Structure-activity relationships of a series of analogues of the RFamide-related peptide 26RFa

Article abstract of DOI:10.1021/jm200418c

26RFa is a new member of the RFamide peptide family that has been identified as the endogenous ligand of the orphan GPCR GPR103. As the C-terminal heptapeptide (26RFa_(20-26)) mimics the action of the native peptide on food intake and gonadotrop

Full text of DOI:10.1021/jm200418c

ARTICLE
pubs.acs.org/jmc
StructureꢀActivity Relationships of a Series of Analogues of the
RFamide-Related Peptide 26RFa
Olivier Le Marec,† Cindy Neveu,† Benjamin Lefranc,† Christophe Dubessy,† Jean A. Boutin,‡
Jean-Claude Do-Rꢀego,§ Jean Costentin,§ Marie-Christine Tonon,† Manuel Tena-Sempere,||
Hubert Vaudry,*,† and Jꢀer^ome Leprince†
†INSERM U982, Laboratory of Neuronal and Neuroendocrine Differentiation and Communication, European Institute for Peptide
Research (IFRMP 23), Cell Imaging Platform (PRIMACEN), University of Rouen, 76821 Mont-Saint-Aignan, France
‡IdRS, 125 Chemin de la Ronde, 78290 Croissy-sur-Seine, France
§Laboratory of Experimental Neuropsychopharmacology, IFRMP 23, University of Rouen, 76183 Rouen, France
ꢀ
Department of Cell Biology, Physiology, and Immunology, University of Cordoba and CIBER Fisiopatologia de la Obesidad y
Nutriciꢀon, 14004 Cordoba, Spain
ABSTRACT: 26RFa is a new member of the RFamide peptide family that
has been identified as the endogenous ligand of the orphan GPCR
GPR103. As the C-terminal heptapeptide (26RFa(20ꢀ26)) mimics the
action of the native peptide on food intake and gonadotropin secretion in
rodents, we have synthesized a series of analogues of 26RFa(20ꢀ26) and
measured their potency to induce [Ca2þ]i mobilization in GR16-
hGPR103-transfected CHO cells. Systematic replacement of each residue
by an alanine (Ala scan) and its D-enantiomer (D scan) showed that the
last three C-terminal residues were very sensitive to the substitutions
while position 23 tolerated rather well both modifications. Most importantly, replacement of Ser23 by a norvaline led to an analogue,
[Nva23]26RFa(20ꢀ26), that was 3-fold more potent than the native heptapeptide. These new pharmacological data, by providing the
first information regarding the structureꢀactivity relationships of 26RFa analogues, should prove useful for the rational design of
potent GPR103 receptor ligands with potential therapeutic application.
’ INTRODUCTION
expressed in extra-hypothalamic regions of the central nervous
system (CNS) and in various peripheral organs including the
kidney, pituitary, testis, adrenal gland, and bone.3,4,13 As a matter
of fact, different studies have shown that 26RFa and/or 43RFa
stimulates LH secretion from rat pituitary explants,14 increases
plasma LH, FSH, and aldosterone concentration in rat,3,14,15
inhibits glucose-induced insulin release from isolated perfused
rat pancreas,16 and evokes a sustained elevation of blood pressure
and heart rate.6 In addition, it has been reported that GPR103
knockout mice suffer from osteopenia and exhibit the character-
istic kyphotic hump of osteoporotic patients.13 These observa-
tions suggest that GPR103 could be the target for novel
therapeutic agents.
The primary structure of the heptapeptide 26RFa(20ꢀ26) has
been fully conserved from fish to mammals,17 suggesting that
the C-terminal region of 26RFa and 43RFa is important for
their biological activity. Indeed, some of the effects of 26RFa
and 43RFa are mimicked by this heptapeptide. In particular,
26RFa(20ꢀ26) enhances food consumption in mice11 and sti-
mulates LH release from rat pituitary gland.14
RFamide-related peptides (RFRPs) constitute a family of
regulatory peptides that share the motif Arg-Phe-NH2 at their
C-terminus.1 We have recently isolated a novel 26-amino acid
RFRP from the frog brain that we have called 26RFa.2 Indepen-
dently, two groups have identified a longer form of 26RFa,
termed QRFP (or 43RFa), and shown that both 26RFa and
43RFa act as natural ligands of the orphan receptor GPR103.3,4
The cDNAs encoding the 26RFa/43RFa precursor have been
cloned in chicken,5 rat,2,3 mouse,3,4 ox,2 and human.2ꢀ4 The
43RFa polypeptide has been purified and characterized from a rat
brain extract,6 and both 26RFa and 43RFa have been isolated
from the human hypothalamus and spinal cord.7 The sequence of
26RFa encompasses a triad of basic amino acids (Arg-Arg-Lys),
suggesting that cleavage of the precursor at this site may generate
the C-terminal heptapeptide 26RFa(20ꢀ26) (Figure 1).
In situ hybridization studies have shown that in the rat and
mouse brain, 26RFa and GPR103 mRNAs are expressed in
hypothalamic nuclei involved in the control of energy home-
ostasis including the ventromedial nucleus, the arcuate nucleus,
and the lateral hypothalamic area.6,8,9 Consistent with these
neuroanatomical observations, it has been found that intracer-
ebroventricular (icv) administration of 26RFa or 43RFa stimu-
lates food intake in mice.1,6,8,10ꢀ12 The GPR103 gene is also
Received: April 7, 2011
Published: May 30, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. Schematic representation of the human precursor of 26RFa, prepro-26RFa, and the other putative RFamide peptides generated by the cleavage
at different basic sites. Basic residues that represent potential cleavages sites and glycine residues that serve for amidation are indicated. SP indicates signal
peptide.
Figure 2. Effect of human and rodent 26RFa-related peptides potentially generated from the 26RFa precursor on [Ca2þ]i mobilization in hGPR103-
transfected cells. (A, B) Representative concentrationꢀresponse curves: 0, h43RFa, 4, r43RFa, O, h26RFa, ], r26RFa, 3, 26RFa(20ꢀ26), [, 9RFa. (C)
Sequence alignments of peptides potentially generated from the human and rat 26RFa precursors and their EC50 calculated from doseꢀresponse curves
similar to those shown in parts A and B. Data are the mean ( SEM of at least three separate experiments.
The aim of the present study was to investigate the struc-
tureꢀactivity relationships of 26RFa in order to design low
molecular weight and lipophilic ligands of GPR103 that could
lead to the development of therapeutic compounds as pre-
viously done with ACE inhibitors.18 We have synthesized a
collection of 26RFa analogues in order to determine the
minimal sequence retaining Ca2þ-mobilizing activity in GR16-
hGPR103-transfected CHO cells and to identify the position-
related structural requirements for agonistic and, possibly,
antagonistic behavior.
’ RESULTS AND DISCUSSION
To compare the potencies and efficacies of all the RFRPs that
may be generated from the human and rat 26RFa precursors
(Figure 1), we have studied the effects of the 26- and 43-amino
acid peptides (h26RFa, r26RFa, h43RFa, r43RFa) as well as the
fully conserved C-terminal heptapeptide 26RFa(20ꢀ26) and the
human-specific peptide 9RFa on [Ca2þ]i in cultured GR16-
hGPR103-transfected CHO cells (Figure 2, Table 1, compounds
1ꢀ5, 16). Concentrationꢀresponse curves showed that rat
26RFa and 43RFa were slightly more potent than their human
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Journal of Medicinal Chemistry
ARTICLE
Table 1. Continued
Table 1. Chemical Data for Compounds 1ꢀ62
HPLC
MS
HPLC
MS
obsdc
compd
peptide
tR (min)a purity (%) calcdb
obsdc
compd
peptide
tR (min)a purity (%) calcdb
50
N-ethyl, N-benzyl-
26RFa(20ꢀ26)
25.0
99.9
933.49 934.63
1
h43RFa
r43RFa
h26RFa
r26RFa
9RFa
22.2
23.2
27.3
23.3
16.4
21.7
19.3
17.7
18.5
18.5
18.3
18.1
18.1
18.2
18.2
19.3
18.6
18.4
14.0
18.3
20.0
19.8
19.1
19.1
15.9
19.2
15.3
20.1
15.6
18.5
18.4
17.8
17.2
18.1
21.0
21.4
20.6
20.3
19.7
20.3
48.6
23.4
18.5
22.2
20.1
26.5
23.7
99.9
99.9
99.9
99.9
99.1
99.9
99.9
99.9
98.9
99.9
98.8
99.9
99.8
99.9
99.0
99.9
99.9
99.9
99.2
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.8
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.0
99.3
4501.19 4501.76
4551.19 4552.10
2830.45 2831.26
2818.45 2819.48
1120.53 1121.50
2585.35 2586.55
2318.19 2319.27
2020.03 2020.99
1648.86 1649.85
1534.82 1535.81
1477.79 1478.79
1314.73 1315.73
1227.70 1228.70
1071.60 1072.60
943.50 944.40
815.41 816.41
758.39 759.47
701.36 702.44
994.46 995.49
1532.80 1533.80
1620.76 1621.70
1904.96 1906.18
829.42 830.38
829.42 830.35
739.38 740.31
799.41 800.39
739.38 740.22
730.34 731.21
739.38 740.44
815.41 816.44
815.41 816.51
815.41 816.41
815.41 816.47
815.41 816.44
841.46 842.50
841.46 842.58
841.46 842.54
841.46 842.36
827.44 828.44
827.44 828.40
813.43 814.43
881.49 882.59
829.42 830.51
2831.43 2833.37
816.39 817.65
3010.54 3011.39
2898.51 2899.39
2
51
52
53
54
55
56
57
58
59
60
61
62
N-benzyl-h26RFa
N-benzyl-26RFa(20ꢀ26)
N-benzyl-h26RFa(1ꢀ25)
N-benzyl-26RFa(20ꢀ25)
Ac-26RFa(20ꢀ26)
Piv-26RFa(20ꢀ26)
Bz-26RFa(20ꢀ26)
Z-26RFa(20ꢀ26)
Adam-26RFa(20ꢀ26)
Fur-26RFa(20ꢀ26)
Tmg-26RFa(20ꢀ26)
pFPha-26RFa(20ꢀ26)
23.7
23.1
22.4
20.1
20.2
23.3
23.5
24.9
28.1
23.7
20.7
26.2
99.9
98.6
99.9
99.9
98.9
99.9
99.9
99.9
99.9
99.9
98.8
99.9
2920.50 2921.63
905.45 906.65
2773.43 2774.33
758.39 759.39
857.42 858.42
899.47 900.47
919.43 920.44
949.44 950.44
977.51 978.52
909.41 910.42
913.49 914.49
951.44 952.45
3
4
5
6
h26RFa(4ꢀ26)
h26RFa(7ꢀ26)
h26RFa(10ꢀ26)
h26RFa(13ꢀ26)
h26RFa(14ꢀ26)
h26RFa(15ꢀ26)
h26RFa(16ꢀ26)
h26RFa(17ꢀ26)
h26RFa(18ꢀ26)
26RFa(19ꢀ26)
26RFa(20ꢀ26)
26RFa(21ꢀ26)
26RFa(22ꢀ26)
h26RFa(8ꢀ16)
h26RFa(4ꢀ17)
h26RFa(1ꢀ16)
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
a Retention time determined by RP-HPLC. b Theoretical monoisotopic
molecular weight. c MHþ value assessed by MALDI-TOF-MS.
counterparts (Figure 2A,B) while the N-terminally elongated
forms (h43RFa and r43RFa) were in the same range of potency
as the corresponding 26RFa forms (Figure 2C). We have
previously shown that in methanol solution the Pro4-Arg17
region of 26RFa adopts a well-defined R-helical structure.19
The slightly higher potency of r26RFa, compared to h26RFa,
can likely be accounted for by the propencity of the Thr7, Ser13,
and Ser14 residues in the rat sequence (compared to the Asn7,
Asn13, and Gly14 residues in the human sequence) to stabilize
this R-helix in lipid bilayer.20 The C-terminal heptapeptide,
26RFa(20ꢀ26), whose sequence is fully conserved from fish to
human,17 was substantially less potent (75-fold) but as effica-
cious as h26RFa to increase [Ca2þ]i (Figures 2B,C). Finally, in
agreement with a previous report,4 we found that 9RFa possesses a
very weak agonistic activity, indicating that this peptide cannot be
considered as an endogenous ligand of GPR103. Conversely, it has
been reported that the affinity of 9RFa for another RFamide
receptor, NPFF2, is similar to that of h26RFa,21 suggesting that
9RFa could activate receptor(s) other than GPR103.
h26RFa(1ꢀ18)
[Ala20]26RFa(20ꢀ26)
[Ala21]26RFa(20ꢀ26)
[Ala22]26RFa(20ꢀ26)
[Ala23]26RFa(20ꢀ26)
[Ala24]26RFa(20ꢀ26)
[Ala25]26RFa(20ꢀ26)
[Ala26]26RFa(20ꢀ26)
[D-Phe22]26RFa(20ꢀ26)
[D-Ser23]26RFa(20ꢀ26)
[D-Phe24]26RFa(20ꢀ26)
[D-Arg25]26RFa(20ꢀ26)
[D-Phe26]26RFa(20ꢀ26)
[Leu23]26RFa(20ꢀ26)
[Nle23]26RFa(20ꢀ26)
[Ile23]26RFa(20ꢀ26)
[Tle23]26RFa(20ꢀ26)
[Val23]26RFa(20ꢀ26)
[Nva23]26RFa(20ꢀ26)
[Abu23]26RFa(20ꢀ26)
[Cha23]26RFa(20ꢀ26)
[hSer23]26RFa(20ꢀ26)
desamide-h26RFa
desamide-26RFa(20ꢀ26)
N,N-dibenzyl-h26RFa
N, N-piperidinyl-
h26RFa
To further investigate the contribution of the N-terminal,
central, and C-terminal regions of the peptide in the [Ca2þ
]
i
mobilizing activity, we have generated downsized analogues of
h26RFa (Table 1, compounds 6ꢀ22). Deletion of three (Table 2,
compound 6), six (Table 2, compound 7), and nine amino acids
(Table 2, compound 8) from the N-terminus did not markedly
affect the biological activity of the peptide, suggesting that the
amino tail and the N-terminal extremity of the R-helix do not play
a crucial role in the biological activity of h26RFa. Additional deletion
of three (Table 2, compound 9), four (Table 2, compound 10),
and five residues (Table 2, compound 11) led to analogues that were
10- to 20-fold less potent than h26RFa (Table 2). Shorter C-terminal
peptides, i.e., 26RFa(16ꢀ26), 26RFa(17ꢀ26), and 26RFa(18ꢀ26)
(Table 2, compounds 12ꢀ14), were approximately 30 times
less potent than h26RFa. Finally, 26RFa(19ꢀ26) and 26RFa(20ꢀ26)
were about 75-fold less potent than h26RFa (Table 2, compounds
15 and 16) while 26RFa(21ꢀ26) and 26RFa(22ꢀ26) were totally
devoid of activity (Table 2, compounds 17 and 18), indicating that
the biological activity of the peptide is harbored in its C-terminal
48
49
N,N-dibenzyl-
27.7
22.9
99.9
98.9
995.50 996.63
883.47 884.59
26RFa(20ꢀ26)
N, N-piperidinyl-
26RFa(20ꢀ26)
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ARTICLE
Table 2. Effects of N-Terminal, Central, and C-Terminal Truncated h26RFa Analogues on Basal [Ca2þ]i and h26RFa-Induced
[Ca2þ]i Increase in CHO-GR16-hGPR103-Transfected Cells
sequence
compd
peptide
h26RFa
5
10
15
20
25
EC50 (nM)a IC50 (nM)a
3
T
S
G
P
P
L
L
G
G
N
N
N
L
L
L
A
A
A
E
E
E
E
E
E
E
E
L
L
L
L
N
N
N
N
N
G
G
G
G
G
G
Y
Y
Y
Y
Y
Y
Y
S
S
S
S
S
S
S
S
R
R
R
R
R
R
R
R
R
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
F
F
F
F
F
F
F
F
F
F
F
F
F
F
S
S
S
S
S
S
S
S
S
S
S
S
S
S
F
F
F
F
F
F
F
F
F
F
F
F
F
F
R
R
R
R
R
R
R
R
R
R
R
R
R
R
F
F
F
F
F
F
F
F
F
F
F
F
F
F
10.4 ( 1.50
43.2 ( 40.3
30.2 ( 11.6
37.5 ( 15.2
95.3 ( 40.7***
185 ( 52**
138 ( 37**
237 ( 57***
282 ( 69***
233 ( 51***
1710 ( 521***
739 ( 149***
NC
6
h26RFa(4ꢀ26)
h26RFa(7ꢀ26)
h26RFa(10ꢀ26)
h26RFa(13ꢀ26)
h26RFa(14ꢀ26)
h26RFa(15ꢀ26)
h26RFa(16ꢀ26)
h26RFa(17ꢀ26)
h26RFa(18ꢀ26)
h26RFa(19ꢀ26)
h26RFa(20ꢀ26)
h26RFa(21ꢀ26)
h26RFa(22ꢀ26)
h26RFa(8ꢀ16)
h26RFa(4ꢀ17)
h26RFa(1ꢀ16)
h26RFa(1ꢀ18)
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
ND
L
L
L
L
A
A
A
A
E
E
E
E
E
E
E
E
L
L
L
L
N
N
N
N
G
G
G
G
Y
Y
Y
Y
S
S
S
S
ND
ND
ND
ND
ND
ND
ND
ND
P
P
P
L
L
L
G
G
G
N
N
N
R
R
T
T
S
S
G
G
K
a Data are the mean ( SEM of at least three distinct experiments. NC, not calculable. ND, not detectable. (//) p < 0.01 and (///) p < 0.001 vs control
(h26RFa) as assessed by MannꢀWitney test.
the affinity of these fragments for GPR103.3 All these data clearly
Table 3. Effects of L-Alanine and D-Residue Substituted
26RFa(20-26) Analogues on Basal [Ca2þ]i and h26RFa-Induced
indicate that the biologically active domain of 26RFa is located in
[Ca2þ]i Increase in CHO-GR16-hGPR103-Transfected Cells
the C-terminal region and that 26RFa(20ꢀ26), which may be
processed in vivo by prohormoneconvertasesandwhosesequence
is fully conserved from fish to mammals, is the shortest analogue
that retains full efficiency to increase [Ca2þ]i in GPR103-trans-
fected cells. 26RFa(20ꢀ26) is also relatively hydrophobic and thus
more suitable for the development of molecules with therapeutic
value. For all these reasons, 26RFa(20ꢀ26) naturally stood out as
the ideal molecular scaffold for the design of GPR103 peptide
ligands with low molecular weight.
The contribution of the amino acid side chains to the biological
activity of 26RFa(20ꢀ26) was evaluated by systematic alanine
replacement of each residue of the peptide sequence (Table 1,
compounds 23ꢀ29).22ꢀ24 Alanine substitution in the N-terminal
region(Table3, compounds23ꢀ26) did not substantially affect the
activity of the peptide (Table 3). In particular, replacement of the
two glycine residues by Ala, which is expected to restrict the
conformational freedom of the peptide backbone at these points,
did not markedly impair the [Ca2þ]i-mobilizing activity of the
peptide (Table 3, compounds 23 and 24). In contrast, L-alanine
substitution in the C-terminal part of the molecule led to analogues
totally devoid of stimulating activity for concentrations ranging from
10ꢀ12 to 10ꢀ5 M (Table 3, compounds 27ꢀ29). Similarly, [Ala24]-,
[Ala25]-, and [Ala26]26RFa(20ꢀ26) did not reduce the h26RFa-
evoked increase in [Ca2þ]i, suggesting that the Phe-Arg-Phe triad is
directly involved in GPR103 activation as already reported for
NPFF and PrRP, which are two other peptides from the RFamide
superfamily.25,26 In order to explore the importance of the orienta-
tion of the amino acid side chains in the biological activity of the
peptide, a series of D-isomer-substituted peptides was synthesized
(Table 1, compounds 30ꢀ34) and the ability of the analogues to
elicit [Ca2þ]i increase was measured (Table 3, compounds
sequence
EC50
(nM)a
IC50
(nM)a
compd
peptide
20 21 22 23 24 25 26
16 26RFa(20ꢀ26)
G G F S F R F 739 ( 149
23 [Ala20]26RFa(20ꢀ26)
24 [Ala21]26RFa(20ꢀ26)
25 [Ala22]26RFa(20ꢀ26)
26 [Ala23]26RFa(20ꢀ26)
27 [Ala24]26RFa(20ꢀ26)
28 [Ala25]26RFa(20ꢀ26)
29 [Ala26]26RFa(20ꢀ26)
30 [D-Phe22]26RFa(20ꢀ26)
31 [D-Ser23]26RFa(20ꢀ26)
32 [D-Phe24]26RFa(20ꢀ26)
33 [D-Arg25]26RFa(20ꢀ26)
34 [D-Phe26]26RFa(20ꢀ26)
A
3350 ( 668**
2985 ( 862**
1333 ( 321
1308 ( 724
ND
A
A
A
s
A
ND
ND
ND
ND
A
ND
A ND
f
ND
4975 ( 2305*
f
ND
ND
ND
ND
ND
ND
r
f
a Data are the mean ( SEM of at least three distinct experiments. ND,
not detectable. (/) p < 0.05 and (//) p < 0.01 vs control (26RFa(20ꢀ26)
as assessed by the MannꢀWitney test.
)
part. Indeed, neither central segments such as h26RFa(8ꢀ16) and
h26RFa(4ꢀ17) nor N-terminal fragments (Table 2, compounds
19ꢀ22) exhibited agonistic or antagonistic activities in GPR103-
transfected cells.
The gradual decline in the biological activity observed with the
N-side truncated analogues has to be correlated to the decrease in
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Table 4. Effect of Ser23 Residue-Substituted 26RFa(20-26)
Analogues on Basal [Ca2þ]i in CHO-Gr16-hGPR103-Trans-
fected Cells
Table 5. Effects of C-Terminally Modified h26RFa and
26RFa(20-26) Analogues on Basal [Ca2þ]i and h26RFa-Induced
[Ca2þ]i Increase in CHO-GR16-hGPR103-Transfected Cells
compd
peptide
EC50 (nM)a
IC50 (nM)a
3
h26RFa
10.4 ( 1.5
739 ( 149
531 ( 319***
ND
16
44
45
46
47
48
49
50
51
52
53
54
26RFa(20ꢀ26)
desamide-h26RFa
desamide-26RFa(20ꢀ26)
N,N-dibenzyl-h26RFa
N,N-piperidinyl-h26RFa
N,N-dibenzyl-26RFa(20ꢀ26)
N,N-piperidinyl-26RFa(20ꢀ26)
N-ethyl,N-benzyl-26RFa(20ꢀ26)
N-benzyl-h26RFa
ND
NC
166 ( 61**
282 ( 134***
ND
1198 ( 286
1088 ( 199
39 ( 5.5
ND
N-benzyl-26RFa(20ꢀ26)
N-benzyl-h26RFa(1ꢀ25)0
N-benzyl-26RFa(20ꢀ25)
ND
ND
67 ( 26**
ND
a Data are the mean ( SEM of at least three distinct experiments. (/) p <
0.05 vs control (26RFa(20ꢀ26)) as assessed by the MannꢀWitney test.
a Data are the mean ( SEM of at least three distinct experiments. NC,
not calculable. ND, not detectable. (//) p < 0.01 and (///) p < 0.001 vs
control (h26RFa) as assessed by the MannꢀWitney test.
30ꢀ34). D-Amino acid replacement at positions 22, 24, 25, and 26
(Table 3, compounds 30, 32ꢀ34) resulted in a complete loss of the
[Ca2þ]i response, while this substitution was rather well tolerated at
position 23 (Table 3, compound 31). Altogether, these data
demonstrate that not only the chemical structure but also the
correct orientation of the side chain of each residue plays a critical
role in the activity of the peptide.
preferences are usually governed by a membrane-mimicking
environment.20 In water and methanol, the C-terminal region
of 26RFa, corresponding to 26RFa(20ꢀ26), does not exhibit a
particular structure.19 However, we assume that in a lipid bilayer
this region adopts a typical conformation that could be strongly
stabilized by uncharged amino acids with short and linear side
chains such as norvaline and 2-aminobutyric acid.
Since the Ser23 residue tolerated rather well the ala- and D-
isomer substitutions, we assumed that this could be a suitable
position to generate superior analogues of 26RFa(20ꢀ26). We
thus designed seven peptides in which the Ser side chain was
replaced by differently branched aliphatic moieties (Table 1,
compounds 35ꢀ43). Substitution of the hydroxymethyl side
chain of Ser by butyl and isobutyl moieties did not modify
the activity of the compounds compared to the native heptapeptide
(Table 4, compounds 36 and 37). In contrast, replacement of the
hydroxymethyl group by a short and hindered sec-butyl
([Leu23]26RFa(20ꢀ26)), tert-butyl ([Tle23]26RFa(20ꢀ26)), or iso-
propyl moiety ([Val23]26RFa(20ꢀ26)) led to compounds with a
2- to 2.5-fold reduced potency (Table 4, compounds 35, 38, and
39). A 2-fold decrease of the potency was also observed with the
[Cha23]26RFa(20ꢀ26) analogue bearing a bulky and nonpolar
methylcyclohexyl moiety (Table 4, compound 42). In contrast,
the presence of a short side chain such as propyl and ethyl enhanced
the activity of the corresponding analogues on [Ca2þ]i (Table 4,
It is well established that C-terminal amidation of peptides
prevents extracellular inactivation by peptidases and often contri-
butes to the biological activity.30ꢀ33 Consistent with these notions,
desamide-h26RFa was at least 50-fold less potent than h26RFa, and
desamide-26RFa(20ꢀ26) was totally devoid of agonistic and antag-
onistic activity (Table 1, compounds 44 and 45). These findings are
in agreement with previous data showing that desamide-h26RFa has
a 400-fold lower affinity for GPR103 than 26RFa.3 Therefore, we
next investigated the contribution of the terminal carboxamide to
the biological activity by using a series of mono- and disubstituted
h26RFa, h26RFa(1ꢀ25), and 26RFa(20ꢀ26) analogues (Tables 1 and
5, compounds 46ꢀ54). The disubstituted analogues, i.e., N,N-
dibenzyl-h26RFa, N,N-piperidinyl-h26RFa, and N-ethyl,N-benzyl-
26RFa(20ꢀ26) (Table 5, compounds 46, 47, and 50), showed a
strong decrease in Ca2þ-mobilizing activity compared to the native
peptide, suggesting that the hydrogen atoms of the primary amide
participate in some interactions either with GPR103 or with a
carboxy function of the peptide. Since the C-terminal extremity of
26RFa presents a random-coil structure in water and methanol, we
assume the presence of intermolecular interactions.19 In support of
this notion, the monosubstituted carboxamide derivative of h26RFa,
N-benzyl-h26RFa, was almost as potent as h26RFa (Table 5,
compound 51). However, N-benzyl-26RFa(20ꢀ26) (Table 5, com-
pound 52) was totally devoid of agonistic or antagonistic activities,
indicating that this analogue was unable to bind to GPR103. This is
an unexpected result if the same mode of action is accepted for both
26RFa and its C-terminal heptapeptide 26RFa(20ꢀ26). It has been
demonstrated that in all mammalian RFRPs the Arg-Phe-NH2 motif
is essential for bioactivity.25 Interestingly, we observed that the
phenylalanine residue can be deleted without loss of activity if
the benzyl side chain is reported to the carboxamide function as in
compounds 40 and 41). In particular, [Nva23]26RFa(20ꢀ26)
,
which exhibited a 3-fold lower EC50 than 26RFa(20ꢀ26), was
the most potent downsized peptide of this series (Table 4,
compound 40). These data suggest that the residue at position
23 may interact with a narrow hydrophobic binding pocket
within the receptor. Finally, the [hSer23]26RFa(20ꢀ26), in which
the Ser hydroxymethyl side chain is elongated by a single
methylene group, was 2 fold more potent than 26RFa(20ꢀ26)
(Table 4, compound 43). This could be accounted for by the
formation of hydrogen bonds, allowing additional forces to anchor
the peptide in the receptor. Alternatively, it is well established
that uncharged amino acids exhibit distinct proclivity for folding
peptides and proteins.27 For instance, Ile and to a lesser extent
Nle have a greater propensity to form a β-structure than the helix
inducers Leu and Ala,28 while Val has been shown to promote a
γ-turn formation in peptide models.29 These conformational
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Journal of Medicinal Chemistry
ARTICLE
Table 6. Effect of N-Terminally Modified 26RFa(20-26) Ana-
logues on Basal [Ca2þ]i in CHO-Gr16-hGPR103-Transfected
Cells
compd
peptidea
EC50 (nM)b
16
55
56
57
58
59
60
61
62
26RFa(20ꢀ26)
739 ( 149
1443 ( 741
2545 ( 694**
722 ( 139
826 ( 166
588 ( 141
695 ( 270
455 ( 76
Ac-26RFa(20ꢀ26)
Piv-26RFa(20ꢀ26)
Bz-26RFa(20ꢀ26)
Z-26RFa(20ꢀ26)
Adam-26RFa(20ꢀ26)
Fur-26RFa(20ꢀ26)
Tmg-26RFa(20ꢀ26)
pFPha-26RFa(20ꢀ26)
Figure 3. Hypothetical model of interaction between 26RFa(20ꢀ26) and
GPR103. The curved lines illustrate the different motifs of the ligand that
potentially interact with GPR103. (A) The bold lines aim at the three
motifs that appear to play a role in receptor activation. (B) The doubled
line designates the serine residue that likely fits into the narrow
hydrophobic pocket. (C) The dashed line illustrates the identified
H-bond interaction. (D) The dotted line points to an unknown
interaction with the GPR103 receptor.
830 ( 292
a Ac, acetyl; Adam, adamantanoyl; Bz, benzoyl; Fur, furoyl; pFPha, p-
fluorophenylacetyl; Piv, pivaloyl; Tmg, tetramethylguanidinyl; Z, ben-
zyloxycarbonyl. b Data are the mean ( SEM of at least three distinct
experiments. (//) p < 0.01 vs control (26RFa(20ꢀ26)) as assessed by the
MannꢀWitney test.
N-benzyl-h26RFa(1ꢀ25) (Table 5, compound 53). Unfortunately,
the truncated N-benzyl-26RFa(20ꢀ25) analogue did not follow the
paradigm (Table 5, compound 54). We thus propose that either the
C-terminal amide favors the formation of at least one hydrogen
bond within the binding pocket of GPR103 or the cavity is too
narrow for harboring sterical hindered moieties. To the best of our
knowledge, only one structureꢀactivity relationship study reports
the chemical modulation of a carboxamide function of a regulatory
peptide, i.e., substance P, and demonstrates the involvement of the
primary amide in the peptideꢀreceptor interaction through hydro-
gen bound in very much the same way as for 26RFa.34
Acylation of the N-terminal extremity of regulatory peptides
affords protection from exopeptidase degradation notably by
dipeptidylaminopeptidases.35,36 Acylation can also modulate the
intrinsic biological activity of peptides. Thus, acetylation is
required for the biological activity of R-MSH whereas it strongly
attenuates the morphinomimetic activity of β-endorphin.37
Therefore, we next examined the effect of N-terminal acylation
of 26RFa(20ꢀ26) on intracellular calcium concentration (Tables 1
and 6, compounds 55ꢀ62). Whatever the physicochemical
feature of the acyl group, i.e., aliphatic or aromatic and small or
bulky, the potency of all N-acylated heptapeptides tested was in
the same range as that of the native peptide, providing further
evidence that the N-terminal region does not participate in the
activation of the GPR103.
26RFa has been shown to exert multiple effects in the CNS.
Thus, icv injection of 26RFa or 43RFa stimulates food
intake,1,6,8,10ꢀ12 activates the hypothalamoꢀpituitaryꢀgonadal
axis,14,15 modulates nociceptive stimuli transmission,38 and in-
creases arterial blood pressure and heart rate in rodents.6 In
addition, the wide distribution of GPR103 mRNAs in peripheral
organs suggests that the 26RFa/GPR103 system may play other
physiological functions.3,4,13 In agreement with this notion, in rat,
43RFa stimulates aldosterone secretion in vivo3 and 26RFa
enhances LH and FSH release in vitro.14 Phenotyping analysis
of GPR103ꢀ/ꢀ mice revealed massive reduction in bone density,
notably in females that exhibit the characteristic kyphotic hump
of osteoporetic patients.13 Altogether, these observations vali-
date the development of GPR103 agonists and antagonists that
could selectively cross or not the bloodꢀbrain barrier for the
treatment of neuroendocrine and metabolic disorders.
In summary, this first structureꢀactivity relationship study of
26RFa revealed that (i) 26RFa is as potent as 43RFa while the
conserved C-terminal heptapeptide 26RFa(20ꢀ26) is 75-fold less
potent, (ii) the Phe-Arg-Phe triad is involved in GPR103
activation, (iii) position 23 of 26RFa(20ꢀ26) can be optimized
using diverse residues for improving GPR103 activation, and
(iv) the C-terminal primary amide is involved in a hydrogen
bound probably with specific residue of GPR103 in contrast to
the N-terminal function of 26RFa(20ꢀ26) (Figure 3). Our data
also demonstrate that a suitable modification of 26RFa(20ꢀ26)
such as Nva23 substitution could open up new opportunities for
the design of potent GPR103 receptor ligands with potential
therapeutic applications.
’ EXPERIMENTAL SECTION
Materials. All Fmoc amino acid residues, Boc-Thr(Bzl)-OH, Boc-
Gly-OH, and Z-Gly-OH were purchased from Senn Chemicals
(Dielsdorf, Switzerland) or Bachem (Weil am Rhein, Germany). The
preloaded 4-hydroxymethylphenoxymethyl-copolystyreneꢀ1%-divinyl-
benzene (HMP) resins, O-benzotriazol-1-yl-N,N,N0,N0-tetramethyluro-
nium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt),
and dichloromethane (DCM) were from Applera-France (Courtaboeuf,
France). Acetonitrile and N-methylpyrrolidinone (NMP) were from
Carlo Erba (Val-de-Reuil, France). The Rink amide 4-methylbenzhy-
drylamine (MBHA) resin was from VWR International (Fontenay-sous-Bois,
France). The preloaded 2-methoxy-4-alkoxybenzyl alcohol resins were
from Bachem. Diisopropylethylamine (DIEA), piperidine, trifluoroacetic
acid (TFA), the nutrient mixture F-12 HAM, L-glutamine, 4-(2-hydro-
xyethyl)-1-piperazine ethanesulfonic acid (HEPES), penicillinꢀstrep-
tomycin solution, probenecid, M2 antibodies, and other reagents were
from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Phenol, Geneticin
(G-418 sulfate), hygromycin B, Hank’s balanced salt solution (HBSS),
and pluronic F-127 were from Gibco Life Technologies (Cergy-Pontoise,
France). Fetal bovine serum was from Lonza (Viviers, Belgium).
Peptide Synthesis. All peptides were synthesized on a 433A Applied
Biosystems peptide synthesizer (Applera-France) using the standard man-
ufacturer’s procedures. C-Terminal amidated analogues, C-terminal N- and
N,N-modified amidated analogues, and C-terminal free analogues of 26RFa
were synthesized (0.1 mmol scale) on a Rink amide MBHA resin, on
preloaded 2-methoxy-4-alkoxybenzyl alcohol resin (Fmoc-Phe-SASRIN or
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Journal of Medicinal Chemistry
ARTICLE
Fmoc-Arg(Pbf)-SASRIN), and on preloaded HMP resin (Fmoc-Ser(tBu)-
HMP, Fmoc-Lys(Boc)-HMP, Fmoc-Arg(Pbf)-HMP), or Fmoc-Phe-
HMP), respectively. All Fmoc-amino acids (1 mmol, 10 equiv) were
coupled by in situ activation with HBTU/HOBt (1.0 mmol/1.0 mmol,
10 equiv) and DIEA (1 mmol, 10 equiv) in NMP. Reactive side chains were
protected as follows, unless otherwise stated: Thr, Ser, and Tyr, tert-butyl
(tBu) ether; Lys, tert-butyloxycarbonyl (Boc) carbamate; Glu, O-tert-butyl
(OtBu) ester; Asn, trityl (Trt) amide; and Arg, pentamethyldihydrobenzo-
furan (Pbf) sulfonylamide.
Establishment of a Cell Line Stably Expressing hGPR103 and
the GR16 Protein. The preparation of CHO-K1-GR16 cells stably
expressing the G-protein R16 subunit has been previously described.41
The cells were maintained in Ham-F12 medium supplemented with
10% (v/v) fetal bovine serum (inactivated at 56 °C for 30 min), 2 mM
glutamine, 500 IU/mL penicillin, and 100 μg/mL streptomycin. The
coding region of the human GPR103 receptor, containing the flag
epitope sequence (DYKDDDDK) at its 30-end, was subcloned into the
pcDNA3.1-neo expression vector (Invitrogen, Cergy Pontoise,
France) and transfected into CHO-K1-GR16 cells using Lipofectamine
as described by the manufacturer (Invitrogen). Stably transfected cells
were selected with Geneticin (800 μg/mL) and tested by immuno-
fluorescence for their ability to bind the M2 antibody.
After completion of the chain assembly, NR-acylation of 26RFa(20ꢀ26)
was performed on the resin by addition of a mixture of acetic anhydride
or acyl chloride/DIEA (0.4 mmol, 4 equiv; 0.5 mmol, 5 equiv) in NMP
at 0 °C for 5 min and at room temperature for 30 min. Reactions were
monitored by Kaiser’s test. Z-26RFa(20ꢀ26) was automatically prepared
one step before completion by addition of Z-Gly-OH.
Ca2þ Mobilization Assays. Changes in intracellular Ca2þ con-
centrations induced by 26RFa analogues in CHO-GR16-hGPR103-
transfected cells were measured on a benchtop scanning fluorometer
Flexstation II (Molecular Devices, Sunnyvale, CA). Briefly, 96-well assay
black plates with clear bottom (Corning International, Avon, France)
were seeded at a density of 40 000 cells/well 24 h prior to assay. For
agonist experiments, cells were loaded with 2 μM Fluo-4AM during 1 h,
washed 3 times, and incubated 30 min with standard HBSS containing
2.5 mM probenecid and 5 mM HEPES. Compounds to be tested were
added at final concentrations ranging from 10ꢀ12 to 10ꢀ5 M (HBSS),
and the fluorescence intensity was measured during 2 min. Antagonist
experiments were conducted using the same protocol except that
compounds were manually injected at 10ꢀ6, 3.16 ꢁ 10ꢀ6, 10ꢀ5, and
3.16 ꢁ 10ꢀ5 M 30 min prior to the injection of the agonist, i.e., h26RFa,
at a single concentration of 10ꢀ7 M. A xenon lamp was used as excitation
source. The wavelengths of excitation (485 nm) and emission (525 nm)
of Fluo-4-AM were selected by two monochromators included in the
device equipped with a bottom reading probe.
The N- and N,N-modified amidated analogues of 26RFa were
prepared by treating Boc-peptidyl-SASRIN with 1% TFA/DCM for
5 min under gentle agitation. After filtration of the resin, the filtrate was
neutralized by addition of pyridine. The treatment was repeated with
further portions of 1% TFA/DCM until the peptide was split off the
resin (TLC; MeOH/H2O/AcOH 1.3/0.15/0.05 v/v/v). The filtrate
was concentrated to 10 mL on a rotary evaporator. The Boc-peptidyl-
OH was in situ activated with HBTU/HOBt (1:1, 1 equiv) and DIEA
(1 equiv). Primary or secondary amine was added dropwise to the stirred
solution, and the mixture was left to stand at 0 °C for 30 min and at room
temperature overnight. The reaction was checked for completion by
TLC (MeOH/H2O/AcOH 1.3:0.15:0.05 v/v/v). The mixture was
evaporated to dryness, dissolved in AcOEt/Et2O (9:1), washed succes-
sively with 5% NaHCO3 (3 ꢁ 10 mL), 5% citric acid (3 ꢁ 10 mL), 5%
NaCl (3 ꢁ 10 mL), and evaporated to dryness.
Peptide and Side Chain Cleavage and Purification. All
peptides were cleaved from the resin and/or deprotected by adding
10 mL of an ice-cold mixture TFA/phenol/H2O/thioanisole/ethane-
dithiol (82.5:5:5:5:2.5, v/v/v/v/v) agitated at 0 °C for 5 min and at
room temperature for 2 h as previously described.39,40 Crude peptides
were purified by semipreparative reversed-phase HPLC (RP-HPLC) on
a Vydac 218TP1022 C18 column (2.2 cm ꢁ 25 cm; Alltech, Templemars,
France) using a linear gradient (10ꢀ50% over 50 min) of acetonitrile/
TFA (99.9:0.1, v/v) at a flow rate of 10 mL/min. Analytical RP-HPLC
analysis, performed on a Vydac 218TP54 C18 column (0.46 cm ꢁ 25 cm,
Alltech), revealed that the purity of all peptides was higher than 98.6%
(Table 1). The authenticity of each peptide was verified by MALDI-
TOF-MS on a Voyager DE-PRO (Applera-France) in the reflector mode
with R-cyano-4-hydroxycinnamic acid as a matrix.
Statistical Analysis. Calcium experiments were performed in
triplicate, and data, expressed as the mean ( SEM, were analyzed with
the Prism software (Graphpad Software, San Diego, CA). The EC50
values were determined from concentrationꢀresponse curves using a
sigmoidal doseꢀresponse fit with variable slope. Differences between
26RFa or 26RFa(20ꢀ26) and analogue activities were analyzed by the
MannꢀWhitney test.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (33) 235-14-6624. Fax: (33) 235-14-6946. E-mail:
hubert.vaudry@univ-rouen.fr.
Cloning of hGPR103 cDNA. The human GPR103 receptor
coding sequence was isolated by RT-PCR from human brain mRNA
(Clontech Laboratories, Palo Alto, CA) using the 50 forward primer (50-
aagcttATGCAGGCGCTTAACATTACC-30, nt 380ꢀ401) and the 30
reverse primer (50-CTCCTTTAGACAGTGGGCATTAAggtacc-30, nt
1653ꢀ1675), which were designed according to GenBank data se-
quence (accession no. NM 198179). Brain mRNA (200 ng) was reverse-
transcribed using oligo(dT)12ꢀ18 and Superscript II reverse transcrip-
tase (Life Technologies). PCR reactions were performed in a volume of
50 μL containing native Pfu buffer, 0.25 mM dNTP, 2 μL of the single-
stranded cDNA preparation, 3ꢁ enhancer solution, 0.4 μM primers, and
2 units of the Pfu native polymerase (Life Technologies) with a 35 cycle
program of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min, and a
pre- and postincubation program of 94 °C for 1 min and 72 °C for 5 min,
respectively. The amplified cDNA was then subcloned into HindIII and
KpnI sites of the pcDNA3.1(þ) vector containing an in-frame M2tag
(DYKDDDDK carboxy-terminal end) after the KpnI site. The sequence
was controlled using dideoxychain termination and automated fluor-
escent dye ABI3730 DNA sequencer (Applied Biosystems).
’ ACKNOWLEDGMENT
This study was supported by INSERM (U982), the PNR-RE,
and the Conseil Rꢀegional de Haute-Normandie. O.L.M. was the
recipient of a fellowship from the Ministꢁere de l’Enseignement
Superieur et de la Recherche. C.N. was the recipient of a
fellowship from the LARC-Neuroscience network. The authors
thank Drs. Marianne Rodriguez, Francis Cogꢀe, and Jean-Pierre
Galizzi for the cloning and construction of the cell line.
’ ABBREVIATIONS USED
Abu, 2-aminobutyric acid; Ac, acetyl; Adam, adamantanoyl; Bz,
benzoyl; Fur, furoyl; MALDI-TOF-MS, matrix-assisted laser de-
sorption ionization time-of-flight mass spectrometry; pFPha, p-
fluorophenylacetyl; Piv, pivaloyl; RP-HPLC, reversed-phase high
performance liquid chromatography; Tle, terleucine; Tmg, tetra-
methylguanidinyl; Z, benzyloxycarbonyl
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Journal of Medicinal Chemistry
ARTICLE
’ ADDITIONAL NOTE
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E.; Tovar, S.; Chartrel, N.; Le Marec, O.; Leprince, J.; Aguilar, E.; Pinilla,
L.; Dieguez, C.; Vaudry, H.; Tena-Sempere, M. Novel role of 26RFa, a
hypothalamic RFamide orexigenic peptide, as putative regulator of the
gonadotropic axis. J. Physiol. 2006, 573, 237–249.
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Curtis, A. E.; Ghatei, M. A.; Bloom, S. R. Pyroglutamylated RFamide
peptide 43 stimulates the hypothalamic-pituitary-gonadal axis via gonado-
tropin-releasing hormone in rats. Endocrinology 2008, 149, 4747–4754.
(16) Egido, E. M.; Hernꢀandez, R.; Leprince, J.; Chartrel, N.; Vaudry,
H.; Marco, J.; Silvestre, R. A. 26RFa, a novel orexigenic neuropeptide,
inhibits insulin secretion in the rat pancreas. Peptides 2007, 28, 725–730.
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Symbols and abbreviations are in accord with recommendations
of the IUPAC-IUB Joint Commission on Biochemical Nomen-
clature: Nomenclature and Symbolism for Amino Acids and
Peptides. Biochem. J. 1984, 219, 345ꢀ373 and A Short Guide to
Abbreviations and Their Use in Peptide Science. J. Pept. Sci.
2003, 9, 1ꢀ8. In our manuscript, all optically active amino acids
are of the L configuration unless otherwise noted.
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