Structural and biological exploration of Phe3-Phe 4-modified endomorphin-2 peptidomimetics

Article abstract of DOI:10.1021/ml400189r

This study reports on our ongoing investigation on hybrid EM-2 analogues, in which the great potential of β-amino acids was exploited to generate multiple conformational modifications at the key positions 3 and 4 of the parent peptide. The effect on the opioid binding affinity was evaluated, by means of ligand stimulated binding assays, which indicated a high nanomolar affinity toward the μ-receptor, with appreciable μ/δ selectivity, for some of the new compounds. The three-dimensional properties of the high affinity μ opioid receptor (MOR) ligands were investigated by proton nuclear magnetic resonance, molecular dynamics, and docking studies. In solution, the structures showed extended conformations, which are in agreement with the commonly accepted pharmacophore model for EM-2. From docking studies on an active form of the MOR model, different ligand-receptor interactions have been identified, thus confirming the ability of active compounds to assume a biologically active conformation.

Full text of DOI:10.1021/ml400189r

Letter
pubs.acs.org/acsmedchemlett
Structural and Biological Exploration of Phe3−Phe4‑Modified
Endomorphin‑2 Peptidomimetics
Giordano Lesma,† Severo Salvadori,‡ Francesco Airaghi,† Thomas F. Murray,§ Teresa Recca,†
Alessandro Sacchetti,*,∥ Gianfranco Balboni,*,⊥ and Alessandra Silvani*,†
†Dipartimento di Chimica, Universita
̀
degli Studi di Milano, via C. Golgi, 19, 20133 Milano, Italy
‡Dipartimento di Scienze Farmaceutiche, Universita
̀
degli Studi di Ferrara, via Fossato di Mortara 17-19, 44100 Ferrara, Italy
§Department of Pharmacology, Creighton University School of Medicine, Omaha, Nebraska 68102, United States
∥Dipartimento di Chimica, Materiali ed Ingegneria Chimica ‘Giulio Natta’, Politecnico di Milano, p.zza Leonardo da Vinci 32, 20133
Milano, Italy
⊥Dipartimento di Scienze della Vita e dell’Ambiente, Universita
̀
degli Studi di Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
S
* Supporting Information
ABSTRACT: This study reports on our ongoing investigation
on hybrid EM-2 analogues, in which the great potential of β-
amino acids was exploited to generate multiple conformational
modifications at the key positions 3 and 4 of the parent
peptide. The effect on the opioid binding affinity was
evaluated, by means of ligand stimulated binding assays,
which indicated a high nanomolar affinity toward the μ-
receptor, with appreciable μ/δ selectivity, for some of the new
compounds. The three-dimensional properties of the high
affinity μ opioid receptor (MOR) ligands were investigated by proton nuclear magnetic resonance, molecular dynamics, and
docking studies. In solution, the structures showed extended conformations, which are in agreement with the commonly
accepted pharmacophore model for EM-2. From docking studies on an active form of the MOR model, different ligand−receptor
interactions have been identified, thus confirming the ability of active compounds to assume a biologically active conformation.
KEYWORDS: Peptidomimetics, opioid receptors, β-amino acids, conformational analysis, docking studies
ost of the biological effects of endogenous opioid
stereochemical spacer, capable of favoring proper spatial
Mpeptides are mediated through activation of three opioid orientation of the aromatic side chain groups.6 In EM-2,
aromatic amino acid side chains exhibit considerable conforma-
tional flexibility, especially the Phe3 aromatic ring, which is free
to adopt a bioactive conformation. Also the C-terminal address
fragment Phe4-NH2 plays an important role in ligand
recognition.7
Several chemical modifications performed on EM-2 have
been described previously, and most of them were focused on
single amino acid substitution.8 However, in recent, multiple
modifications in EM-2-based drug design are going to
constitute the major interest.9−13 Indeed, apart the changes in
binding properties, most of these additional modifications tend
to shift the pharmacokinetics and pharmacodynamics positively,
facilitating a more feasible potential application of ligands for
clinical purposes.
receptors designated μ, δ, and κ in the peripheral or central
nervous system.1 One of the endogenous peptide ligands for
the μ opioid receptor (MOR), endomorphin-2 (Tyr-Pro-Phe-
Phe-NH2, EM-2, Figure 1), exhibits high affinity and
extraordinarily high selectivity relative to δ-opioid and κ-opioid
receptor systems. This tetrapeptide has a strong antinociceptive
effect on acute pain, similar to that of morphine, and it is also
more effective than the majority of the opioid peptides against
neuropathic pain even at low doses. However, as found in the
case of most short linear natural peptides, native EM-2 lacks
critical therapeutic characteristics such as bioavailability,
duration of action, and oral activity.2 Since MOR is proven
to be the major target of analgesics3 because of its particular
physiological characteristics, development of synthetic EM-2
analogues is of great importance.
Research efforts are focused on the design of analogues and
peptidomimetics in order to improve EM-2 properties as
potential drug as well as to better understand the key structural
and conformational features on which receptor recognition and
binding are based.4,5 It is well-known that the amino acid Pro
represents a crucial factor in determining the structural and
conformational properties of EM-2 analogues, acting as a
Aiming to help the definition of privileged structures
exclusively associated with bioactivity, we recently investigated
the biological activity and the conformational requirements of
hybrid EM-2 analogues, in which the C-terminal Phe3−Phe4
Received: May 17, 2013
Accepted: July 11, 2013
Published: July 11, 2013
© 2013 American Chemical Society
795
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ACS Medicinal Chemistry Letters
Letter
Figure 2. Molecular structure of the β-dipeptidomimetics 8−14 used
to replace the native Phe3−Phe4-based residue in EM-2.
Tia-methyl esters were obtained starting from N-Cbz-β2-hPhe
methyl ester and from N-Cbz-β3-hPhe methyl ester, respec-
tively, by means of TFA-catalyzed Pictet−Spengler condensa-
tion, followed by Cbz removal. Peptide coupling was better
performed by means of BOP-Cl and TEA in CH2Cl2 in the case
of constrained amino acids derivatives (compounds 8, 9, and
12) and by means of EDC, HOBt, and DIPEA in CH2Cl2 in all
other cases (compounds 10, 11, 13, and 14).
Figure 1. Molecular structure of EM-2 and analogues 1−7.
dipeptide is simultaneously substituted by various β-peptidomi-
metic elongated structures. In some cases, we could observe the
maintenance of opioid affinity and we also could highlight
common binding modes for active compounds, confirming the
necessity for specific binding interactions, for instance, between
Asp 147 and Tyr1, as evidenced by docking studies.14 As
recently outlined,15 peculiar advantages are connected with the
employment of heterogeneous (mixed α and β) backbones in
peptidomimetic chemistry, such as a high stability toward
hydrolytic enzymes, more possibility of chemical diversity and,
in most cases, more predictable secondary structures, each of
which offers a potentially distinctive way to project sets of side
chains in space and to generate preferential conformations.16,17
Being aware that the potential of such strategic substitutions
were not fully explored in the key Phe3−Phe4 fragment of EM-
2, we went on in our research project, and we report here on
the EM-2 analogues given in Figure 1.
They are obtained by replacing the Phe amino acids in
positions 3 and 4 with various different β-homologues residues,
characterized as β2- or β3-aa’s, according to Seebach’s
nomenclature,18 and in some cases endowed with skeletal
constraints in the fourth residue. They were thought able to
deeply alter the tetrapeptide backbone conformation and thus
modulate in new ways the bioactivity of the reference EM-2
peptide. Their synthesis, opioid receptor binding affinities and
selectivities, NMR and molecular dynamics conformational
behavior, and binding modes from docking are here reported.
Orthogonally protected intermediates 8−14 (Figure 2) were
synthesized from the key precursors β3-hPhe and β2-hPhe
amino acids and their constrained Tbac (2,3,4,5-tetrahydro-1H-
benzo[c]azepine-4-carboxylic acid) and Tia (2-(1,2,3,4-tetrahy-
droisoquinolin-3-yl)acetic acid) derivatives. Preparation of N-
Boc-β3-hPhe and β3-hPhe methyl ester was accomplished
following the protocol of stereospecific classical homologa-
tion19 of the corresponding L-αPhe, by means of the Kolbe
reaction. N-Boc-β2-hPhe and β2-hPhe methyl ester could be
obtained in both the enantiomeric forms following an efficient
protocol developed by Gellman.20 The constrained Tbac- and
With the dipeptidomimetic scaffolds 8−14 in our hands, EM-
2 analogues 1−7 were obtained by conversion of the terminal
methyl ester to the corresponding primary amide, followed by
standard peptide coupling chemistry and isolation of the target
compounds as trifluoroacetate salts. All new products were
1
characterized by H NMR and HR-MS data.
Amides 1−7 have been evaluated for their affinity and
selectivity for μ and δ opioid receptors in radioligand binding
assays under standard conditions, using cloned receptors stably
expressed on Chinese hamster ovary (CHO) cells, and
[3H]DPDPE (cyclo[D-Pen2,DPen5]enkephalin) and
[3H]DAMGO ([D-Ala2,NMePhe4,glyol5-enkephalin), respec-
tively, as the radioligands. The data are summarized in Table 1.
All compounds 1−7 exhibit a very good nanomolar affinity
toward the μ-opioid receptor, with appreciable μ/δ selectivity
in most cases. The highest affinity, coupled with notable
selectivity, is shown by peptidomimetics 3 and 4, reaching the
low nanomolar scale as the parent EM-2. It should be noted
that such a finding is indeed remarkable for EM-2 analogues
Table 1. Opioid Receptor Binding Affinities and Selectivities
of EM-2 Analogues 1−7
selectivity
affinity Ki (nm)
compd
Kiμ /Kiδ
Kiδ /Kiμ
[3H]DAMGO (μ)
[3H]DPDPE (δ)
EM-2
0.002
0.03
639
33
5.3 0.4 (4)
21 1.4 (3)
14 1.2 (3)
5.5 0.18 (4)
3.3 0.15 (5)
20 1.9 (3)
19 1.4 (3)
101 8.4 (3)
3385 309 (3)
695 21.4 (3)
187 14.5 (4)
1347 112 (3)
463 38.5 (3)
1650 14 (3)
2798 (3)
1
2
3
4
5
6
7
0.075
0.004
0.007
0.012
0.007
0.011
13
245
140
83
147
9
922 10.0 (4)
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with multiple structural modifications in the Phe−Phe portion,
comprising amino acid homologation and variation upon the
configuration of the incorporated β2hPhe-NH2 at the fourth
position.
and 4 seems to go hand in hand with an essentially extended
conformation, such as that adopted as a result of β3-hPhe-β2-
hPhe insertion in place of the third and fourth EM-2 residues.
To evaluate the pharmacophoric ability of our analogues 3
and 4, we decided to explore their conformational behavior by
means of computational tools, focusing our attention on the
intramolecular distances outlined by the pharmacophore model,
assembled for bioactivity and MOR selectivity. It suggests as
ideal the key distance of 10−13 Å (C distance) between the
aromatic rings of residues 1 and 3. In addition, two other
distances concerning the message domain have been
considered conclusive for activity, namely, the Tyr1 N−Tyr1
O distance (about 8 Å) and the Tyr1 N−Phe3 ring distance
(about 7 Å).25,26
First a conformational analysis was performed, then the
lowest energy conformers were submitted to a 10 ns molecular
dynamics at 300 K to test their conformational stability.
Calculations were executed on the Tyr1 protonated form of 3
and 4 in water and in DMSO. The conformations were also
clustered according to folded or extended conformations by
measuring the interatomic Cα distance between the first and
the fourth residue. Results showed a preference for folded
conformations in water for the two ligands, both after Monte
Carlo conformational analysis (MC) and molecular dynamics
(MD) simulations. In DMSO, a substantial equilibrium
between folded and extended conformations is established,
with a slight preference for the latter. The pharmacophore A, B,
and C distances were measured and averaged on all found
conformations for both compounds. A good agreement of all
values with the proposed model could be observed for water
environment, while lower C distances have been measured in
DMSO. In both environments, comparison between com-
pounds 3 and 4 highlights a greater value of the C distance for
compound 4, accordingly with its slightly higher activity.
In order to better evaluate the binding orientations and to
study the key ligand−receptor interactions involved in the
molecular recognition process with the μ opioid receptor
(MOR), most active compounds 3 and 4 were submitted to
docking studies. Recently the crystal structure of the mouse
MOR with the irreversible morphinan antagonist β-funaltrex-
amine (β-FNA) has been reported.27 Since it can be supposed
that the reported structure was referred to an inactive form of
the receptor, we rather decided to use an active form of the
MOR model as described by Mosberg.28 The ligands were
docked in to the MOR model flexibly with the Molegro Virtual
Docker software (Figure 3).
The conformationally constrained mimics 1, 2, and 5 are not
as active as 3 and 4, disclosing how deep modifications in the
key address fragment (C-terminal Phe4-NH2), leading to an
appreciable reduction of the conformational freedom, would
result in a decrease in affinity. Finally, the low activity of
peptidomimetic 7 clearly highlights the detrimental effect of β2-
homologation at Phe3, which alters significantly the pharmaco-
phore distances21 of the aromatic ring in the message fragment
(N-terminal tripeptide unit). This date confirms once again that
the proper aromatic ring distance and spatial orientation of the
third residue is highly influencing the binding process,
discriminating a potent lead from a much less active derivative.
Structural studies, in particular 1D and 2D proton NMR,
molecular dynamics, and docking analysis, have been performed
in order to fully investigate the three-dimensional properties of
synthesized analogues (see Supporting Information). At
present, a large number of data have been reported on the
conformational analysis of endomorphins, but conclusions are
rather contradictory as both extended and folded structures
have been suggested as the bioactive conformation. For
instance, the importance of the Tyr1−Pro2 amide bond
conformation of EM-2 in its bioactive form is a controversial
and maybe an overstressed problem. In fact, if the opioid
receptor protein selects its ligand by conformational selection
in a dynamic environment, the conformation of a certain amide
bond alone cannot be a strict determinant for a stable ligand−
receptor interaction. However, relying on total energy measure-
ments, it was recently proposed that the trans isomer of EM-2
and analogues could be the mainly bioactive form and that the
cis isomer is mainly an artifact under the solution conditions.22
We performed NMR studies in DMSO-d6 solution, as this
solvent is considered a good physical approximation of
transport fluids environments23 and is claimed to be a better
approximation for the mechanical and electrostatic environ-
ment of binding to the MOR than D2O is.24 Moreover, being a
good hydrogen bond acceptor, it may reveal intrinsic
conformational preferences, mimicking at the same time the
physical circumstances of receptor−ligand interactions. The
spectra show the presence of two conformers (cis and trans
with respect to the Tyr−Pro peptide bond) for all compounds.
A higher prevalence of the trans conformers (trans/cis ratio
ranging from 1 for most compounds to 4 for 7) has been
observed according to the intensities of OH peaks in 1H NMR
spectra.
Both compound 3 and 4 displayed high negative MolDock
score (−260 and −339, respectively), indicating a strong
favorable interaction of the ligands with the receptor, with a
lower energy for 4, thus in accordance with biological activity
results. Most of the interactions found to be relevant for the
EM-2 activity are observed in the binding mode of 3 and 4. The
Tyr1 residue is deeply placed in the binding site providing
strong interactions with the receptor. An H-bond/electrostatic
interaction between the protonated amino group and the
carboxylate moiety of the Asp147 residue is present, and the
Tyr-OH is involved in an H-bond with the nitrogen of His297.
A π−π interaction with Trp293 is established. Further H-bonds
are formed with Tyr148 and Glu229. Also a stabilizing
interaction with Lys 233 is established. Other important
interactions are found between lipophilic regions of the ligand
and Met151, Phe152, Ile296, Val300, and Ile322. The binding
mode of 4 showed additional π−π interactions between Tyr1
From relevant inter-residue ROE interactions, some common
conformational behaviors can be highlighted within all
compounds. In particular, the Tyr1 aromatic ring seems to be
always folded over the Pro2 segment (ROE between Pro H-δ
and various Tyr protons), and in most cases, the NH of the β-
hPhe3 interacts with the Pro’s H-α. Constrained compounds 1
and 2 present additional ROE contacts, involving the β2-hTbac4
residue from one side, and the benzylic or H-α protons of β3-
hPhe3, on the other side, and clearly underlining a folding
between the third and fourth residue. No significant inter-
residue ROE interaction could be observed for linear
derivatives, revealing preferentially extended conformations.
The only exception regards compound 6 where the presence of
multiple ROE contacts would suggest a prominent folding of
the overall structure. Back to biological results, high activity of 3
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Thanks to these outcomes, peptidomimetics 3 and 4 may
validate further study for the development of a peptide
analgesic based on the EM-2 sequence.
Even if caution must be still used in providing key structural
parameters for bioactivity versus opioid receptors, which exist
as dynamic entities that can occupy multiple conformations, we
believe that the recent report of the crystal structure of the
mouse MOR and the joint investigation of the receptor and its
ligands will soon provide conclusive evidence with regard to the
structural features of the biologically active form of EM-2 and
analogues.
ASSOCIATED CONTENT
■
Figure 3. Binding mode of peptides 3 (a) and 4 (b) as obtained by
docking simulations. Hydrogen bonds are represented as yellow
dashed lines.
S
* Supporting Information
Detailed procedure for the synthesis of all compounds and their
characterization; copy of NMR spectra for all compounds;
radioligand binding assay; and computational details. This
material is available free of charge via the Internet at http://
pubs.acs.org.
and Trp293 and Phe152 and Tyr148. The terminal amide
moiety is involved with a range of H-bonds: the NH2 group is
bonded with Glu229, and the carbonyl oxygen interacts with
both the backbone nitrogen of Phe221 and the OH of Thr220.
These strong H-bond interactions in the recognized C-terminal
address domain, could be related to the higher affinity showed
by peptide 4. It is worth to notice that these results are in
agreement with site-directed mutagenesis studies, which already
highlighted the importance of some residues as Asp147,
Tyr148, Glu229, and His297.4
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: alessandro.sacchetti@polimi.it (A.Sa.); gbalboni@
unica.it (G.B.); alessandra.silvani@unimi.it (A.Si.).
Notes
The authors declare no competing financial interest.
As a further consideration, some relevant analogies with the
binding pocket of the recently reported crystal structure of the
(β-FNA)-MOR complex can also be highlighted. Some of the
residues most interacting with β-FNA (Asp147, Tyr148,
Met151, Ile296, Glu229, Lys233, Trp293, His 297, Val300,
and Ile322) were also found to be in close contact with 3 and 4,
thus confirming the validity of our model. Comparison of
binding modes of less active compounds confirmed that the
sequence β3hPhe3−β2hPhe4−NH2 is critical for the effective-
ness of the ligands. In fact, when a β2hPhe3 is present
(compound 7) the binding is less effective due to the lack of the
H-bond with Tyr148; in the case of β3hPhe4−NH2 substitution
(compound 6), a conformational change in the terminal residue
make not possible the important H-bond interaction with
Glu229. The same considerations can be done for compounds
1, 2, and 5, thus confirming the hypothesis that constraining
the phenyl ring of the C-terminal residue is detrimental to the
activity of the peptides.
In conclusion, a small collection of EM-2 analogues were
synthesized, by incorporating various β-dipeptidomimetics to
replace the native Phe3−Phe4-based residue in the reference
compound. In two cases, the multiple structural modifications
resulted in highly active and selective analogues, thanks to the
retention of the structural properties required for MOR
binding.
On the basis of NMR analysis and molecular dynamics
studies, the solution structures of the selected high affinity
MOR ligands 3 and 4 were found to show prevalent extended
conformations and to be in full agreement with the commonly
accepted pharmacophore model of EM-2. Since conformational
studies on isolated molecules in the absence of their receptor
do not necessarily provide unambiguous information on the
bioactive structure, these data were complemented with
docking studies. Different intramolecular interactions have
been identified, which confirm the ability of active compounds
3 and 4 to assume a biologically active correct conformation.
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