Structure-activity study at positions 3 and 4 of human neuropeptide S

Article abstract of DOI:10.1016/j.bmc.2008.08.073

Neuropeptide S (NPS) has been identified as the endogenous ligand of a previously orphan receptor now named NPSR. Previous studies demonstrated that the N-terminal sequence Phe²-Arg³-Asn⁴ of the peptide is crucial for biological activity. Here, we report on a focused structure-activity study of Arg³ and Asn⁴ that have been replaced with a series of coded and non-coded amino acids. Thirty-eight human NPS analogues were synthesized and pharmacologically tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated the following NPS position 3 structure-activity features: (i) the guanidine moiety and its basic character are not crucial requirements, (ii) an aliphatic amino acid with a linear three carbon atom long side chain is sufficient to bind and fully activate NPSR, (iii) the receptor pocket allocating the position 3 side chain can accommodate slightly larger side chains at least to a certain degree [hArg, Arg(NO₂) or Arg(Me)₂ but not Arg(Tos)]. Position 4 seems to be more sensitive to amino acids replacement compared to position 3; in fact, all the amino acid replacements investigated produced either an important decrease of biological activity or generated inactive derivatives suggesting a pivotal role of the Asn⁴ side chain for NPS bioactivity.

Full text of DOI:10.1016/j.bmc.2008.08.073

Bioorganic & Medicinal Chemistry 16 (2008) 8841–8845
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity study at positions 3 and 4 of human neuropeptide S
a
b
a
b,*
a
a
Valeria Camarda , Claudio Trapella , Girolamo Calo’ , Remo Guerrini , Anna Rizzi , Chiara Ruzza ,
b
b
c
a
b
Stella Fiorini , Erika Marzola , Rainer K. Reinscheid , Domenico Regoli , Severo Salvadori
Department of Experimental and Clinical Medicine, Section of Pharmacology and Neuroscience Center, University of Ferrara, and National Institute of Neuroscience, Italy
Department of Pharmaceutical Sciences and Biotechnology Center, University of Ferrara, Via Fossato di Mortara 19, 44100 Ferrara, Italy
Department of Pharmaceutical Sciences, University of California Irvine, 360 Med Surge 2, Irvine, CA 92697, USA
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2008
Revised 21 August 2008
Accepted 28 August 2008
Available online 31 August 2008
Neuropeptide S (NPS) has been identified as the endogenous ligand of a previously orphan receptor now
2
3
4
named NPSR. Previous studies demonstrated that the N-terminal sequence Phe -Arg -Asn of the peptide
is crucial for biological activity. Here, we report on a focused structure–activity study of Arg3 and Asn4
that have been replaced with a series of coded and non-coded amino acids. Thirty-eight human NPS ana-
logues were synthesized and pharmacologically tested for intracellular calcium mobilization using
HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated the following
NPS position 3 structure–activity features: (i) the guanidine moiety and its basic character are not crucial
requirements, (ii) an aliphatic amino acid with a linear three carbon atom long side chain is sufficient to
bind and fully activate NPSR, (iii) the receptor pocket allocating the position 3 side chain can accommo-
Keywords:
Neuropeptide S
Bioassay
Structure–activity study
Peptide synthesis
2 2
date slightly larger side chains at least to a certain degree [hArg, Arg(NO ) or Arg(Me) but not Arg(Tos)].
Position 4 seems to be more sensitive to amino acids replacement compared to position 3; in fact, all the
amino acid replacements investigated produced either an important decrease of biological activity or
4
generated inactive derivatives suggesting a pivotal role of the Asn side chain for NPS bioactivity.
Ó 2008 Elsevier Ltd. All rights reserved.
1
. Introduction
of NPSR is consistent with the biological effects elicited by supraspi-
nal administration of NPS in rodents, namely anxiolytic like ef-
1
,4,5
1
1,5
Neuropeptide S (NPS, human primary sequence SFRNGVGT
fects,
activity,
arousal promoting action,
and inhibition of food intake.
stimulation of locomotor
,5–8
8–10
GMKKTSFQRAKS) has been identified as the endogenous ligand of
the G protein coupled receptor now named NPSR. In cells express-
ing the recombinant NPSR, NPS produced concentration dependent
increases of calcium levels and cAMP formation, suggesting trans-
ductional mechanisms involving Gq and Gs. NPSR mRNA is widely
expressed throughout the nervous system while the NPS precursor
mRNA is strongly expressed only in the locus coeruleus area and in
a few other nuclei of the brainstem.1 The anatomical distribution
Up to now the involve-
1
ment of NPSR in the in vivo actions of NPS has been demonstrated
only for the locomotor stimulant action using the non-peptide NPSR
6
antagonist SHA 68. The development of selective ligands for NPSR is
2
of paramount importance for investigating the functions and roles of
this novel neurotransmitter system in central nervous system phys-
iology and pathology. To this aim, we started a systematic investiga-
tion of NPS chemical and conformational requirements for NPSR
,3
2
binding and activation. First, in line with previous observations ,
2
3
4
we identified the N-terminal sequence Phe -Arg -Asn as the most
Abbreviations: BSA, bovine serum albumin; cAMP, cyclic adenosine monophos-
phate; DMF, N,N-dimethylformamide; DMEM, Dulbecco’s modified Eagles’s med-
ium; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HATU, [O-(7-azabenzotriazol-1-yl)-
7
important structural component for biological activity , a finding
confirmed by other researchers. Second, we focussed on the chem-
ical requirements of Phe of NPS demonstrating that this position is
important for both receptor binding and activation. Third, we per-
formed a conformation–activity relationship study suggesting
that helicity can be tolerated in the C-terminal part of NPS but not
around Gly , a result which is only in part in line with the nascent he-
1
1
2
1
,1,3,3-tetramethyluronium hexafluorophosphate]; HBSS, Hank’s balanced salt
solution; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HEK, human
embryonic kidney; HPLC, high performance liquid chromatography; mRNA, mes-
senger ribonucleic acid; Ndm, 3-(dimethylcarbamoyl)-2-aminopropanoic acid;
NMM, 4-methylmorpholine; Nmm, 3-(methylcarbamoyl)-2-aminopropanoic acid;
NPS, neuropeptide S; NPSR, NPS receptor; Ova, 2-amino-4-oxo-pentanoic acid; tBu,
tert-butyl; TFA, trifluoroacetic acid; SAR, structure–activity relationship; SAH 68, 3-
oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-
benzylamide.
12
1
3
7
11
lix spanning residues 5 through 13 proposed by Bernier et al.
In this paper, we present results obtained from a SAR study per-
3
4
formed by replacing Arg and Asn with several coded and non-
coded amino acid residues. 38 novel NPS analogues were synthes-
*
Corresponding author. Tel.: +39 0532 455 988; fax: +39 0532 455953.
E-mail address: r.guerrini@unife.it (R. Guerrini).
0
968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.073

8
842
V. Camarda et al. / Bioorg. Med. Chem. 16 (2008) 8841–8845
ised and pharmacologically evaluated in a calcium mobilization as-
say using HEK293 cells stably expressing mouse NPSR
Tables 1 and 2 summarize data obtained investigating the SAR
requirements of NPS positions 3 and 4, respectively. In line with
2
,12
(
HEK293mNPSR) and the fluorometric imaging plate reader FlexSta-
previous results
increased intracellular calcium levels in a concentration dependent
manner showing high potency (pEC50 ꢀ8) and efficacy (Emax
.5-fold over the basal values).
For investigating the chemical features required in positions 3
, in the calcium mobilization assay NPS
tion II.
ꢀ
2
2
. Results and discussion
and 4 of NPS for NPSR interaction, we adopted a classical approach
in which the original amino acid residues were replaced with
coded and non-coded amino acids. The substitution of Arg3 with
aromatic residues (compounds 1–3) produced a drastic loss of bio-
Compounds 1–38 and the reference compound human NPS
were prepared with good yield by solid phase peptide synthesis.
For the synthesis of compounds 35, 37 and 38, the commercially
unavailable amino acids Nmm and Ndm were synthesized in good
yield by amidation of the Fmoc-Asp-OtBu side chain with mono or
dimethyl amine followed by TFA deprotection of the Ca carboxylic
function. The non-coded amino acid Ova was synthesized and
3
logical activity; the best compound ([His ]NPS) being more than
1
00-fold less potent than the natural peptide. Similar results were
obtained with non-aromatic lipophilic residues (compounds 4–5).
The introduction in position 3 of residues with hydrophilic neutral
1
4
15
Fmoc protected following procedures reported in the literature.
(
1
compounds 6–11), basic (compound 12) or acidic (compounds
3–14) character produced low potency or inactive NPS analogues.
The chemical formulae of the non-coded amino acids employed in
this study are displayed in Figure 1.
3
Among these, only [Lys ]NPS displayed a potency value similar to
H N
2
NH2
H N
2
H N
2
COOH
H N
2
COOH
H N
COOH
H N
2
COOH
H N
COOH
2
2
Orn (15)
Nva (16, 36)
Abu (17)
Dab (18, 34)
Dap (19)
NH2
NH
NH2
N H
N
O
HN
N
HN
HN
HN
HN
H N
COOH
H N
2
COOH
H N
COOH
H N
COOH
2
2
2
Cit (20)
hArg (21)
Arg(CH ) Asym (22)
Arg(CH ) Sym (23)
3
2
3 2
O
O N
CF3
2
S
N
NH O
H N
2
HN
O
NH
HN
HN
N
H N
2
COOH
H N
2
COOH
H N
COOH
H N
2
COOH
2
Arg(NO ) (24)
Arg(Tos) (25)
Lys(Tfa) (26)
2-Pal (27)
2
N
N
O
O
O
N
H
N
H N
COOH
H N
2
COOH
H N
COOH
H N
2
COOH
H N
2
COOH
2
2
3-Pal (28)
4-Pal (29)
Ova (35)
Nmm (37)
Ndm (38)
Figure 1. Chemical formula of the non-coded amino acids employed in this study.

V. Camarda et al. / Bioorg. Med. Chem. 16 (2008) 8841–8845
8843
3
Table 1
Effects of NPS and [X ]NPS analogues in HEK293 cells expressing the mNPSR
that harbors the Arg side chain. However, this same pocked does
not tolerate an acidic side chain as demonstrated by the lack of
3
3
No. Compound
pEC50 (CL95%
)
Emax ± SEM
activity of [Glu ]NPS. Moreover, the data obtained by the replace-
ment Arg/Gln suggested that basicity in position 3 is not crucial for
bioactivity.
NPS
[Phe ]NPS
[Tyr ]NPS
[His ]NPS
[Leu ]NPS
[Val ]NPS
7.92 (7.83–8.01)
Crc incomplete: at 10
Crc incomplete: at 10
5.81 (5.20–6.42)
Crc incomplete: at 10
Crc incomplete: at 10
6.06 (5.97–6.15)
Crc incomplete: at 10
6.43 (5.97–6.89)
5.90 (5.71–6.09)
Crc incomplete: at 10
6.89 (6.76–7.02)
7.74 (7.70–7.78)
Crc incomplete: at 10
Crc incomplete: at 10
7.50 (7.24–7.76)
267 ± 10%
273 ± 18%
272 ± 20%
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
l
l
M 46 ± 6%
M: 73 ± 4%
3
The shortening of the Lys side chain by one carbon atom (com-
pound 15) appears to be well tolerated producing a peptide only 2-
fold less potent. In addition, the elimination of the primary amino
function (compound 16) produced a modest reduction of biological
activity corroborating the hypothesis that basicity in position 3 is
not a stringent requirement for NPSR interaction. Further shorten-
ing of the side chain (compound 17) reduced peptide potency
while the addition on the ethyl moiety of an amino function, as
in Dab (compound 18), produced an NPS analog with similar po-
tency as compound 16. Finally, the elimination of a methylene
group from the side chain of 18 (compound 19) produced negligi-
ble changes in biological activity. Collectively, these data indicate
that the length of the side chain of the amino acid in position 3
and its linear shape seem to be more important for biological activ-
ity than its basic character. Surprisingly, the transformation of the
guanidine function into urea (compound 20) generated a peptide
10-fold less potent than NPS. Since compound 20 is also less potent
than compound 12 or 15, it is probable that the reduction of NPSR
binding is due to the urea carbonyl (C@O), rather than to its non-
basic character.
3
3
lM 36 ± 9%
3
lM 129 ± 30%
3
[Thr ]NPS
3
[Ser ]NPS
l
M 175 ± 19%
M 161 ± 33%
3
[Cys ]NPS
[Gly ]NPS
[Asn ]NPS
[Gln ]NPS
[Lys ]NPS
[Asp ]NPS
[Glu ]NPS
[Orn ]NPS
256 ± 26%
264 ± 7%
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
l
3
295 ± 21%
290 ± 17%
3
3
l
l
M 73 ± 24%
M 89 ± 3%
3
3
318 ± 11%
207 ± 23%
218 ± 17%
207 ± 32%
196 ± 17%
285 ± 11%
288 ± 16%
281 ± 15%
272 ± 10%
288 ± 17%
3
[Nva ]NPS
[Abu ]NPS
[Dab ]NPS
6.87 (5.95–7.79)
6.29 (5.43–7.15)
6.69 (6.12–7.26)
6.62 (6.32–6.87)
6.78 (6.45–7.11)
7.97 (7.70–8.24)
3
3
3
[Dap ]NPS
3
[Cit ]NPS
3
[hArg ]NPS
[Arg(Me)
[Arg(Me)
[Arg(NO
[Arg(Tos) ]NPS
[Lys(Tfa) ]NPS
[2’-Pal ]NPS
[3’-Pal ]NPS
[4’-Pal ]NPS
3
2
(Asym)]NPS 7.65 (7.52–7.78)
3
3
2
(Sym)]NPS
7.50 (7.40–7.60)
7.66 (7.49–7.83)
2
) ]NPS
3
Crc incomplete: at 10
7.07 (6.60–7.54)
Crc incomplete: at 10
Crc incomplete: at 10
Crc incomplete: at 10
lM 104 ± 16%
3
231 ± 18%
3
3
An increase in length of the side chain of Arg (compound 21) or
l
l
l
M 19 ± 4%
M 42 ± 11%
M 154 ± 17%
3
its methylation (compounds 22 and 23) are completely tolerated.
3
3
The elimination of the basic character of the Arg side chain ob-
tained with compound 24 was tolerated while that obtained with
compound 25 was not. Collectively these results corroborate the
hypothesis that the guanidine moiety and its basicity are not par-
ticularly relevant for biological activity. In addition, the steric hin-
drance of the guanidine moiety does not seem to represent a
particularly stringent chemical requirement since it can be en-
larged (as in compounds 22–24) without loss of potency. However,
this does not apply for moieties as bulky as tosyl since compound
25 was found inactive.
pEC50: the negative logarithm to base 10 of the molar concentration of an agonist
that produces 50% of the maximal possible effect.
CL95%: 95% confidence limits.
E
max: the maximal effect elicited by the agonist expressed as % over the baseline.
SEM: standard error of the mean.
crc: concentration response curve.
Table 2
4
Effects of hNPS and [X ]hNPS analogues in HEK293 cells expressing the mNPSR
The elimination of the Lys side chain basicity (compound 26)
produced a 3-fold decrease of activity compared to 12; this could
be due, at least in part, to the increase of the side chain steric hin-
drance, similar to what observed with compound 25. Finally we in-
serted in position 3 amino acids with a pyridine nucleus as side
chain, which combines in the same moiety aromatic character
and a nitrogen atom able to accept hydrogen bond (compounds
No.
Compound
NPS
pEC50 (CL95%
)
E
max ± SEM
234 ± 12%
M 146 ± 6%
M 118 ± 19%
253 ± 10%
210 ± 19%
M 169 ± 18%
7.99 (7.65–8.33)
4
30
31
32
33
34
35
36
37
38
[Asp ]NPS
Crc incomplete: at 10
Crc incomplete: at 10
6.99 (6.86–7.12)
l
l
4
[Phe ]NPS
4
[Thr ]NPS
4
[Gln ]NPS
6.25 (5.65–6.85)
4
[Dab ]NPS
Crc incomplete: at 10
Crc incomplete: at 10
Crc incomplete: at 10
Crc incomplete: at 10
Crc incomplete: at 10
l
l
l
l
l
4
[Ova ]NPS
M 136 ± 17%
M 240 ± 39%
M 188 ± 18%
M 89 ± 12%
2
7–29). In line with previous findings (see compounds 1–3) this
4
[Nva ]NPS
4
chemical substitution was not tolerated, corroborating the evi-
dence that aromaticity in position 3 is detrimental for NPSR
binding.
[Nmm ]NPS
4
[Ndm ]NPS
pEC50: the negative logarithm to base 10 of the molar concentration of an agonist
that produces 50% of the maximal possible effect.
CL95%: 95% confidence limits.
4
Data obtained substituting Asn in NPS with a limited series of
coded and non-coded amino acids are summarized in Table 2. The
replacement of position 4 with amino acids with acidic (compound
E
max: the maximal effect elicited by the agonist expressed as % over the baseline.
SEM: standard error of the mean.
crc: concentration response curve.
3
0) or aromatic (compound 31) side chains produced inactive
13
derivatives. Previous findings indicated that aromaticity in posi-
tion 4 is detrimental for biological activity since [His ]NPS was
4
found more than 100 fold less potent than NPS. A 10-fold reduction
of potency has been obtained by replacing Asn with Thr (com-
pound 32) while the Asn related amino acid Gln (compound 33)
produced a larger decrease of biological activity (55-fold). To inves-
tigate in details the contribution of the primary amide function of
3
that of native NPS. These results, obtained by exchanging Arg with
coded amino acids, suggest that position 3 does not tolerate substi-
3
tution with aromatic and aliphatic branched residues. The Arg
replacement with hydrophilic residues with short side chain pro-
duced in general low potency peptides (compounds 6-10). Inter-
estingly, while the substitution of Arg3 with Gln (compound 11)
reduced peptide potency only by 10-fold, exchanges to Asn or
Glu lead to inactive analogs. These results, together with the inac-
4
Asn to biological activity, we substituted either alone (compound
34 and 35) or in combination (compound 36) the C@O and –NH
groups with –CH and –CH , respectively. A profound loss of bio-
2
2
3
logical activity (>1000-fold) was observed with all these analogs
suggesting a pivotal role of the amide function of the Asn4 side
chain for biological activity. In compounds 37 and 38 this moiety
was mono- or dimethylated generating, also in this case, inactive
3
tivity of [Val ]NPS, indicate that a linear 3-carbon atom moiety of
the amino acid side chain, which is similar in both Arg and Gln,
but not in Val or Ala,7,11 is important for binding the NPSR pocket

8
844
V. Camarda et al. / Bioorg. Med. Chem. 16 (2008) 8841–8845
peptides. These latter modifications reduced or eliminated the
hydrogen bond donor properties of the amide function and at the
same time progressively increased the steric hindrance of position
obtained using a Hypersil BDS C18 column (4.6 Â 250 mm, 5
particle size) with solvent A: 35 mM NaH PO (pH 2.1) and solvent
B: 59 mM NaH PO (pH 2.1)-acetonitrile (60:40 v/v). The column
lm
2
4
2
4
4
. The relative importance of these factors in the loss of NPSR bind-
was perfused at a flow rate of 1 mL/min with a linear gradient from
5% to 60% B over 25 min. All analogues showed >95% purity when
monitored at 220 nm. Molecular weights of compounds were
determined by a mass spectrometer ESI Micromass ZMD-2000, val-
ues are expressed as MH . The analytical properties of NPS ana-
logues are reported in Table 3.
ing can not be clearly unraveled. Collectively, these results, to-
7
,11
gether with previous Ala- and
D
-amino acid scan indications
,
underline the importance of Asn4 for the activity of NPS and sug-
gest strict chemical requirements of the agonist binding pocket
in NPSR allocating the side chain of this residue.
+
4
.4. Calcium mobilization experiments
3
. Conclusions
2
HEK293mNPSR cells were generated as previously described and
maintained in DMEM medium supplemented with 10% fetal bovine
serum, 2 mM -Glutamine, Hygromycin (100 mg/L), and cultured
at 37 °C in 5% CO humidified air. HEK293mNPSR cells were prepared
for calcium mobilization experiments as previously described.
In the present SAR study, we investigated the chemical require-
ments of NPS position 3 (Arg) and 4 (Asn) for NPSR binding. Results
related to position 3 suggest that (i) the guanidine moiety and its
basic character are not crucial requirements, (ii) an aliphatic amino
acid with a linear three carbon atom long side chain is sufficient to
bind and fully activate NPSR, (iii) the receptor pocket allocating the
side chain of position 3 can accommodate slightly larger side
L
2
12
Concentrated solutions (1 mM) of NPS and related peptides were
made in bidistilled water and kept at À20 °C. Serial dilutions were
carried out in HBSS/HEPES (20 mM) buffer (containing 0.02% BSA
fraction V). After placing both plates (cell culture and master plate)
into the fluorometric imaging plate reader FlexStation II (Molecular
Devices, Sunnyvale, CA), fluorescence changes were measured at
room temperature (ꢀ22 °C). On-line additions were carried out in
2 2
chains at least to a certain degree [hArg, Arg(NO ) or Arg(Me)
but not Arg(Tos)]. Position 4 seems to be more sensitive to amino
acids replacement compared to position 3; in fact, all the amino
acid replacements investigated produced an important decrease
of biological activity or generated inactive derivatives suggesting
4
a volume of 50 lL/well.
a pivotal role of the Asn side chain for NPS bioactivity.
4
4
. Experimental
Table 3
Analytical properties of the NPS analogues
.1. General information
at
bMH+
No.
Abbreviated names
R
I
II
Calculated
Found
Amino acids, protected amino acids, resins for solid phase syn-
hNPS
11.88
11.32
10.67
10.82
11.40
11.44
10.76
11.14
11.83
11.12
10.42
10.98
10.86
10.84
11.04
10.06
11.77
11.33
10.87
10.08
10.15
10.20
10.60
11.00
10.11
13.62
13.18
11.30
11.90
10.89
9.63
19.42
19.95
18.86
19.07
20.15
19.11
19.06
18.87
19.27
19.55
18.38
19.42
19.04
18.99
19.52
17.94
19.38
19.12
19.06
18.00
18.03
18.11
18.86
19.28
18.07
22.02
21.96
19.46
19.35
19.39
11.79
13.88
13.32
13.79
11.64
13.28
10.78
12.01
11.82
2188.5
2179.5
2195.5
2169.5
2145.5
2131.5
2133.4
2119.4
2134.4
2089.4
2146.4
2160.5
2160.5
2147.4
2161.4
2146.5
2131.5
2117.4
2132.5
2118.5
2189.5
2202.5
2215.5
2215.5
2233.5
2342.7
2256.5
2180.5
2180.5
2180.5
2189.5
2221.6
2175.5
2202.6
2174.5
2186.5
2173.6
2202.5
2216.6
2188.2
2178.9
2194.3
2168.7
2144.7
2131.0
2133.2
2118.9
2135.0
2087.9
2145.1
2159.5
2158.9
2146.8
2160.6
2147.1
2131.4
2116.4
2132.0
2117.6
2190.1
2201.8
2216.0
2216.2
2188.9
2342.4
2256.4
2180.2
2180.2
2180.2
2189.2
2222.0
2175.6
2203.2
2175.0
2186.2
2173.8
2202.2
2216.8
thesis and chemicals were purchased from Bachem, Novabiochem,
Fluka or Chem-Impex International. Stock solutions (1 mM) of pep-
tides were made in distilled water and kept at À20 °C until use. All
other reagents were from Sigma (Poole, UK) or Merck (Darmstadt,
Germany) and were of the highest purity available.
3
1
2
3
4
5
6
7
8
9
[Phe ]NPS
3
[Tyr ]NPS
3
[His ]NPS
3
[Leu ]NPS
3
[Val ]NPS
3
[Thr ]NPS
3
[Ser ]NPS
4
.2. General procedures for the solid phase peptide synthesis
[Cys ]NPS
3
3
[Gly ]NPS
3
10
[Asn ]NPS
All peptides were synthesised following the procedures previ-
3
ously reported in details12 using Fmoc/t-butyl chemistry with a
11
[Gln ]NPS
3
1
1
2
3
[Lys ]NPS
Syro XP multiple peptide synthesizer (MultiSynTech GmbH, Witten
Germany). Fmoc-Ser(tBu)-4-Benzyloxybenzyl Alcohol resin was
3
[Asp ]NPS
3
14
15
16
[Glu ]NPS
3
used for the synthesis of all peptides. The peptides were cleaved
[Orn ]NPS
3
from the resin using reagent B16; after filtration of the exhausted
[Nva ]NPS
3
1
1
7
8
[Abu ]NPS
resin, the solvent was concentrated in vacuo, and the residue
was triturated with ether.
3
[Dab ]NPS
3
19
[Dap ]NPS
3
20
21
22
23
24
[Cit ]NPS
3
[hArg ]NPS
[Arg(Me)
[Arg(Me)
[Arg(NO
[Arg(Tos) ]NPS
[Lys(Tfa) ]NPS
[2’-Pal ]NPS
[3’-Pal ]NPS
4
.3. Peptide purification and analytical determinations
3
2
(Asym)]NPS
(Sym)]NPS
3
3
2
Crude peptides were purified by preparative reversed-phase
2
) ]NPS
3
HPLC using a Waters Delta Prep 4000 system with a Jupiter column
m spherical particle size). The col-
25
26
2
2
2
3
3
C
18 (250 Â 30 mm, 300 A, 15
l
3
7
8
9
0
umn was perfused at a flow rate of 25 mL/min with a mobile phase
containing solvent A (10%, v/v, acetonitrile in 0.1% TFA), and a lin-
ear gradient from 0% to 50% of solvent B (60%, v/v, acetonitrile in
3
3
[4’-Pal ]NPS
4
[Asp ]NPS
4
0
.1% TFA) over 25 min for the elution of peptides. Analytical HPLC
analyses were performed on a Beckman 116 liquid chromatograph
equipped with a Beckman 166 diode array detector. Analytical pur-
31
[Phe ]NPS
11.07
10.87
11.18
9.51
10.91
8.64
4
32
33
34
35
[Thr ]NPS
4
[Gln ]NPS
4
[Dab ]NPS
R
ity and retention time (t ) of the peptides were determined using
4
[Ova ]NPS
4
two different HPLC conditions.
36
37
[Nva ]NPS
4
Retention time I was obtained using a Luna C18 column
[Nmm ]NPS
[Ndm ]NPS
9.79
9.65
4
38
(
(
4.6 Â 100 mm, 3
lm particle size) with the above solvent system
solvents A and B) programmed at a flow rate of 0.5 mL/min using a
a
tR is the retention time determined by analytical HPLC.
The mass ion (MH ) was obtained by electro spray mass spectrometry.
b
+
linear gradient from 0% to 65% B over 25 min. Retention time II was

V. Camarda et al. / Bioorg. Med. Chem. 16 (2008) 8841–8845
8845
4
.5. Data analysis and terminology
2. Reinscheid, R. K.; Xu, Y. L.; Okamura, N.; Zeng, J.; Chung, S.; Pai, R.; Wang, Z.;
Civelli, O. J. Pharmacol. Exp. Ther. 2005, 315, 1338.
3.
Xu, Y. L.; Gall, C. M.; Jackson, V. R.; Civelli, O.; Reinscheid, R. K. J. Comp. Neurol.
007, 500, 84.
The data were expressed as mean ± SEM of at least four inde-
2
pendent experiments made in duplicate. Maximum change in fluo-
rescence, expressed in percent of baseline fluorescence, was used
to determine agonist responses. Non-linear regression analysis
using GraphPad Prism software (v.4.0) allowed logistic iterative fit-
ting of the resultant responses and the calculation of agonist
potencies and maximal effects. Agonist potencies are given as
pEC50 (the negative logarithm to base 10 of the molar concentra-
tion of an agonist that produces 50% of the maximal possible ef-
fect). Differences in maximal effects between ligands were
statistically analyzed via one-way analysis of variance followed
by the Dunnett’s test for multiple comparisons.
4. Leonard, S. K.; Dwyer, J. M.; Sukoff Rizzo, S. J.; Platt, B.; Logue, S. F.; Neal, S. J.;
Malberg, J. E.; Beyer, C. E.; Schechter, L. E.; Rosenzweig-Lipson, S.; Ring, R. H.
Psychopharmacology (Berl.) 2008, 197, 601.
5.
6.
7.
Rizzi, A.; Vergura, R.; Marzola, G.; Ruzza, C.; Guerrini, R.; Salvadori, S.; Regoli,
D.; Calo, G. Br. J. Pharmacol. 2008, 154, 471.
Okamura, N.; Habay, S. A.; Zeng, J.; Chamberlin, A. R.; Reinscheid, R. K. J.
Pharmacol. Exp. Ther. 2008, 325, 893.
Roth, A. L.; Marzola, E.; Rizzi, A.; Arduin, M.; Trapella, C.; Corti, C.; Vergura, R.;
Martinelli, P.; Salvadori, S.; Regoli, D.; Corsi, M.; Cavanni, P.; Calo, G.; Guerrini,
R. J. Biol. Chem. 2006, 281, 20809.
8.
Smith, K. L.; Patterson, M.; Dhillo, W. S.; Patel, S. R.; Semjonous, N. M.; Gardiner,
J. V.; Ghatei, M. A.; Bloom, S. R. Endocrinology 2006, 30, 30.
9. Beck, B.; Fernette, B.; Stricker-Krongrad, A. Biochem. Biophys. Res. Commun.
005, 332, 859.
2
10. Cline, M. A.; Godlove, D. C.; Nandar, W.; Bowden, C. N.; Prall, B. C. Comp.
Biochem. Physio.l A Mol. Integr. Physiol. 2007, 148, 657.
Acknowledgment
11. Bernier, V.; Stocco, R.; Bogusky, M. J.; Joyce, J. G.; Parachoniak, C.; Grenier, K.;
Arget, M.; Mathieu, M. C.; O’Neill, G. P.; Slipetz, D.; Crackower, M. A.; Tan, C. M.;
Therien, A. G. J. Biol. Chem. 2006, 281, 24704.
This work was supported by funds from the Italian Ministry of
the University (Cofin 2006 Grant to S.S. and G.C.).
1
2. Camarda, V.; Trapella, C.; Calo, G.; Guerrini, R.; Rizzi, A.; Ruzza, C.; Fiorini, S.;
Marzola, E.; Reinscheid, R. K.; Regoli, D.; Salvadori, S. J. Med. Chem. 2008, 51,
655.
1
3. Tancredi, T.; Guerrini, R.; Marzola, E.; Trapella, C.; Calo, G.; Regoli, D.;
References and notes
Reinscheid, R. K.; Camarda, V.; Salvadori, S.; Temussi, P. A. J. Med. Chem.
2
007, 50, 4501.
1
.
Xu, Y. L.; Reinscheid, R. K.; Huitron-Resendiz, S.; Clark, S. D.; Wang, Z.; Lin, S. H.;
Brucher, F. A.; Zeng, J.; Ly, N. K.; Henriksen, S. J.; de Lecea, L.; Civelli, O. Neuron
1
1
1
4. Carpino, L. A.; Han, G. A. J. Org. Chem. 1972, 37, 3404.
5. Marcaurelle, L. A.; Bertozzi, C. A. Tetrahedron Lett. 1998, 39, 7279.
6. Sole’, N. A.; Barany, G. J. Org. Chem. 1992, 57, 5399.
2
004, 43, 487.