Discovery of dipeptides with high affinity to the specific binding site for substance P1-7

Article abstract of DOI:10.1021/jm901352b

Substance P 1-7 (SP1-7, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) is the major bioactive metabolite of substance P. The interest in this heptapeptide originates from the observation that it modulates, and in certain cases opposes the effects of the parent peptide, e.g., the nociceptive effect. The μ-opioid receptor agonist endomorphin-2 (EM-2, H-Tyr-Pro-Phe-Phe-NH₂) has been found to also interact with the specific binding site of SP ₁-₇ with only a 10-fold lower affinity compared to the native peptide. Considering the smaller size of EM-2 compared to the target heptapeptide, it was selected as a lead compound in the development of low-molecular-weight ligands to the SP₁-₇ binding site. An alanine scan and truncation study led to the unexpected discovery of the dipeptide H-Phe-Phe-NH₂ (K_i = 1.5 nM), having equal affinity as the endogenous heptapeptide SP₁-₇. Moreover, the studies show that the C-terminal phenylalanine amide is crucial for the affinity of the dipeptide.

Full text of DOI:10.1021/jm901352b

J. Med. Chem. 2010, 53, 2383–2389 2383
DOI: 10.1021/jm901352b
Discovery of Dipeptides with High Affinity to the Specific Binding Site for Substance P1-7
†
Rebecca Fransson, Milad Botros, Christian Skold, Fred Nyberg, Gunnar Lindeberg, Mathias Hallberg, and
‡
†
‡
†
‡
€
,†
€
Anja Sandstrom*
†Department of Medicinal Chemistry, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden, and
‡Department of Pharmaceutical Biosciences, Uppsala University, Box 591, SE-751 24 Uppsala, Sweden
Received September 11, 2009
Substance P 1-7 (SP1-7, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) is the major bioactive metabolite of
substance P. The interest in this heptapeptide originates from the observation that it modulates, and in
certain cases opposes the effects of the parent peptide, e.g., the nociceptive effect. The μ-opioid receptor
agonist endomorphin-2 (EM-2, H-Tyr-Pro-Phe-Phe-NH2) has been found to also interact with the
specific binding site of SP1-7 with only a 10-fold lower affinity compared to the native peptide.
Considering the smaller size of EM-2 compared to the target heptapeptide, it was selected as a lead
compound in the development of low-molecular-weight ligands to the SP1-7 binding site. An alanine
scan and truncation study led to the unexpected discovery of the dipeptide H-Phe-Phe-NH2 (Ki =
1.5 nM), having equal affinity as the endogenous heptapeptide SP1-7. Moreover, the studies show that
the C-terminal phenylalanine amide is crucial for the affinity of the dipeptide.
Introduction
Although the actions of SP1-7 are well-known, no explicit
receptor for this peptide fragment has yet been identified.
However, specific binding sites for SP1-7 in mouse and rat
spinal cord and in the ventral tegmental area of the rat brain
have been found.14-16 SP1-7 displayed greater affinity tothese
binding sites than SP and other related degradation frag-
ments, e.g., SP1-6, SP1-8, and SP1-9 (100-2000 times lower).
Moreover, established ligands [Sar9, Met(O2)11]-SP, R396,
and senktide of the NK-1, NK-2, and NK-3 receptors,
respectively, showed weak or negligible affinity toward the
SP1-7 binding sites. Negligible binding of SP1-7 to any of the
known tachykinin and μ-opioid receptors indicates the occur-
rence of a specific binding site for SP1-7, which most probably
is a specific receptor according to data reported for this
bioactive fragment.14,15
Recent published data by Zhou et al. indicates that the
attenuating effect on morphine withdrawal by SP1-7 might be
related to the σ receptor system because a σ receptor agonist
could reverse the SP1-7 related effect observed in the study.17
However, the same σ receptor agonist showed weak binding
affinity to the SP1-7 binding site, which indicates that SP1-7 is
modulating the effect seen for the σ receptor system rather
than working directly as a ligand for the σ receptor.
Historically, the identification of receptors (mostly G-protein
coupled receptors, GPCRs) combined with the development
of nonpeptide antagonists and agonists have been crucial for
the elucidation of the functional role of neuropeptides.18,19
Because they are encoded in the genome, the function of a
neuropeptide and its role in the mechanism behind diseases
can bestudied by knockout techniques in transgene animals.20
By nature, the specific biological effects of metabolites of
genome-encoded peptides cannot be studied by such a techni-
que. Thus, metabolically stable and selective nonpeptide
ligands (or peptidomimetics) of neuropeptide metabolites
are essential pharmacological tools for such studies.
The undecapeptide substance P (SPa), discovered in 1931,
was thefirstmammalianneuropeptide tobeidentified.1 SP is a
neurotransmitter and neuromodulator at G-protein coupled
neurokinin-1 (NK-1) receptors and is involved in a variety
of processes in different tissues. In the past, its role in pain
transmission in the central nervous system (CNS) has attrac-
ted the most attention.2 However, lately, interest has focused
on the involvement of SP in mental disorders like depression3
and anxiety.4 Despite worldwide efforts toward development
of SP related drugs, aprepitant (MK-869, approved as an
antiemetic agent) remains the only NK-1 receptor antagonist
on the market.5
Neuropeptides are in many cases degraded into bioactive
fragments with similar or modified biological activity. Addi-
tionally, it seems that the degradation products often exert a
modulatory effect on the response of the parent peptide.6
Substance P is cleaved enzymatically into at least five meta-
bolites, of which the N-terminal heptapeptide SP1-7 (H-Arg-
Pro-Lys-Pro-Gln-Gln-Phe-OH) is the major fragment.7,8
SP1-7 has been shown not only to modulate several actions
ofSPbut also opposeseveral effects of SP, e.g., the nociceptive
effect,9 inflammatory effect,10 and the potentiating effect on
opioid withdrawal symptoms.11,12 In addition, SP1-7 has also
been shown to promote memory function.13
*To whom correspondence should be addressed. Phone: þ46-18-
4714957. Fax: þ46-18-4714474. E-mail: Anja.Sandstrom@orgfarm.uu.se.
a Abbreviations: SP, substance P; NK-1, neurokinin-1; CNS, central
nervous system; NK-2, neurokinin-2; NK-3, neurokinin-3; GPCR,
G-protein coupled receptor; SAR, structure-activity relationship; EM-2,
endomorphin-2; EM-1, endomorphin-1; FHDoE, focused hierarchical
design of experiments; Phg, phenylglycine; Tyr(OMe), O-methyltyrosine;
Phe(2-Me), 2-methylphenylalanine; Thi, 2-thienylalanine; Cha, cyclohexyl-
alanine; Phe(3-F), 3-fluorophenylalanine; tciz, theoretical chemically in-
tuitive z-scale; ACE, angiotensin-converting enzyme; Boc, tert-butoxycar-
bonyl; NKB, neurokinin B. Abbreviations used for amino acids are
according to the rules of the IUPAC-IUB in Jones, J. H. J. Pept. Sci.
2003, 9, 1-8.
We aim at developing metabolically stable SP1-7 mimetics
to be used as research tools for the identification of the SP1-7
r
2010 American Chemical Society
Published on Web 02/23/2010
pubs.acs.org/jmc

2384 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Fransson et al.
target (for example a receptor) and for functional studies in
complex animal models. In the long term, such compounds
could be of pharmaceutical interest, i.e., as analgesics, anxio-
lytics, or anti-inflammatory drugs. Previously, we reported a
thorough structure-activity relationship (SAR) study of
SP1-7 involving an alanine-scan, truncations, and C- and
N-terminal modifications of the heptapeptide.21 It was demon-
strated that the C-terminal part of the heptapeptide and
especially the phenylalanine in position 7 is most essential
for binding. Moreover, C-terminal amidation potentiated the
ligands 5-10-fold; an effect also observed in an in vivo study
where the amidated analogue of SP1-7 was shown to reduce
opioid withdrawal signs in rats more effectively than the
native heptapeptide.17 The SAR study further led to the
identification of the tripeptide H-Gln-Gln-Phe-NH2 (Ki =
1.9 nM) showing equal affinity as the parent heptapeptide
(Ki=1.6 nM). The tripeptide constitutes a good starting point
toward low-molecular-weight drug-like compounds.
Table 1. Analytical Data and Purities of Peptides 1-20
compd
molecular mass
M þ Hþ
purity (%)a
purity (%)
1
571.3
479.3
545.3
495.3
495.3
572.3
408.2
311.2
164.1
312.2
353.2
311.2
311.2
311.2
297.2
277.2
355.2
317.1
297.2
297.2
335.2
572.3
480.3
546.3
496.3
496.3
573.3
409.1
312.1
165.0
313.1
354.1
312.1
312.1
312.1
298.1
278.1
356.2
318.1
298.1
298.1
336.2
99.8
98.5
>99.9b
>99.9b
>99.9b
>99.9b
>99.9b
>99.9b
>99.9c
>99.9b
2
3
99.8
99.6
4
5
99.7
6
>99.9
>99.9
>99.9
7
8
9d
10
11
12
13
14
15
16
17
18
19(I)
19(II)
20
>99.9
>99.9
98.4
96.2c
>99.9c
>99.9c
>99.9c
99.4c
99.8c
99.7c
>99.9c
>99.9c
>99.9c
>99.9c
>99.9c
>99.9
99.3
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
These results indirectly explained a previous observation by
Botros et al. that the tetrapeptide endomorphin-2 (EM-2,
H-Tyr-Pro-Phe-Phe-NH2), having a C-terminal phenylala-
nine amide, is a relative efficient binder to the SP1-7 binding
site with only a 10-fold reduction in affinity compared to
a The purity was determined by RP-HPLC with a H2O/MeCN
gradient with 0.1% TFA and UV detection at 220 nm using the column:
ACE 5 C18 (50mm ꢀ 4.6 mm). b The purity was determined by RP-
HPLC using the column: ACE 5 Phenyl (50 mm ꢀ 4.6 mm). c The purity
was determined by RP-HPLC using the column: Thermo Hypersil
Fluophase RP (50 mm ꢀ 4.6 mm). d The peptide was of analytical grade
from commercial source and tested without any further purification.
SP1-7.
15 EM-2 together with endomorphin-1 (EM-1, H-Tyr-
Pro-Trp-Phe-NH2) were identified as late as 1997 and con-
stitute the most potent endogenous ligands for the μ-opioid
receptors.22 Even though EM-1 contains a C-terminal pheny-
lalanine, it does not possess any significant affinity to the
SP1-7 binding site, suggesting the presence of a discriminating
binding pocket not able to fit a tryptophan adjacent to the
phenylalanine. The SAR of EM-1 and EM-2 for the affinity to
the μ-opioid receptors is rather well-explored, and in this case
the N-terminal amine and the phenolic tyrosine part are
crucial.23-25 Thus, an alanine scan of EM-2, examining the
importance of each amino acid residue for the μ-opioid
receptor interaction, showed that the substitutions of tyrosine
and proline for an alanine reduced affinity 20000- and 5000-
fold, respectively. However, when substituting the two
C-terminal phenylalanines, the affinity dropped only 400-
and 100-fold, respectively.23
We felt prompted to perform a SAR study of EM-2 toward
its binding to the SP1-7 binding site. Thus, we herein present
the results from an alanine scan and truncation study of EM-2
and report on the discovery of the dipeptide H-Phe-Phe-NH2,
found to have higher affinity to the SP1-7 binding site than the
parent tetrapeptide EM-2 and equal affinity as the heptapep-
tidic endogenous ligand SP1-7. The structural and stereo-
chemical requirements for the binding of H-Phe-Phe-NH2
have been further studied via small dipeptide libraries based
on this lead.
Figure 1. Inhibition of [3H]-SP1-7 binding by selected representa-
tive peptides. Data represent three independent experiments in
triplicate as mean ( SEM.
To determine the importance of the EM-2 amino acid side
chains for the binding to the SP1-7 binding site, each amino
acid residue was sequentially substituted for an alanine. The
three N-terminal amino acid residues (tyrosine, proline, and
the internal phenylalanine) could be substituted for an
alanine without significantly affecting the affinity (cf. 1 with
2, 3, and 4). However, the same substitution of the C-ter-
minal phenylalanine resulted in a dramatic loss of affinity
(see compound 5). These results prompted the synthesis and
development of N-terminally truncated analogues 7-9.
While the removal of the N-terminal tyrosine was well
tolerated, the removal of the two N-terminal amino acids
resulted in a 6-fold higher binding affinity (8) compared to
the parent peptide 1. However, further truncation to the
single phenylalanine amide 9 was deleterious for the affinity.
To assess the influence of the C-terminal primary amide,
carboxylic acid derivatives 6 and 10 were prepared. Intro-
duction of a carboxylic acid in the tetrapeptide 6 resulted in a
3-fold drop in binding affinity, while this modification in
the corresponding dipeptide gave an inactive compound.
Results
Affinity Studies toward the SP1-7 Binding Site. Peptides
1-20 were prepared using solid-phase peptide synthesis. The
purities determined by RP-HPLC (at 220 nm) were g96%.
In Table 1, the analytical data and the purities of the
synthesized peptides are shown. The affinity of these to the
SP1-7 binding site was determined in a binding assay using
spinal cord membrane from Sprague-Dawley rats and
radioactive [3H]-SP1-7 as a tracer. Competition experiments
were performed at six different concentrations, as illustrated
in Figure 1. The Ki values of 1-20 are reported in Table 2.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2385
N-terminal acetylation of the dipeptide was accompanied
with a 10-fold decrease in affinity. The preferred configura-
tion of the two phenylalanines of H-Phe-Phe-NH2 was
determined via the evaluation of the four L and D-variants
8 and 12-14. The natural L,L configuration gave the highest
binding affinity.
To investigate the effect on affinity from small variations
in side chain size and polarity, six new dipeptide analogues of
8 were designed using the focused hierarchical design of
experiments method (FHDoE).26 FHDoE is a statistical
molecular design method for selecting a diverse set of
analogues based on an active peptide. Each peptide in the
set is designed to retain a high resemblance to the active
peptide, thus increasing the probability of obtaining new
active compounds. Using the FHDoE method, the six dipep-
tides 15-20 shown in Table 2 (for an illustration of the used
amino acids see Figure 2) were selected and prepared. Three
of the new dipeptides were inactive (15, 17, and 20, Ki >
10000 nM) and the other three (16, 18, and 19) had signifi-
cantly lower affinity compared to 8 as shown in Table 2.
Human NK-1 and NK-3 Receptor Binding of H-Phe-Phe-
NH2. The possible binding of H-Phe-Phe-NH2 (8) to the
human neurokinin receptors NK-1 and NK-3 was studied.
The affinity of the compound was tested at a single concen-
tration of 10 μM in agonist radioligand binding assays
relying on displacement of [Sar9, Met(O2)11]-SP from NK-1
receptors and [MePhe7]-NKB from NK-3 receptors, respec-
tively. Compound 8 showed neither affinity for the NK-1
receptor nor for the NK-3 receptor.
Discussion
The design of low-molecular-weight drug-like molecules as
mimetics of bioactive peptides aims at eliminating problems
inherent to the peptide structure such as low bioavailability
and rapid degradation by proteolytic enzymes. Nonpeptide
ligands have in the past worked as valuable tools in the
investigation of neuropeptide receptors and their endogenous
ligands.19 Specifically, nonpeptide receptor antagonists have
played an important role in understanding the physiological
function of neuropeptides.18
In previous studies, it was found that the tetrapeptide EM-2
interacted with the SP1-7 site with only a 10-fold reduction in
affinity. Furthermore, a few other Tyr-Pro-containing opioid
related neuropeptides such as Tyr-MiF-1, β-casomorphin,
and morphiceptin showed moderate affinity to the SP1-7
binding site.15 Considering the smaller size of EM-2 compared
with the target heptapeptide, it was chosen as a starting point
for further development of low-molecular-weight SP1-7-mi-
metics. A systematic survey of the influence of each amino
acid of EM-2for affinity tothe SP1-7 binding site was initiated
via an alanine scan and truncation study.
The results from the alanine scan and the N-terminal
truncation study of EM-2 were conclusive and comparable
with the previous SAR study of SP1-7. Thus, the nature of the
three N-terminal amino acids is not important for binding
because these could be replaced by alanine without significant
change in affinity (cf. 1 with 2-4). In fact, the presence of the
N-terminal Tyr-Pro sequence reduces the binding strength
because its removal affords a dipeptide with 6-fold higher
affinity (8, Ki = 1.5 nM) as compared with EM-2 (1, Ki =
8.7 nM). This terminated early speculation about the involve-
ment of the Tyr-Pro sequence in binding to the SP1-7 binding
site.15 The C-terminal phenylalanine is required for strong
binding to this site, as seen by the 170-fold drop in affinity
(compared with EM-2) for the tetrapeptide 5, having an
alanine instead of phenylalanine.
Table 2. Ki Values of EM-2 Derivatives and Phe-Phe Analogues for
Inhibition of [3H]-SP1-7 Binding to Rat Spinal Cord Membrane
compd
sequence
Ki ( SEM (nM)
SP1-7 H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH
1 (EM-2) H-Tyr-Pro-Phe-Phe-NH2
1.6 ( 0.1
8.7 ( 0.1
Alanine Substituted and Terminally Modified Peptides
2
3
4
5
6
H-Ala-Pro-Phe-Phe-NH2
H-Tyr-Ala-Phe-Phe-NH2
H-Tyr-Pro-Ala-Phe-NH2
H-Tyr-Pro-Phe-Ala-NH2
H-Tyr-Pro-Phe-Phe-OH
11.5 ( 0.1
10.2 ( 0.3
9.4 ( 0.1
1460 ( 15
30.2 ( 1.7
Truncated Peptides
7
8
9
H-Pro-Phe-Phe-NH2
H-Phe-Phe-NH2
H-Phe-NH2
10.9 ( 0.7
1.5 ( 0.1
5028 ( 31
Terminally Modified Phe-Phe Peptides
H-Phe-Phe-OH
Ac-Phe-Phe-NH2
10
11
>10000
18.5 ( 1.7
Phe-Phe Analogues
(L)-Phe-(D)-Phe-NH2
12
13
14
540 ( 20
64 ( 3
175 ( 13
(D)-Phe-(D)-Phe-NH2
(D)-Phe-(L)-Phe-NH2
Phe-Phe Analogues Having Noncoded Amino Acids
(FHDoE Generated)
However, this single phenylalanine is not sufficient for
binding as can be observed for 9 with almost 600-times weaker
binding affinity than EM-2. N-Terminal acetylation
(dipeptide 11) provides a 10-fold affinity reduction compared
to the dipeptide amide 8, suggesting that an interaction,
possibly a hydrogen bond or an electrostatic interaction with
the free amine at the second (N-terminal) phenylalanine, is
lost. The C-terminal primary amide is also essential for the
affinity; the corresponding tetrapeptide carboxylic acid (6)
15
16
17
18
19a
H-Phe-Phg-NH2
H-Leu-Phe-NH2
>10000
10.2 ( 1.0
>10000
251 ( 4
(I) 2247 ( 115
(II) 182 ( 7
>10000
H-Tyr(OMe)-Phe(2-Me)-NH2
H-Phe-Thi-NH2
H-Phg-Phe-NH2
20
H-Cha-Phe(3-F)-NH2
a I and II: either of the diastereomers (L-Phg-L-Phe-NH2, D-Phg-L-
Phe-NH2).
Figure 2. Formulas of the side chains of coded and noncoded amino acids used for the synthesis of FHDoE generated analogues.

2386 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Fransson et al.
has more than three times lower affinity. The same substitu-
tion in the dipeptide series results in a completely inactive
carboxylic acid compound (10). The total loss of binding is
more drastic than expected because the corresponding car-
boxylic acid tetrapeptide 6 still exhibits an affinity of 30 nM.
Possibly, the C-terminal carboxylic acid and the C-terminal
primary amide induce different conformations of the dipep-
tide. Altogether, is seems that a dipeptide with a free R-amino
group and with a C-terminal phenylalanine amide fits well
into a small discrete binding pocket for the C-termini of SP1-7
and EM-2. The penultimate phenylalanine is of less impor-
tance because the affinity remains unchanged in the tetrapep-
tide having alanine in this position.
whether SP1-7 and the ligands tested here indeed target the
NK-3 receptor. The possible binding of 8 to NK-1 and NK-3
receptors was therefore evaluated. However, no binding was
achieved at a concentration of 10 μM. In addition, two
independent research groups have found that established
NK-3 agonists, i.e., the [Pro7]NKB and senktide, are inactive
at the SP1-7 binding site,14,15 which further strengthens our
hypothesis of anexplicit, not yet isolated receptor withspecific
features for the SP1-7 fragment.
One interesting aspect of our results is that 8 is a naturally
occurring cleavage product from the first step in the catabo-
lism of EM-2.31 In vivo, EM-2 is cleaved by dipeptidylpepti-
dase IV at the Pro2-Phe3 peptide bond and the dipeptides
generated are then hydrolyzed into amino acids by an amino-
peptidase. It is enticing to propose that some of the bioactivity
exerted by SP1-7 might be attributed to the dipeptide acting at
the SP1-7 binding site because co-occurrence of SP and EM-2
in the dorsal spinal cord has been established.32
The newly identified dipeptide lead 8 was further studied.
The stereochemical requirements for binding to the SP1-7
binding site were evaluated by synthesis of the three remaining
L and D variants 12, 13, and 14. Obviously, the preferred
configuration of the two phenylalanines in 8 is the natural
L,L configuration. Of the other variants, the D,D compound 13
showed the highest affinity although 40-times lower than the
L,L compound. For the D,L and the L,D dipeptides (12 and 14),
the affinity was significantly reduced (120- and 360-fold,
respectively). Thus, it seems that the mutual arrangement of
the two phenylalanine side chains is important. It would be of
interest to study these compounds for functional activity as
well because there are several examples where an L to D switch
of an amino acid within a neuropeptide results in antagonistic
properties. However, such functional assays are still awaited.
To investigate the effects of structural modifications of 8 on
binding affinity, the FHDoE method was used to design a
small series of dipeptides with similar physicochemical prop-
erties as 8. Because the FHDoE method yields relatively
conservative modifications, the probability of obtaining
high-affinity peptides should be rather high. However, all
dipeptides selected using the FHDoE method were either
inactive (Ki > 10 000 nM) or had considerably less affinity
than 8. This indicates that modifications of the dipeptide side
chains are not well tolerated. It appears that the C-terminal
phenylalanine is the one most sensitive toward modification
because shortening of the side chain to give phenylglycine (15)
resulted in inactivity. Furthermore, replacement of the phenyl
ring for the bioisosteric thiophene ring as in compound 18
reduced the affinity 170-fold. ortho-Methyl and meta-fluoro
substituents on the phenyl moiety gave inactive compounds
(17 and 20). However, in these cases, the N-terminal amino
acid was also changed which complicates the interpretation.
The pocket for the side chain of the N-terminal amino acid
seems to be less discriminating because the replacement of
phenylalanine by leucine was rather well accepted, albeit with
a 7-fold reduction in affinity (cf. 16 with 8).
Conclusion
A systematic evaluation of the affinity of peptide ligands
originating from the tetrapeptide lead EM-2 toward the SP1-7
binding site has been performed. Thus, we were able to
identify the dipeptide H-Phe-Phe-NH2 with equal affinity as
the endogenous heptapeptide ligand SP1-7 and higher affinity
than the tetrapeptide lead EM-2. Further SAR studies suggest
that the C-terminal phenylalanine amide is crucial for binding
and that the L-configuration gives the preferred side chain
arrangement. The results also indicate that the binding pocket
interacting with the side chain of the N-terminal amino acid is
more tolerant for changes. Conversion of the dipeptide lead
H-Phe-Phe-NH2 into stable nonpeptide ligands is in progress.
H-Phe-Phe-NH2 and nonpeptide ligandsthereofwillbeexplo-
red further in functional animal studies to achieve a more
thorough understanding of the physiological function of SP1-7
.
Experimental Section
General Methods. Preparative RP-HPLC was performed on
a system equipped with a Zorbax SB-C8 column (150 mm ꢀ
21.2 mm) or a 10 μm Vydac C18 column (250 mmꢀ22 mm), in
both cases with UV detection at 230 nm. Analytical RP-HPLC-
MS was preformed on a Gilson-Finnigan ThermoQuest AQA
system (Onyx monolithic C18 column, 50 mmꢀ4.6 mm; MeCN/
H2O gradient with 0.05% HCOOH) in ESI mode, using UV
(214 and 254 nm) and MS detection. The purity of each of the
peptides was determined by RP-HPLC using the columns ACE
5 C18 (50 mmꢀ4.6 mm) and ACE 5 Phenyl (50 mmꢀ4.6 mm) or
Thermo Hypersil Fluophase RP (50 mmꢀ4.6 mm) with a H2O/
MeCN gradient with 0.1% TFA and UV detection at 220 nm.
All peptides showed purity above 95%.
NMR spectra were recorded on a Varian Mercury plus
1
spectrometer (1H at 399.8 MHz and 13C at 100.5 MHz or H
In summary, we have been able to identify a dipeptide
H-Phe-Phe-NH2 with affinity in the nanomolar range to
the binding site of the endogenous heptapeptide SP1-7. We
are optimistic about the future conversion of this lead into
stable nonpeptide ligands. In fact, dipeptides have previously
served as successful starting points for the elegant design and
development of orally bioavailable nonpeptides, e.g., antago-
nists to the NK-1, NK-2, and NK-3 receptors.27 Several
angiotensin-converting enzyme (ACE) inhibitors on the drug
market can be seen as modified dipeptides,28 and the dipeptide
Boc-Phe-Phe-NH2 was the lead for the development of
PD161182, a nonpeptide NK-3 receptor selective antagonist.
29,30 Apart from the N-protecting Boc-group, it is identical to
our dipeptide lead 8, a fact that of course raises the question
at 399.9 MHz and 13C at 100.6 MHz) at ambient temperature.
Chemical shifts (δ) are reported in ppm referenced indirectly to
TMS via the solvent residual signal. Exact molecular masses
were determined on a Micromass Q-Tof2 mass spectrometer
equipped with an electrospray ion source at the Department
of Pharmaceutical Biosciences, Uppsala University, Sweden.
Amino acid analyses were performed at the Department of
Biochemistry and Organic Chemistry, Uppsala University,
Sweden. All chemicals and solvents were of analytical grade
from commercial sources.
General Synthesis of Peptides 1-6. The peptides 1-6 were
synthesized with a Symphony instrument (Protein Technologies,
Inc., Tucson, AZ) using standard Fmoc chemistry as earlier
described.21 Cleavage and purification were carried out as

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2387
described below. The reported yields are based on the loading
of the starting resin and with correction for peptide content
according to amino acid analysis.
from male Sprague-Dawley rats. The rats (Alab AB, Sollentuna,
Sweden), weighing 200-250 g, were housed in groups of four in
air-conditioned rooms (22-23 °C and a humidity of 50-60%)
under an artificial light-dark cycle and had free access to food
and water. Prior to tissue sampling, the rats were allowed to
adapt to the laboratory environment for 1 week. The animals
(n = 15) were killed by decapitation, and the spinal cords were
rapidly removed and quickly frozen. Tissues were then kept at
-80 °C until analyzed. The animal experiment was approved by
the local ethical committee in Uppsala, Sweden.
The frozen spinal cords weighing approximately 250-
300 mg/animal were thawed and placed on ice before being
homogenized for 30 s in 30 volumes of ice-cold 50 mM Tris-
HCl buffer (pH 7.4), containing 5 mM KCl and 120 mM NaCl,
using a Polytron homogenizer. The homogenate was then
centrifuged at 40000g for 20 min at 4 °C, and the supernatant
was discarded. The resulting pellet was resuspended in 30 volumes
of ice-cold 50 mM Tris-HCl buffer (pH 7.4), containing 300 mM
KCl and 10 mM EDTA. Following incubation on ice for 30 min,
the sample was centrifuged at 40000g for 20 min at 4 °C. The
pellet obtained was diluted and homogenized in 30 volumes
of ice-cold 50 mM Tris-HCl, containing 0.02% BSA, 5 mM,
EDTA, 3 mM MnCl2, and 40 μg bacitracin and recentrifuged
twice as described above. The final pellet was resuspended and
homogenized in 5 volumes of ice-cold 50 mM Tris-HCl buffer
(pH 7.4) and immediately frozen at -80 °C until used in binding
studies. The protein concentration of the membrane suspension
was determined according to the method of Lowry33 using
bovine serum albumin as protein standard.
Radioligand Binding Assay. Assessment of the binding affinity
for the various compounds analyzed in this study was carried
out using the analogue [3H]-SP1-7 as tracer. Assays were per-
formed in tubes containing 50 μL of spinal cord membrane
suspension (200 μg protein) and 0.9 nM of [3H]-SP1-7 (specific
activity: 3.11 TBq/mmol (84 Ci/mmol)) in a final volume of
0.5 mL 50 mM Tris binding buffer (pH 7.4), containing, 3 mM
MnCl2, 0.2% BSA, and peptidase inhibitors (40 μg/mL baci-
tracin, 4 μg/mL leupeptin, 2 μg/mL aprotinin, and 4 μg/mL
phosphoramidon). The amounts of total and unspecific binding
in percent of the total radioactivity added were approximately
7% and 1.7%, respectively. The competition experiments were
conducted at six different concentrations varying between
0.01 nM and 1 μM of unlabeled compounds. Nonspecific
binding was determined in the presence of 1 μM SP1-7. Samples
were incubated for 60 min at 4 °C before being terminated by
rapid filtration under vacuum with a Brandel 24-sample cell
harvester through Whatman GF/C glass fiber filters treated
with a solution containing 50 mM Tris (pH 7.4), 0.3% poly-
ethylenimine (PEI), and 0.5% Triton X-100 at 4 °C overnight.
Filters were washed twice with 3 mL of cold 50 mM Tris-HCl
(pH 7.4) complemented with 0.1 mg/mL BSA and 3 mM MnCl2.
The filters were air-dried for about 60 min before the bound
radioactivity was determined using a liquid scintillation counter
(Beckman LS 6000IC) at 63% efficiency in 5 mL of counting
scintillant. The specific binding was determined as the difference
between total and unspecific binding. All assays were run
in triplicates, and each assay was repeated at least three times
at different days. Data and statistics from the competition
experiments were analyzed in the GraFit program (Erithacus
Software, UK).
Peptide 8 (H-Phe-Phe-NH2). To an ice-cold solution of
Z-Phe-OSu (200 mg, 0.505 mmol) and the hydrochloride salt
of H-Phe-NH2 (111 mg, 0.555 mmol) in EtOAc (15 mL) N,N-
diisopropylethylamine (DIEA, 97 μL, 0.555 mmol) was added
and the mixture stirred at room temperature. After 1.5 h, an
additional portion of DIEA (97 μL, 0.555 mmol) was added and
the mixture was stirred overnight. The precipitate was filtered
off and dried under vacuum to yield Z-Phe-Phe-NH2 (131 mg) as
a white solid. The crude product was analyzed by LC-MS and
found to be adequate for further reaction.
The Z-protected product was dissolved in MeOH:CHCl3 (1:1,
10 mL) in a round-bottom flask and Pd/C (10%, 33 mg, 0.032
mmol) was added. The flask was sealed with a septum and
purged, first with nitrogen and then with hydrogen gas. The
reaction mixture was stirred under hydrogen atmosphere over-
night and was then filtered and evaporated. The product was
purified by RP-HPLC as described below to yield H-Phe-Phe-
NH2 in 19 mg (20%). HPLC purity: C18 column >99.9%,
phenyl column >99.9%. LC/MS (M: 311.2): 312.1 (M þ Hþ).
1H NMR (CD3OD) δ 2.96 (dd, J=8.5, 13.8 Hz, 1H), 3.01 (dd,
J=8.6, 14.3 Hz, 1H), 3.14 (dd, J=6.2, 13.8 Hz, 1H), 3.26 (dd,
J=5.5, 14.3 Hz, 1H), 4.08 (J=5.5, 8.6 Hz, 1H), 4.66 (dd, J=6.2,
8.5 Hz, 1H), 7.17-7.39 (m, 10H). 13CNMR(CD3OD) δ38.6, 39.1,
55.5, 56.0, 127.9, 128.9, 129.5, 130.2, 130.3, 130.5, 135.5, 138.2,
169.5, 175.3. Amino acid analysis: Phe, 2.00 (70% peptide).
General Synthesis of Peptides 7 and 10-20. Coupling. The
peptides 7 and 10-20 were synthesized manually from Rink
Amide MBHA resin (0.66 mmol/g) or Phe-2-chlorotrityl resin
(0.85 mmol/g) in 2 mL disposable syringes fitted with porous
polyethylene filters. Standard Fmoc conditions were used, and
the Fmoc protecting group was removed by treatment with 20%
piperidine in DMF (2ꢀ1.5 mL, 2 þ 10 min) and the polymer was
washed with DMF (6 ꢀ 1.5 mL, 6 ꢀ 1 min). Coupling of the
appropriate amino acid, Fmoc-AA-OH (4 equiv) was per-
formed in DMF (1.5 mL) using N-[(1H-benzotriazole-1-yl)-
(dimethylamino)methylene]-N-methylmethanaminium hexafluo-
rophosphate N-oxide (HBTU, 4 equiv) in the presence of DIEA
(8 equiv). The resin was washed with DMF (5 ꢀ 1.5 mL, 5 ꢀ
1 min) and subsequently deprotected and washed as described
above. After completion of the coupling cycle, the resin was also
washed with several portions of DMF, CH2Cl2, and MeOH
before it was dried in in vacuo.
Cleavage. The final peptide was cleaved from the resin by
treatment with triethylsilane (100 μL) and 95% aqueous tri-
fluoroacetic acid (TFA, 1.5 mL) followed by agitation for 2 h at
room temperature. The resin was filtered off and washed with
TFA (2ꢀ0.3-0.5 mL). The filtrate was collected in a centrifuge
tube and concentrated in a stream of nitrogen. Cold diethyl
ether (e7 mL) was used to precipitate the product, which
was collected by centrifugation, washed with cold diethyl ether
(e3 ꢀ 7 mL), and dried.
Purification. The crude peptide was dissolved in MeCN/0.1%
aqueous TFA, filtered through a 0.45 μm nylon membrane and
purified in 1-2 runs by RP-HPLC. Selected fractions were
analyzed by RP-HPLC and RP-HPLC-MS, and those contain-
ing pure product were pooled and lyophilized. The reported
yields are based on the loading of the starting resin and with
correction for peptide content according to amino acid analysis.
[2,4-DehydroPro]SP1-7. The precursor peptide for tritium-
labeling [2,4-DehydroPro]SP1-7 was prepared by standard solid-
phase peptide synthesis techniques using Fmoc/t-butyl protec-
tion and purified as described above. Tritium labeling of the
precursor was performed by Amersham Biosciences (Cardiff,
UK) and resulted in 370 MBq (10 mCi) of [3H]-SP1-7 with a
specific activity of 3.11 TBq/mmol (84 Ci/mmol).
In Vitro Pharmacology: Human NK-1 and NK-3 Receptor
Binding Assays. This study was performed at Cerep (France)
according to literature.34,35 One concentration (10 μM) of 8 was
tested. [Sar9, Met (O2)11]-SP was used as control agonist for the
NK-1 receptor, and [MePhe7]-NKB was used as control agonist
for the NK-3 receptor.
Library Design using Focused Hierarchical Design of Experi-
ments (FHDoE). A dipeptide library was designed using the
FHDoE method, focused toward 8. The method was directly
adopted from the original article by Muthas et al.26 in which
Animal Experiment and Membrane Preparation. The prepara-
tions of receptor membranes were conducted using spinal cords

2388 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Fransson et al.
both the dipeptide design matrix and the principal properties for
113 R-amino acids are reported. The principal properties for the
R-amino acids are obtained as tcizR1 and tcizR2 values and are
mainly related to amino acid side chain size and polarity,
respectively. A set of amino acids similar to phenylalanine was
selected by considering only amino acids with tcizR1 and tcizR2
values similar to phenylalanine. The range was set to -1.23 to
1.93 for tcizR1 and -2.28 to 0.2 for tcizR2 (corresponding to
(25% of the total tcizR1 and tcizR2 span, respectively, centered
on phenylalanine). The final selection within each list of possible
amino acids used for substitution of Phe in each analogue was
based on in-house availability. A more detailed description
of the FHDoE method can be found in the Supporting Infor-
mation.
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Acknowledgment. We gratefully acknowledge the financial
support from the Swedish Research Council and the Knut and
Alice Wallenberg Foundation. We thank Anna Carlsson for
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FHDoE method. This material is available free of charge via the
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