Synthesis, biological evaluation and structural analysis of novel peripherally active morphiceptin analogs

Article abstract of DOI:10.1016/j.bmc.2016.02.034

Morphiceptin (Tyr-Pro-Phe-Pro-NH₂), a tetrapeptide amide, is a selective ligand of the μ-opioid receptor (MOR). This study reports the synthesis and biological evaluation of a series of novel morphiceptin analogs modified in positions 2 or/and

Full text of DOI:10.1016/j.bmc.2016.02.034

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and structural analysis of novel
peripherally active morphiceptin analogs
Anna Adamska a, Alicja Kluczyk b, Maria Camilla Cerlesi c, Girolamo Calo c, Anna Janecka a, Attila Borics d,
⇑
a Department of Biomolecular Chemistry, Faculty of Medicine, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland
b Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, Poland
c Department of Medical Sciences, University of Ferrara, Via Fossato di Mortara 17/19, 44121 Ferrara, Italy
d Institute of Biochemistry, Biological Research Centre of Hungarian Academy of Sciences, Temesvári krt. 62, Szeged H-6726, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Morphiceptin (Tyr-Pro-Phe-Pro-NH2), a tetrapeptide amide, is a selective ligand of the
l
-opioid receptor
Received 13 January 2016
Revised 23 February 2016
Accepted 24 February 2016
Available online xxxx
(MOR). This study reports the synthesis and biological evaluation of a series of novel morphiceptin ana-
logs modified in positions 2 or/and 4 by introduction of 4,4-difluoroproline (F2Pro) in or configuration.
L
D
Depending on the fluorinated amino acid configuration and its position in the sequence, new analogs
behaved as selective full MOR agonists showing high, moderate, or relatively low potency. The most
potent analog, Tyr-F2Pro-Phe-D-F2Pro-NH2, was also able to activate the j-opioid receptor (KOR),
Keywords:
Solid-phase peptide synthesis
Binding studies
Opioid receptors
Hot-plate test
although with low potency. Docking studies and the comparison of results with the high resolution crys-
tallographic structure of a MOR-agonist complex revealed possible structure–activity relationships of this
compound family.
Ó 2016 Elsevier Ltd. All rights reserved.
Gastrointestinal transit
Molecular dynamics
Docking
1. Introduction
morphiceptin and its analogs encourages further studies of this
group of opioid peptides, whose therapeutical possibilities have
A number of milk protein fragments has been shown to behave
as opioid receptor ligands, able to address opioidergic systems. One
class of such opioid peptides that show some preference for the l-
not been fully appreciated yet.
Morphiceptin was one of the first opioid peptides, of which
structure was investigated in detail to explain its affinity to the
opioid receptor (MOR)1,2 is the group of b-casomorphins, originat-
ing from the milk protein, b-casein as a product of proteolytic frag-
mentation. A tetrapeptide amide Tyr-Pro-Phe-Pro-NH2, known as
morphiceptin, shows considerable opioid potency and MOR selec-
tivity.3 Morphiceptin, among other opioid peptides, elicits strong
supraspinal antinociception when given intracerebroventricularly
(i.c.v.), in other words directly to the central nervous system
(CNS). This effect is not observed after peripheral administration,
due to the relatively rapid degradation of morphiceptin, resulting
in its short duration of action and limited delivery to the CNS.
However, morphiceptin analogs have been proposed as peripheral
agents for the treatment of diarrhea. Subcutaneous (s.c.) adminis-
tration of a potent morphiceptin analog, Tyr-Pro-NMePhe-Pro-
NH2, inhibited diarrhea and decreased gastrointestinal transit in
mice.4 The advantage of such agents is the lack of the central
effects, such as analgesia and sedation. Peripheral selectivity of
MOR. The L
-configuration of Pro2 was found vital for manifestation
of opioid activity.5 The phenolic OH and the protonated free amine
group of Tyr1 and the aromatic side chain of Phe3 in a well-defined
relative spatial arrangement were proposed to facilitate high affin-
ity MOR binding, in which pose of the Pro2 residue acts as a stere-
ochemical spacer responsible for the correct orientation of
pharmacophoric groups.6 However, later studies comparing sev-
eral MOR ligands of various affinities did not confirm the necessity
of an intrinsic tendency of MOR ligands to adopt such spatial
arrangement.7 Instead, many parallel studies revealed that apart
from possessing the necessary pharmacophores, the high propen-
sity of a ligand to form bent backbone structure8–12 or maintain
high degree of conformational flexibility7 may result in high affin-
ity binding to the MOR. In the past, notable emphasis was put on
the role of cis–trans isomerization of the peptide bond preceding
Pro2 in opioid peptides. As both exclusively cis5 and trans peptide
ligands13 were shown to bind to the MOR with high affinity, this
structural property was proved to be of little relevance with regard
to MOR activity. The X-ray crystallographic structure of both, the
⇑
Corresponding author.
E-mail address: borics.attila@brc.mta.hu (A. Borics).
http://dx.doi.org/10.1016/j.bmc.2016.02.034
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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A. Adamska et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
agonist-14 and antagonist-bound MOR15 has been published
recently, which provided answer to numerous questions regarding
morphiceptin were tested in vitro in radioligand receptor binding
assays and calcium mobilization-based functional tests. Conforma-
tional preferences of the new ligands were studied by performing
molecular dynamics (MD) simulations. In order to reveal atomistic
details of specific interactions between the new ligands and the
MOR, docking studies were performed.
ligand structure and activity. Nevertheless, the design of
a
MOR-agonist peptide with pharmaceutical properties appropriate
for therapeutic application remains a challenge.
Unique physico-chemical properties of fluorine, such as small
size and high electronegativity, seem to be of special advantage
in drug design. Fluorinated compounds show higher bioavailability
and metabolic stability. One of the major effects of fluorination is a
modulation of acidity of a parent compound which can strongly
influence binding affinity and pharmacokinetic properties of an
analog. While substitution of fluorine for a hydrogen atom results
in minor steric alterations, electrostatic interactions in a molecule
may lead to significant conformational changes.16 So far, only a few
fluorinated amino acids have been introduced into the analogs of
opioid peptides.17–19
2. Materials and methods
2.1. Peptide synthesis
Most of the chemicals were purchased from Sigma Aldrich. Pro-
tected amino acids were purchased from NovaBiochem or Bachem.
MBHA Rink-Amide peptide resin (100–200 mesh, 0.6 mmol/g) was
obtained from NovaBiochem. Fmoc-protected L- and D-F2Pro was
purchased from TriMen Chemicals (Lodz, Poland). Peptides were
synthesized by a standard solid-phase procedure, using technique
for Fmoc-protected amino acids on MBHA Rink-Amide resin as
described earlier.20 20% piperidine in DMF was used for the depro-
tection of Fmoc groups and TBTU was employed as a coupling
In the search for novel morphiceptin analogs with improved
pharmacological profile we have synthesized a series of analogs
modified in positions 2 or/and 4 by introduction of 4,4-difluoro-
Pro (F2Pro) in
L or D configuration (Fig. 1). Novel analogs of
Figure 1. Structure of the studied fluorinated derivatives 1–4 and their parent compound, morphiceptin.
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A. Adamska et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Figure 2. Specifically bound, low energy docked complexes of morphiceptin (B), 1 (C), 2 (D), 3 (E) and 4 (F) in comparison with the crystallographic structure of a MOR-bound
morphinan agonist BU42 (A).
agent. Simultaneous deprotection and cleavage from the resin was
accomplished by treatment with TFA/TIS/water (95:2.5:2.5) for 3 h
at room temperature.
gradient of 0–100% B over 50 min at a flow rate of 1 ml/min was
used for the analysis. Identity of the synthesized peptides was
characterized by high resolution mass spectroscopy, using Bruker
micrOTOF-Q mass spectrometer (Bruker Daltonics, Bremen,
Germany) with electrospray ionization (ESI-MS).
Crude peptides were purified by preparative RP-HPLC on a
Vydac C18 column (10
l
m, 22 ꢀ 250 mm) equipped with a Vydac
guard cartridge. The solvent system of 0.1% TFA in water (A)/80%
acetonitrile in water containing 0.1% TFA (B) and a linear gradient
of 0–100% B over 15 min was used. The purity of the final peptides
was verified by analytical HPLC employing a Vydac C18 column
2.2. Receptor binding assays
Receptor binding assays were performed according to the mod-
ified method described by Misicka et al.,21 using brain homoge-
nates of adult male Wistar rats (for MOR and d-opioid receptor,
(5
l
m, 4.6 ꢀ 250 mm) and the solvent system of 0.1% TFA in water
(A)/80% acetonitrile in water containing 0.1% TFA (B). A linear
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A. Adamska et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
DOR) or adult male Dunkin Hartley guinea pigs (for
j
-opioid recep-
The evolution of secondary structure of the peptides along each
trajectory was analyzed with the STRIDE algorithm28 and Perl
scripts written in-house. The analysis programs of the GROMACS
5.0.4 program suite was used for the measurement of the C(1)–
tor, KOR). The opioid receptor binding affinities for MOR, DOR and
KOR were determined by radioligand competition analysis using
[3H]DAMGO, [3H][Ile5,6]deltorphin-2 and [3H]nor-BNI, respectively.
Three independent experiments for each assay were carried out in
duplicate. The data were analyzed by a nonlinear least square
regression analysis computer program Graph Pad PRISM 6.0
(Graph Pad Software Inc., San Diego, USA).
C (2)–C (3)–N(4) virtual dihedral angle and the distance between
a
a
the terminal C atoms. A bend structure was assigned when this
a
dihedral angle was between ꢁ80° and 80° and the distance was
less than 10.0 Å.29
2.3. Calcium mobilization assay
2.5. Docking studies
Chinese Hamster Ovary (CHO) cells stably co-expressing human
recombinant MOR or KOR and the C-terminally modified Gaqi5 and
The crystallographic structure of the active murine MOR (PDB
code: 5C1M)13 was used as docking target after missing side chains
were added. Dockings were performed with the Autodock 4.2 soft-
ware. Side chains in contact with the bound ligand, observed in the
crystal complex of MOR and BU72 were kept flexible as well as all
CHO cells co-expressing the human recombinant DOR receptor and
the GaqG66Di5 chimeric protein were generated as previously
described.22–24
Agonist potencies were given as pEC50 representing a negative
logarithm of the molar concentration of an agonist that produces
50% of the maximal possible effect. Concentration response curves
were fitted with the four parameter logistic nonlinear regression
model:
U, w
and v1 ligand torsions. Blind docking of morphiceptin and
derivatives 1–4 were performed using the Lamarckian genetic
algorithm in a 80 Å ꢀ 80 Å ꢀ 80 Å grid volume, large enough to
cover the whole receptor region accessible from the extracellular
side. The spacing of grid points was set at 0.375 Å and 1000 dock-
ings were done for all ligands. The resultant ligand–receptor com-
plexes were clustered and ranked according to the corresponding
binding free energies. In silico inhibitory constants were calculated
Emax ꢁ baseline
Effect ¼ baseline þ
50ꢁXÞꢂn
1 þ 10ðlogEC
according to the following equation:
DG = RTlnKi. The pool of
where X is the agonist concentration and n is the Hill coefficient.
ligand–receptor complexes was reduced by excluding the bound
states in which specific, conserved ligand–receptor interactions
observed in the crystallographic structures of the MOR com-
plexes14,15 were not present.
Ligand efficacy was expressed as intrinsic activity (a) calculated
as the Emax of the ligand to Emax of the standard agonist ratio. Curve
fittings were performed using GraphPad PRISM 6.0 (GraphPad Soft-
ware Inc., San Diego, USA).
2.4. MD simulations
3. Results and discussion
3.1. Peptide synthesis
MD simulations of compounds 1–4 were started from extended,
energy minimized geometries and executed using the GROMACS
5.0.4 program package and the AMBER ff03 force field parameter
set.25 Parameters for unnatural amino acid residues were supple-
mented from the generalized Amber force field (gAFF)26 and partial
charges were determined at the HF/6-31G(d) level using the
restrained electrostatic potential (RESP) method with the same
force field parameters as described above. Each starting structure
was immersed in a cubic box (35 Å ꢀ 35 Å ꢀ 35 Å) of pre-equili-
brated TIP3P27 water molecules. Solvent molecules were removed
from the box when the distance between any atom of the solute
and solvent molecules was less than the sum of their van der Waals
radii. Protonated N-termini of peptides were neutralized by replac-
ing solvent molecules by Cl-ions at a position with the most favor-
able electrostatic potential. All systems were then subjected to
1000 steps of steepest descent, followed by 1000 steps conjugate
gradient energy minimization with 0.001 kJ molꢁ1 convergence
criteria. In order to allow the solvent density to equilibrate,
0.5 ns NVT MD simulations at 300 K were performed while the
position of the solute was fixed in the center of the box with a force
constant of 1000 kJ molꢁ1 Å2 on each heavy atom. Subsequently,
200.5 ns NPT MD simulations were performed for the four pep-
tides, each at constant temperature (300 K) and pressure (1 bar),
with the following parameters: the time step was set to 2 fs, the
LINCS algorithm was used to constrain all bonds to their correct
lengths, temperature was regulated with the v-rescale algorithm
with a coupling constant of 0.1 ps, constant pressure was main-
tained using isotropic scaling with a relaxation constant of 1.0 ps
and 4.5 ꢀ 10ꢁ5 barꢁ1 isothermal compressibility. Non-bonded
interactions were calculated using the PME method with all cut-
off values set at 12 Å. The coordinates were stored after every
1000 steps to yield a total of 100,000 sampled conformations for
each trajectory, after excluding the first 0.5 ns of equilibration.
Peptides were synthesized by the conventional solid-phase pro-
cedure on the MBHA Rink Amide resin, using techniques for Fmoc-
protected amino acids. High resolution mass spectrometry (ESI-
MS) confirmed the identity of all synthesized peptides (see Supple-
mentary material). RP-HPLC analyses of the final purified products
indicated purity of 97% or greater (Table 1).
3.2. Opioid receptor binding studies
Opioid receptor binding affinities of peptides for the MOR, DOR
and KOR were determined by radioligand competition analysis
using [3H]DAMGO, [3H][Ile5,6]deltorphin-2 and [3H]nor-BNI,
respectively. The IC50 values were determined from logarithmic
dose–displacement curves, and the values of the inhibitory
Table 1
Physicochemical data of new opioid peptide analogs
b
No. Sequence
Formula
m/z [M+H]+a
Calcd Obsd
HPLC tR
(min)
1
2
3
4
Tyr-
Pro-NH2
Tyr-Pro-Phe-
F2Pro-NH2
Tyr-F2Pro-Phe-
F2Pro-NH2
Tyr-F2Pro-Phe-
F2Pro-NH2
D
-F2Pro-Phe-
C28H34F2N5O5 558.252 558.252 14.10
C28H34F2N5O5 558.252 558.271 14.22
C28H32F4N5O5 594.233 594.244 14.56
C28H32F4N5O5 594.233 594.247 14.99
D-
D-
a
Mass was measured using ESI-MS.
b
tR with Vydac C18 column (5
lm, 4.6 ꢀ 250 mm) using the solvent system of
0.1% TFA in water (A) and 80% acetonitrile in water containing 0.1% TFA (B) and a
linear gradient of 0–100% solvent B over 50 min, with the flow rate 1 ml/min.
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A. Adamska et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
constant (Ki) of peptides were calculated according to the equation
of Cheng and Prusoff30 are shown in Table 2. Morphiceptin, as
expected, displayed only mild affinity for MOR but high selectivity
for this receptor. Among the new analogs, only 2 and 4 with
ligands, determined in the absence of the receptor do not rule
out the possibility of high affinity binding and receptor activation,
but on the other hand do not provide basis for the explanation of
in vitro experimental results.
D
-configured F2Pro in position 4 were able to bind to MOR. Analog
4, with two fluorinated Pro residues ( , respectively) showed
L
,
D
3.5. Docking studies
subnanomolar affinity for MOR and very weak for KOR, although
considering that [3H]nor-BNI is also very weak MOR ligand, the
measured KOR affinity may be argued at this point. As compared
Configuration inversion and/or fluorination of the Pro residues
in morphiceptin in this study were disproved to result in such a
significant change of conformational preferences that could be
held responsible for the observed differences in bioactivity. In
order to gain explanation, the aim was set to study the specific
interactions formed by the receptor bound ligands. Dockings of
morphiceptin and compounds 1–4 were performed to investigate
how configuration inversion and fluorination of the Pro residues
of morphiceptin affect interactions with the MOR. It is known for
most docking algorithms that they may present low energy bound
structures even for compounds which do not show any affinity for
the receptor in experiments.32 Such occurrence of false positive
hits is usually even higher in the case of ligands with high confor-
mational flexibility.
Blind, flexible docking of morphiceptin and analogs 1–4 to the
MOR resulted in low energy complexes in which all the ligands
were located in the previously described binding cavity. The Ki val-
ues calculated for the lowest binding free energy complexes suggest
high affinity binding of these ligands in general (Table 2). However,
the relative receptor–ligand orientation in the bound ligands did
not reflect any structural specificity. In order to exclude the non-
specifically bound ligand conformations from analysis, the low
energy ligand–receptor complexes were inspected for the presence
of previously described interactions. A salt bridge between the pos-
itively charged amino groups of opioid ligands and Asp147 of the
mouse MOR was shown to be essential both by structure–activity
studies10 and site-directed mutagenesis of the receptor.33 The pres-
ence of this interaction was confirmed in the crystallographic com-
plexes.14,15 Furthermore, the tertiary amine group and the phenolic
OH group of the MOR bound morphinan agonist BU72 were found
to adopt an alignment identical to that of the MOR bound morphi-
nan antagonist b-FNA, suggesting that regardless of functional
properties of ligands, this structural arrangement is a requisite of
high affinity binding. The above mentioned functional groups of
morphinan ligands are analogous to the phenolic OH and free
amine pharmacophores of Tyr1 in opioid peptides. Consequently,
docked ligand–receptor complexes in which the conserved salt
bridge and correct alignment of pharmacophores were not present,
including some of the lowest energy ones, were considered as non-
specifically bound and excluded from further analysis.
to the earlier described analog, Tyr-Pro-Phe-D-Pro-NH2 (IC50 =
4.3 nM),31 it seems that fluorination increased MOR affinity by an
order of magnitude.
3.3. Functional assay
The pharmacological profiles of peptides were characterized
in vitro at all three opioid receptors in calcium mobilization
assays.24 The calculated agonist potencies (pEC50) and efficacies
(a) of the analogs are summarized in Table 3. Dermorphin, DPDPE,
and dynorphin A were used as the reference agonists for calculat-
ing intrinsic activity at the MOR, DOR, and KOR, respectively.
Results of this study demonstrated that all novel compounds
behaved as selective full agonists of MOR showing high (4), moder-
ate (2), or relatively low (3) potency. Compound 4 is also able to
activate the KOR with low potency (MOR/KOR selectivity 174 fold),
which suggests that the measured low KOR binding affinity of this
analog is not an artifact emerging from the applied experimental
conditions.
3.4. MD simulations
MD simulations were performed to investigate the relationship
between the MOR binding properties and conformational prefer-
ences of compounds 1–4. Results of trajectory analysis are summa-
rized in Table 4. All morphiceptin analogs were found to adopt
various
c and b-turns with similar frequency as it was observed
for the parent compound morphiceptin in a previous study.7 As a
result of a tertiary amide in position 4 of the sequences, no b-turn
types stabilized through a 1 4 hydrogen bond were found. Nei-
ther any of the identified specific secondary structural elements,
nor their population were found to correlate with MOR affinity.
The tendency of compounds 1–4 to form bent backbone structure,
which was indicated previously to be advantageous for MOR bind-
ing was found to be significantly lower than that of the parent
compound. However, it was also reported earlier that ligands
which are not likely to form bent structure in solution, but main-
tain considerable conformational flexibility, such as DAMGO, the
prototypical ligand of the MOR, can still bind to the receptor with
high affinity.7 In summary, conformational preferences of these
Lowest binding free energy complexes of the specifically bound
fluorinated analogs 1–4 and their parent compound, in comparison
Table 2
In vitro and in silico opioid receptor binding data of fluorinated morphiceptin analogs
No.
Sequence
Kia (nM)
In silico Kib (nM)
MOR non-specificc
MOR
DOR
KOR
MOR
specificd
Tyr-Pro-Phe-Pro-NH2 (morphiceptin)
36.39
>1000
13.28
105.63
0.37
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
385.90
0.72
1.99
0.48
1.52
0.81
21.44
121.35
35.91
103.18
1.73
1
2
3
4
Tyr-
Tyr-Pro-Phe-
Tyr-F2Pro-Phe-F2Pro-NH2
Tyr-F2Pro-Phe- -F2Pro-NH2
D-F2Pro-Phe-Pro-NH2
D-F2Pro-NH2
D
a
Binding affinity values determined by competitive displacement of the selective radioligands, [3H]DAMGO (for MOR) and [3H][Ile5,6]deltorphin-2 (for DOR), using rat
brain membranes, and [3H]nor-BNI (for KOR) using guinea pig brain membranes.
b
Theoretical inhibitory constants were calculated from binding free energies resulting from docking studies using the following equation:
DG = RTlnKi.
c
Overall lowest binding free energy complexes.
Lowest binding free energy complexes which possess the conserved receptor–ligand interactions.
d
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A. Adamska et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 3
Effects of reference agonists and compounds assessed at human recombinant opioid receptors coupled with calcium signaling via chimeric G proteins
Compound
MOR
DOR
KOR
pEC50 (CL95%
8.40 (8.12–8.68)
Inactivea
6.67 (6.17–7.17)
6.13 (5.86–6.39)
Inactive
7.69 (7.41–7.97)
6.40 (6.15–6.66)
8.33 (8.03–8.64)
)
a
SEM
pEC50 (CL95%
)
a
SEM
pEC50 (CL95%
)
a SEM
Dermorphin
DPDPE
Dynorphin A
Morphiceptin
1
2
3
4
1.00
6.43 (5.95–6.91)
7.77 (7.38–8.16)
7.73 (7.46–8.00)
1.03 0.07a
1.00
Inactivea
Inactivea
0.83 0.10a
0.96 0.04
0.99 0.04a
8.82 (8.62–9.02)
Inactive
1.00
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Crc incomplete
Inactive
1.06 0.11
1.03 0.08
1.04 0.06
6.09 (5.92–6.27)
0.84 0.06*
Inactive means that the compound was inactive up to 1 lM.
Crc incomplete means that maximal effects could not be determined due to the low potency of the compounds. Dermorphin, DPDPE and dynorphin A were used as reference
agonists for calculating intrinsic activity at MOR, DOR, and KOR receptor, respectively.
a
These data are from Camarda and Calò.24
p <0.05 according to one way ANOVA followed by the Dunnett’s test for multiple comparisons.
*
Table 4
among the studied compounds as morphiceptin and analogs 2–4
were more likely to leave this polar network intact (Fig. 2D) or
extend it by contributing their C-terminal amide group (Fig. 2BEF).
Frequency of canonical secondary structural elements identified by STRIDE analysis
and the overall occurrence of bent backbone structure
D
configuration of Pro4/F2Pro4 may provide a further advantage in
Secondary
structural element
Residues involved
Populationa (%)
this model by placing the apolar Pro4/F2Pro4 side chain away from
1
2
3
4
the polar network (Fig. 2F).
Type IV b-turn
Type VIII b-turn
Tyr1-Pro4/F2Pro4
Tyr1-Pro4/F2Pro4
Tyr1-Phe3
1.63 3.59
1.75
1.96
1.89
3.59
—
3.62
Inverse
Inverse
Classic
Inverse
c
c
-turn
-turn
—
10.08 12.85 16.05
4. Conclusion
Phe3-Pro4/F2Pro4-NH2
Phe3-Pro4/F2Pro4-NH2
Tyr1-Phe3, Phe3-Pro4/
F2Pro4-NH2
5.39
—
—
—
2.58
—
7.01
—
1.39
—
1.60
—
c
-turn
Substitution of the Pro2 and Pro4 residues of morphiceptin by
c
-turns
L- and/or D-F2Pro resulted in a series of analogs with diverse bioac-
Inverse + classic
turns
c
-
Tyr1-Phe3, Phe3-Pro4/
—
—
—
0.44
6.86
tivity a new, high affinity MOR-selective peptide ligand 4 was pre-
sented. The solution conformational preferences of these
compounds in aqueous media were found to be highly similar, sug-
gesting that the observed differences in MOR affinity may be
attributed to different ligand receptor contacts and specific inter-
actions rather than conformational selection. The recently pub-
lished high resolution structure of the active and inactive MOR
proved to be a solid independent platform for the evaluation of
receptor bound geometries of peptide ligands obtained from dock-
ing. Using these structures as a reference facilitated the explana-
tion of the structure–activity relationships of the studied
compounds. Previous suggestions for the MOR-bound ligand struc-
ture such as bent backbone and trans conformation of the Tyr1 side
chain were confirmed. In addition to the well described interac-
tions with the conserved Asp147 and the polar network formed
by Tyr148, Lys233 and His297 of the receptor, Pro2 and F2Pro2 of
the studied compounds were found to locate in a cavity of
F2Pro4-NH2
Bend structureb
Any possible
8.73 8.12
4.85
a
Population fraction of the total conformational ensemble generated by MD
simulations.
Bent structure was assigned based on the analysis of the N(1)–C (2)–C (3)–C(4)
virtual dihedral angle and the distance between the terminal C -atoms.
b
a
a
a
to the crystallographic structure of a MOR-bound morphinan ago-
nist are shown in Figure 2. Theoretical Ki values calculated for
these complexes fairly reproduce the experimentally observed val-
ues and their differences, providing basis for structural explanation
of receptor affinity. In general, trans conformation of the Tyr1 side
chain was found to furnish the desired alignment of pharma-
cophores described above and apart from one exception (Fig. 2D),
ligands were found to bind in a bent backbone structure (Fig. 2).
This observation confirms hypotheses included in previous phar-
macophore models.10 Pro2 and F2Pro2 of the studied compounds
were observed to occupy the same, mainly hydrophobic cavity
formed by residues Trp133, Val143 and Ile144, as the pendant phenyl
group of BU72 in the crystal structure of the active MOR. This sug-
gests, that fluorination of Pro2 may give rise to the strength of
hydrophobic interactions between the ligand and the receptor.
Furthermore, it gives explanation for lower receptor affinities
observed for endomorphin analogs with hydroxyproline substitu-
hydrophobic character, lined by residues Trp133, Val143 and Ile144
.
Substitution of Pro2 to the more hydrophobic derivative F2Pro2
appears to be beneficial for the formation of stronger ligand–recep-
tor interactions. In general, our results indicate that observations of
specific MOR–ligand interactions in experimental and in silico
complex models could constitute the basis of the future design of
MOR ligands with fine-tuned chemical structure and optimal
effectiveness.
tion in this position of the sequence.34,35 The
D configuration of
Acknowledgements
F2Pro2 in 1 was shown to have a deleterious effect on receptor
binding which is also in agreement with previous observations.5,36
However, inspecting receptor–ligand complexes in Figure 2 it is
apparent that Pro2/F2Pro2 could interact with the same hydropho-
This work was supported by Hungarian-Polish Non-Govern-
mental Cooperation Programme. Research of A.B. has been sup-
ported by the János Bolyai Research Scholarship of the Hungarian
Academy of Sciences.
bic side chains of the binding cavity in both L and D configuration.
Apparently, the LDLL configuration pattern of compound 1 results in
the projection of the hydrophobic Phe3 side chain into the pocket
region lined by Tyr148, Lys233 and His297 (Fig. 2C) and disrupting
the water molecule assisted polar network essential for the proper
function of the receptor.14,37 Analog 1 is unique in this sense
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.02.034.
Please cite this article in press as: Adamska, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.034

A. Adamska et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
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