.3 μM almost completely inhibited
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Novel kisspeptin phosphinic peptides
Fig 3. Stimulation of the phosphorylation of ERK1/2 by KP-10, PKPS (A) or PKPR (B). HEK293 cells transiently
expressing kisspeptin receptor were starved for 20 hours. The cells were then treated with vehicle (vehicle1, 0.02% (v/v)
propylene glycol or vehicle2, 0.02% (v/v) DMSO), 100 nM KP-10 (□) or increasing concentrations of PKPS or PKPR
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Novel kisspeptin phosphinic peptides
(■) for 3 minutes. Western blot analyses were done using monoclonal anti-phospho-ERK1/2 and anti-β-actin
antibodies. Representative western blot and densitometric quantitation are shown. The data represent the mean fold-
changes over the basal of the vehicle-treated control SD of three independent experiments. ÃÃ, P < 0.01; ÃÃÃ
P < 0.005, compared with the basal.
,
https://doi.org/10.1371/journal.pone.0195089.g003
the activities of both MMP-2 and MMP-9 (Fig 7). PKPR at a final concentration of 10 μM
inhibited the activity of MMP-2 by 35.98% 10.22 (P <0.001, Fig 7A). However, PKPS and a
lower concentration of PKPR did not inhibit the activity of MMP-2. Moreover, none of the KP
phosphine peptides inhibited the activity of MMP-9 (Fig 7B).
Fig 4. Effect of PKPS and PKPR on KP-10-induced phosphorylation of ERK1/2. MCF-7 cells transiently expressing
kisspeptin receptor were serum-starved for 20 hours. The cells were then treated with vehicle (0.02% (v/v) DMSO; black bars),
10 μM PKPS (white bars) or PKPR (grey bars) for 30 minutes. After that, vehicle (0.02% (v/v) propylene glycol, labelled–KP-
10) or 100 nM KP-10 (labelled + KP-10) was added for a further 10-minute stimulation. Western blot analyses were carried
out using monoclonal anti-phospho-ERK1/2 and anti-β-actin antibodies. Representative western blot (upper) and
densitometric quantitation (bottom) are shown. The data represent the mean fold-changes over the basal treated with
vehicle SD of three independent experiments. Ã, P < 0.05, compared with the basal treated with vehicle.
https://doi.org/10.1371/journal.pone.0195089.g004
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Fig 5. Binding of Sulfo-Cy5-KP-18 to kisspeptin receptor. HEK293 cells were transiently transfected with kisspeptin
receptor and cultured for 48 hours. The cells were then incubated with increasing concentrations of Sulfo-Cy5-KP-18
at 4˚C for 4 hours. Total binding of Sulfo-Cy5-KP-18 was measured by fluorescence intensity after washing off free
ligand. Non-specific binding was determined in the presence of 10 μM KP-10. The mean fold-changes over the basal of
the fluorescence intensity SD from three independent experiments were calculated and plotted. Specific binding was
calculated by subtracting non-specific binding from the total. Total binding (ꢀ; dash curve), non-specific binding (□;
dot curve) and specific binding (▲; solid curve).
https://doi.org/10.1371/journal.pone.0195089.g005
Discussion
KPs and their cognate receptor play important roles in the regulation of cancer metastasis and
reproduction and, therefore, are important targets for therapeutic intervention. However, nat-
ural KPs are cleaved rapidly by various serum-containing proteases especially MMPs, and,
therefore, are metabolically unstable [22–24]. In order to explore the potential development of
KP analogues with improved metabolic stability, especially with inhibition to MMPs, two KP
phosphinic peptides containing a replacement of the peptide bond between Gly51-Leu52 with a
phosphinic acid moiety were synthesized based on the primary sequence of KPs (Figs 1 and 2).
The agonistic activity of the KP phosphinic peptides on the kisspeptin receptor was assessed
by measurement of their stimulation on the phosphorylation of ERK1/2. The assay was carried
out on HEK293 cells transiently expressing kisspeptin receptor, given that HEK293 cells have
high transfection efficiency for cDNA and low basal activity of ERK1/2 phosphorylation. In
addition, HEK293 cells were reported to express kisspeptin receptor naturally which regulates
embryonic kidney morphogenesis and glomerular development [39], although no kisspeptin
receptor binding and activity were detected in our cells. The results showed that PKPR acti-
vated kisspeptin receptor at 10 μM (Fig 3B), indicating that it possesses the capability to bind
and activate kisspeptin receptor. By contrast, its enantiomer (PKPS) did not stimulate ERK1/2
phosphorylation (Fig 3A), indicating that the pseudo-Leu residue is stereochemically essential
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Novel kisspeptin phosphinic peptides
Fig 6. Effect of KP phosphinic peptides on Sulfo-Cy5-KP-18-induced internalisation of kisspeptin receptor. MCF-
7 cells transiently expressing kisspeptin receptor were co-incubated with 50 nM of Sulfo-Cy5-KP-18 and vehicle
(control), different concentrations of KP-10 (black bars), PKPS (white bars) or PKPR (grey bars) respectively for 30
minutes. The internalisation of Sulfo-Cy5-KP-18 was normalised as percentage of that (control) measured in the
absence of KP-10 (vehicle only). The data represent the mean percentage-changes of four independent experiments. Ã,
P < 0.05; ÃÃ, P < 0.01; ÃÃÃ, P < 0.005, compared with the control.
https://doi.org/10.1371/journal.pone.0195089.g006
for kisspeptin receptor binding and activation. This is consistent with the observation that
individual substitution of the last five amino acids at C-terminus of KP-10 with the corre-
sponding D-enantiomer leads to the complete loss of the agonistic activities of the peptides
(Niida et al., 2006).
Since the introduction of a dipeptide isostere structure to biologically functional peptides
may alter their original bioactivity [32], the kisspeptin receptor-antagonistic activity of PKPS
and PKPR was also examined by detecting their inhibition of KP-10-induced phosphorylation
of ERK1/2. The results showed that neither PKPS nor PKPR inhibited the KP-10-induced
phosphorylation of ERK1/2 (Fig 4). Further examination of the effect of KP phosphinic pep-
tides on the fluorescence-labelled agonist-induced internalization of kisspeptin receptor was
carried out using a similar method described by Tomita and colleagues [42]. Introduction of
Cy5.5 to the N-terminal amine of KP-10 leads to the fluorescence-labelled ligand being func-
tionally inactive [41]. Here, finding that the linkage of a water-soluble Sulfo-Cy5 to the N-ter-
minal amine of KP-18 retains the peptide binding capability with kisspeptin receptor (Fig 5).
However, a significant receptor binding of the fluorescence-labelled ligand could only be
detected at 50 nM or higher concentrations of Sulfo-Cy5-KP-18. A similar result was reported
for tetramethylrhodamine and rhodamine green-labelled KP-14 or KP-52 [43]. The develop-
ment of the functional Sulfo-Cy5-KP-18 allowed us to determine potential binding of the KP
phosphinic peptides via their inhibition to Sulfo-Cy5-KP-18-induced receptor internalisation.
Co-incubation of KP-10 at concentrations ꢁ 50 nM with Sulfo-Cy5-KP-18 significantly inhib-
ited the internalisation of Sulfo-Cy5-KP-18 with a maximum inhibition of about 22%. How-
ever, no inhibition was observed for KP phosphinic peptides, even at a high concentration of
5 μM, indicating that PKPS and PKPR may not bind to kisspeptin receptor or bind to the
receptor with a relatively low affinity. In combination with the functional results of KP
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Fig 7. Effect of KP phosphinic peptides on MMP-2 (A) or MMP-9 (B) enzyme activity. The inhibition assays on
MMPs were conducted using MMP colorimetric drug discovery kits in 96-well microtiter plates as per the
manufacturer’s instructions. A final concentration of 10 μM PKPS, 10 μM PKPR, 1 μM PKPR, or 1.3 μM NNGH
(positive control) as indicated were respectively added to the assay mixture containing MMP-2 or MMP-9 enzyme.
The maximum activity (control) was measured in the absence of inhibitors. The data represent the mean percentage-
changes over the control SD from three independent experiments. ÃÃÃÃ, P < 0.001 compared with the control.
https://doi.org/10.1371/journal.pone.0195089.g007
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Novel kisspeptin phosphinic peptides
phosphinic peptide-induced ERK1/2 phosphorylation, we propose that PKPS does not bind to
kisspeptin receptor, while PKPR binds with kisspeptin receptor at a relatively low affinity, as
stimulation of HEK293 cells transiently expressing kisspeptin receptor by 10 μM PKPR leads
to phosphorylation of ERK1/2.
In order to check if introduction of a non-hydrolysable ~[P(O)(OH)CH2]~ group at the
cleavage site of KPs leads to inhibition of the peptides to zinc metalloproteases, their ability to
inhibit the activity of MMP-2 and MMP-9 was further evaluated. A significant inhibition of
PKPR at 10 μM on MMP-2 enzyme activity was observed (Fig 7A) while PKPS had no effect
on MMP-2 activity, indicating the specificity of PKPR to MMP-2. By contrast, both PKPS and
PKPR did not inhibit the activity of MMP-9 (Fig 7B), indicating that PKPR is a selective inhib-
itor toward MMP-2. In addition, the result also indicates that PKPR may form a stable com-
plex with MMP-2 and, thus, may be resistant to MMP-2-mediated hydrolysis. Currently, most
inhibitors of zinc-metalloproteases, such as MMPs, are designed based on the usage of a strong
zinc-chelating group, such as carboxylate and hydroxamic groups [34]. Although these pep-
tides provide high potency, they exhibit low selectivity, indicating that their inhibition may
mainly rely on the interaction between the zinc-chelating group and the zinc ion. In contrast,
the phosphoryl group of phosphinic peptides is a weak zinc-chelating group and, as a conse-
quence, only the peptides that allow optimal and sufficient intermolecular interactions with
the enzyme active sites will provide inhibition. Moreover, phosphinic peptides mimic the
structure of the substrate in the transition state [33, 34]. Thus, phosphinic peptides may pro-
vide an opportunity to develop potent and selective inhibitors for zinc-metalloproteases which
are involved in promoting cancer metastasis. PKPR presented here may, hence, serve as a lead
compound for further modification to develop a potent and selective inhibitor of MMP-2.
In conclusion, our studies propose a new option to develop novel KP phosphinic analogues,
such as PKPR, which activate kisspeptin receptor and inhibit the activity of MMPs, by replac-
ing the peptide bond between Gly51 and Leu52 with a phosphinic acid moiety. PKPR may serve
as a lead for further development of a metabolically stable peptide analogue for hormone
replacement therapies and/or of a potent anti-metastasis agent targeting both kisspeptin recep-
tor and MMPs.
Supporting information
S1 Fig. Time-course of the phosphorylation of ERK1/2 induced by KP-10 in HEK293 (A)
and MCF-7 (B) cells. Cells transiently expressing kisspeptin receptor were starved for 20
hours. The cells were then treated with vehicle (0.02% (v/v) propylene glycol) or 100 nM KP-
10 for different times as indicated. Western blot analyses were carried out using monoclonal
anti-phospho-ERK1/2 and anti-β-actin antibodies. Representative western blot and densito-
metric analysis are shown. The data represent the mean fold-change over basal (vehicle treated
control) SD of three independent experiments. ÃÃ, P < 0.01; ÃÃÃÃ, P < 0.001, compared with
the basal.
(TIF)
S2 Fig. Entire blots of the representative western blots plotted in Fig 3. HEK293 cells
expressing kisspeptin receptor were stimulated by PKPS (A) or PKPR (B) and the phosphory-
lation of ERK1/2 was measured by western blotting.
(TIF)
S3 Fig. Entire blots of the representative western blots plotted in Fig 4. Effect of PKPS and
PKPR on KP-10-induced phosphorylation of ERK1/2.
(TIF)
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Novel kisspeptin phosphinic peptides
S4 Fig. Entire blots of the representative western blots plotted in S1 Fig. Phosphorylation of
ERK1/2 stimulated by KP-10 in HEK293 (A) and MCF-7 (B) cells.
(TIF)
Author Contributions
Conceptualization: Magdalini Matziari, Zhi-Liang Lu.
Funding acquisition: Zhi-Liang Lu.
Investigation: Xiaoyang Zhang, Yixin Xie.
Methodology: Xiaoyang Zhang.
Project administration: Zhi-Liang Lu.
Supervision: David Fernig, Rong Rong, Jia Meng, Zhi-Liang Lu.
Writing – original draft: Xiaoyang Zhang, Magdalini Matziari.
Writing – review & editing: David Fernig, Zhi-Liang Lu.
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