Influence of polar side chains modifications on the dual enkephalinase inhibitory activity and conformation of human opiorphin, a pain perception related peptide

Article abstract of DOI:10.1016/j.bmcl.2015.09.071

The dual inhibitory action of the pain related peptide opiorphin (H-Gln-Arg-Phe-Ser-Arg-OH) against neutral endopeptidase (NEP) and aminopeptidase N (AP-N) was further investigated by a SAR study involving minor modifications on the polar side chains of Arg residues and glycosylation with monosaccharides at Ser. None of them exerted dual or individual inhibitory potency superior than opiorphin. However, the correlations deduced offer further proof for the key role of these residues upon the binding and bioactive conformational stabilization of opiorphin. NMR conformational studies on the glycopeptides suggest that they are still very flexible compounds that may attain their respective bioactive conformations.

Full text of DOI:10.1016/j.bmcl.2015.09.071

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5190–5193
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Influence of polar side chains modifications on the dual
enkephalinase inhibitory activity and conformation of human
opiorphin, a pain perception related peptide
Mònica Rosa a, Filipa Marcelo b, Luis P. Calle c, Catherine Rougeot d, Jesús Jiménez-Barbero c,e
,
Gemma Arsequell a, Gregorio Valencia a,
⇑
a Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), E-08034 Barcelona, Spain
b UCIBIO, REQUIMTE Faculdade Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal
c CIC bioGUNE, Bizkaia Technological Park, E-48160 Derio, Spain
d Institut Pasteur-Unité de Biochimie Structurale et Cellulaire/URA2185—CNRS, Paris Cedex 15 75724, France
e Ikerbasque, Basque Foundation for Science, E-48013 Bilbao, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 July 2015
Revised 28 September 2015
Accepted 29 September 2015
Available online 14 October 2015
The dual inhibitory action of the pain related peptide opiorphin (H-Gln-Arg-Phe-Ser-Arg-OH) against
neutral endopeptidase (NEP) and aminopeptidase N (AP-N) was further investigated by a SAR study
involving minor modifications on the polar side chains of Arg residues and glycosylation with monosac-
charides at Ser. None of them exerted dual or individual inhibitory potency superior than opiorphin.
However, the correlations deduced offer further proof for the key role of these residues upon the binding
and bioactive conformational stabilization of opiorphin. NMR conformational studies on the glycopep-
tides suggest that they are still very flexible compounds that may attain their respective bioactive
conformations.
Keywords:
Opiorphin analogs
SAR study
Neutral endopeptidase
AP-N
Ó 2015 Elsevier Ltd. All rights reserved.
Glycopeptides pain
The amplification of the natural analgesic effect of the endoge-
nous opioid peptides Met- and Leu-enkephalin by inhibition of
their enzymatic inactivation has long been considered a design
principle that may let to alternative analgesics with intermediate
potencies between the nonsteroidal anti-inflammatory drugs and
traditional opiates like morphine.1,2 Because of the lack of authen-
tic structural information on the target enzymes, the small
molecule inhibitors thus developed, from which thiorphan is an
early example, have reached little success as analgesics.3 Up to
date, only acetorphan is clinically used as antidiarrheal (Racecado-
trilÒ) owing to its antisecretory activity.4 Although there is still a
long way to go, the discovery of the human peptide regulator, opi-
orphin (H-Gln-Arg-Phe-Ser-Arg-OH) which is a dual physiological
inhibitor of the human ectoenkephalinases, neutral endopeptidase
(NEP, EC 3.4.24.11) and aminopeptidase N, (AP-N, EC 3.4.11.2) has
opened new perspectives.5 Indeed, opiorphin exerts a number of
in vivo analgesic and neurological effects that are specifically
mediated throughout endogenous enkephalin-related activation
of opioid pathways in animal models.6–14 These facts make
opiorphin a promising template for the design of a new class of
peptide drug candidates for pain modulation.
To learn about the structural requirements of this inhibitory
substrate and in search of functional derivatives with improved
dual inhibitory potency we had previously conducted a first SAR
study of opiorphin and developed a computational model of its
active conformations against NEP and AP-N. It was found, for
instance, that some modifications on the Phe3 position of opiorphin
increased AP-N inhibition while depleting NEP activity but none of
the analogs was a more potent dual inhibitor.15The computational
models of the bioactive conformations for NEP and APN fit to a sort
of distorted ‘S’ and ‘C’ shapes, respectively. These very unrelated
configurations emphasize the complexity of designing dual-acting
NEP/AP-N inhibitors owing to the inborn flexibility of this small
peptide.16
Parallel efforts have been devoted to search for functional ana-
logs of opiorphin with superior resistance to circulating peptidases
and permeation properties across membranes to improve the
ADME profile. In this case a peptidomimetic product [Cys-(CH2)6-
Gln-Arg-Phe-Ser(O-(CH2)7CH3)-Arg] having a cysteine residue as
a zinc chelating group, a polyethylene linker and a hydrophobic
C8 alkyl chain residue was found to hold an increased metabolic
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2015.09.071
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

M. Rosa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5190–5193
5191
stability while retaining full dual biological activities of
opiorphin.17
using specific fluorescent synthetic peptide substrates21–23 (SI for
details). The potencies of the peptides expressed as IC50 values
are shown in Table 1. Compounds showing EC50 values of over
The aims of this work were to better understand the dual inhi-
bitory activity of opiorphin and to provide further support to our
computational model that postulates that the residues 2, 4 and 5
play a key role in binding and maintaining the bioactive conforma-
tions. Accordingly, in this follow up narrow SAR study on opiorphin
we have examined the role of the guanidinium side chain on Arg2
and Arg5 positions and of the primary alcohol on the side chain of
Ser4 by simply substituting the amino acids in these positions by
very close common amino acid homologs. Other more elaborated
substitutions based on amino acid mimics were not considered
owing the lack of a precise prediction tool to select them. In addi-
tion and because the Ser4 position tolerates drastic modifications
such as conjugation to fatty chain hydrophobic residues17 it
seemed of interest to investigate the influence of adding residues
of opposite nature such as hydrophilic simple monosaccharides
by O-glycosylation. This choice was also made in view of our expe-
rience on opioid glycopeptides18 and because a natural potent dual
inhibitor of these enzymes, namely, phosphoramidon (IC50 = 140 -
nM for AP-N and 2 nM for NEP) shows a monosaccharide residue
(rhamnose) on its molecule.19
Six new positional analogs of amino acids at 2, 4 and 5 of opior-
phin and three O-glycoside analogs at position 4, using either
GlcNAc or GalNAc monosaccharides which are listed in Figure 1,
were prepared. These peptides and glycopeptides were synthe-
sized by following manual solid phase peptide synthesis proce-
dures using Fmoc protocols and commercial building blocks. The
glycosyl amino acids building blocks were also prepared in our lab-
oratory following already published procedures20 (see SI for
details).
100
l
M were considered inactive while values of EC50 > 70
lM
were indicative of very poor inhibitors and EC50 > 50
l
M of active
compounds. A common feature from all these analogs is that they
lose the dual inhibitory properties of opiorphin upon the substitu-
tions assayed.
The substitution of any of the two Arg residues by Lys is detri-
mental for AP-N inhibitory potency but not for NEP. Contrarily,
when any Arg residue is substituted by Cit, the NEP activity was
completely lost, while some AP-N activity still remained. This fact
suggests that both Arg2 and Arg5 have equivalent participation and
importance for both maintaining the bioactive conformations
against NEP and AP-N and binding to the active site of these
enzymes. Also, because Arg for Lys substitution (guanidinium for
methylene amine) strongly modifies the steric volume of the side
chain, but not its net positive charge, and Arg for Cit replacement
(guanidinium for carbamic amide) keeps the volume but not the
charge, the data from Table 1 strongly suggest that the active con-
formation of opiorphin for NEP is not very sensitive to the chemical
nature of these Arg polar groups, but still requires two net positive
charges. Conversely, AP-N is highly selective for the guanidinium
functional group and at most only tolerates to lose one of them.
These observations are in good agreement with our own bioac-
tive conformations previously deduced by computational tools.16
Thus, from the ‘C’-shaped AP-N calculated conformation, it is evi-
dent that the roles of both Arg2 and Arg5 along with Ser4 seem cru-
cial for keeping the shape of the peptide. In contrast, for the ‘S’-
shaped NEP conformation, only Arg2 seems required to fix the scaf-
fold in the required conformation, thus leaving Arg5, Ser4 and Phe3
in a suitable conformation for making stabilizing contacts with the
enzyme. Therefore, from this new data, the role of the guanidinium
residues looks essential for AP-N and, to a less extent, for NEP.
The role of the primary alcohol function on Ser4 on the biolog-
ical activity of opiorphin was investigated by the isosteric substitu-
These opiorphin analogs were tested in vitro for their inhibitory
properties against both soluble human recombinant NEP and AP-N
(devoid of their respective N-terminal cytosol and transmembrane
fragments) by means of a continuous FRET fluorometric assay
tions of Ser by Cys or D-Ser and by replacing Ser by a more hindered
H
Thr secondary alcohol side chain (an analog we have already
reported in Ref. 16). None of the analogs were active against NEP
which is an indication of the importance of Ser4 in providing the
appropriate intermolecular contacts of opiorphin with the enzyme
rather than controlling its bioactive conformation. On the other
hand, AP-N tolerates the isosteric changes (Ser by Cys much better
N
NH2
NH
OH
O
O
O
H
N
H
H2N
N
N
H
N
H
OH
NH
O
O
than Ser by D-Ser) but not a secondary alcohol on the side chain.
H2N
O
This is in agreement with our proposed model where Ser4 together
with Arg2 and Arg5 are key residues for maintaining the bioactive
conformation of opiorphin for the AP-N binding site.16 In summary,
HN
NH2
OPIORPHIN
H-Gln-Arg2-Phe-Ser4-Arg5-OH
OPIORPHIN ANALOGS
H-Gln-Xaa2-Phe-Xaa4-Xaa5-OH
Table 1
Inhibition of AP-N and NEP by opiorphin and its positional analogs on positions 2, 4
and 5 and by opiorphin glycopeptides
H2N
HN
O
NH2
OH
OH
SH
OH
OH
OH
#
Peptides
IC50
IC50
AP-N (lM)
NEP (lM)
H2N
H2N
H2N
OH
O
O
Cys4
OH
1
2
3
4
5
6
7
8
Opiorphin
8.1 0.1
30
3
O
Thr4
H2N
H2N
-Ser4]-opiorphin
85
20
5
4
>100
>100
>100
50
35
90 14
>100
>100
>100
>100
D-Ser4
O
[D
O
[Cys4]-opiorphin
a[Thr4]-opiorphin
[Lys2]-opiorphin
[Lys5]-opiorphin
[Cit2]-opiorphin
[Cit5]-opiorphin
Lys2
Cit2 or 5
OH
OH
>100
>70
>70
O
O
HO
HO
7
5
HO
HO
OH
O
AcHN
AcHN
HO
HO
OH
O
O
O
22
62
32
52
3
NHAc
OH
7
3
7
OH
H2N
[Ser4(
[Ser4(b-GlcNAc)]-opiorphin
[Thr4(
-GalNAc)]-opiorphin
a-GalNAc)]-opiorphin
H2N
Ser(O-α-D-GalNAc)
H2N
9
10
11
O
O
O
Thr(O- α-D-GalNAc) Ser(O-β-D-GlcNAc)
a
>100
a
[Thr4]-opiorphin was already prepared and reported by us (see Ref. 16) and is
Figure 1. Opiorphin sequence and amino acids used to prepare positional analogs
here included for reference comparison.
on positions 2, 4 and 5.

5192
M. Rosa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5190–5193
the integrity of Ser4 seems essential for NEP and less demanding
for AP-N activity but not to the point to tolerate a slightly more
hindered Thr polar group (Table 1).
experimentally derived ensemble of these glycopeptides in solu-
tion. The simulations were accomplished using the AMBER 12
package37 with the ff12SB38 and Glycam0639 force fields. The
cluster analysis, in terms of energy was accomplished using the
ptraj module integrated in AMBER 12 package.
In spite of these results and on the light of previous data
outlined above showing that bold modifications at Ser4, that is,
etherification by octyl alcohol, are tolerated and even increase
the biological activity of opiorphin,17 we have further examined
this position. As described for other biologically active pep-
tides24–28 and according to our experience on peptide glycosyla-
tion,18,29 glycoconjugation seemed an interesting choice because
is in opposition to the hydrophobic substitutions already tested.
In doing so, naturally occurring GlcNAc- and GalNAc-glycosylation
on the Ser4 residues30–32 of opiorphin were chosen. Since the Ser4
for Thr substitution inactivates opiorphin,16 the Thr glycosylated
analog was also synthesized and tested as negative control.
The simulations demonstrated that both glycopeptides are
intrinsically disordered. However, two major populated clusters,
defined by A and B, were found for both Ser4(O-
a-GalNAc),
Ser4(O-b-GlcNAc) opiorphin analogs (Figs. 2 and 3). Glycosylation
induced alterations to the native ‘C’- and ‘S’-shaped conformation
of the natural opiorphin. However, these small alterations in the
peptide (and Ser4) conformation upon glycosylation are still com-
patible with ‘C’-shaped bioactive peptide conformations required
for AP-N inhibitions (cluster A in Figs. 2 and 3). In fact, a similar
conclusion may be extracted from the fact that the
D
-Ser4 and
The Thr4(
inactive against both NEP and AP-N. The Ser4(
a
-O-GalNAc)-opiorphin glycopeptide was totally
-O-GalNAc) and
Cys4 analogs also keep activity. They still may adopt the necessary
bioactive conformation at the AP-N binding site, without a major
entropy or enthalpy penalty. In the case of the alternative
‘S’-shaped bioactive conformation required for NEP activity, the
observed small conformational alterations upon glycosylation do
not preclude that the glycopeptides adopt this bioactive conforma-
tion. However, it is very likely that the observed impeded binding
inhibition it is mostly due to the presence and bulkiness of the
sugar moieties attached to the Ser4 residue rather than to confor-
mational effects, as predicted by our proposed computational
model.16
a
Ser4(b-O-GlcNAc) glycoside analogs also lost the NEP inhibitory
activity, although still retained some of AP-N activity. These obser-
vations comply with our computer model were Ser4 along with
Phe3 and Arg5 of opiorphin seem to be directly involved in binding
events with NEP making its inhibitory activity highly structure
dependent. Besides, Ser4 and Phe3 mostly seem to participate in
the less demanding tasks of maintaining the bioactive conforma-
tion for APN, thus structural changes on Ser4 are showing a weaker
effect on activity.
It is a well-known fact that short glycopeptides, as those
reported herein, mainly adopt disordered conformations in water
solution and show a high degree of adaptability to their recep-
tors.33 This means that the sugar residue has a limited influence
on the conformation of the peptide chain being the conformational
effects mainly observed on the attachment residue. This is a rele-
vant fact when seeking transport improvement or protease resis-
tance on peptide pharmacophores without destruction of
receptor affinity and selectivity by glycosylation strategies.34 To
examine if these general structural features apply to opiorphin
and to analyze if subtle modifications in the dynamic behavior of
the peptide chain upon glycosylation could take place, NMR
parameters (chemical shifts, coupling constants and NOEs) were
measured for the different glycoopiorphins and compared to those
deduced for opiorphin. Accordingly, the values of the experimental
In conclusion, none of the compounds tested is a dual inhibitor
showing individual NEP and APN activities lower than those of opi-
orphin, therefore, residues Arg2, Arg5 and Ser4 are key for AP-N and
NEP inhibitory activities. Concerning Arg2 and Arg5, both have
equivalent participation and importance for maintaining the bioac-
tive conformations of opiorphin against NEP and AP-N and for
binding with the active site of these enzymes. The active conforma-
tion of opiorphin for NEP is not very sensitive to the chemical nat-
ure of these Arg polar groups, but still requires two net positive
charges. Conversely, AP-N is highly selective for these two guani-
dinium functional groups and at most only tolerates to lose one
of them. In general, it can be said that the role of the guanidinium
residues looks essential for AP-N and, to a less extent, for NEP inhi-
bitory activity. Concerning Ser4 modifications, the integrity of this
residue seems essential for NEP and less demanding for AP-N activ-
ity but not to the point to tolerate a slightly more hindered Thr
polar group. Glycosylation of Ser4 was fully detrimental for NEP
while retaining some APN inhibitory activity. The NMR conforma-
tional studies with these glycoopiorphins have suggested that
although glycosylation induces certain rigidity, these compounds
3JH
coupling constants that define the orientation of Ser4 sug-
a
,Hb
gested alterations in the
v
1 torsion angle of Ser4 upon glycosyla-
tion. Thus, for the parent opiorphin, the coupling constants 3JH
a
,
and 3JH
were 5.5 Hz and 12.8 Hz respectively, suggesting
Hb2
a,Hb3
a well-defined gauche-type relationship of the side chain of Ser4.
In contrast, glycosylation at Ser4 produced intermediate values
for both 3JH
(around 6 Hz). This fact evidences the existence
,Hb
a
of conformational averaging around
v1, with the concomitant
increase of flexibility. This result is in agreement with other
reported
v1 values related to O-Ser substitutions by either GlcNAc
or GalNAc.35,36
Regarding NOEs examination, besides the typical sequential
contacts and although very few inter-residual cross peaks could
be recorded, the presence of glycosylation induced the increase
of the number of detected NOEs in the peptide chain (more than
8 and up to 20 additional NOE cross peaks). This observation
suggests that glycosylation causes a more structured peptide (see
SI Figs. S5 and S6), although still highly flexible.
Concerning the conformation around the glycosidic linkage, for
both Ser4(O- -GalNAc), Ser4(O-b-GlcNAc) opiorphin analogs,
a
strong NOEs were measured between the H1 sugar anomeric pro-
ton and both Hb of the vicinal Ser residue (see SI Figs. S7 and S8). A
20-ns molecular dynamics simulations with time-averaged
restraints (MD-tar), for Ser4(O- -GalNAc), Ser4(O-b-GlcNAc)
a
Figure 2. Representative model structure of Ser4(O-b-GlcNAc)-opiorphin
opiorphin peptides were carried out in explicit water to get the
(glycoopiorphin 10) obtained after MD simulations. (A) Cluster A; (B) cluster B.

M. Rosa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5190–5193
5193
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This work was supported by a Grant from the Fundació La
Marató de TV3 (Pain, Project Reference: 070430-31-32-33) Spain.
Mònica Rosa acknowledges a fellowship (Grant AP2009-2534
Formación de Profesorado Universitario) from MINECO Spain.
Filipa Marcelo thanks to FCT-Portugal for the post-doctoral
research Grant (SFRH/BPD/65462/2009).
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.09.
071.
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