Structure-activity relationship study of opiorphin, a human dual ectopeptidase inhibitor with antinociceptive properties

Article abstract of DOI:10.1021/jm2012112

Toward developing new potential analgesics, this first structure-activity relationship study of opiorphin (H-Gln-Arg-Phe-Ser-Arg-OH), a human peptide inhibiting enkephalin degradation, was performed. A systematic Ala scanning proved that Phe³ is a key residue for neprilysin and aminopeptidase N (AP-N) ectoenkephalinase inhibition. A series of Phe³-halogenated analogues revealed that halogen bonding based optimization strategies are not applicable to this residue. Additional substituted Phe³ derivatives showed that replacing l-Phe³ for d-Phe³ increased the AP-N inhibition potency by 1 order of magnitude. NMR studies and molecular mechanics calculations indicated that the improved potency may be due to CH-π stacking interactions between the aromatic ring of d-Phe³ and the Hγ protons of Arg². This structural motif is not possible for the native opiorphin and may be useful for the design of further potent and metabolically stable analogues.

Full text of DOI:10.1021/jm2012112

Article
pubs.acs.org/jmc
Structure−Activity Relationship Study of Opiorphin, a Human Dual
Ectopeptidase Inhibitor with Antinociceptive Properties
Monica Rosa,† Gemma Arsequell,† Catherine Rougeot,‡ Luis P. Calle,§ Filipa Marcelo,§ Marta Pinto,∥
̀
Nuria B. Centeno,∥ Jesus Jimenez-Barbero,§ and Gregorio Valencia*,†
́
́
†Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain
‡Institut Pasteur-Unite
de Biochimie Structurale et Cellulaire/URA2185CNRS, Paris, France
§Chemical and Physical Biology, Centro de Investigaciones Biolog
icas (CIB-CSIC), Madrid, Spain
́
́
∥Computer-Assisted Drug Design Laboratory, Research Group on Biomedical Informatics (GRIB), IMIM-Universitat Pompeu Fabra,
Barcelona, Spain
S
* Supporting Information
ABSTRACT: Toward developing new potential analgesics,
this first structure−activity relationship study of opiorphin (H-
Gln-Arg-Phe-Ser-Arg-OH), a human peptide inhibiting
enkephalin degradation, was performed. A systematic Ala
scanning proved that Phe3 is a key residue for neprilysin and
aminopeptidase N (AP-N) ectoenkephalinase inhibition. A
series of Phe3-halogenated analogues revealed that halogen
bonding based optimization strategies are not applicable to this
residue. Additional substituted Phe3 derivatives showed that
replacing L-Phe3 for D-Phe3 increased the AP-N inhibition
potency by 1 order of magnitude. NMR studies and molecular
mechanics calculations indicated that the improved potency
may be due to CH−π stacking interactions between the aromatic ring of D-Phe3 and the Hγ protons of Arg2. This structural motif
is not possible for the native opiorphin and may be useful for the design of further potent and metabolically stable analogues.
information available for AP-N is more limited and reduced to
three X-ray crystal structures corresponding to complexes of a
small-molecule inhibitor with an AP-N variant from Escherichia
coli.3 Regrettably, human AP-N has an overall sequence identity
of only 13.6% with the one fom E. coli.3a Even though
homology models of human AP-N have been recently
developed for designing nonpeptidic inhibitors, the authors
accept that authentic structural information on the human
variant would be necessary for building more accurate and
useful models.4 In spite of these caveats, the analogies of the
well-studied bacterial protease thermolysin with NEP have long
been used for the early designs of selective as well as dual and
transition-state inhibitors of NEP and AP-N using secondary
structure prediction and molecular modeling algorithms.5 One
of the first examples of such compounds is the selective NEP
inhibitor thiorphan, which has led to drugs with antisecretory
activity such as the antidiarrheal racecadotril (acetorphan).6
The difficulty in formulating a dual inhibitor arises from the
obvious necessity to find optimal interactions for two zinc
peptidases that, despite their similarity, show different
specificities. Nevertheless, mixed inhibitory products such as
RB101, which is prepared by covalently linking a potent AP-N
INTRODUCTION
■
The need for compounds with intermediate analgesic activity
between nonsteroidal anti-inflammatory drugs (NSAIDs) and
opioids has fueled the interest in enzyme inhibitors that protect
endogenous opioid neuropeptides. One goal has been to
potentiate the natural analgesic effect of the endogenous
peptides Met- and Leu-enkephalin by inhibiting their enzymatic
degradation. Owing to the complementary roles of both
neprilysin [neutral endopeptidase (NEP), EC 3.4.24.11] and
aminopeptidase N (AP-N; EC 3.4.11.2) in enkephalin
inactivation, the design of dual inhibitors of these ectopepti-
dases seems an ideal approach.1 Both NEP and AP-N are
membrane-bound zinc metalloenzymes that belong to the M13
(NEP) and M1 (AP-N) peptidase families. Although they share
a common catalytic domain, differences in the spatial
arrangement of the catalytic residues and of their surroundings
cause NEP to function as a carboxypeptidase that cleaves
substrates at the N-terminal side of hydrophobic amino acids
such as Phe, Leu, and Met, while AP-N releases the N-terminal
amino acids from peptides that are not substituted α-amino
acids. To date, both enzymes have escaped full experimental
structural elucidation. Thus, the X-ray crystal structure of the
extracellular domain (Asp52−Trp749) complexed with six
different small-molecule inhibitors is the only structural
information available for NEP.2 Conversely, the structural
Received: September 13, 2011
Published: January 8, 2012
© 2012 American Chemical Society
1181
dx.doi.org/10.1021/jm2012112 | J. Med. Chem. 2012, 55, 1181−1188

Journal of Medicinal Chemistry
Article
Scheme 1. Opiorphin, Ala Scan Opiorphin Analogues, and Halogenated [Phe3]-Opiorphin Analogues
with an NEP inhibitor, have been proposed.7 A further step in
this process has been the design of single-molecule compounds
such as RB3001 that are true dual inhibitors.8 Unfortunately,
none of them have yet reached clinical applications in pain
conformational studies in solution and molecular mechanic
calculations.
RESULTS AND DISCUSSION
■
To gain a first insight into the most influential residues in NEP
and AP-N inhibitory activity of human opiorphin, five
analogues corresponding to a traditional Ala scan were
synthesized (Scheme 1). The resulting peptides, as well as
the other peptide derivatives reported in this paper, were
prepared by solid-phase peptide synthesis techniques using
standard Fmoc protocols and purified by reversed-phase liquid
chromatography using a VersaFlash flash chromatography
system. All the compounds were evaluated for potential
inhibitory activity in vitro using commercial recombinant
human NEP and AP-N soluble enzymes and synthetic
fluorogenic substrates.14−16 The monitoring of substrate
hydrolysis in the presence of several concentrations of inhibitor
provided kinetic plots such as those depicted in Figure 1 for the
[Ala1]-opiorphin analogue.
management.
This search for analgesics that modulate endogenous
enkephalin circulating levels has received a new impulse due
to the recent discovery and isolation from mammalian sources
of the peptides spinorphin (LVVYPWT)9 and sialorphin
(QHNPR).10 These peptides, together with their human
counterpart opiorphin (QRFSR),11 which was isolated from
human saliva, act as endogenous NEP and AP-N inhibitors. It
has been proved that opiorphin induces antinociceptive effects
in various rodent models of morphine-sensitive pain by
activating endogenous enkephalin-related opioid pathways,
thus demonstrating that the natural catabolism of enkephalins
is physiologically regulated.11,12 This, in turn, supports the
therapeutic concept that analgesia may be amenable to
enkephalin-induced potentiation by using molecules derived
from naturally occcuring peptides such as opiorphin.13 In a first
exploration of this concept and toward the design of potential
small-molecule peptidomimetic drugs, we have used a “ligand-
based” approach to circumvent the lack of reliable structural
information on these enzyme targets. Therefore, we here
present our results on an Ala substitution scanning study that
has revealed that Phe3 is a crucial residue for opiorphin
inhibitory activity toward both NEP and AP-N ectoenzymes.
This, in turn, prompted us to conduct the first SAR study of
opiorphin that is focused on Phe3. The results show that
halogen atoms placed at the aromatic function of Phe3 are not
able to induce additional stabilizing interactions with either of
the two enzymes by halogen bonding. Most notably, we have
found that a D-Phe for L-Phe substitution induces a 1 order of
magnitude increase in AP-N inhibition potency, which can be
explained by CH−π stacking interactions between the aromatic
ring of Phe3 and the Hγ protons of Arg2, as unveiled by NMR
From these kinetic plots of enzyme inhibition, the
corresponding dose-dependent inhibition curves were calcu-
lated for every compound and the two enzymes (Figure 2).
Activity values expressed as EC50 have been extrapolated for
these curves. Compounds showing EC50 values of over 100 μM
were considered inactive, and compounds showing EC50 values
over 70 μM very poor inhibitors.
As seen in Table 1, all the Ala-substituted analogues were
inferior dual inhibitors compared to the parent opiorphin. Only
the Ala for Arg2 substitution produced an analogue slightly
more potent for NEP than the parent peptide, but it was almost
inactive for AP-N. The most interesting compound is 4, which
is a poor inhibitor for AP-N (IC50 over 70 μM) and is totally
inactive against NEP (IC50 over 100 μM), conditions that are
unique among the other compounds of Table 1. To us, 4 being
the simultaneously least active compound of the series is a good
indication of the relevant role played by the aromatic residue of
Phe3 in the interactions of opiorphin with both its targets, NEP
1182
dx.doi.org/10.1021/jm2012112 | J. Med. Chem. 2012, 55, 1181−1188

Journal of Medicinal Chemistry
Article
Figure 2. Dose-dependent inhibition by opiorphin (1), [D-Phe]-
opiorphin (15), and [p-F-D-Phe]-opiorphin (11) of Ala-MCA
substrate hydrolysis by human AP-N. Each point represents the
percentage of inhibition calculated as percentage of specific velocity
without inhibitor − velocity in the presence of inhibitor/velocity
without inhibitor. The opiorphin analogue concentration (μM) is
plotted on a log scale.
Figure 1. Fluorescence monitoring of Ala-MCA substrate hydrolysis
by AP-N in the presence and absence of the [Ala1]-opiorphin
analogue. According to the conditions of initial velocity measurement,
the kinetics of appearance of the fluorescent signal (RFU) were
directly proportional to the rate of hydrolysis of the substrates by AP-
N. The background rate of substrate autolysis representing the
fluorescent signal obtained in the absence of the enzyme was
subtracted to calculate the initial velocities [RFU (relative fluorescent
units)/min].
Table 1. Inhibition of hAP-N and hNEP Activities by
Opiorphin and Ala Analogues
hAP-N IC50
hNEP-endoP
peptide compound
sequence
SD (μM) IC50 SD (μM)
and AP-N, and therefore was considered a good target for an
SAR study.
1
opiorphin
H-Gln-Arg-Phe-
Ser-Arg-OH
8.1 0.1
30
3
First, given the critical role of Phe3 in the inhibitory potency
of opiorphin toward NEP and AP-N, and that this aromatic side
chain residue is a suitable structural motif for the introduction
of halogen atoms, we sought to improve the inhibitory
properties of opiorphin by applying the halogen bonding
rationale. This approach is based on the ability of halogen
atoms to function as general, effective, and reliable sites for
directing molecular recognition processes.17 The fact that about
half of the molecules currently entering high-throughput
screening tests are halogenated highlights the importance of
this concept for drug design.18 Thus, a suitable set of
commercial Phe building blocks with halogen atoms in their
side chains were chosen and the corresponding opiorphin
analogues prepared and tested (Scheme 1).
2
[Ala1]-
opior-
phin
[Ala2]-
opior-
phin
[Ala3]-
opior-
phin
[Ala4]-
opior-
phin
H-Ala-Arg-Phe-
Ser-Arg-OH
37
3
3
>70
3
4
5
6
H-Gln-Ala-Phe-
Ser-Arg-OH
65
12
1
H-Gln-Arg-Ala-
Ser-Arg-OH
>70
47
≫100
H-Gln-Arg-Phe-
Ala-Arg-OH
2
39
33
2
[Ala5]-
opior-
phin
H-Gln-Arg-Phe-
Ser-Ala−OH
>70
2
To further examine the role of Phe3 in opiorphin activity, a
second series of analogues, in which Phe3 was substituted by
Phe isomers, derivatives, and mimics that have been chosen
because they are commonly used for SAR studies of other
naturally occurring peptides19,20 and their corresponding
building blocks are commercially available, were prepared and
tested against both NEP and AP-N activities (Scheme 2). A
further criterion for this selection was to preliminarily see if
substitutions possibly leading to enzymatic resistance such as L
by D and α by β changes on the configuration of this amino acid
have an effect on the biological activity of opiorphin. The Bpa
As Table 2 reflects, none of the seven halogenated
compounds assayed (7−13) were more potent dual NEP and
AP-N inhibitors than the parent opiorphin. Moreover, in the
homologous series of halogenated analogues at the para
position (10, 12, 13), activities did not gradually increase
with the atomic weight of the halogen atom (F vs Cl vs I). A
similar situation is suggested by the shortest ortho-halogenated
series (7, 8) (F vs Br). This evidence supports that ortho and
para halogenation of Phe3 does not generate new stabilizing
interactions mediated by halogen bonds between opiorphin and
the two enzymes. Fluorination at meta (9) and perfluorination
of Phe3 (14) totally prevent enzymatic inhibition.
1183
dx.doi.org/10.1021/jm2012112 | J. Med. Chem. 2012, 55, 1181−1188

Journal of Medicinal Chemistry
Article
Table 2. Inhibition of hAP-N and hNEP Activities by
Opiorphin and Halogenated Phe3 Analogues
Table 3. Inhibition of hAP-N and hNEP Activities by
Opiorphin and Other Phe3 Analogues
hNEP-
hNEP-endoP
IC50 SD
(μM)
hAP-N IC50 endoP IC50
hAP-N IC50
SD (μM)
peptide
name
compound
SD (μM)
SD (μM)
name
compound
1
opiorphin
H-Gln-Arg-Phe-
Ser-Arg-OH
8.1 0.1
30
3
3
2
1
opiorphin
H-Gln-Arg-Phe-
Ser-Arg-OH
8.1 0.1
30
3
7
8
[o-F-Phe3]-
H-Gln-Arg-(o-F)
Phe-Ser-Arg-OH
44
3
4
48
15 [D-Phe3]-
H-Gln-Arg-D-Phe-
Ser-Arg-OH
0.5 0.1
0.5 0.1
≫100
opiorphin
opiorphin
[ o-Br-Phe3]-
H-Gln-Arg-(o-Br)
Phe-Ser-Arg-OH
65
30
11 [p-F-D-Phe3]-
H-Gln-Arg-(p-F)-
D-Phe-Ser-Arg-
OH
>100
opiorphin
opiorphin
9
[m-F-Phe3]-
H-Gln-Arg-(m-F)
Phe-Ser-Arg-OH
>100
>100
>100
>100
opiorphin
16 [D-β-Phe3]-
H-Gln-Arg-D-β-
Phe-Ser-Arg-OH
>70
≫100
[p-F-Phe3]-
H-Gln-Arg-(p-F)
Phe-Ser-Arg-OH
13.5 0.5
0.5 0.1
opiorphin
10
11
12
13
14
opiorphin
17 [Homophe3]-
H-Gln-Arg-
Homophe-Ser-
Arg-OH
54 10
70 10
[p-F-D-Phe3]-
H-Gln-Arg-(p-F)-D-
Phe-Ser-Arg-OH
opiorphin
opiorphin
18 [Phg3]-
H-Gln-Arg-Phg-
Ser-Arg-OH
10
64
45
30
3
5
4
3
≫100
70 10
>100
[p-Cl-Phe3]-
H-Gln-Arg-(p-Cl)
Phe-Ser-Arg-OH
48
30
5
3
70 10
>100
opiorphin
opiorphin
19 [Tyr3]-
H-Gln-Arg-Tyr-
Ser-Arg-OH
[p-I-Phe3]-
H-Gln-Arg-(p-I)
Phe-Ser-Arg-OH
opiorphin
opiorphin
20 [p-Me-Phe3]-
H-Gln-Arg-(p-Me)
Phe-Ser-Arg-OH
[2,3,4,5,6-
pentafluoro-
Phe3]-
H-Gln-Arg-
(2,3,4,5,6-
pentafluoro)Phe-
Ser-Arg-OH
≫100
>100
opiorphin
21 [Bpa3]-
H-Gln-Arg-Bpa-
Ser-Arg-OH
>100
opiorphin
opiorphin
22 [Nphe3]-
H-Gln-Arg-Nphe-
Ser-Arg-OH
≫100
≫100
Scheme 2. [Phe3]-Opiorphin Analogues
opiorphin
dispersed than those containing the natural L-Phe residue. In a
more quantitative analysis, the chemical shift indexes (CSIs)21
were calculated, which measure the deviation of the different
backbone α protons (Hα) from the typical random coil values
(Figure S1 and Table S3, Supporting Information). However,
the most remarkable structural differences between the
opiorphin analogues and the natural compound were observed
when comparing the D-Phe versus L-Phe analogues due to the
different β-sheet-forming propensity of Arg2. The chemical-
shift-based evidence for these differences was obtained by
3
measuring the values of the key coupling constant JNH,Hα for
Arg2 on these epimeric peptides. Thus, for instance, the value of
3JNH,Hα measured for [p-F-Phe3]-opiorphin was 6.9 Hz, while
for the corresponding [D-Phe3]-opiorphin analogue it decreased
to 5.4 Hz. An additional difference concerning the Arg2 residues
was that the Hγ protons of the D-Phe peptides were
significantly shielded with respect to their L-Phe3 counterparts
(Table S1, Supporting Information). In particular, the R2 Hγ2
and Hγ3 protons of [p-F-D-Phe3]-opiorphin appeared shielded
at a higher field, −0.38 and −0.52 ppm, respectively, than those
of the L-Phe analogue ([p-F-Phe3]-opiorphin).
substitution (21) was screeened in view of future photoaffinity
labeling experiments.
As seen from their EC50 values (Table 3), none of the eight
compounds (15−21) are dual NEP and AP-N inhibitors. Also,
small structural changes (i.e., Homophe, Phg, and Tyr for Phe3)
(17, 18, 19) drastically affected NEP inhibition while still
preserving some AP-N inhibition. The most notable finding
was that [D-Phe3]-opiorphin is 1 order of magnitude more
potent than opiorphin in AP-N inhibition. This substitution
even tolerates para fluorination, as the resulting compound
displays similar AP-N inhibitory potency.
At this point, taking into account this experimental evidence,
molecular mechanics calculations were performed to construct
a 3D model. Thus, 3D structures for the natural opiorphin
peptide and its corresponding [D-Phe3]-opiorphin analogue
were generated by employing a conformational search protocol
with Macromodel,22 as integrated in the Maestro package.23 As
expected from their small size, different minima were found for
these two peptides. However, for the D-Phe3 derivative, the
global minimum presented spatial proximity between the D-
Phe3 aromatic ring and the Hγ protons of Arg2, a fact that
supports the experimental chemical shift evidence (Figure 3).
This geometry is also in agreement with the experimental
coupling constants that define the orientation of D-Phe3, that is,
To rationalize these observations from a structural
perspective, solution NMR studies were then performed.
Using standard 2D NMR methods, chemical shift assignments
for the natural peptide opiorphin and for a subset of the
analogues were collected (Table S2, Supporting Information).
Analysis of these data mainly served to verify the different
tendencies of natural [L-Phe3]-opiorphin with respect to its D-
Phe3 analogue. On the other hand, no significant differences
were appreciated between the fluorine-containing derivatives
and the parent compound.
3
3JHα,Hβ2 and JHα,Hβ3 (4.6 and 11.3 Hz, respectively). Only in
this calculated orientation does the aromatic ring adopt the
proper presentation to provide CH−π stacking interactions
with the Hγ protons at the arginine side chain. The observed
1
On a first inspection, the H NMR spectra of the peptides
containing D-Phe showed that the 1H signals were more
1184
dx.doi.org/10.1021/jm2012112 | J. Med. Chem. 2012, 55, 1181−1188

Journal of Medicinal Chemistry
Article
experimental NOE contacts. In particular, the 3D structure
proposed for the synthetic [D-Phe3]-opiorphin analogue
satisfactorily explains three interresidual contacts (NH-F3/
NH-S4, NH-R5/Hβs-S4, and NH-F3/Hβs-R2). In contrast, the
NOE pattern observed for natural opiorphin permits the
postulation of a different spatial orientation of these residues.
The most noticeable differences between both molecules are
observed for the NH-R5/Hβs-S4 and NH-F3/NH-S4 NOEs.
In summary, the different spatial architectures of these two
peptides, which originate from the relative presentations of Arg2
and Phe3 residues, translate into the rather distinct electrostatic
potential energy surface plots of these molecules that may be at
the origin of the differential properties discussed above.
CONCLUSIONS
■
An alanine scanning study of opiorphin has provided evidence
that an Ala3 for Phe3 substitution produces the least active
analogue, thus indicating the relevant role that this aromatic
residue may play in opiorphin binding activity and specificity
toward both targets, NEP and AP-N. In an attempt to apply the
halogen bonding rationale to improve the biological properties
of opiorphin through Phe3 halogenation, we found that ortho
and para halogenation of the Phe3 aromatic ring does not result
in opiorphin activity potentiation by new halogen bonding
stabilizing interactions. Also, fluorination at meta and
perfluorination completely block its enzyme inhibition activity.
A further preparation of a series of substituted Phe3 derivatives
has shown that substitution of L-Phe3 by D-Phe3 increases the
AP-N inhibition potency of opiorphin by 1 order of magnitude
while depleting its NEP inhibition activity. Comparative
conformational studies in solution by NMR and molecular
mechanics calculations have shown that the observed AP-N
inhibition potency enhancement may be due to CH−π stacking
interactions between the aromatic ring of Phe3 and the Hγ
protons of Arg2, which can take place only in the D-Phe3
analogue. This first SAR study on opiorphin has failed to render
a more potent opiorphin-based dual NEP and AP-N inhibitor
but has identified a strategy to boost its AP-N inhibition
potency by simultaneously providing analogues more metabol-
ically stable than the native peptide.
Figure 3. Model structure of the [D-Phe3]-opiorphin peptide (15)
showing the eventual interaction between the Hγ protons of Arg2 (in
green) and D-Phe3 (in red).
nuclear Overhauser effect (NOE) contacts also support the
prevalent existence of this conformation, defined by a χ1(NH−
Cα−Cβ−Car) value of around −60°.
In contrast to the D-Phe3 analogue, key NMR experimental
data such as coupling constants and NOEs for the native
opiorphin are consistent with the existence of a different
conformation for the corresponding side chain of Phe3, with a
χ1(NH−Cα−Cβ−Car) value of around 180°. Notably, this
geometry does not allow the formation of CH−π stacking
interactions with the Hγ protons of Arg2. According to
molecular mechanics calculations, this undetected conforma-
tion, which would permit the π-cation, would be strongly
3
destabilized in energy terms. Moreover, the expected JHα,Hβ2
3
and JHα,Hβ3 coupling constants for this geometry should have
small values, in contrast with those observed experimentally
(one small and one large value of 9.6 Hz, respectively).
Finally, the 3D structures that in our hands best fit the
experimental NMR data for opiorphin and its corresponding D-
Phe3 analogue are presented in Figure 4. These conformers
were validated by the presence of a significant number of
Figure 4. Electrostatic potential energy surface plots for the modified [D-Phe3]-opiorphin peptide (15) (left) and the natural opiorphin peptide (1)
(right).
1185
dx.doi.org/10.1021/jm2012112 | J. Med. Chem. 2012, 55, 1181−1188

Journal of Medicinal Chemistry
Article
(Germany). Alanine-MCA (Ala-MCA), a fluorogenic substrate for
measuring aminopeptidase activity, was purchased from Sigma.
Measurement of hNEP-Ectopeptidase Activity. Using a black half-
area 96-well microplate, the standard reaction mixture consisted of the
enzyme in 100 mM Tris−HCl, pH 7, containing 200 mM NaCl (100
μL final volume). The substrate was added after preincubation for 10
min at 28 °C, and the kinetics of appearance of the fluorescent signal
(RFU) were directly analyzed over 40 min at 28 °C using a fluorimeter
microplate reader (monochromator Infinite 200-Tecan) at 320 and
420 nm excitation and emission wavelengths, respectively.
Measurement of hAP-N-Ectopeptidase Activity. The standard
reaction mixture consisted of the enzyme in 100 mM Tris−HCl, pH
7.0 (100 μL final volume). The Ala-MCA substrate was added after
preincubation for 10 min at 28 °C, and the kinetics of appearance of
the signal were monitored for 40 min at 28 °C by using the fluorimeter
reader at 380 nm excitation and 460 nm emission wavelengths.
EXPERIMENTAL SECTION
■
Materials. All amino acid building blocks, coupling reagents, and
derivatized resins (Fmoc-Ala Wang resin, Fmoc-Arg(Pmc) Wang
resin) were purchased from Novabiochem AG. ACS grade organic
solvents and other reagents such as sodium dodecyl sulfate-d25 were
purchased from Sigma-Aldrich. All other reagents used were of
analytical grade.
Peptide Synthesis. Opiorphin (1) and the peptide analogues 2−
22 were synthesized manually by using standard N -Fmoc solid-phase
α
methodology on either Fmoc-Ala-prederivatized Wang resin or Fmoc-
Arg(Pmc)-prederivatized Wang resin.24 N -Fmoc-protected amino
α
acids (3 equiv) were used. The side chain protecting groups used to
build the peptide sequences were the following: Trt for Gln and Asn, t-
Bu for Ser and Thr, Boc for Lys, and Pmc for Arg. The Fmoc-
Arg(Pmc) Wang resin (1 equiv) was placed in a glass peptide synthesis
column with a frit on the bottom and swollen in DMF for 1 h. The
amide couplings were effected by DIC (3 equiv) and HOBt (6 equiv).
In the case of Fmoc-Gln(Trt)OH, couplings were effected using
PyBOP (3 equiv), HOBt (3 equiv), and DIEA (6 equiv). Each
coupling was performed manually in this peptide synthesis column
using DMF as a solvent under reciprocal oscillating agitation. The
coupling efficiencies were monitored by the Kaiser ninhydrin test. The
Fmoc groups were removed with a solution of 20% piperidine in DMF
(1 × 9 min). The deprotected resin was washed with DMF, DCM, and
then DMF. After assembly of the peptide sequence, a cocktail of TFA/
TIS/H2O (95:2.5:2.5) was used to remove the side chain protecting
groups and to cleave the peptide from the resin. The crude peptides
were precipitated with ice-cold tert-butyl methyl ether, filtered,
redissolved in water, and lyophilized. The final peptides were purified
on a reversed-phase (C-18) column with the VersaFlash flash
chromatography system using a H2O−ACN gradient. The homoge-
neity and identity of the final peptides were determined by analytical
RP-HPLC and HRMS on a Waters UPLC-ESI/TOF system. All the
obtained final peptides showed >98% purity. The purified peptides
were characterized by high-resolution mass spectroscopy, TLC,
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR, HRMS, FRET fluorescence monitoring, and NMR
data of opiorphin and its corresponding analogues. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +34934006113. Fax: +34 932045904. E-mail:
gregorio.valencia@iqac.csic.es.
ACKNOWLEDGMENTS
■
This work was supported by a grant from the Fundacio
de TV3 (Pain, Project Reference 070430-31-32-33). We thank
́
Marato
́
F. J. Canada for helpful discussions. J.J.-B. acknowledges
̃
1
analytical HPLC, and H NMR (see the Supporting Information).
financial support from the Ministerio de Ciencia e Innovacion
́
NMR Spectroscopy. All experiments were recorded in H2O/D2O
(90:10) on a Bruker Avance 600 MHz instrument equipped with a
triple-channel cryoprobe and at 278 K. NMR assignments were
accomplished using standard 2D TOCSY experiments at different
mixing times (20 and 60 ms) and 2D NOESY experiments (300 ms).
The resonance of 2,2,3,3-tetradeuterio-3-(trimethylsilyl)propionic acid
(TSP) was used as a chemical shift reference in the 1H NMR
experiments [δ(TSP) = 0 ppm].
Initial NMR experiments for the natural peptide opiorphin were
carried out to optimize the experimental conditions. The same
methodology was then applied to the other analogues containing Phe3
amino acid modifications.
Structure Determination. Molecular mechanics were conducted
using Macromodel 9.6,22 as implemented in version 8.5.110 of the
Maestro suite,23 using OPLS-2005* 25 as the force field. The starting
coordinates for conformational search calculations (OPLS-2005 as the
force field) were those obtained after energy minimization for both L-
and D-Phe3 analogues. The continuum GB/SA solvent model26 was
employed, and the general PRCG (Polak−Ribiere conjugate gradient)
method for energy minimization was used. A conformational search
protocol was then performed, using the Monte Carlo torsional
sampling (MCMM) method, with the same force field and
minimization conditions.
Determination of Biological Activities: Measurement of NEP
and AP-N Activities Using Fluorimetric Assays. Reagents.
Recombinant human NEP and human AP-N (devoid of their
respective N-terminal cytosol and transmembrane segment) were
purchased from R&D Systems and used as pure sources of
ectopeptidases. Abz-dRGL-(EDDnp) FRET peptide (Abz = o-
aminobenzoic acid; EDDnp = N-(2,4-dinitrophenyl)ethylenediamine),
that is, an internally quenched fluorescent substrate specific for NEP-
endopeptidase activity, was synthesized by Thermo-Fisher Scientific
(Spain) (Grant CTQ2009-08536). F.M. thanks FCT-Portugal
for a postdoctoral research grant (SFRH/BPD/65462/2009).
M.R. acknowledges a fellowship (Grant AP2009-2534,
Formacion
of Education.
́
de Profesorado Universitario) from the Ministry
ABBREVIATIONS USED
■
Abz, o-aminobenzoic acid; β-D-Phe, (S)-3-amino-3-phenyl-
propionic acid or D-β-phenylalanine; Boc, tert-butoxycarbonyl;
ACN, acetonitrile; Bpa, p-benzoylphenylalanine; CSI, chemical
shift index; DCM, dichloromethane; DIC, N,N′-diisopropylcar-
bodiimide; DIEA, N,N-diisopropylethylamine; DMF, N,N-
dimethylformamide; Dnp, 2,4-dinitrophenyl; Fmoc, (fluoren-
9-ylmethoxy)carbonyl; Fmoc-L-Ala Wang resin, Nα-Fmoc-L-
alanine 4-(benzyloxy)benzyl ester polymer-bound; Fmoc-L-
Arg(Pmc) Wang resin, Nα-Fmoc-Nδ-Pmc-L-arginine 4-
(benzyloxy)benzyl ester polymer-bound; hAP-N, human
aminopeptidase N; hNEP, human neprilysin; Homophe,
homophenylalanine; MCMM, Monte Carlo torsional sampling
method; MCA, methoxycoumarin; NMR, nuclear magnetic
resonance; Nphe, N-benzylglycine; NOESY, nuclear Over-
hauser enhancement spectroscopy; Phg, phenylglycine; Pmc,
2,2,5,7,8-pentamethylchroman-6-sulfonyl; PyBOP, (benzotria-
zol-1-yloxy)trispyrrolidinophosphonium hexafluorophosphate;
rmsd, root-mean-square deviation; RFU, relative fluorescence
unit; RP-LC, reversed-phase liquid chromatography (Versa-
Flash flash chromatography system); SAR, structure−activity
relationship; SPPS, solid-phase peptide synthesis; t-Bu, tert-
butyl; TFA, trifluoroacetic acid; TIS, triisopropylsilane;
1186
dx.doi.org/10.1021/jm2012112 | J. Med. Chem. 2012, 55, 1181−1188

Journal of Medicinal Chemistry
Article
(9) (a) Yamamoto, Y.; Ono, H.; Ueda, A.; Shimamura, M.;
Nishimura, K.; Hazato, T.. Spinorphin as an endogenous inhibitor of
enkephalin-degrading enzymes: roles in pain and inflammation. Curr.
Protein Pept. Sci. 2002, 3, 587−599. (b) Nishimura, K.; Hazato, T.
Isolation and identification of an endogenous inhibitor of enkephalin-
degrading enzymes from bovine spinal cord. Biochem. Biophys. Res.
Commun. 1993, 194, 713−719.
(10) Rougeot, C.; Messaoudi, M.; Hermitte, V.; Rigault, A. G.;
Blisnick, T.; Dugave, C.; Desor, D.; Rougeon, F. Sialorphin, a natural
inhibitor of rat membrane-bound neutral endopeptidase that displays
analgesic activity. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 8549−8554.
(11) Wisner, A.; Dufour, E.; Messaoudi, M.; Nejdi, A.; Marcel, A.;
Ungeheuer, M. N.; Rougeot, C. Human opiorphin, a natural
antinociceptive modulator of opioid-dependent pathways. Proc. Natl.
Acad. Sci. U.S.A. 2006, 103, 17979−17984.
TOCSY, total correlation spectroscopy; Trt, trityl; TSP, 2,2,3,3-
tetradeuterio-3-(trimethylsilyl)propionic acid27
REFERENCES
■
(1) (a) Roques, B. P. Novel approaches to targeting neuropeptide
systems. Trends Pharmacol. Sci. 2000, 21, 475−483. (b) Noble, F;
Roques, B. P. Protection of endogenous enkephalin catabolism as
natural approach to novel analgesic and antidepressant drugs. Expert
Opin. Ther. Targets 2007, 11, 145−159.
(2) (a) Oefner, C.; D’Arcy, A.; Hennig, M.; Winkler, F. K.; Dale, G.
E. Structure of human neutral endopeptidase (Neprilysin) complexed
with phosphoramidon. J. Mol. Biol. 2000, 296, 341−349. (b) Oefner,
́
C.; Roques, B. P.; Fournie-Zaluski, M. C.; Dale, G. E. Structural
analysis of neprilysin with various specific and potent inhibitors. Acta
Crystallogr., D: Biol. Crystallogr. 2004, 60, 392−396. (c) Oefner, C.;
Pierau, S.; Schulz, H.; Dale, G. E. Structural studies of a bifunctional
inhibitor of neprilysin and DPP-IV. Acta Crystallogr., D: Biol.
Crystallogr. 2007, 63, 975−981. (d) Sahli, S.; Frank, B.; Schweizer,
(12) (a) Tian, X. Z.; Chen, J.; Xiong, W.; He, T.; Chen, Q. Effects
and underlying mechanisms of human opiorphin on colonic motility
and nociception in mice. Peptides 2009, 30, 1348−1354. (b) Rougeot,
C.; Robert, F.; Menz, L.; Bisson, J. F.; Messaoudi, S. Systemically
active human opiorphin is a potent yet non-addictive analgesic without
drug tolerance effects. J. Physiol. Pharmacol. 2010, 61, 483−490.
(c) Javelot, H. M.; Messaoudi, S.; Garnier, S.; Rougeot, C. Human
opiorphin is a naturally occurring antidepressant acting selectively on
enkephalin-dependent delta-opioid pathways. J. Physiol. Pharmacol.
2010, 61, 355−362. (d) Yang, Q. Z.; Lu, S. S.; Tian, X. Z.; Yang, A.
M.; Ge, W. W.; Chen, Q. The antidepressant-like effect of human
opiorphin via opioid-dependent pathways in mice. Neurosci. Lett. 2011,
489, 131−135.
W. B.; Diederich, F.; Blum-Kaelin, D.; Aebi, J. D.; Bohm, H. J. Second-
̈
generation inhibitors for the metalloprotease neprilysin based on
bicyclic heteroaromatic scaffolds: synthesis, biological activity, and X-
ray crystal-structure analysis. Helv. Chem. Acta 2005, 88, 731−750.
(3) (a) Ito, K.; Nakajima, Y.; Onohara, Y.; Takeo, M.; Nakashima, K.;
Matsubara, F.; Ito, T.; Yoshimoto, T. Crystal structure of amino-
peptidase N (proteobacteria alanyl aminopeptidase) from Escherichia
coli and conformational change of methionine 260 involved in
substrate recognition. J. Biol. Chem. 2006, 281, 33664−33676.
́
(b) Fournie-Zaluski, M. C.; Poras, H.; Roques, B. P.; Nakajima, Y.;
(13) Rougeot, C. Opiorphin peptide derivatives as potent inhibitors
of enkephalin-degrading ectopeptidases. PCT/EP2009/054171, April
4, 2009; WO/2009/124948, Oct 15, 2009.
Ito, K.; Yoshimoto, T. Structure of aminopeptidase N from Escherichia
coli complexed with the transition-state analogue aminophosphinic
inhibitor PL250. Acta Crystallogr., D: Biol. Crystallogr. 2009, 65, 814−
822.
(14) (a) Medeiros, M. A.; Franca̧ , M. S.; Boileau, G.; Juliano, L.;
Carvalho, K. M. Specific fluorogenic substrates for neprilysin (neutral
endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and
metalloproteases. Braz. J. Med. Biol. Res. 1997, 30, 1157−1162.
(b) Barros, N. M.; Campos, M.; Bersanetti, P. A.; Oliveira, V.; Juliano,
M. A.; Boileau, G.; Juliano, L.; Carmona, A. K. Neprilysin
carboxydipeptidase specificity studies and improvement in its
detection with fluorescence energy transfer peptides. Biol. Chem.
2007, 388, 447−455.
(4) Albrecht, S.; Al-Lakkis-Wehbe, M.; Orsini, A; Defoin, A.; Pale, P.;
Salomon, E.; Tarnus, C.; Weibel, J. M. Amino-benzosuberone: a novel
warhead for selective inhibition of human aminopeptidase-N/CD13.
Bioorg. Med. Chem. 2011, 19, 1434−1449.
(5) (a) Matthews, B. W. Structural basis of thermolysin and related
zinc peptidases. Acc. Chem. Res. 1988, 21, 333−340. (b) Roques, B. P.
Zinc metallopeptidases: active site structure and design of selective and
mixed inhibitorsnew approaches in the search for analgesics and
antihypertensives. Biochem. Soc. Trans. 1999, 21, 678−685. (c) Roques,
(15) Rougeot C. Method for identifying BPLP and opiorphin
agonists or antagonists. PCT/EP2009/050567, Jan 19, 2009; WO/
2009/090265, July 23, 2009.
́ ́
B. P.; Noble, F.; Dauge, V.; Fournie-Zaluski, M. C.; Beaumont, A.
Neutral endopeptidase 24.11: structure, inhibition, and experimental
and clinical pharmacology. Pharmacol. Rev. 1993, 45, 87−146.
(6) (a) Matheson, A. J.; Noble, S. Racecadotril. Drugs 2000, 59, 829−
835. (b) Spillantini, M. G.; Geppetti, P.; Fanciullacci, M.; Michelacci,
S.; Lecomte, J. M.; Sicuteri, F. In vivo 'enkephalinase' inhibition by
acetorphan in human plasma and CSF. Eur. J. Pharmacol. 1986, 125,
147−150. (c) Reggiani, A.; Carenzi, A.; Frigeni, V.; Della Bella, D.
Effect of bestatin and thiorphan on [Met5]enkephalin-Arg6-Phe7-
induced analgesia. Eur. J. Pharmacol. 1984, 105, 361−364.
(16) (a) Mantle, D.; Hardy, M. F.; Lauffart, B.; McDermott, J. R.;
Smith, A. I.; Pennington, R. J. Purification and characterization of the
major aminopeptidase from human skeletal muscle. Biochem. J. 1983,
211, 567−573. (b) Abid, K.; Rochat, B.; Lassahn, P. G.; Stocklin, R.;
̈
Michalet, S.; Brakch, N.; Aubert, J. F.; Vatansever, B.; Tella, P.; De
Meester, I.; Grouzmann, E. Kinetic study of neuropeptide Y (NPY)
proteolysis in blood and identification of NPY3−35: a new peptide
generated by plasma kallikrein. J. Biol. Chem. 2009, 284, 24715−24724.
(17) (a) Parisini, E.; Metrangolo, P.; Pilati, T.; Resnati, G.; Terraneo,
G. Halogen bonding in halocarbon-protein complexes: a structural
survey. Chem. Soc. Rev. 2011, 40, 2267−227. (b) Hardegger, L. A.;
Kuhn, B.; Spinnler, B.; Anselm, L.; Ecabert, R.; Stihle, M.; Gsell, B.;
Thoma, R.; Diez, J.; Benz, J.; Plancher, J. M.; Hartmann, G.; Banner,
D. W.; Haap, W.; Diederich, F. Systematic investigation of halogen
bonding in protein-ligand interactions. Angew. Chem., Int. Ed. 2011, 50,
314−318.
(7) (a) Jutkiewicz, E. M. RB101-mediated protection of endogenous
opioids: potential therapeutic utility? CNS Drug Rev. 2007, 13, 192−
́
205. (b) Fournie-Zaluski, M. C.; Coric, P.; Turcaud, S.; Lucas, E.;
Noble, F.; Maldonado, R.; Roques, B. P. “Mixed inhibitor−prodrug” as
a new approach toward systemically active inhibitors of enkephalin-
degrading enzymes. J. Med. Chem. 1992, 35, 2474−2481. (c) Roques,
́ ́
B. P.; Noble, F.; Dauge, V.; Fournie-Zaluski, M. C.; Beaumont, A.
Neutral endopeptidase 24.11: structure, inhibition, and experimental
(18) Ibrahim, M. A. Molecular mechanical study of halogen bonding
in drug discovery. J. Comput. Chem. 2011, 32, 2564−2574.
(19) Guerrini, R.; Calo, G.; Bigoni, R.; Rizzi, D.; Rizzi, A.; Zucchini,
́
and clinical pharmacology. Pharmacol. Rev. 1993, 45, 87−146.
(8) (a) Chen, H.; Noble, F.; Mothe,
Danascimento, S.; Roques, B. P.; George, P.; Fournie-
́
A.; Meudal, H.; Coric, P.;
Zaluski, M. C.
́
M.; Varani, K.; Hashiba, E.; Lambert, D. G.; Toth, G.; Borea, P. A.;
Salvadori, S.; Regoli, D. Structure−activity studies of the Phe(4)
residue of nociceptin(1−13)-NH(2): identification of highly potent
agonists of the nociceptin/orphanin FQ receptor. J. Med. Chem. 2001,
44, 3956−3964.
Phosphinic derivatives as new dual enkephalin degrading enzyme
inhibitors: synthesis, biological properties, and antinociceptive
activities. J. Med. Chem. 2000, 43, 1398−1408. (b) Chen, H.; Noble,
F.; Coric, P.; Fournie-Zaluski, M. C.; Roques, B. P. Aminophosphinic
inhibitors as transition state analogues of enkephalin-degrading
enzymes: a new class of central analgesics. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 12028−12033.
(20) Shao, X.; Gao, Y.; Zhu, C.; Liu, X.; Yao, J.; Cui, Y.; Wang, R.
Conformational analysis of endomorphin-2 analogs with phenylalanine
1187
dx.doi.org/10.1021/jm2012112 | J. Med. Chem. 2012, 55, 1181−1188

Journal of Medicinal Chemistry
Article
mimics by NMR and molecular modeling. Bioorg. Med. Chem. 2007,
15, 3539−3547.
(21) Wishart, D. S.; Bigam, C. G.; Holm, A.; Hodges, R. S.; Sykes, B.
D. 1H, 13C and 15N random coil NMR chemical shifts of the common
amino acids. I. Investigations of nearest-neighbor effects. J. Biomol.
NMR 1995, 5, 67−81.
(22) MacroModel, version 9.6; Schrodinger, LLC: New York, 2008.
̈
(23) Maestro, a Powerful All-Purpose Molecular Modeling Environment,
version 8.5; Schrodinger, LLC: New York, 2008.
̈
(24) Chan, W. C.; White, P. D. In Fmoc Solid Phase Peptide Synthesis:
A Practical Approach; Chan, W.C., White, P.D., Eds.; Oxford
University Press: Oxford, U.K., 2000; pp 41−76.
(25) Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. Development
and testing of the OPLS all-atom force field on conformational
energetics and properties of organic liquids. J. Am. Chem. Soc. 1996,
118, 11225−11236.
(26) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T..
Semianalytical treatment of solvation for molecular mechanics and
dynamics. J. Am. Chem. Soc. 1990, 112, 6127−6129.
(27) Abbreviations used for amino acids and designation of peptides
follow the rules of the IUPAC-IUB Commission of Biochemical
Nomenclature in J. Biol. Chem. 1972, 247, 977−983.
1188
dx.doi.org/10.1021/jm2012112 | J. Med. Chem. 2012, 55, 1181−1188