Development of novel G-protein-coupled receptor 54 agonists with resistance to degradation by matrix metalloproteinase

Article abstract of DOI:10.1021/jm800930w

Kisspeptin-GPR54 signaling is involved in the suppression of cancer metastasis and regulation of hormonal secretion. Recently, matrix metalloproteinase mediated deactivation of kisspeptins through hydrolysis of the Gly-Leu peptide bond has been reported.

Full text of DOI:10.1021/jm800930w

J. Med. Chem. 2008, 51, 7645–7649
7645
Development of Novel G-Protein-Coupled Receptor 54 Agonists with Resistance to Degradation
by Matrix Metalloproteinase
Kenji Tomita,† Shinya Oishi,*,† Hiroaki Ohno,† Stephen C. Peiper,‡ and Nobutaka Fujii*,†
Graduate School of Pharmaceutical Sciences, Kyoto UniVersity, Sakyo-ku, Kyoto 606-8501, Japan, and Anatomy and Cell Biology, Thomas
Jefferson UniVersity, Philadelphia, PennsylVania 19017
ReceiVed July 24, 2008
Kisspeptin-GPR54 signaling is involved in the suppression of cancer metastasis and regulation of hormonal
secretion. Recently, matrix metalloproteinase mediated deactivation of kisspeptins through hydrolysis of
the Gly-Leu peptide bond has been reported. In the present report, GPR54 agonistic peptides having several
nonhydrolyzable Gly-Leu dipeptide isosteres were designed and synthesized. (E)-Alkene- and hydroxyeth-
ylene-type isostere-containing analogues maintained the original activity with higher stability in murine
serum and resistance to MMP-9-mediated cleavage.
Table 1. Sequences of Kisspeptins and Pentapeptide GPR54 Agonist
Introduction
GPR54a is a Gq-protein-coupled receptor,1,2 which was
isolated as an orphan receptor sharing significant homology with
galanin receptors.3 GPR54 is paired with endogenous kisspeptins
(kisspeptin-54 has also been designated metastin),1,2,4 although
galanin is not recognized by this receptor. Kisspeptins, which
are generated by proteolytic cleavage of a precursor, belong to
the RF-amide peptide family and share the amino acid sequence
of the C-terminal decapeptide, which is highly conserved
between human and mouse.5,6 High levels of kisspeptin expres-
sion have been observed in placenta, while GPR54 expression
is high in the pancreas and placenta.4
Recent studies demonstrated that the kisspeptin-GPR54
system is involved in placentation and the onset of
puberty.7Plasma concentration of kisspeptin is significantly
increased in pregnancy, which returns to nearly the baseline
level after delivery.8 GPR54 stimulation in the hypothalamus
Peptide
Sequence
Kisspeptin-54
H-Gly-Thr-Ser · · ·Tyr-Asn-Trp-Asn-Ser-Phe-
Gly-Leu-Arg-Phe-NH2
H-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-
Phe-NH2
Kisspeptin-10
1
4-fluorobenzoyl-Phe-Gly-Leu-Arg-Trp-NH2
mediated by suppressed expression of matrix metalloproteinases
(MMPs), which contribute to cell invasion.18,19 Thus, kisspeptins
may be useful as inhibitory peptides against cancer metastasis.
We recently identified a pentapeptide 1 as a potent GPR54
agonist by structure-activity relationship studies on kiss-
peptin-10,20,21 which is the C-terminal decapeptide fragment
of kisspeptins (Table 1). Kisspeptin-10 exerts the most potent
receptor binding affinity among kisspeptins reported so far.4 It
has been reported that kisspeptins are inactivated by the cleavage
of their Gly-Leu peptide bond in the C-terminal region by
MMPs.22 Since kisspeptins and peptide 1 share a common
sequence (Phe-Gly-Leu-Arg) of the MMP-mediated cleavage
site, peptide 1 would be also deactivated by MMP-mediated
digestion. We envisioned that substitution of the Gly-Leu
dipeptide moiety in peptide 1 with an appropriate dipeptide
isostere would afford resistance to enzymatic degradation with
maintenance of GPR54 agonistic activity, as well as the potential
for additional inhibitory activity against MMPs. This could
provide novel antimetastasis agents targeting two molecules,
GPR54 and MMPs. Since it is possible that the introduction of
a dipeptide isostere structure to bioactive peptides may lead to
loss of the original bioactivity,23 several Gly-Leu dipeptide
isosteres were analyzed for facile identification of appropriate
amide equivalents. For efficient synthesis of a series of Gly-
Leu dipeptide isosteres, (E)-alkene dipeptide isostere (EADI)
was converted into several isosteres by modification of the olefin
moiety. In this manuscript, preparation and biological evaluation
of peptidomimetics containing a Gly-Leu dipeptide isostere for
GPR54 agonists are described.
and
pi-
tuitary prompts the release of gonadotropin-releasing hor-
mone (GnRH),9 which is required for the onset of puberty.10
Furthermore, it is known that the mutation in GPR54 results
in idiopathic hypogonadotrophic hypogonadism (IHH).11-13
These reports indicate the therapeutic potential of GPR54
ligands for disorders of endocrine secretion.
Alternatively, the kisspeptin-GPR54 system was originally
discovered in oncology research because a precursor protein of
kisspeptins is encorded by the metastasis suppresser gene KiSS-
1.14 The motility of several cancer cell lines expressing GPR54
was found to be attenuated in the presence of kisspeptins.4,15,16
Although the mechanism by which kisspeptin-GPR54 signaling
blocked cancer metastasis, a recent report indicated that GPR54
activation suppressed metastasis-related CXCL12 (SDF-1)-
CXCR4 signaling.17 Moreover, antimetastasis activity is also
* To whom correspondence should be addressed. Phone: +81-75-753-
4551. Fax: +81-75-753-4570. E-mail: for S.O., soishi@pharm.kyoto-u.ac.jp;
for N.F., nfujii@pharm.kyoto-u.ac.jp.
†
Kyoto University.
Thomas Jefferson University.
Results and Discussion
‡
a Abbreviations: GPR54, G-protein-coupled receptor 54; MMP, matrix
metalloproteinase; Fmoc, 9-fluorenylmethoxycarbonyl; SPPS, solid-phase
peptide synthesis; GnRH, gonadotropin-releasing hormone; IHH, idiopathic
hypogonadotrophic hypogonadism; EADI, (E)-alkene dipeptide isostere;
Mts, mesitylenesulfonyl; DIC, N,N′-diisopropylcarbodiimide; HOAt, N-hy-
droxy-7-azabenzotriazole.
Chemistry. Synthesis of Gly-Leu type EADI 5 was initiated
from D-serine 2 as chiral educt (Scheme 1), which was converted
into a chiral aziridine 3 in three steps.24 After DIBAL-H
treatment of 3, (Z)-selective Horner-Wadsworth-Emmons
reaction25 of the resulting aldehyde gave (Z)-enoate 4 with the
10.1021/jm800930w CCC: $40.75
2008 American Chemical Society
Published on Web 11/14/2008

7646 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Scheme 1. Synthesis of Hydrocarbon-Type Dipeptide Isosteresa
Brief Articles
Table 2. Bioactivities and Stability of Peptide Analogues
a Reagents and conditions: (a) DIBAL-H, -78 °C, 30 min; (b) (o-
MePhO)2P(O)CH2CO2t-Bu, NaI, DBU, -78 °C, 1 h, then -40 °C, 11 h;
(c) i-BuCu(CN)MgCl·2LiCl, -78 °C, 30 min; (d) Pd/C, H2, room temp,
14 h; (e) DBU, room temp, 20 h; (f) 1 M TMSBr-thioanisole/TFA, room
temp, 1 d; (g) Fmoc-OSu, Et3N, room temp, 3 h.
Scheme 2. Synthesis of Allyl Alcohol-Type Dipeptide Isosteresa
a IC50 values indicate the concentration needed for 50% inhibition of
b
receptor binding of [125I]kisspeptin-15. QIC values are calculated as QIC
) IC50(compound)/IC50(kisspeptin-10). c EC50 values mean the concentration
needed for 50% of the full agonistic activity induced by 1 µM kisspeptin-10.
d Half-life in murine serum. e Kisspeptin-10 was completely digested within
1 h.
Scheme 3. Synthesis of Peptidomimetics 24a,b and 25a,b
Containing Hydroxyethylene-Type Isosterea
a Reagents and conditions: (a) m-CPBA, room temp, 20 h; (b) NaH, room
temp, 2 h; (c) K2CO3, CH3OH, 50 °C, 5.5 h; (d) 1 M TMSBr-thioanisol/
TFA, room temp, 1 d; (e) Fmoc-OSu, Et3N, room temp, 3 h; (f) TESCl,
imidazole, 4 °C, 4 h.
concomitant formation of small amount of (E)-isomer (E/Z )
1:4). Employment of 4 in the reaction with organocuprate
afforded desired Gly-Leu type EADI 5 as a single product in
90% yield.26 The absolute configuration at the R-position in 5
can be determined by measurement of circular dichroism.27
A
negative n f π* Cotton effect (∆ε ) -2.77 at 225.4 nm in
isooctane) indicated the (R)-configuration (L-configuration) at
the R-isobutyl group of 5. Reduction or isomerization of double
bond in 5 gave other dipeptide isosteres 6 and 7. The
simultaneous removal of N-mesitylenesulfonyl (Mts) and tertiary
butyl groups of dipeptide isosteres 5-7 with 1 M TMSBr-
thioanisole/TFA followed by protection of the resulting amino
groups afforded the Fmoc-protected dipeptide isosteres 8-10,
respectively.
Next, dipeptide isosteres containing a γ-hydroxy group were
prepared from EADI 5 (Scheme 2). Epoxidation of 5 with
m-CPBA gave a mixture of the diastereomers of desired
epoxides 11 and 12 (11/12 ) 3:1). After separation of these
a Reagents and conditions: (a) H2, Pd(OAc)2, room temp, 3 h.
diastereomers by column chromatography, each epoxide was
treated with base to afford allyl alcohols 13 and 14.28-30
Stereochemistry of the γ-position of 13 and 14 was determined
by transformation into the ꢀ-aziridinyl-R,ꢀ-enoates (see Sup-
porting Information). Deprotection of 13 and 14 followed by
sequential protections of the resulting amino and hydroxy groups
gave the Fmoc-protected isosteres 17 and 18.

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7647
Figure 1. Stability evaluation of GPR54 agonists 1, 19, and 25b by treatment with (a) MMP-9 and (b) murine serum: 1 (b), 19 (0), 25b (]),
kisspeptin-10 (2). Kisspeptin-10 was completely digested in murine serum within 1 h.
Five Fmoc-protected Gly-Leu dipeptide isosteres 8-10, 17,
and 18 in hand were applied to Fmoc-based solid-phase peptide
synthesis (SPPS) for the mimetics of GPR54 agonist 1. All
isosteres were smoothly introduced into the peptide on Rink-
amide resin with N,N′-diisopropylcarbodiimide (DIC) and
N-hydroxy-7-azabenzotriazole (HOAt). After completion of the
peptide elongation, the desired peptides 19-23 were yielded
(Table 2) as major products by release from the solid support
followed by HPLC purification. Further hydrogenation of the
olefin in 22 and 23 using H2/Pd(OAc)2 provided hydroxyeth-
ylene-type isostere-containing peptides 24a,b and 25a,b as
mixtures of diastereomers,30,31 which were separated by HPLC
(Scheme 3). Configuration of the R-carbon of 24a,b and 25a,b
bearing an isobutyl group was determined using reference
peptides, which were prepared through a separate approach using
an Evans chiral auxiliary (see Supporting Information).
Biological Evaluation and Structure-Activity Relation-
ship for GPR54 Ligands. The bioactivity of peptides 19-25
is summarized in Table 2. Initially, we evaluated GPR54 binding
affinity of all peptide analogues by competitive binding assay
using [125I]kisspeptin-15.4 We also assessed GPR54 agonistic
activity (EC50) of the compounds, which exerted high binding
affinity, by FLIPR assay.32 Since biological values of the
reference kisspeptin-10 varied among assay plates, relative
bioactivities were calculated [QIC ) IC50(compound)/IC50(kiss-
peptin-10)].
MMP-9 was significantly lower than the rapid digestion of
kisspeptin-10. MMP-2-mediated digestion gradually cleaved the
Gly-Leu peptide bond in kisspeptin-10 as reported,22 while
parent peptide 1 was resistant to the treatment with MMP-2
(see Supporting Information). Isostere-containing peptides were
quite stable in the presence of both enzymes (Figure 1a and
Supporting Information). These results indicate that introduction
of Gly-Leu isosteres and downsizing from kisspeptin-10 were
beneficial to the resistance against MMPs-mediated digestion.
Furthermore, the stability of peptide analogues in murine
serum was investigated. Kisspeptin-10 was completely digested
within 1 h. A small amount of residual peptide 1 was observed
after treatment for 12 h. On the other hand, half-lives (t1/2) of
isostere-containing peptides were significantly longer than that
of the parent peptide 1 by at least 4-fold (Table 2, Figure 1b,
and Supporting Information), indicating that introduction of the
Gly-Leu dipeptide isostere successfully contributed to the im-
proved stability of GPR54 agonist peptides. However, slow
degradation of isostere-containing peptides in murine serum was
still observed, implying the possible presence of alternative
cleavage sites within GPR54 agonists by some enzymes.
Next, their inhibitory activities for MMP-2 and -9 were
evaluated. None of the peptides exhibited significant inhibition
of either enzyme at 10 µM. These findings suggest that a series
of C-terminal short peptides and peptidomimetics of kisspeptins
are not recognized by the catalytic domain of MMPs.
Among a series of peptidomimetics 19-25, EADI-containing
analogue 19 showed the highest binding affinity [QIC(19) ) 1.0],
which is slightly more potent than the parent, peptide 1 [QIC(1)
) 7.3]. The constrained structure resulting from the double bond
within EADI contributes to the high binding affinity, which is
in contrast to the lower binding affinity of a flexible analogue
20 [QIC(20) ) 10]. On the other hand, R,ꢀ-unsaturated analogues
(21-23) are not potent ligands for GPR54, which may be caused
by loss of the chirality at the isostere R-position. This is
consistent with the lower binding affinity of the D-Leu-
containing analogue 26 [QIC(26) ) 240].20,32 The binding
affinities of the analogues containing a hydroxyethylene-type
isostere was dependent on the stereochemistry at the R- and
γ-positions of the isostere. A higher binding affinity was also
observed in 25b [QIC(25b) ) 2.5] with an (2R,4S)-configuration.
Similarly, peptide 24b, which contains an L-Leu isosteric unit,
was more potent than the D-Leu-containing congener 24a. These
findings suggest that the L-configuration of Leu is needed for
the bioactivity. Moreover, the ability of the peptides showing
high binding affinities to activate GPR54 was also evaluated.
All peptides functioned as full agonists, and the bioactivity
showed a positive correlation with the binding affinity.33
Stability of Isostere-Containing GPR54 Ligands in MMPs
and Serum and the Inhibitory Activity against MMPs. The
stability of GPR54 agonists against digestion by MMPs was
evaluated. Cleavage of the Gly-Leu peptide bond in 1 by
Conclusions
In this study, several Gly-Leu dipeptide isosteres were
prepared using EADI 5 as a common key intermediate. The
resulting isosteres were utilized for improvement of the stability
of a GPR54 agonist, peptide 1, against inactivation by peptidases
such as MMP-9. Two isostere-containing peptides 19 and 25b
showed resistance to degradation by MMP-2 and -9, with
maintenance of the bioactivity for GPR54. Furthermore, these
peptides are more stable in murine serum than the parent peptide
1. Information from this research is useful for development of
novel GPR54 ligands having in vivo stability.
Experimental Section
tert-Butyl (2R,3E)-2-Isobutyl-5-[N-(2,4,6-trimethylphenylsul-
fonyl)amino]pent-3-enoate (5). To a stirred solution of CuCN (108
mg, 1.20 mmol) and LiCl (102 mg, 2.40 mmol) in dry THF (2.4
mL) at -78 °C was added by syringe i-BuMgCl (2.0 M THF
solution, 600 µL, 1.20 mmol). The mixture was allowed to warm
to 4 °C and was stirred at this temperature for 30 min. A solution
of ester 4 (105 mg, 0.300 mmol) in dry THF (3 mL) was added
dropwise to the above reagent at -78 °C with stirring, and the
stirring was continued for 30 min followed by quenching at -78
°C with 6 mL of a mixture of saturated NH4Cl solution and 28%
NH4OH solution (1:1). After concentration of the mixture, the
residue was extracted with Et2O, and the extract was washed with
brine and dried over MgSO4. The solvent was removed under

7648 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Brief Articles
reduced pressure, and the residue was purified by flash chroma-
tography over silica gel with n-hexane-EtOAc (5:1) to give the ꢀ,γ-
enoate 5 (110 mg, 90% yield) as a colorless solid: mp 51-53 °C;
[R]2D3 -22.0 (c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.84
(d, J ) 6.3 Hz, 3H), 0.87 (d, J ) 6.3 Hz, 3H), 1.17-1.26 (m, 1H),
1.42 (s, 9H), 1.45-1.54 (m, 2H), 2.30 (s, 3H), 2.63 (s, 6H), 2.88
(ddd, J ) 8.5, 7.3, 7.3 Hz, 1H), 3.51 (dd, J ) 6.1, 6.1 Hz, 2H),
4.43 (t, J ) 6.1 Hz, 1H), 5.41 (dt, J ) 15.4, 6.1 Hz, 1H), 5.55 (dd,
J ) 15.4, 8.5 Hz, 1H), 6.96 (s, 2H); 13C NMR (100 MHz, CDCl3)
δ 20.9, 22.3, 22.4, 23.0, 25.6, 28.0, 41.4, 44.5, 47.7, 80.6, 126.7,
132.0, 132.7, 133.6, 139.0, 142.2, 173.3. Anal. Calcd for
C22H35NO4S: C, 64.51; H, 8.61; N, 3.42. Found: C, 64.39; H, 8.33;
N, 3.48.
Measurement of [Ca2+]i Using FLIPR Technology.32 GPR54/
CHO cells (3.0 × 104 cells per 200 µL/well) were inoculated in
10% dFBS/DMEM onto a 96-well plate for FLIPR analysis (Black
Plate Clear Bottom, Coster, Inc.), followed by incubation at 37 °C
overnight in 5% CO2. After the medium was removed, 100 µL of
the pigment mixture was dispensed into each well of the plate,
followed by incubation at 37 °C for an hour in 5% CO2. Then 1
mM peptide in DMSO was diluted with HANKS/HBSS containing
2.5 mM probenecid, 0.2% BSA, and 0.1% CHAPS. The dilution
was transferred to a 96-well plate for FLIPR analysis (V-Bottom
plate, Coster, Inc.; hereafter referred to as a sample plate). After
completion of the pigment loading onto the cell plate, the cell plate
was washed 4 times with wash buffer (2.5 mM probenecid in
HANKS/HBSS) using a plate washer. After the washing, 100 µL
of wash buffer was left. The cell plate and the sample plate were
set in FLIPR (Molecular Devices, Inc.), and 0.05 mL of a sample
from the sample plate was automatically transferred to the cell plate.
(2R,3E)-5-[N-(9-Fluorenylmethoxycarbonyl)amino]-2-isobu-
tylpent-3-enoic Acid (8). The ester 5 (819 mg, 2.00 mmol) was
dissolved in 1 M TMSBr-thioanisole/TFA (40 mL) at room
temperature, and the mixture was stirred for 1 day at this
temperature. Concentration under reduced pressure gave an oily
residue, which was poured into ice-cold dry diethyl ether. The
resulting pellet was collected by centrifugation and washed with
ice-cold dry diethyl ether, which was dissolved in water (10 mL).
Et3N (1.65 mL, 12.0 mmol) and Fmoc-OSu (670 mg, 2.00 mmol)
in CH3CN (10 mL) were successively added to the above solution
at 4 °C. After being stirred for 3 h at room temperature, the reaction
was quenched with 1 N HCl at 4 °C. After concentration under
reduced pressure, the resulting residue was extracted with EtOAc.
The extract was washed with 1 N HCl and brine and dried over
MgSO4. Concentration under reduced pressure was followed by
flash chromatography over silica gel with n-hexane/EtOAc (2:1)
containing 1% acetic acid. The eluent was wash with brine and
dried over MgSO4 followed by concentration to give the Fmoc-
protected δ-amino acid 8 (400 mg, 51% yield in two steps) as a
colorless solid: mp 146-148 °C; [R]2D4 -30.8 (c 1.01, CHCl3); 1H
NMR (400 MHz, CDCl3) δ 0.88 (d, J ) 6.3 Hz, 3H), 0.91 (d, J )
6.3 Hz, 3H), 1.35-1.47 (m, 1H), 1.52-1.70 (m, 2H), 3.04-3.17
(m, 1H), 3.67-3.88 (m, 2H), 4.21 (t, J ) 6.6 Hz, 1H), 4.41 (d, J
) 6.6 Hz, 2H), 4.79-4.91 (m, 1H), 5.48-5.68 (m, 2H), 7.30 (dd,
J ) 7.6, 7.3 Hz, 2H), 7.39 (dd, J ) 7.3, 7.3 Hz, 2H), 7.58 (d, J )
7.3 Hz, 2H), 7.75 (d, J ) 7.6 Hz, 2H); 13C NMR (100 MHz, CDCl3)
δ 22.1, 22.5, 25.5, 41.1, 42.6, 46.7, 47.2, 66.7, 120.0, 125.0, 127.0,
127.7, 129.2, 130.0, 141.3, 143.9, 156.2, 179.7. HRMS (FAB), m/z
calcd for C24H28NO4 (MH+) 394.2013, found 394.2014.
Peptide 19. Isostere 8 (120 mg, 0.3 mmol) was employed in
Fmoc-based SPPS (see Supporting Information). The peptide 19
was yielded as a TFA salt (33 mg, 36% yield from Rink amide
resin): [R]2D3 -17.5 (c 0.22, CH3OH); HRMS (FAB), m/z calcd for
C42H53FN9O5 (M + H+) 782.4148, found 782.4163.
Peptide 23. Isostere 18 (160 mg, 0.3 mmol) was employed in
Fmoc-based SPPS. The peptide 23 was yielded as a TFA salt (48
mg, 53% yield from Rink amide resin): [R]2D1 -22.3 (c 0.15,
CH3OH); HRMS (FAB), m/z calcd for C42H53FN9O6 (M + H+)
798.4097, found 798.4109.
Peptides 25a and 25b. To a solution of the peptide 23 (18 mg,
0.020 mmol) in CH3OH (2 mL) was added Pd(OAc)2 (4.5 mg, 0.020
mmol), and the mixture was stirred for 3 h under H2 at room
temperature. The mixture was filtered through a pad of Celite, and
the filtrate was concentrated under reduced pressure. The crude
product was purified by preparative HPLC to afford the expected
peptides 25a (5.4 mg, 30% yield) and 25b (2.9 mg, 16% yield).
25a: [R]2D2 +3.6 (c 0.08, CH3OH); HRMS (FAB), m/z calcd for
C42H55N9O6F (M + H+) 800.4254, found 800.4271. 25b: [R]2D3
+10.7 (c 0.11, CH3OH); HRMS (FAB), m/z calcd for C42H55FN9O6
(M + H+) 800.4254, found 800.4250.
Measurement of Binding Affinity. Assasys were performed as
previously described.4 Membrane fraction was prepared using
homogenizing buffer (10 mM NaHCO3, 2 mM EGTA, 0.2 mM
MgCl2, protease inhibitors, pH 7.4) and stored in 50% glyc-
erol-50% homogenizing buffer at -20 °C. Kisspeptin-15 was
labeled with 125I-Na using lactoperoxidase and purified to a carrier-
free single peak.
Acknowledgment. We thank Takeda Pharmaceutical Co.
Ltd. for the evaluation of biological activity for GPR54 of a
series of compounds. This work is supported by a Grant-in-
Aid for Scientific Research and Molecular Imaging Research
Program from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan. K.T. is grateful for Research
Fellowships from the JSPS for Young Scientists.
Supporting Information Available: Experimental procedures,
characterization, and HPLC and bioassay data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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