Replacement of Thr32 and Gln34 in the C -terminal neuropeptide y fragment 25-36 by cis -cyclobutane and cis -cyclopentane β-amino acids shifts selectivity toward the Y4 receptor

Article abstract of DOI:10.1021/jm4008505

Neuropeptide Y (NPY) and pancreatic polypeptide (PP) control central and peripheral processes by activating the G protein coupled receptors Y _xR (x = 1, 2, 4, 5). We present analogs of the C-terminal fragments 25-36 and 32-36 of NPY and PP containing (1R,2S)-cyclobutane (βCbu) or (1R,2S)-cyclopentane (βCpe) β-amino acids, which display exclusively Y₄R affinity. In particular, [βCpe³⁴]-NPY-(25-36) is a Y₄R selective partial agonist (EC₅₀ 41 ± 6 nM, E_max 71%) that binds Y₄R with a K_i of 10 ± 2 nM and a selectivity >100-fold relative to Y₁R and Y₂R and >50-fold relative to Y₅R. Comparably, [Y ³², βCpe³⁴]-NPY(PP)-(32-36) selectively binds and activates Y₄R (EC₅₀ 94 ± 21 nM, E_max 73%). The NMR structure of [βCpe³⁴]-NPY-(25-36) in dodecylphosphatidylcholine micelles shows a short helix at residues 27-32, while the C-terminal segment R³³βCpe³⁴R³⁵Y ³⁶ is extended. The biological properties of the βCbu- or βCpe-containing NPY and PP C-terminal fragments encourage the future application of these β-amino acids in the synthesis of selective Y ₄R ligands.

Full text of DOI:10.1021/jm4008505

Article
pubs.acs.org/jmc
Replacement of Thr32 and Gln34 in the C‑Terminal Neuropeptide Y
Fragment 25−36 by cis-Cyclobutane and cis-Cyclopentane β‑Amino
Acids Shifts Selectivity toward the Y4 Receptor
Łukasz Berlicki,†,‡,# Melanie Kaske,§,# Raquel Gutierrez-Abad,†,∥ Gunther Bernhardt,§ Ona Illa,∥
́
̈
Rosa M. Ortuno,∥ Chiara Cabrele,⊥ Armin Buschauer,*,§ and Oliver Reiser*,†
̃
†Institute of Organic Chemistry, University of Regensburg, Universitatsstrasse 31, 93053 Regensburg, Germany
‡Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze Wyspian
̈
skiego 27, 50370
̇
Wrocław, Poland
§Institute of Pharmacy, University of Regensburg, Universitatsstrasse 31, 93053 Regensburg, Germany
̈
∥Departament de Química, Universitat Auton
̀
oma de Barcelona, 08193 Cerdanyola del Valles
⊥Department of Molecular Biology, University of Salzburg, Billrothstrasse 11, 5020 Salzburg, Austria
̀
, Spain
S
* Supporting Information
ABSTRACT: Neuropeptide Y (NPY) and pancreatic polypep-
tide (PP) control central and peripheral processes by activating
the G protein coupled receptors YxR (x = 1, 2, 4, 5). We present
analogs of the C-terminal fragments 25−36 and 32−36 of NPY
and PP containing (1R,2S)-cyclobutane (βCbu) or (1R,2S)-
cyclopentane (βCpe) β-amino acids, which display exclusively
Y4R affinity. In particular, [βCpe34]-NPY-(25−36) is a Y4R
selective partial agonist (EC50 41 6 nM, Emax 71%) that binds
Y4R with a Ki of 10 2 nM and a selectivity >100-fold relative
to Y1R and Y2R and >50-fold relative to Y5R. Comparably, [Y32,
βCpe34]-NPY(PP)-(32−36) selectively binds and activates Y4R (EC50 94 21 nM, Emax 73%). The NMR structure of [βCpe34]-
NPY-(25−36) in dodecylphosphatidylcholine micelles shows a short helix at residues 27−32, while the C-terminal segment
R33βCpe34R35Y36 is extended. The biological properties of the βCbu- or βCpe-containing NPY and PP C-terminal fragments
encourage the future application of these β-amino acids in the synthesis of selective Y4R ligands.
function21 and gastrointestinal regulation.22 Recently, Y4R-
INTRODUCTION
■
preferring agonists have been proposed as antiobesity
agents.23−27 Moreover, Y4R is involved in the control of
emotional and stress-related behavior.28,29 For example,
similarly to Y2R knockout animals, Y4R knockout mice have
shown attenuated anxiety and depression states.30−32 Impor-
tantly, Y4R knockout does not impair object memory, which,
instead, was observed in the case of Y2R deletion. For this
reason, the Y4R subtype might become an interesting target for
drugs regulating behavioral homeostasis.32 Finally, the Y5R
subtype, which is expressed in the hippocampus, induces food
intake and is involved in the control of seizures.33,34
Potent and selective agonists and antagonists are available for
Y1R, Y2R, and Y5R subtypes, whereas the number of known
selective Y4R ligands is very low.3,10 Two acylguanidines, N-{[3-
(1H-imidazol-4-yl)propylamino](amino)methylene}-3-cyclo-
hexylpropanamide (UR-AK49) and N-(amino(3-(pyridin-2-
yl)propylamino)methylene)-3,3-diphenylpropanamide (UR-
PI284), were identified as very weak Y4R antagonists (Ki
Neuropeptide Y (NPY) and its closely related analogs
pancreatic polypeptide (PP) and peptide YY (PYY) are
neurohormones that activate G protein coupled receptors,
designated as NPY receptors.1−3 NPY, which was first isolated
from porcine brain (pNPY),4,5 is one of the most abundant
neuropeptides in the central nervous system. PP and PYY were
isolated from pancreas and intestine, respectively.6,7 In humans,
the central and peripheral biological effects of the members of
the NPY family are mediated by four receptor subtypes,
designated as Y1, Y2, Y4, and Y5 receptors (YxR, x = 1, 2, 4,
5).8−10 The Y1R subtype is mainly expressed in blood vessels,
the cerebral cortex, thalamus, and amygdala, mediates vaso-
constriction,11 and is involved, for example, in anxiolysis12 and
the regulation of food intake.13 It has attracted special attention
due to its expression in various tumor entities such as human
breast cancer.14−16 The Y2R subtype is found in sympathetic
and parasympathetic nerve fibers, the hippocampus, the
intestine, and certain blood vessels and is implicated in
memory retention,17 epilepsy, and regulation of neuro-
transmitter release.18−20 The Y4R subtype is expressed in the
heart, intestine, colon, and pancreas and is related to cardiac
Received: June 8, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm4008505 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
values in the two-digit micromolar range).35,36 Potent Y4R
antagonists are not reported at all. The Y4R subtype is the only
Y receptor preferring PP to NPY and PYY. Several peptides
with agonistic activity at Y4R are known, but none of them is
selective. For example, 3 (GR23118, GW1229, or 1229U91),37
the decapeptide [WR-Nva-RY]2-NH2 (4),38 and the oxidized
form of CWR-Nva-RY-NH2 (5)38 act as agonists at Y4R and as
antagonists at Y1R (Figure 1). A series of dimeric pentapeptides
Y4R-preferring peptides. The use of such unnatural building
blocks as constituents of peptides is attractive since it can
greatly improve the stability of the resulting peptide ligands
toward proteases. On the basis of our experience with
cyclobutane- and cyclopentane-based amino acids, we used
the following three cis-configured building blocks: (1R,2S)-2-
aminocyclobutanecarboxylic acid (βCbu), (1R,3S)-3-amino-
2,2-dimethylcyclobutanecarboxylic acid (γCbu), and (1R,2S)-
2-aminocyclopentanecarboxylic acid (βCpe) (Figure 2).
Figure 2. Chemical structures of βACC42 and the β- and γ-amino acids
used in this work for α-amino acid replacement in the C-terminal
fragment 25−36 of hNPY and hPP.
RESULTS AND DISCUSSION
Figure 1. Amino acid sequences of human NPY and hPP and
structures of 3 and several dimeric pentapeptides reported in the
literature26,37,38 as Y4R agonists. Identical residues in the sequences of
hNPY and hPP and the corresponding amino acids in peptides 3−7
are represented by bold letters. The most important residues of hNPY
for high Y4R affinity, as suggested by a previous Ala-scan study,3 are in
red. Dpr = 2,3-diaminopropionic acid. Nva = norvaline.
■
Design. It was shown recently that C-terminal fragments of
NPY (25−36 or 28−36) are valuable scaffolds for NPY
receptor ligands with different subtype selectivity upon
appropriate amino acid substitutions.42,48 In particular, non-
conservative replacement of T32 and Q34 may significantly
modify the NPY receptor selectivity profile. Examples are Aib32,
tolerated only at Y5R,49 P34, tolerated at Y1R, Y4R, and Y5R but
not at Y2R,3 L34, tolerated at Y4R,3 and the constrained β-amino
acid (1R,2R,3R)-βACC, which has been used to develop the
Y1R ligands [βACC34]-NPY-(25−36), [βACC32,34]-NPY-(25−
36), and [R31, βACC32,34]-NPY-(25−36).42 In the present
work, the peptide design was based on the NPY C-terminal
fragment 25−36. Additionally, considering the preference of
hPP for Y4R, the hPP fragment 25−36 was applied as a
putatively superior scaffold compared to the C-terminal
dodecapeptide of NPY toward the development of Y4R
selective ligands. However, as hPP contains the air-sensitive
residue methionine at position 30, we used the analog
containing norleucine (Nle30) instead of methionine, in order
to avoid spontaneous peptide oxidation. Furthermore, the
hNPY and hPP peptide scaffolds were truncated to the C-
terminal five residues 32−36 to explore the downsizing of the
ligand to the minimal structure (Table 1). We focused on the
replacement of positions 34 and/or 32 in hNPY and hPP and
designed new analogs of hNPY-(25−36) and hPP-(25−36) as
well as of hNPY-(32−36) and hPP-(32−36) containing cyclic
building blocks, namely, the cyclobutane β- and γ-amino acids
(1R,2S)-βCbu and (1R,3S)-γCbu, the latter being the smallest
possible cyclic γ-amino acid, and the cyclopentane β-amino acid
(1R,2S)-βCpe (Figure 2 and Table 1).
(6a−c) exhibits agonistic activity only at Y4R, but it also binds
to Y1R (Figure 1).26 The related compound 7 shows high
affinity and agonistic activity at Y4R (Ki 0.05 nM, EC50 14.8
nM), but it is also an agonist at Y1R (Ki 7.5 nM, EC50 240
nM).26 Obviously, the design of compounds discriminating
between Y1R and Y4R is challenging.
The natural peptide hPP remains by and large unsurpassed as
an Y4R ligand in terms of affinity and agonistic potency (Ki 0.53
nM, EC50 11 nM).35,36 However, it can also activate Y1R and
Y5R.39 In contrast, NPY is a highly potent agonist of Y1R, Y2R,
and Y5R and a moderate agonist of Y4R.36,39
Comparison of the sequences of hNPY and hPP reveals an
identity of 47%; in particular, the segment 25−36 is
characterized by identical amino acids and conservative
exchanges (H/R, L/M, I/L), with the exception of position
34, which is occupied by Q in hNPY and P in hPP (Figure 1).
On the basis of a previous Ala-scan study3 of pNPY, which
differs from hNPY only at position 17 (L instead of M), the
residues that are important for Y4R binding are located in the
C-terminal region and are conserved in hPP as well (Y20,27,36
,
R25,33,35, T32).
Along with our work on the synthesis of cyclic β- and γ-
amino acids and their use for the development of bioactive
peptide mimics and foldamers,40−47 we previously succeeded in
Synthesis. Solid-phase peptide-chain assembly was carried
out manually or on an automated peptide synthesizer, using
Fmoc chemistry and a double-coupling procedure with HBTU/
HOBt/DIPEA. As NPY and PP are C-terminally amidated
peptides, the syntheses were performed on a Rink-amide
MBHA polystyrene/1% divinylbenzene resin. All Nα-Fmoc-
amino acids were employed with the following side-chain
conferring Y1R affinity (Ki 37
20 nM) to the non-Y1R-
binding NPY fragment 25−36 by replacing Q34 with
(1R,2R,3R)-3-(methoxycarbonyl)cyclopropane β-amino acid
(βACC).42 In the present work we report on an approach to
shift the selectivity profile of the C-terminal fragments of hNPY
and hPP by introducing cyclic unnatural amino acids, aiming at
B
dx.doi.org/10.1021/jm4008505 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
the corresponding benzyl ester. The Boc group was then
cleaved with HCl, followed by the coupling of Fmoc-R(Pmc)-
OH with PyBOP/DIPEA and, finally, by hydrogenation for the
removal of the benzyl group.
After peptide-chain assembly the N-terminus was acetylated,
then the peptides were simultaneously cleaved from the resin
and deprotected by using TFA with 10% scavengers. The crude
products were precipitated from ether, recovered by
centrifugation, and purified by preparative RP-HPLC (>95%
purity, as determined by analytical RP-HPLC).
Pharmacological Activity. The NPY receptor binding
profiles of all synthesized peptides were determined by flow
cytometry, in accordance to previously described proce-
dures,35,56−58 on HEL cells expressing Y1R, CHO cells
expressing Y2R or Y4R, and HEC-1B cells expressing Y5R, by
displacement of the fluorescent ligands Cy5-[K4]hPP35 (for
Y4R) or Cy5-pNPY56 (for Y1R, Y2R, Y5R) (Table 2 and Figure
3).57,58
The functional Y4R activity of the ligands with nanomolar
affinity was determined in the GTPase assay on Sf9 cell
membranes expressing human Y4R, Gαi2, and Gβ1γ2.36 This
Y4R assay was performed in the agonist mode and, for selected
compounds (8, 10, 13, 16), also in the antagonist mode.
However, all investigated peptides revealed only agonistic
activity (Figure 4). Potent Y4R antagonists are not available to
confirm a Y4R mediated response. Therefore, control experi-
ments were performed on membranes devoid of Y4R, only
expressing the G proteins (Gαi2 and Gβ1γ2), in order to exclude
receptor-independent G protein activation. In contrast to
mastoparan, which is a direct G protein activator59−61 and was
thus used as positive control, the peptides did not promote
GTPase activity at the concentrations of interest (data not
shown).
Table 1. Amino Acid Sequences of the Synthesized Peptides
compd
no.
a
a
name
sequence
8
NPY-(25−36)
Ac-RHYINLITRQRY-NH2
Ac-RHYINLITRLRY-NH2
Ac-RHYINLITR⬟RY-NH2
Ac-RHYINLITR■RY-NH2
Ac-RHYINLITR⧫RY-NH2
Ac-RHYINLI⬟R⬟RY-NH2
Ac-RHYINLR■R■RY-NH2
Ac-RHYINLI⧫R⧫RY-NH2
Ac-RRYINXLTRPRY-NH2
Ac-RRYINXLTR⬟RY-NH2
Ac-RRYINXLTR■RY-NH2
Ac-RRYINNLTR■RY-NH2
Ac-TRLRY-NH2
9
[L34]-NPY-(25−36)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
[⬟34]-NPY-(25−36)
[■34]-NPY-(25−36)
[⧫34]-NPY-(25−36)
[⬟32,34]-NPY-(25−36)
[R31, ■32,34]-NPY-(25−36)
[⧫32,34]-NPY-(25−36)
[X30]-hPP-(25−36)
[X30, ⬟34]-hPP-(25−36)
[X30, ■34]-hPP-(25−36)
[N30, ■34]-hPP-(25−36)
[L34]-NPY(PP)-(32−36)
[⬟34]-NPY(PP)-(32−36)
[Y32, L34]-NPY(PP)-(32−36)
[Y32, ⬟34]-NPY(PP)-(32−36)
Ac-TR⬟RY-NH2
Ac-YRLRY-NH2
Ac-YR⬟RY-NH2
a
X, Nle; ■, βCbu; ⧫, γCbu; ⬟, βCpe.
protections: Tr for N and H; t-Bu for Y, T, and D; and Pbf or
Pmc for R. (1R,2S)-βCpe was prepared according to a
procedure reported in the literature50 and successively Fmoc-
protected at the β-amino group.50,51 Nγ-Fmoc-protected
(1R,3S)-γCbu was synthesized from Nγ-Cbz-γCbu52,53 upon
Cbz removal and introduction of the Fmoc group. (1R,2S)-2-
Aminocyclobutanecarboxylic acid (βCbu) was coupled in the
form of the α,β-dipeptide Fmoc-R(Pmc)-βCbu-OH, in order to
avoid base-mediated Nβ-Fmoc cleavage that results in ring-
opening. By following synthetic protocols developed in our
laboratory for the corresponding β-aminocyclopropanecarbox-
ylic acids,54 this α,β-dipeptide was prepared starting from Nβ-
Boc-βCbu-OH. Briefly, Nβ-Boc-βCbu-OH55 was converted to
Table 2. Binding Data of the Synthesized Peptides at Human Y1R, Y2R, Y4R, and Y5R Subtypes and hY4R Agonistic Activity
a
b
a
a
a
hY4R (CHO)
hY4R (Sf9)
EC50 (nM)
hY1R
hY2R
hY5R
compound
pNPY
Ki (nM)
Emax (%)
Ki (nM)
Ki (nM)
Ki (nM)
c
d
d
e
f
e
1a
8
282 39
417 42
56
0.5 0.2
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
0.8 0.2
1506 65
>1000
2.2 0.8
>1000
>1000
>500
NPY-(25−36)
>1000
530 160
100 24
56
9
[L34]-NPY-(25−36)
157 47
100
71
10
11
12
13
14
15
2
[⬟34]-NPY-(25−36)
[■34]-NPY-(25−36)
[⧫34]-NPY-(25−36)
[⬟32,34]-NPY-(25−36)
[R31, ■32,34]-NPY-(25−36)
[⧫32,34]-NPY-(25−36)
hPP
10
72
2
7
41
6
>1000
122 28
67
>1000
>1000
>1000
>500
g
g
>1000
nd
nd
>1000
37
35
7
48 29
66 26
49
76
>1000
2
>1000
>1000
>1000
17 1.5
>1000
>500
g
g
>1000
nd
nd
>1000
0.53 0.1
11
53
2
8
100
70
70
74
70
97
59
97
73
440 74
>1000
>5000
16
17
18
19
20
21
22
23
[X30]-hPP-(25−36)
75
14
93
70
2
7
4
9
>1000
[X30, ⬟34]-hPP-(25−36)
[X30, ■34]-hPP-(25−36)
[N30, ■34]-hPP-(25−36)
[L34]-NPY(PP)-(32−36)
[⬟34]-NPY(PP)-(32−36)
[Y32, L34]-NPY(PP)-(32−36)
[Y32, ⬟34]-NPY(PP)-(32−36)
51 18
281 89
>1000
217 28
708 107
>1000
284 46
76 21
>1000
>1000
>1000
>1000
>1000
>1000
>1000
490 90
509 160
50 17
291 48
451 241
>1000
>1000
>1000
>1000
65
9
>1000
>1000
105 42
94 21
>1000
>1000
a
Flow cytometry binding assay using Cy5-[K4]hPP (3 nM, Kd = 5.62 nM) at CHO-hY4R-Gqi5-mtAEQ cells or Cy5-pNPY (5 nM, Kd = 4.4−5.5 nM)
at HEL-hY1R, CHO-hY2R, and HEC-1B-Y5R cells. Data represent mean values SEM of two or three independent experiments performed in
b
triplicate. Functional [γ-33P]GTPase assay at Sf9 cell membranes expressing hY4R-Gαi2-Gβ1γ2-RGS4; the maximal response (Emax) of hPP was set to
100%, and the intrinsic activities of other compounds were referred to this value. Data represent mean values SEM of two or three independent
c
d
e
f
g
experiments performed in duplicate. Reference 35. Reference 36. Reference 56. Reference 57. Not determined.
C
dx.doi.org/10.1021/jm4008505 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 3. Binding curves of hPP (2) and the synthesized peptides 9−11, 13, 14, and 16−23 on human Y4R obtained by flow cytometry on CHO-
hY4R-Gqi5-mtAEQ cells using Cy5-[K4]hPP (3 nM) as fluorescent ligand and compound 3 (1 μM) to determine unspecific binding; log c on the x-
axis refers to the decadic logarithm of the molar ligand concentration.
Whereas literature reports that NPY-(25−36) retains Y2R
binding affinity in the nanomolar range, as determined in
radioligand binding assays on LN319 cells (IC50 132 nM),62
rabbit kidney (Ki 10.2 nM),63 and SMS-KAN cell membranes
(Ki 7.1 nM),63 a submicromolar Ki value was not confirmed for
compound 8 by flow cytometry using CHO cells expressing
hY2R (Table 2). Y4R binding and partial agonism of 8 were
weak. By contrast, compound 16 was a Y4R partial agonist (Ki
nM (13)]. In contrast, compared to analog 11 containing the
monosubstitution βCbu34, compound 14 containing two βCbu
units (βCbu32,34) and R31 instead of I31 was superior by a factor
of 2 both in Y4R affinity (Ki 35
2 vs 72
7 nM) and
agonistic potency. Previously, we observed an analogous
behavior for the two βACC-containing Y1R ligands
[βACC34]-NPY-(25−36) and [R31, βACC32,34]-NPY-(25−36):
the latter binds almost 2-fold stronger to Y1R (Ki of 29 13 vs
50 10 nM) and gains Y5R affinity (Ki of 118 vs 617 nM).42
The C-terminal NPY(PP) pentapeptides 20 and 21
containing L34 or βCpe34, respectively, retained Y4R affinity
(Ki about 0.5 μM) and acted as agonists in the submicromolar
range. A second modification, namely, the replacement of T32
by the sterically more demanding Y (compound 22), enhanced
Y4R binding affinity and agonistic potency by a factor of almost
10 and 5, respectively. Compound 22 can be considered the
monomeric form of the covalent dimer 7 (Figure 1), which has
been previously reported by Balasubramaniam et al.26 as an
agonist with picomolar affinity and nanomolar potency at Y4R,
and with nanomolar affinity and potency at Y1R, as mentioned
in the Introduction. Replacement of L34 with βCpe (23) was
well-tolerated, causing only a minor decrease in agonistic
potency [EC50 65 9 nM (22) vs 94 21 nM (23)]. To the
best of our knowledge, these two analogs of NPY(PP)32−36
with the double substitution [Y32, L34] (22) or [Y32, βCpe34]
(23) represent the smallest Y4R selective peptide agonists with
75
2 nM, EC50 53
8 nM with 70% maximal response
compared to hPP). Interestingly, contrarily to compound 8,
compound 9 not only displayed moderate Y4R binding (Ki of
157 47 nM) but also full agonistic activity (EC50 100 24
nM). However, the presence of an aliphatic α-residue at
position 34 (i.e., P or L) favors Y4R binding but it is not a
guarantee of Y4R selectivity; indeed, the NPY fragment 25−36
containing L34 and an additional replacement at position 32 (Y
in place of T) was reported to be a Y2R ligand (IC50 12 nM).64
The monosubstituted hNPY and hPP analogs containing
βCpe34 (10, 17) displayed about 7-fold higher affinity and were
3−5-fold more potent Y4R agonists than the corresponding
analogs containing βCbu34 (11, 18). The introduction of a
second βCpe unit at position 32 (13) was accompanied by an
about 4-fold decrease in affinity (Ki from 10 2 to 37 7 nM)
and by a reduction of the maximal response (from 71% to 49%)
in the functional assay, whereas the agonistic potency was not
significantly affected [EC50 from 41 6 nM (10) to 48 29
D
dx.doi.org/10.1021/jm4008505 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 4. Y4R agonistic activity of hPP (2) and the synthesized peptides 8−11, 13, 14, and 16−23 obtained in steady-state GTPase assays36 at Sf9
cell membranes expressing hY4R, Gαi2, and Gβ1γ2. The increase in GTPase activity is referred to the maximal response induced by hPP (100 nM);
log c on the x-axis refers to the decadic logarithm of the molar ligand concentration.
nanomolar potency (EC50 values 65 nM and 94 nM,
respectively) known so far.
NOESY spectra. The identification of a high number of
sequential and medium-range interproton contacts (Figure 6)
indicates that the peptide adopts a well-defined three-
dimensional structure. Contacts of type Hα-HN (i, i + 2),
Hα-HN (i, i + 3), and Hα-HN (i, i + 4) over the segment Y27-
T32 strongly support the presence of a helical structure in this
region.
Molecular dynamics calculation with simulated annealing
protocol and constraints derived from the NMR measurements
(42 NOE contacts including 22 nonsequential ones, table S3,
Supporting Information) gave a family of 50 structures: 20
structures with the lowest energy are shown in Figure 7C.
RMSD values for the backbone heavy atoms go from 1.03 Å for
βCpe34 to 2.35 Å for Y36, supporting the existence of a well-
defined conformation. The fragment from Y27 to T32 is helical,
whereas the C-terminal part is extended.
The previously reported NMR solution structures of full-
length pNPY67 and bovine PP (bPP)68 bound to DPC micelles
display an amphipathic helix that extends up to Y36 or T32,
respectively (Figure 7A,B). Although it is not surprising that the
C-terminal motif of the Y4R α,β-peptide agonist 10 structurally
resembles that of bPP (with P34) much more than that of pNPY
(with Q34), it is worthwhile to note that the C-terminal
tetrapeptide R33βCpe34R35Y36-NH2 is quite rigid (RMSD of
2.35 Å for Y36), whereas the α-amino acid segment 32−36 of
bPP is rather flexible (RMSD of 5.0 Å for Y36). We suggest that
the rigid C-terminal α,β-peptide agonist mimics the con-
formation of the Y4R-bound native ligand PP and shifts the
NPY receptor subtype selectivity toward the Y4R.
Conformation of the Y4R Selective Peptide Ligands. In
order to gain insights into the conformation of the Y4R selective
ligands, the peptides were investigated by CD and NMR
spectroscopy. The peptides were flexible in phosphate buffer
(100 mM, pH 7), as indicated by the presence of a negative CD
band below 200 nm and weak CD contribution in the region
205−230 nm (Figure 5). However, upon addition of 30% TFE,
which is a well-known structure-inducing cosolvent, the α-
peptides 8 and 9 adopted a well-defined helical structure, as
shown by the presence of the CD maximum around 195 nm
and of two minima close to 208 and 220 nm (Figure 5A).
Instead, the α-peptide 16, which contains the helix breaker P at
position 34, and the α,β-peptides were significantly less helical,
as shown by the weak intensity of the band at 220 nm as well as
by a slight blue-shift of the band at 208 nm. This suggests that
the cyclic β-amino acid at position 34 acts as helix breaker,
similarly to P. Accordingly, the α,β-peptides containing two β-
amino acids at positions 32 and 34 were even less helical than
the parent analogs with one β-amino acid at position 34, as
indicated by the weakening of the negative shoulder around
220 nm and the disappearance of the positive CD band around
195 nm (Figure 5A). Therefore, peptide 16 and the α,β-
peptides are likely to build a short N-terminal helix (probably
both α-helix and 310-helix65 that coexist) which propagates until
the helix-breaking P or β-residue.
NMR Solution Structure of Compound 10. It was
suggested that the members of the NPY family bind first to the
plasma membrane and then to the receptor.66 Therefore, we
investigated the NMR solution structure of the more potent
Y4R α,β-peptide agonist (10) in membrane-mimicking micelles
of deuterated dodecylphosphatidylcholine (DPC-d38). Assign-
ment of the crucial resonances was done by using TOCSY and
CONCLUSIONS
■
We have shown that βCbu and βCpe are suitable building
blocks to obtain short peptides with high Y4R selectivity and
agonistic activity. In particular, the NPY fragment 25−36
E
dx.doi.org/10.1021/jm4008505 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 7. Comparison of the solution NMR structures of (A) pNPY
(PDB ID 1F8P),67 (B) bPP (PDB ID 1LJV),68 and (C) compound 10.
For pNPY and bPP, only the C-terminal segment 25−36 is shown for
reasons of clarity. The ensemble of the 20 lowest energy structures is
presented by tubes and one structure is drawn by stick representation.
agonistic activity in the nanomolar range. This suggests that
downsizing of peptidic structures is a promising strategy to
refine the structure−activity relationships of subtype-selective
NPY receptor ligands. This may also pave a way to Y4R
antagonists.
EXPERIMENTAL SECTION
■
General. pNPY, hPP, [K4]-hPP, and the Cy5-labeled analogs of
pNPY and [K4]-hPP were prepared in our laboratory as described
previously.35,56 Compound 3 was a gift from Dr. A. J. Daniels, Glaxo
Wellcome Inc.. (1R,2S)-βCpe was synthesized by following a protocol
reported in the literature49 and protected at the amino group with the
Fmoc group.50,51 Unless otherwise indicated, chemicals, buffers, and
reagents were purchased from Merck (Darmstadt, Germany). HEPES
and bovine serum albumin (BSA) were purchased from Serva
(Heidelberg, Germany). FCS was obtained from Biochrom (Berlin,
Germany), Cy5-succinimidyl ester from Amersham Biosciences (Little
Chalfont, UK). Rink-amide MBHA resin (loading 0.45 mmol/g),
Fmoc-protected amino acids, HBTU, HOBt, DIPEA, and NMP were
purchased from IRIS Biotech (Marktredwitz, Germany). Piperidine,
thioanisole, and ethane-1,2-dithiol were from Sigma Aldrich (Munich,
Germany). DMF, DCM, and diethyl ether were obtained from Acros
Organics (Geel, Belgium). Preparative HPLC peptide purification was
performed using a Phenomenex Luna C18 column (100 Å, 250 mm ×
21.2 mm) with an Agilent 1100 LC system (flow rate 21 mL/min).
The elution system consisted of two eluents: (A) 0.0059% TFA in
water and (B) acetonitrile. Analytical HPLC of purified peptides was
performed on C18 column (100 Å, 100 mm × 2 mm) with an Agilent
1100 LC system coupled with a ThermoQuest Finnigan TSQ 7000
mass spectrometer using 0.1% formic acid in water and 0.1% formic
acid in acetonitrile as eluents. High-resolution mass spectra were
measured with a Waters LCT Premier XE spectrometer (electrospray
ionization). Stock solutions of the peptides (1−2 mM) for binding and
functional studies were prepared in H2O (Millipore) and stored at
−20 °C.
Peptide Synthesis. The α-peptides and the α,β(γ)-peptides
containing βCpe or β(γ)Cbu were obtained using manual or
automated solid phase peptide synthesis on a Syro I synthesizer
(Multisyntech, Witten, Germany). Rink amide MBHA resin (loading
0.45 mmol/g) was used. Double couplings (2 × 40 min) with 4 equiv
of Fmoc-amino acid/HOBt/HBTU and 8 equiv of DIPEA were
applied. Fmoc cleavage was performed with piperidine in DMF (40%
for 3 min and 20% for 10 min). After N-terminal acetylation with
acetic anhydride and DIPEA (10 equiv each) in DMF for 30 min, the
peptides were side-chain deprotected and simultaneously cleaved from
the resin by using the mixture TFA/TIS/water (90/5/5, v/v/v) for 3
h. Precipitation from ice-cold diethyl ether and recovery by
centrifugation at 3 °C for 5 min gave the crude products that were
purified by preparative reverse-phase HPLC. The lyophilized peptides
were >95% pure, based on analytical HPLC, and were identified by
mass spectrometry (Table S1, Supporting Information).
Figure 5. CD spectra of the synthesized peptides 8−10, 13, 16, and
17. Peptide concentration was 100 μM in phosphate buffer (100 mM,
pH 7) with or without 30% TFE.
Figure 6. Summary of sequential and medium-range NOE signals for
compound 10 in a membrane-mimicking environment of DPC-d38
micelles (1.5 mM 10, 150 mM DPC-d38 in H2O/D2O, 9:1 mixture).
The thickness of the lines indicates the different intensities of the NOE
cross-peaks.
containing βCpe at position 34 (10) is superior to NPY as an
Y4R (partial) agonist and only about 4-fold less potent than the
native Y4R agonist, hPP, in the GTPase assay. The
conformation of the Y4R α,β-peptide agonist in the presence
of PDC micelles shows the presence of a short N-terminal helix
spanning residues 27−32, followed by the C-terminal
tetrapeptide R33βCpe34R35Y36-NH2 in an extended conforma-
tion, which is apparently fulfilling the structural requirement for
selective Y4R binding and activation. Further, searching for
small-size peptide ligands, we have found that the modified C-
terminal NPY(PP) motif 32−36, XRZRY, with X/Z equal to Y/
L or Y/βCpe, is sufficient for selective Y4R binding and
F
dx.doi.org/10.1021/jm4008505 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Cell Culture. Except for HEL (human erythroleukemia) cells,
which constitutively express functional Y1R and are widely accepted as
a standard model for such receptor subtype, all other cells were
genetically engineered to stably express the receptor of interest. HEL
cells expressing hY1R,69 HEC-1-B-hY5R cells,70 and CHO-hY2R/-hY4R
cells35,57 were maintained as described elsewhere. HEL cells
(suspension) were passaged in RPMI with FCS (5%). HEC-1-B-Y5R
cells were cultured in EMEM with FCS (10%) and G418 (400 μg/
mL) as described previously.70 CHO cells expressing Y2R or Y4R were
grown in Ham’s F12 medium supplemented with FCS (10%) and
G418 (400 μg/mL), hygromycin (400 μg/mL), and zeocin (200 μg/
mL).35,57
Flow Cytometric Binding Assay. The peptides were investigated
for binding to the Y4R and the other receptor subtypes (Y1R, Y2R, and
Y5R) by flow cytometry using Cy5-[K4]hPP35 (3 nM) or Cy5-pNPY56
(5 nM), respectively, as fluorescent receptor ligands. For all
experiments, HEPES buffer (25 mM, pH 7.4) containing CaCl2·
2H2O (2.5 mM) and MgCl2 (1.5 mM) was supplemented with bovine
serum albumin (BSA, 1%) and bacitracin (0.1 mg/L). All binding
assays were performed in a final volume of 500 μL. Saturation binding
assays were performed in the presence of increasing concentrations of
peptides. Unspecific binding was determined by addition of an excess
(1 μM) of either compound 3 for Y4R or pNPY for Y1R, Y2R, and Y5R
(pNPY is only a weak binder for Y4R). The samples were incubated in
“siliconized” (Sigmacote, Sigma-Aldrich, Taufkirchen, Germany)
Eppendorf reaction vessels at rt for 90−120 min and analyzed by a
FacsCaliburTM flow cytometer (Becton Dickinson, Heidelberg,
Germany). The procedure and the instrument settings were described
in detail previously.35,56−58
of 1.0 mm path length. Measurements were performed at room
temperature scanning a wavelength range from 260 nm to 190 nm.
The bandwidth was 1.0 nm, the sensitivity 20 mdeg, and the scan
speed 20 nm/min. The background spectrum (phosphate buffer or the
phosphate buffer/TFE mixture) was subtracted from each spectrum.
The compounds were dissolved at a concentration of 100 μM in
phosphate buffer (pH 7) or phosphate buffer/TFE mixture (70:30 v/
v). The peptide concentrations were determined by UV spectroscopy
by using the extinction coefficient of Y at 280 nm (1490 M−1 cm−1).
The molar ellipticity is expressed as the mean residue ellipticity [θ]R in
deg cm2 dmol−1.
NMR Measurements. All measurements were performed on a
Bruker 600 Avance spectrometer equipped with a cryo-probe and
operating at 600.25 MHz (1H). The peptide sample was 1.5 mM in
H2O/D2O (90:10, v/v) mixture containing DPC-d38 (150 mM). 1D-
NMR, COSY, TOCSY, and NOESY spectra were recorded at 310 K.
The TOCSY experiment was performed with homonuclear Hartman−
Hahn transfer with the MLEV17 sequence, 80 ms mixing time, and
eight scans. The NOESY experiment was done with a mixing time of
200 ms and 32 scans. For all the two-dimensional spectra 2K time
domain points (F2) and 512 increments (F1) were applied.
Structure Calculation. Cross-peaks in the NOESY spectrum were
integrated by using the TopSpin 2.0 program (Bruker). Upper limits of
interproton distances were calculated from measured volume integrals
(plus 10%). Molecular dynamics calculation using simulated annealing
protocol with NMR restraints (20 sequential and 22 nonsequential
contacts) was performed using the XPLOR-NIH program.72,73
ASSOCIATED CONTENT
* Supporting Information
■
GTPase Assay. The steady state GTPase activity assay was
essentially performed as described previously,36,61,71 except for using
[γ-33P]GTP instead of [γ-32P]GTP.
S
MS data of the peptides; NMR signal assignments and
nonsequential NOE contacts for structure calculation of 10;
synthetic schemes for Fmoc-R(Pmc)-(1R,2S)-βCbu-OH and
Membrane Preparation. Sf9 cells were infected with a dilution of
high-titer hY4R and Gαi2/Gαo baculovirus stocks. After culturing for
24−48 h, the cells were lysed using 1 mM ethylenediaminetetraacetic
acid (EDTA) (Merck, Darmstadt, Germany), 0.2 mM phenyl-
methylsulfonyl fluoride (Sigma, Munich, Germany), 10 μg/mL
benzamide (Sigma, Munich, Germany), and 10 μg/mL leupeptin
(Calbiochem, Darmstadt, Germany) as protease inhibitors in 10 mM
Tris-HCl (pH 7.4), followed by multiple homogenization and
centrifugation steps. For storing at −80 °C, the membranes were
suspended in binding buffer (12.5 mM MgCl2, 1 mM EDTA, and 75
mM Tris-HCl, pH 7.4) at 0.5−1.0 mg protein/mL.
Assay Protocol. Membranes were thawed, sedimented, and
resuspended in Tris-HCl (10 mM, pH 7.4). The assay tubes contained
Sf9 membranes expressing hY4R-Gαi2/Gαo fusion proteins (10 μg
protein/tube in 20 μL), 1.0 mM MgCl2, 0.1 mM EDTA, 0.1 mM ATP,
100 nM GTP, 0.1 mM adenylyl imidodiphosphate, 5 mM creatine
phosphate, 40 μg creatine kinase, 0.2% (w/v) BSA in Tris-HCl (50
mM, pH 7.4, 50 μL), and Y4R agonists at various concentrations (10
μL). After incubation of the reaction mixtures (80 μL) at 25 °C for 2
min, 20 μL of [γ-33P]-GTP (0.1 μCi/tube) was added. All stock and
work solutions of [γ-33P]-GTP were prepared in Tris-HCl (20 mM,
pH 7.4). Reactions were terminated by addition of 900 μL of slurry
consisting of 5% (w/v) activated charcoal and NaH2PO4 (50 mM, pH
2.0). Charcoal-treated reaction mixtures were centrifuged for 7 min at
room temperature at 15 000g, 600 μL of the fluid supernatant of the
reaction mixtures was removed, and 33Pi was determined by liquid
scintillation counting in a LS6500 liquid scintillation counter
(Beckman-Coulter, Munich, Germany) using Optiphase Supermix
(Perkin-Elmer, Rodgau, Germany). Enzyme activities were corrected
for spontaneous hydrolysis of [γ-33P]GTP, which was determined in
tubes containing all components mentioned above plus a very high
concentration of unlabeled GTP (1 mM). In the presence of Sf9
membranes, this high excess of unlabeled GTP prevents enzymatic
hydrolysis of [γ-33P]GTP due to competition with GTP. Spontaneous
hydrolysis of [γ-33P]GTP amounted to <1% of the total amount of
radioactivity added.36,71
1
Nγ-Fmoc-(1R,3S)-γCbu-OH; H and 13C NMR spectra of the
new cyclobutane derivatives. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*A.B.: phone, +49 941 9434827; e-mail, Armin.Buschauer@
chemie.uni-regensburg.de.
■
*O.R.: phone, +49 941 9434631; e-mail, Oliver.Reiser@
chemie.uni-regensburg.de.
Author Contributions
#These authors contributed equally. The manuscript was
written through contributions of all the authors. All authors
have given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by funds from 7th Framework
Programme of European Commission within the ’People’
Specific Programme, the Intra-European Fellowships (grant no
CANPY), the DFG-ANR project RE-948/9-1, and initiated by
the Graduate Training Program (Graduiertenkolleg) GRK1910
of the Deutsche Forschungsgemeinschaft. Financial support
́ ́
from the Spanish Direccion General de Investigacion
(CTQ2010-15408) and Generalitat de Catalunya, Spain
(2009SGR-733) is gratefully acknowledged.
ABBREVIATIONS USED
■
βACC, (1R,2R,2R)-3-(methoxycarbonyl)cyclopropanecarboxy-
lic acid; Aib, aminoisobutyric acid; bPP, bovine pancreatic
polypeptide; βCbu, (1R,2S)-2-aminocyclobutanecarboxylic
CD Measurements. Spectra were recorded with a JASCO
spectropolarimeter (model J-710/720 or J-815) using a quartz cuvette
G
dx.doi.org/10.1021/jm4008505 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(19) Illes, P.; Finta, E. P.; Nieber, K. Neuropeptide Y potentiates via
Y2-receptors the inhibitory effect of noradrenaline in rat locus
coeruleus neurones. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1993,
348, 546−548.
(20) Colmers, W. F.; Klapstein, G. J.; Fournier, A.; St-Pierre, S.;
Treherne, K. A. Presynaptic inhibition by neuropeptide Y in rat
hippocampal slice in vitro is mediated by a Y2 receptor. Br. J.
Pharmacol. 1991, 102, 41−44.
(21) Smith-White, M. A.; Herzog, H.; Potter, E. K. Cardiac function
in neuropeptide Y Y4 receptor-knockout mice. Regul. Pept. 2002, 110,
47−54.
(22) Lin, S.; Shi, Y. C.; Yulyaningsih, E.; Aljanova, A.; Zhang, L.;
Macia, L.; Nguyen, A. D.; Lin, E. J.; During, M. J.; Herzog, H.;
Sainsbury, A. Critical role of arcuate Y4 receptors and the
melanocortin system in pancreatic polypeptide-induced reduction in
food intake in mice. PLoS ONE 2009, 4, e8488.
(23) Kamiji, M. M.; Inui, A. Neuropeptide Y receptor selective
ligands in the treatment of obesity. Endocr. Rev. 2007, 28, 664−684.
(24) Kamiji, M. M.; Inui, A. NPY Y2 and Y4 receptors selective
ligands: Promising anti-obesity drugs? Curr. Top. Med. Chem. 2007, 7,
1734−1742.
(25) Balasubramaniam, A.; Joshi, R.; Su, C.; Friend, L. A.; James, J. H.
Neuropeptide Y (NPY) Y2 receptor-selective agonist inhibits food
intake and promotes fat metabolism in mice: Combined anorectic
effects of Y2 and Y4 receptor-selective agonists. Peptides 2007, 28,
235−240.
acid; γCbu, (1R,3S)-3-amino-2,2-dimethyl-cyclobutanecarbox-
ylic acid; βCpe, (1R,2S)-2-aminocyclopentanecarboxylic acid;
Cy5, cyanine 5; DIPEA, N,N-diisopropylethylamine; DPC,
dodecylphosphocholine; HBTU, O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate; hNPY,
human neuropeptide Y; HOBt, 1-hydroxybenzotriazole; hPP,
human pancreatic polypeptide; Pbf, 2,2,4,6,7-pentamethyldihy-
drobenzofuran-5-sulfonyl; Pmc, 2,2,5,7,8-pentamethylchro-
mane-6-sulfonyl; pNPY, porcine neuropeptide Y; PyBOP,
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluoro-
phosphate; PYY, peptide YY.
REFERENCES
■
(1) Balasubramaniam, A. A. Neuropeptide Y family of hormones:
Receptor subtypes and antagonists. Peptides 1997, 18, 445−457.
(2) Cerda-Reverter, J. M.; Larhammar, D. Neuropeptide Y family of
peptides: Structure, anatomical expression, function, and molecular
evolution. Biochem. Cell Biol. 2000, 78, 371−392.
(3) Cabrele, C.; Beck-Sickinger, A. G. Molecular characterization of
the ligand−receptor interaction of the neuropeptide Y family. J. Pept.
Sci. 2000, 6, 97−122.
(4) Tatemoto, K. Isolation and characterization of peptide YY (PYY),
a candidate gut hormone that inhibits pancreatic exocrine secretion.
Proc. Natl. Acad. Sci. U. S. A. 1982, 79, 2514−2518.
(5) Tatemoto, K.; Carlquist, M.; Mutt, V. Neuropeptide YA novel
brain peptide with structural similarities to peptide YY and pancreatic
polypeptide. Nature 1982, 296, 659−660.
(26) Balasubramaniam, A.; Mullins, D. E.; Lin, S.; Zhai, W.; Tao, Z.;
Dhawan, V. C.; Guzzi, M.; Knittel, J. J.; Slack, K.; Herzog, H.; Parker,
E. M. Neuropeptide Y (NPY) Y4 receptor selective agonists based on
NPY(32−36): Development of an anorectic Y4 receptor selective
agonist with picomolar affinity. J. Med. Chem. 2006, 49, 2661−2665.
(27) Li, J.-B.; Asakawa, A.; Terashi, M.; Cheng, K.-C.; Chaolu, H.;
Zoshiki, T.; Ushikai, M.; Sheriff, S.; Balasubramaniam, A.; Inui, A.
Regulatory effects of Y4 receptor agonist (BVD-74D) on food intake.
Peptides 2010, 31, 1706−1710.
(6) Kimmel, J. R.; Hayden, L. J.; Pollock, H. G. Isolation and
characterization of a new pancreatic polypeptide hormone. J. Biol.
Chem. 1975, 250, 9369−9376.
(7) Tatemoto, K.; Mutt, V. Isolation of two novel candidate
hormones using a chemical method for finding naturally occurring
polypeptides. Nature 1980, 285, 417−418.
(8) Balasubramaniam, A. Clinical potentials of neuropeptide Y family
of hormones. Am. J. Surg. 2002, 183, 430−434.
(28) Morales-Medina, J. C.; Dumont, Y.; Quirion, R. A possible role
of neuropeptide Y in depression and stress. Brain Res. 2010, 1314,
194−205.
(9) Lin, S.; Boey, D.; Herzog, H. NPY and Y receptors: Lessons from
transgenic and knockout models. Neuropeptides 2004, 38, 189−200.
(29) Edelsbrunner, M. E.; Painsipp, E.; Herzog, H.; Holzer, P.
Evidence from knockout mice for distinct implications of neuro-
peptide-Y Y2 and Y4 receptors in the circadian control of locomotion,
exploration, water and food intake. Neuropeptides 2009, 43, 491−497.
(30) Painsipp, E.; Herzog, H.; Holzer, P. Evidence from knockout
mice that neuropeptide-Y Y2 and Y4 receptor signalling prevents long-
term depression-like behaviour caused by immune challenge. J.
Psychopharmacol. 2010, 24, 1551−1560.
(10) Walther, C.; Morl, K.; Beck-Sickinger, A. G. Neuropeptide Y
̈
receptors: Ligand binding and trafficking suggest novel approaches in
drug development. J. Pept. Sci. 2011, 17, 233−246.
(11) Grundemar, L.; Jonas, S. E.; Morner, N.; Hogestatt, E. D.;
Wahlestedt, C.; Hakanson, R. Characterization of vascular neuro-
peptide Y receptors. Br. J. Pharmacol. 1992, 105, 45−50.
(12) Wahlestedt, C.; Pich, E. M.; Koob, G. F.; Yee, F.; Heilig, M.
Modulation of anxiety and neuropeptide Y-Y1 receptors by antisense
oligodeoxynucleotides. Science 1993, 259, 528−531.
(31) Tasan, R. O.; Lin, S.; Hetzenauer, A.; Singewald, N.; Herzog, H.;
Sperk, G. Increased novelty-induced motor activity and reduced
depression-like behavior in neuropeptide Y (NPY)-Y4 receptor
knockout mice. Neuroscience 2009, 158, 1717−1730.
(32) Painsipp, E.; Wultsch, T.; Edelsbrunner, M. E.; Tasan, R. O.;
Singewald, N.; Herzog, H.; Holzer, P. Reduced anxiety-like and
depression-related behavior in neuropeptide Y Y4 receptor knockout
mice. Genes Brain Behav. 2008, 7, 532−542.
(33) Marsh, D. J.; Hollopeter, G.; Kafer, K. E.; Palmiter, R. D. Role of
the Y5 neuropeptide Y receptor in feeding and obesity. Nat. Med.
1998, 4, 718−721.
(34) Marsh, D. J.; Baraban, S. C.; Hollopeter, G.; Palmiter, R. D. Role
of the Y5 neuropeptide Y receptor in limbic seizures. Proc. Natl. Acad.
Sci. U. S. A. 1999, 96, 13518−13523.
(35) Ziemek, R.; Schneider, E.; Kraus, A.; Cabrele, C.; Beck-
Sickinger, A. G.; Bernhardt, G.; Buschauer, A. Determination of affinity
and activity of ligands at the human neuropeptide Y Y4 receptor by
flow cytometry and aequorin luminescence. J. Recept. Signal Transduct.
2007, 27, 217−233.
(36) Pop, N.; Igel, P.; Brennauer, A.; Cabrele, C.; Bernhardt, G.;
Seifert, R.; Buschauer, A. Functional reconstitution of human
neuropeptide Y (NPY) Y2 and Y4 receptors in Sf9 insect cells. J.
Recept. Signal Transduct. 2011, 31, 271−285.
(13) Pedrazzini, T.; Seydoux, J.; Kunstner, P.; Aubert, J. F.;
Grouzmann, E.; Beermann, F.; Brunner, H. R. Cardiovascular
response, feeding behavior and locomotor activity in mice lacking
the NPY Y1 receptor. Nat. Med. 1998, 4, 722−726.
(14) Khan, I. U.; Zwanziger, D.; Bohme, I.; Javed, M.; Naseer, H.;
Hyder, S. W.; Beck-Sickinger, A. G. Breast-cancer diagnosis by
neuropeptide Y analogues: From synthesis to clinical application.
Angew. Chem., Int. Ed. 2010, 49, 1155−1158.
(15) Korner, M.; Reubi, J. C. NPY receptors in human cancer: A
̈
review of current knowledge. Peptides 2007, 28, 419−425.
(16) Memminger, M.; Keller, M.; Lopuch, M.; Pop, N.; Bernhardt,
G.; von Angerer, E.; Buschauer, A. The neuropeptide y y(1) receptor:
A diagnostic marker? Expression in mcf-7 breast cancer cells is down-
regulated by antiestrogens in vitro and in xenografts. PLoS ONE 2012,
7, e51032.
(17) Flood, J. F.; Morley, J. E. Dissociation of the effects of
neuropeptide Y on feeding and memory: Evidence for pre- and
postsynaptic mediation. Peptides 1989, 10, 963−966.
(18) Potter, E. K.; Mitchell, L.; McCloskey, M. J.; Tseng, A.;
Goodman, A. E.; Shine, J.; McCloskey, D. I. Pre- and postjunctional
actions of neuropeptide Y and related peptides. Regul. Pept. 1989, 25,
167−177.
H
dx.doi.org/10.1021/jm4008505 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(37) Daniels, A. J.; Matthews, J.; Slepetis, R. J.; Jansen, M.; Viveros,
O. H.; Tadapalli, A.; Harrington, W.; Heyer, D.; Landavazo, A.; Laban,
J. J.; Spaltenstein, A. High-affinity neuropeptide Y receptor antagonists.
Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 9067−9071.
(38) Parker, E. M.; Babij, C. K.; Balasubramaniam, A.; Burrier, R. E.;
Guzzi, M.; Hamud, F.; Mukhopadhyay, G.; Rudinski, M. S.; Tao, Z.;
Tice, M.; Xia, L.; Mullins, D. E.; Salisbury, B. G. GR231118
(1229U91) and other analogues of the C-terminus of neuropeptide
Y are potent neuropeptide Y Y1 receptor antagonists and neuropeptide
Y Y4 receptor agonists. Eur. J. Pharmacol. 1998, 349, 97−105.
(39) Lindner, D.; Stichel, J.; Beck-Sickinger, A. G. Molecular
recognition of the NPY hormone family by their receptors. Nutrition
2008, 24, 907−917.
(40) Berlicki, L.; Pilsl, L.; Weber, E.; Mandity, I. M.; Cabrele, C.;
Martinek, T. A.; Fulop, F.; Reiser, O. Unique alpha,beta- and
alpha,alpha,beta,beta-peptide foldamers based on cis-beta-aminocyclo-
pentanecarboxylic acid. Angew. Chem., Int. Ed. 2012, 51, 2208−2212.
(41) De Pol, S.; Zorn, C.; Klein, C. D.; Zerbe, O.; Reiser, O.
Surprisingly stable helical conformations in alpha/beta-peptides by
incorporation of cis-beta-aminocyclopropane carboxylic acids. Angew.
Chem., Int. Ed. 2004, 43, 511−514.
(42) Koglin, N.; Zorn, C.; Beumer, R.; Cabrele, C.; Bubert, C.;
Sewald, N.; Reiser, O.; Beck-Sickinger, A. G. Analogues of neuro-
peptide Y containing beta-aminocyclopropane carboxylic acids are the
shortest linear peptides that are selective for the Y1 receptor. Angew.
Chem., Int. Ed. 2003, 42, 202−205.
(53) Aguilera, J.; Moglioni, A. G.; Moltrasio, G. Y.; Ortuno, R. M.
Stereodivergent and efficient synthesis of the first bis(cyclobutane)
gamma-dipeptides. Tetrahedron: Asymmetry 2008, 19, 302−308.
(54) Beumer, R.; Bubert, C.; Cabrele, C.; Vielhauer, O.; Pietzsch, M.;
Reiser, O. The synthesis of diastereo- and enantiomerically pure beta-
aminocyclopropanecarboxylic acids. J. Org. Chem. 2000, 65, 8960−
8969.
(55) Izquierdo, S.; Rua, F.; Sbai, A.; Parella, T.; Alvarez-Larena, A.;
Branchadell, V.; Ortuno, R. M. (+)- and (−)-2-aminocyclobutane-1-
carboxylic acids and their incorporation into highly rigid beta-peptides:
Stereoselective synthesis and a structural study. J. Org. Chem. 2005, 70,
7963−7971.
(56) Schneider, E.; Mayer, M.; Ziemek, R.; Li, L.; Hutzler, C.;
Bernhardt, G.; Buschauer, A. A simple and powerful flow cytometric
method for the simultaneous determination of multiple parameters at
G-protein-coupled receptor subtypes. ChemBioChem 2006, 7, 1400−
1409.
(57) Ziemek, R.; Brennauer, A.; Schneider, E.; Cabrele, C.; Beck-
Sickinger, A. G.; Bernhardt, G.; Buschauer, A. Fluorescence- and
luminescence-based methods for the determination of affinity and
activity of neuropeptide Y(2) receptor ligands. Eur. J. Pharmacol. 2006,
551, 10−18.
(58) Schneider, E.; Keller, M.; Brennauer, A.; Hoefelschweiger, B. K.;
Gross, D.; Wolfbeis, O. S.; Bernhardt, G.; Buschauer, A. Synthesis and
characterization of the first fluorescent nonpeptide NPY Y1 receptor
antagonist. ChemBioChem 2007, 8, 1981−1988.
(59) Higashijima, T.; Burnier, J.; Ross, E. M. Regulation of Gi and Go
by mastoparan, related amphiphilic peptides, and hydrophobic amines.
Mechanism and structural determinants of activity. J. Biol. Chem. 1990,
265, 14176−14186.
(60) Higashijima, T.; Uzu, S.; Nakajima, T.; Ross, E. M. Mastoparan,
a peptide toxin from wasp venom, mimics receptors by activating
GTP-binding regulatory proteins (G proteins). J. Biol. Chem. 1988,
263, 6491−6494.
(61) Seifert, R.; Lee, T. W.; Lam, V. T.; Kobilka, B. K. Reconstitution
of beta2-adrenoceptor-GTP-binding-protein interaction in Sf9 cells–
high coupling efficiency in a beta2-adrenoceptor-G(s alpha) fusion
protein. Eur. J. Biochem. 1998, 255, 369−382.
(62) Beck-Sickinger, A. G.; Jung, G. Structure−activity relationships
of neuropeptide Y analogues with respect to Y1 and Y2 receptors.
Biopolymers 1995, 37, 123−142.
(63) Rist, B.; Zerbe, O.; Ingenhoven, N.; Scapozza, L.; Peers, C.;
Vaughan, P. F.; McDonald, R. L.; Wieland, H. A.; Beck-Sickinger, A. G.
Modified, cyclic dodecapeptide analog of neuropeptide Y is the
smallest full agonist at the human Y2 receptor. FEBS Lett. 1996, 394,
169−173.
(64) Leban, J. J.; Heyer, D.; Landavazo, A.; Matthews, J.; Aulabaugh,
A.; Daniels, A. J. Novel modified carboxy terminal fragments of
neuropeptide Y with high affinity for Y2-type receptors and potent
functional antagonism at the Y1-type receptor. J. Med. Chem. 1995, 38,
1150−1157.
(65) Formaggio, F.; Peggion, C.; Crisma, M.; Kaptein, B.;
Broxterman, Q. B.; Mazaleyrat, J. P.; Wakselman, M.; Toniolo, C.
Recent contributions of electronic circular dichroism to the
investigation of oligopeptide conformations. Chirality 2004, 16,
388−397.
(66) Pedersen, S. L.; Bhatia, V. K.; Jurt, S.; Paulsson, J. F.; Pedersen,
M. H.; Jorgensen, R.; Holst, B.; Stamou, D.; Vrang, N.; Zerbe, O.;
Jensen, K. J. Improving membrane binding as a design strategy for
amphipathic peptide hormones: 2-Helix variants of PYY3-36. J. Pept.
Sci. 2012, 18, 579−587.
(43) Fernandez, D.; Torres, E.; Aviles, F. X.; Ortuno, R. M.; Vendrell,
J. Cyclobutane-containing peptides: Evaluation as novel metal-
locarboxypeptidase inhibitors and modelling of their mode of action.
Bioorg. Med. Chem. 2009, 17, 3824−3828.
(44) Gorrea, E.; Pohl, G.; Nolis, P.; Celis, S.; Burusco, K. K.;
Branchadell, V.; Perczel, A.; Ortuno, R. M. Secondary structure of
̃
short beta-peptides as the chiral expression of monomeric building
units: A rational and predictive model. J. Org. Chem. 2012, 77, 9795−
9806.
(45) Torres, E.; Gorrea, E.; Burusco, K. K.; Da Silva, E.; Nolis, P.;
Rua, F.; Boussert, S.; Diez-Perez, I.; Dannenberg, S.; Izquierdo, S.;
Giralt, E.; Jaime, C.; Branchadell, V.; Ortuno, R. M. Folding and self-
assembling with beta-oligomers based on (1R,2S)-2-aminocyclobu-
tane-1-carboxylic acid. Org. Biomol. Chem. 2010, 8, 564−575.
(46) Torres, E.; Gorrea, E.; Da Silva, E.; Nolis, P.; Branchadell, V.;
Ortuno, R. M. Prevalence of eight-membered hydrogen-bonded rings
in some bis(cyclobutane) beta-dipeptides including residues with trans
stereochemistry. Org. Lett. 2009, 11, 2301−2304.
(47) Gorrea, E.; Nolis, P.; Torres, E.; Da Silva, E.; Amabilino, D. B.;
Branchadell, V.; Ortuno, R. M. Self-assembly of chiral trans-
cyclobutane-containing beta-dipeptides into ordered aggregates.
Chemistry 2011, 17, 4588−4597.
(48) Zwanziger, D.; Bohme, I.; Lindner, D.; Beck-Sickinger, A. G.
̈
First selective agonist of the neuropeptide Y1-receptor with reduced
size. J. Pept. Sci. 2009, 15, 856−866.
(49) Cabrele, C.; Langer, M.; Bader, R.; Wieland, H. A.; Doods, H.
N.; Zerbe, O.; Beck-Sickinger, A. G. The first selective agonist for the
neuropeptide YY5 receptor increases food intake in rats. J. Biol. Chem.
2000, 275, 36043−36048.
(50) Davies, S. G.; Ichihara, O.; Lenoir, I.; Walters, I. A. S.
Asymmetric synthesis of (−)-(1R,2S)-cispentacin and related cis- and
trans-2-amino cyclopentane- and cyclohexane-1-carboxylic acids. J.
Chem. Soc., Perkin Trans. 1 1994, 1411−1415.
(51) Lapatsanis, L.; Milias, G.; Froussios, K.; Kolovos, M. Synthesis
of N-2,2,2-(Trichloroethoxycarbonyl)-L-amino acids and N-(9-fluo-
renylmethoxycarbonyl)-L-amino acids involving succinimidoxy anion
as a leaving group in amino-acid protection. Synthesis 1983, 671−673.
(52) Gutierrez-Abad, R.; Carbajo, D.; Nolis, P.; Acosta-Silva, C.;
Cobos, J. A.; Illa, O.; Royo, M.; Ortuno, R. M. Synthesis and structural
study of highly constrained hybrid cyclobutane-proline gamma,gam-
ma-peptides. Amino Acids 2011, 41, 673−686.
(67) Bader, R.; Bettio, A.; Beck-Sickinger, A. G.; Zerbe, O. Structure
and dynamics of micelle-bound neuropeptide Y: Comparison with
unligated NPY and implications for receptor selection. J. Mol. Biol.
2001, 305, 307−329.
(68) Lerch, M.; Gafner, V.; Bader, R.; Christen, B.; Folkers, G.;
Zerbe, O. Bovine pancreatic polypeptide (bPP) undergoes significant
changes in conformation and dynamics upon binding to DPC micelles.
J. Mol. Biol. 2002, 322, 1117−1133.
I
dx.doi.org/10.1021/jm4008505 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(69) Martin, P.; Papayannopoulou, T. HEL cells: A new human
erythroleukemia cell line with spontaneous and induced globin
expression. Science 1982, 216, 1233−1235.
(70) Moser, C.; Bernhardt, G.; Michel, J.; Schwarz, H.; Buschauer, A.
Cloning and functional expression of the hNPY Y5 receptor in human
endometrial cancer (HEC-1B) cells. Can. J. Physiol. Pharmacol. 2000,
78, 134−142.
(71) Preuss, H.; Ghorai, P.; Kraus, A.; Dove, S.; Buschauer, A.;
Seifert, R. Constitutive activity and ligand selectivity of human, guinea
pig, rat, and canine histamine H2 receptors. J. Pharmacol. Exp. Ther.
2007, 321, 983−995.
(72) Schwieters, C. D.; Kuszewski, J. J.; Tjandra, N.; Clore, G. M.
The Xplor-NIH NMR molecular structure determination package. J.
Magn. Reson. 2003, 160, 65−73.
(73) Schwieters, C. D.; Kuszewski, J.; Clore, G. M. Using Xplor-NIH
for NMR molecular structure determination. Progr. NMR Spectrosc.
2006, 48, 47−62.
J
dx.doi.org/10.1021/jm4008505 | J. Med. Chem. XXXX, XXX, XXX−XXX