Characterization of the receptor binding residues of kisspeptins by positional scanning using peptide photoaffinity probes

Article abstract of DOI:10.1016/j.bmcl.2013.02.098

Kisspeptins, endogenous peptide ligands for GPR54, play an important role in GnRH secretion. Since in vivo administration of kisspeptins induces increased plasma LH levels, GPR54 agonists hold promise as therapeutic agents for the treatment of hormonal se

Full text of DOI:10.1016/j.bmcl.2013.02.098

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2628–2631
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Bioorganic & Medicinal Chemistry Letters
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Characterization of the receptor binding residues of kisspeptins
by positional scanning using peptide photoaffinity probes
a
a
a
a
Ryosuke Misu a, Shinya Oishi a, , Shohei Setsuda , Taro Noguchi , Masato Kaneda , Hiroaki Ohno ,
⇑
Barry Evans b, Jean-Marc Navenot b, Stephen C. Peiper b, Nobutaka Fujii a,
⇑
a Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
b Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Kisspeptins, endogenous peptide ligands for GPR54, play an important role in GnRH secretion. Since
in vivo administration of kisspeptins induces increased plasma LH levels, GPR54 agonists hold promise
as therapeutic agents for the treatment of hormonal secretion diseases. To facilitate the design of novel
potent GPR54 ligands, residues in kisspeptins that involve in the interaction with GPR54 were investi-
gated by kisspeptin-based photoaffinity probes. Herein, we report the design and synthesis of novel kiss-
peptin-based photoaffinity probes, and the application to crosslinking experiments for GPR54-expressing
cells.
Received 1 February 2013
Accepted 22 February 2013
Available online 4 March 2013
Keywords:
GPCR
GPR54
Ó 2013 Elsevier Ltd. All rights reserved.
Kp-54
Kp-14
photoaffinity probe
Kisspeptins, proteolytic products of the propeptide encoded by
the Kiss1 gene, have been identified as an endogenous ligand of G
protein-coupled receptor 54 (GPR54).1–5 Full-length 54-residue
kisspeptin (Kp-54) and endogenous short peptides [kisspeptin-14
(Kp-14) and kisspeptin-13 (Kp-13)] share the C-terminal consen-
sus sequence of the RF-amide peptide family, -Arg-Phe-NH2.1 The
kisspeptin-GPR54 signaling complex plays a vital role in gonado-
tropin secretion via stimulation of gonadotropin-releasing hor-
mone (GnRH) release.6–16 The in vivo administration of
kisspeptins and derivatives with potent GPR54 agonistic activity
induces an increase in the plasma LH level.17,18 Thus, GPR54 ago-
nists are expected to be potential pharmaceutical agents for disor-
ders of hormone secretion.
In 2001, Ohtaki et al. reported that the C-terminal 10-residue
peptide [kisspeptin-10 (Kp-10)] was the minimal bioactive se-
quence of kisspeptins. Although Kp-10 has been reported to exert
antimetastatic effects via GPR54 activation,4,19 Kp-10 was found
to be rapidly degraded in serum.20 Recently, several biostable Kp-
10 analogs have been developed.20–23 For example, TAK-488 with
good peptidase resistance retains highly potent GPR54 agonistic
activity that is comparable to Kp-10.22 We have also identified a
pentapeptide-based GPR54 agonist, FTM080, through down-sizing
studies of Kp-10.24,25 Further structure-activity relationship stud-
ies of FTM080 using
a series of peptidomimetics identified
FTM145 with high biological stabilities.26
These previous studies demonstrated the requirement for the
C-terminal region of kisspeptins in direct receptor binding and
activation of GPR54. However, for the design of more potent
GPR54 ligands, elucidation of the contact residues in kisspeptins
and the recognition modes for GPR54 are required. A positional
scanning approach of kisspeptin-based photoaffinity probes was
used to identify residues that interact with GPR54. Reported herein
is the development and application of novel photoaffinity probes
derived from two endogenous kisspeptins, Kp-54 and Kp-14.
Initially, we designed Kp-54-based probes that contain a photo-
reactive group to form a covalent bond onto GPR54 and a biotin tag
to detect labeled GPR54.27–29 Since the C-terminal region of kiss-
peptins is required for binding to GPR54,4,24–26 the biotin group
was conjugated at the N-terminus of Kp-54. A panel of ligands
was created with the photoreactive functional group conjugated
Abbreviations: GPR54, G-protein coupled receptor 54; Kp-54, kisspeptin-54; Kp-
14, kisspeptin-14; Kp-13, kisspeptin-13; Kp-10, kisspeptin-10; GnRH, gonadotro-
pin-releasing hormone; NPFFRs, neuropeptide FF receptors; PEG, polyethylene
glycol; Fmoc-SPPS, Fmoc-based solid phase peptide synthesis; HRP, horseradish
peroxidase.
to the Cys thiol group at the N-terminal Ser5, Ser10, Gln15, Ser20
,
Arg25, Pro30, Leu35, or Lys40 locations with Cys replacements
(designated S5C, S10C, Q15C, S20C, R25C, P30C, L35C, or K40C,
respectively) (Fig. 1). Each residue in the C-terminal sequence,
Tyr45-Leu52, was also modified with the photoreactive group
(designated Y45C, N46C, W47C, N48C, S49C, F50C, G51C, and
⇑
Corresponding authors. Tel.: +81 75 753 4551; fax: +81 75 753 4570.
E-mail addresses: soishi@pharm.kyoto-u.ac.jp (S. Oishi), nfujii@pharm.kyoto-
u.ac.jp (N. Fujii).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.098

R. Misu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2628–2631
2629
with the findings from our previous alanine scanning experiments
of Kp-10, in which substitution of these residues resulted in signif-
icant reductions in the agonistic activity.25 The modification of
these residues with a photoaffinity functional group may interfere
with crucial interactions necessary for binding to GPR54. Taken to-
gether, we have identified six Kp-54-based photoaffinity probes
with modification of residues upstream of the C-terminal neces-
sary residues for GPR54 binding, suggesting that this region
(Pro30-Trp47) in Kp-54 may contribute to the secondary site(s)
for binding to GPR54.
We next used this approach for the development of photo-
affinity probes using Kp-14. On the basis of the structure-activity
relationships obtained by the Kp-54-based probe studies, we de-
signed three photoaffinity probes with an N-terminal biotin and
a photoreactive group at Tyr5, Asn6 or Trp7 in Kp-14 (Fig. 1). These
modified residues correspond to Tyr45, Asn46 and Trp47 in Kp-54,
which were successfully modified with the photoreactive group
in the functional Kp-54-based probes. However, contrary to our
expectation, no labeling of GPR54 was observed using these probes
(Fig. 3A). We assumed that streptavidin binding to the probes in
the Western blot analysis was inhibited because the biotin tag
was attached directly to the bioactive Kp-14 sequence without
any linker.30
To avoid the possible reduced accessibility of streptavidin, three
probes with a polyethylene glycol (PEG)27 linker between biotin
and the Kp-14 sequence (Y5C, N6C and W7C) were designed and
synthesized. Among these pegylated Kp-14-based probes, cross-
linking of W7C to GPR54 was observed by Western blot analysis
using HRP-conjugated streptavidin (Fig. 3A). The binding specific-
ity of these Kp-14-derived probes for GPR54 was also confirmed
Figure 1. Design of Kp-54- and Kp-14-based photoaffinity probes for GPR54 and
the structure of photoaffinity reagent 1.
L52C). All the probes retained the indispensable C-terminal
RF-amide substructure (-Arg53-Phe54-NH2).
Peptide chains of Kp-54 derivatives were constructed by stan-
dard Fmoc-based solid-phase peptide synthesis (Fmoc-SPPS) on
NovasynÒ TGR resin. The amino acid at the modification site for
the photoreactive functional group was substituted with a Cys res-
idue. The N-terminal biotin was attached by the standard coupling
protocol. After final deprotection and cleavage from the resin, the
photoreactive group-conjugated maleimide 1 was attached onto
the Cys thiol group in the biotinylated Kp-54 peptides. RP-HPLC
purification afforded the expected Kp-54 peptides, which were
identified with ESI-MS (Table S1).
Using this panel of Kp-54-based probes, the crosslinking exper-
iment was carried out for HEK293 cells stably expressing GPR54.
After incubation with the probes, the cells were exposed to UV
light through a 300 nm long pass filter for 30 s. The cell lysates
were separated by SDS–PAGE and the biotin on the kisspeptin
crosslinked to GPR54 was subsequently detected by probing Wes-
tern blots with horseradish peroxidase (HRP)-conjugated strepta-
vidin. When using six probes (P30C, L35C, K40C, Y45C, N46C and
W47C), a 65-kDa band was detected (Fig. 2A). In competitive bind-
ing experiments with these six probes in the presence of unlabeled
Kp-10, this 65-kDa band completely disappeared (Fig. 2B), indicat-
ing the specific detection of GPR54. Of note, the probes with mod-
ification at the N-terminus (S5C, S10C, Q15C, S20C or R25C) did not
form a covalent crosslink to GPR54, presumably because these res-
idues are not located in sufficiently close proximity to directly
interact with the receptor. In addition, when using the probes with
modification at a C-terminal residue (N48C, S49C, F50C, G51C or
L52C), crosslinks to GPR54 were not observed. This is consistent
by
(Fig. 3B).
Hormonal secretions are regulated by multiple interactions be-
a competitive binding experiment with unlabeled Kp-10
tween a number of neuropeptides/peptide hormones and recep-
tors. Recently, we and others reported that short kisspeptin
peptides such as Kp-14 and Kp-10 activate two neuropeptide FF
receptors (NPFFRs: NPFFR1/GPR147 and NPFFR2/GPR74),31,32
which possibly function(s) as negative regulator(s) of GnRH secre-
tion.33–35 Although the roles in reproductive physiology for the
kisspeptin-NPFFR pairs have not been clearly defined, Kp-54 and
the proteolytically processed short peptides may contribute to
some physiological functions including GnRH release from the
median eminence via an unknown interaction network(s).36–39
These Kp-14-based probes would be useful for the photoaffinity
labeling experiments to identify previously unrecognized target(s)
of kisspeptins.27–29
It has been reported that the GPR54 protein (42.5 kDa) contains
three N-linked glycosylation sites within the N-terminal region.3
Figure 2. Photocrosslinking experiment of GPR54 with Kp-54-based probes. (A) GPR54-expressing HEK293 cells were incubated with Kp-54-based photoaffinity probes,
followed by UV irradiation. Biotin on the crosslinked GPR54 was detected by Western blot analysis. (B) GPR54-expressing HEK293 cells were exposed to a potential Kp-54-
based probe (P30C, L35C, K40C, Y45C, N46C and W47C; 500 nM) in the presence (+) or absence (À) of unlabeled Kp-10 (10
lM) for the competitive experiments.

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R. Misu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2628–2631
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
02.098.
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To further rationalize the identification of GPR54 using Kp-54- and
Kp-14-based photoaffinity probes, deglycosylation experiments of
the crosslinked 65 kDa protein were carried out. After UV light
exposure in the presence of W47C or W7C (500 nM) for photo-
crosslinking, the cell lysates were subjected to deglycosylation
treatment using peptide N-glycosidase (PNGase F). SDS–PAGE
separation followed by Western blot analysis demonstrated that
the 65 kDa band was shifted to a mass indicative of a protein
that had undergone removal of N-linked oligosaccharide chains
(Fig. 4). This observation provides further evidence that the labeled
protein with the probes was GPR54.
In conclusion, we have designed and synthesized photoaffinity
probes based on two endogenous kisspeptin sequences (Kp-54
and Kp-14). Six Kp-54-based probes (P30C, L35C, K40C, Y45C,
N46C and W47C) and one Kp-14-based probe (W7C) clearly de-
tected GPR54 on living cells in a specific manner, suggesting these
residues constitute the secondary interactive sites of Kp-54 and
Kp-14 for receptor binding to GPR54. These probes should be
promising tools to identify the distributions of possible kisspeptin
receptors, including GPR54.
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This work was supported by Grants-in-Aid for Scientific Re-
search and Platform for Drug Discovery, Informatics, and Structural
Life Science from the Ministry of Education, Culture, Sports, Sci-
ence, and Technology of Japan; and Research Program on Innova-
tive Technologies for Animal Breeding, Reproduction, and Vaccine
Development (REP-2003) from the Ministry of Agriculture, For-
estry and Fisheries of Japan. R.M. is grateful for Research Fellow-
ships from the JSPS for Young Scientists.

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