Discovery of amphipathic dynorphin A analogues to inhibit the neuroexcitatory effects of dynorphin A through bradykinin receptors in the spinal cord

Article abstract of DOI:10.1021/ja501677q

We hypothesized that under chronic pain conditions, up-regulated dynorphin A (Dyn A) interacts with bradykinin receptors (BRs) in the spinal cord to promote hyperalgesia through an excitatory effect, which is opposite to the well-known inhibitory effect of opioid receptors. Considering the structural dissimilarity between Dyn A and endogenous BR ligands, bradykinin (BK) and kallidin (KD), this interaction could not be predicted, but it allowed us to discover a potential neuroexcitatory target. Well-known BR ligands, BK, [des-Arg¹⁰, Leu⁹]-kallidin (DALKD), and HOE140 showed different binding profiles at rat brain BRs than that previously reported. These results suggest that neuronal BRs in the rat central nervous system (CNS) may be pharmacologically distinct from those previously defined in non-neuronal tissues. Systematic structure-activity relationship (SAR) study at the rat brain BRs was performed, and as a result, a new key structural feature of Dyn A for BR recognition was identified: amphipathicity. NMR studies of two lead ligands, Dyn A-(4-11) 7 and [des-Arg⁷]-Dyn A-(4-11) 14, which showed the same high binding affinity, confirmed that the Arg residue in position 7, which is known to be crucial for Dyn As biological activity, is not necessary, and that a type I B-turn structure at the C-terminal part of both ligands plays an important role in retaining good binding affinities at the BRs. Our lead ligand 14 blocked Dyn A-(2-13) 10-induced hyperalgesic effects and motor impairment in in vivo assays using na?ve rats. In a model of peripheral neuropathy, intrathecal (i.th.) administration of ligand 14 reversed thermal hyperalgesia and mechanical hypersensitivity in a dose-dependent manner in nerve-injured rats. Thus, ligand 14 may inhibit abnormal pain states by blocking the neuroexcitatory effects of enhanced levels of Dyn A, which are likely to be mediated by BRs in the spinal cord.

Full text of DOI:10.1021/ja501677q

Article
pubs.acs.org/JACS
Open Access on 04/17/2015
Discovery of Amphipathic Dynorphin A Analogues to Inhibit the
Neuroexcitatory Effects of Dynorphin A through Bradykinin
Receptors in the Spinal Cord
Yeon Sun Lee,† Dhanasekaran Muthu,† Sara M. Hall,† Cyf Ramos-Colon,† David Rankin,‡ Jackie Hu,‡
Alexander J. Sandweiss,‡ Milena De Felice,‡ Jennifer Yanhua Xie,‡ Todd W. Vanderah,‡ Frank Porreca,‡
Josephine Lai,‡ and Victor J. Hruby*,†
†Department of Chemistry and Biochemistry and ‡Department of Pharmacology, The University of Arizona, Tucson, Arizona 85721,
United States
S
* Supporting Information
ABSTRACT: We hypothesized that under chronic pain
conditions, up-regulated dynorphin A (Dyn A) interacts with
bradykinin receptors (BRs) in the spinal cord to promote
hyperalgesia through an excitatory effect, which is opposite to
the well-known inhibitory effect of opioid receptors.
Considering the structural dissimilarity between Dyn A and
endogenous BR ligands, bradykinin (BK) and kallidin (KD),
this interaction could not be predicted, but it allowed us to
discover a potential neuroexcitatory target. Well-known BR
ligands, BK, [des-Arg10, Leu9]-kallidin (DALKD), and
HOE140 showed different binding profiles at rat brain BRs than that previously reported. These results suggest that neuronal
BRs in the rat central nervous system (CNS) may be pharmacologically distinct from those previously defined in non-neuronal
tissues. Systematic structure−activity relationship (SAR) study at the rat brain BRs was performed, and as a result, a new key
structural feature of Dyn A for BR recognition was identified: amphipathicity. NMR studies of two lead ligands, Dyn A-(4−11) 7
and [des-Arg7]-Dyn A-(4−11) 14, which showed the same high binding affinity, confirmed that the Arg residue in position 7,
which is known to be crucial for Dyn A’s biological activity, is not necessary, and that a type I β-turn structure at the C-terminal
part of both ligands plays an important role in retaining good binding affinities at the BRs. Our lead ligand 14 blocked Dyn A-
̈
(2−13) 10-induced hyperalgesic effects and motor impairment in in vivo assays using naive rats. In a model of peripheral
neuropathy, intrathecal (i.th.) administration of ligand 14 reversed thermal hyperalgesia and mechanical hypersensitivity in a
dose-dependent manner in nerve-injured rats. Thus, ligand 14 may inhibit abnormal pain states by blocking the neuroexcitatory
effects of enhanced levels of Dyn A, which are likely to be mediated by BRs in the spinal cord.
analogues can produce motor impairment, paralysis, and
enhanced sensitivity to sensory stimuli.3−7 These effects are
not blocked by naloxone, and thus are nonopioid in nature.
It has been proposed that after nerve injury, up-regulated
Dyn A in the spinal cord may interact directly with spinal
bradykinin receptors (BRs) to promote neuropathic pain.8,9
Considering the lack of structural similarity between Dyn A and
the endogenous ligands for the BRs, bradykinin (BK) and
kallidin (KD), this interaction between Dyn A and BR could
not be predicted but allowed us to identify a putative direct
neuroexcitatory target. Here, we test the therapeutic potential
of a pharmacological intervention of Dyn A at spinal BRs in
chronic pain states by developing BR antagonists based on the
prototypic structures of Dyn A.
INTRODUCTION
Dynorphin A (Dyn A, Figure 1) is a known endogenous opioid
peptide along with enkephalin and endorphin, and is
■
Figure 1. The structures of Dyn A and Dyn A-(2−13).
characterized by its high affinity for mu (μ), delta (δ), and
kappa (κ) opioid receptors. The high affinity of Dyn A for the
opioid receptors is mainly due to the N-terminal tyrosine,
because des-tyrosyl fragments show very low binding affinity at
opioid receptors.1 In vivo, Dyn A is degraded rapidly upon
release to the des-tyrosyl analogue by aminopeptidases in the
synapse to terminate Dyn A’s agonist action at the opioid
receptors.2 Dyn A inhibits smooth muscle contractility, which is
characteristic of opioid agonists and is blocked by naloxone. It
is also well documented that Dyn A and its des-tyrosyl
Received: October 10, 2013
Published: April 17, 2014
© 2014 American Chemical Society
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Table 1. Binding Affinities of Dyn A (H-Tyr1-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln17-OH)
Fragments and Dyn A-(4−11) Analogues at BRs in Rat Brain
a
BR , [3H]DALKD
b
no.
ligand
log [IC50]
IC50 (nM)
1
Dyn A-(3−6)
Dyn A-(3−7)
Dyn A-(3−8)
Dyn A-(3−9)
Dyn A-(3−10)
Dyn A-(3−11)
Dyn A-(4−11)
Dyn A-(5−11)
Dyn A-(5−12)
Dyn A-(2−13)
Dyn A-(3−13)
Dyn A-(4−13)
Dyn A-(5−13)
−6.02 0.08
−5.01 0.10
−5.63 0.27
−6.11 0.09
−6.09 0.28
−6.88 0.08
−6.86 0.06
−6.55 0.06
−5.15 0.09
−6.78 0.09
−6.50 0.07
−6.41 0.13
−6.33 0.16
−6.71 0.11
−5.19 0.20
−6.92 0.28
−6.20 0.15
−6.44 0.13
−6.67 0.15
−6.86 0.20
−6.84 0.12
−6.86 0.12
−6.85 0.08
−6.70 0.29
−6.35 0.18
−6.96 0.20
−6.93 0.08
−7.12 0.11
960
9800
2300
780
810
130
140
280
7100
170
320
390
470
190
6500
120
630
360
210
140
150
140
140
200
450
110
120
76
2
3
4
5
6
7
8
9
c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
c
H-Phe-Leu-Arg-Ile-Arg-Pro-Lys-OH
H-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-NH2
Ac- Phe-Leu-Arg-Ile-Arg-Pro-Lys-OH
Ac- Phe-Leu-Arg-Ile-Arg-Pro-DLys-OH
Ac-DPhe-Leu-Arg-Ile-Arg-Pro-Lys-OH
H-Phe-Leu-Arg-Ile-Arg-Pro-Arg-OH
Ac- Phe-Leu-Arg-Ile-Arg-Pro-Arg-OH
Ac-DPhe-Leu-Arg-Ile-Arg-Pro-Arg-OH
H-Phe-Nle-Arg-Nle-Arg-Pro-Arg-OH
Ac-Phe-Nle-Arg-Nle-Arg-Pro-Arg-OH
Ac-DPhe-Nle-Arg-Nle-Arg-Pro-Arg-OH
H-Phe-Ala-Arg-Ala-Arg-Pro-Arg-OH
H-Phe-Leu-Arg-Arg-Ile-Arg-Arg-Pro-Lys-OH
H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH
H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu-OH
c
BK
DALKD
a
b
Competition assays were carried out at pH 7.4 using rat brain membranes. Logarithmic values determined from the nonlinear regression analysis
c
of data collected from at least two independent experiments in duplicate. 10: log [IC50] = −7.29 0.21, IC50 = 51 nM. 14: log [IC50] = −6.85
0.04, IC50 = 140 nM. BK: log [IC50] = −7.81 0.17, IC50 = 15 nM at pH 6.8 in competition assays using [3H]BK, and transfected HEK 293 cells
expressing the human B2R.
terminus its affinity was reduced 15-fold (9, IC50 = 7100 nM).
However, subsequent truncation of a Leu residue at the C-
terminus recovered or even increased its binding to the
receptor (8, IC50 = 280 nM). This distinct SAR at the C-
terminus was maintained during the successive truncations of
C-terminal residues, and this result confirmed the important
role of a positive charge for the C-terminal amino acid residues
in receptor recognition.
On the other hand, consecutive truncations of the N-terminal
amino acid residues to position 4 did not affect their affinities to
the receptor. The truncation of two amino acid residues, Gly
and Phe, in 6 and 10 resulted in negligible reduction of the
affinities to the receptor. These results suggest that the N-
terminal part of the Dyn A is remote and thus not involved in
binding to the BRs. This is an important feature of SAR to
understand how Dyn A analogues recognize the receptor. The
positive charge of the C-terminal basic amino acid residue may
be mainly involved in electrostatic interactions with the
receptor.
RESULTS AND DISCUSSION
■
Structure−Activity Relationships (SAR) Study. The key
step in the rational design of BR antagonists is to identify the
pharmacophore of Dyn A that directly interacts with the BRs as
an agonist and then to refine the structure for the binding site
by examining the effects of different substituents to obtain an
antagonist.10 First, each amino acid at the C-terminal or N-
terminal position of Dyn A-(2−13) (10), which has been
reported to have neuroexcitatory nonopioid effects through
BRs in the spinal cord,1,7,8 was truncated in sequence and the
resulting fragments were tested for their binding to BRs against
[3H][des-Arg10,Leu9]-kallidin ([3H]DALKD) or [3H]BK in rat
brain membranes. As a result, a key pharmacophore of Dyn A
for the BRs was identified as well as distinct SAR (Table 1).
Dyn A-(4−11) (7), which has the same range of binding
affinity (IC50 = 140 nM) as 10 (IC50 = 170 nM), was identified
as a good pharmacophore for the BRs. Also, the SAR results
revealed that the BR recognition predominantly depends on the
basicity of the C-terminal amino acid residue.
Further SAR studies on the minimum pharmacophore 7 were
performed to distinguish the receptor interaction and to
increase the potency and in vivo stability. Although two Arg
residues at positions 6 and 7 have been known to be important
for the biological activity of Dyn A,11,12 on the basis of our
primary SAR results and the amphipathic properties of Dyn A
The Dyn A fragments 4, 6, and 13, which have a C-terminal
basic amino acid such as Lys or Arg, showed higher binding
affinities than those fragments (3, 5, and 9) with a C-terminal
hydrophobic amino acid such as Ile, Pro, or Leu. For example,
Dyn A-(5−13) (13) showed moderate affinity (IC50 = 470 nM)
at the receptor, and after truncation of a Lys residue at the C-
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structure, it did not seem to be necessary to retain two Arg
residues for binding to BRs. Therefore, the Arg residue at
position 7 was deleted, and interestingly the resulting ligand 14
retained high binding affinity (IC50 = 190 nM) to the BRs. The
removal of the Arg residue at position 7 did not affect binding
affinity at all. This is remarkable because in general, deleting
one amino acid in the middle of a bioactive sequence typically
causes significant topographical changes and a different
biological profile.
from rat brain BR binding sites differ significantly from that
using guinea pig ileum (GPI) where both BK and HOE140 had
high affinity (IC50 = 3.5 nM and 0.43 nM, respectively) similar
to that previously reported.13−16 Thus, in the rat central
nervous system, we may be targeting a neuronal BR that is
pharmacologically distinct from that previously defined in non-
neuronal tissues. Furthermore, this neuronal BR exhibits affinity
for Dyn A in the nanomolar range.
For comparison, selected Dyn A ligands that showed good
affinities at rat brain BRs were tested for their binding affinities
in transfected human embryonic kidney (HEK) 293 cells
expressing the human B2R or B1R. Ligands 10 (IC50 = 51 nM),
14 (IC50 = 140 nM), and other ligands (see Supporting
Information) exhibited a similar range of binding affinities for
the HEKB2R as for the rat brain BRs (Table 1). However, none
of the Dyn A ligands showed affinities at the HEKB1R where
their affinities are >10000 nM. These results suggest that Dyn A
ligands are selective for B2R over B1R.
As discussed earlier, the C-terminal basic amino acid residue
plays an important role in BR recognition. If the receptor
recognition is mainly through electrostatic interactions with
positive charges of the side chain group of a basic amino acid
residue, the amidation of a C-terminal acid group can be a
useful modification to increase in vivo stability in the same
range of binding affinity. For this purpose, the C-terminal acid
was amidated in 15, but the modified ligand had greatly
reduced binding affinity (IC50 = 6500 nM). All other amidated
ligands (see Supporting Information) exhibited very low
binding affinities in the micromolar range, and this confirms
the role of a C-terminal acid in the recognition of the BR.
In contrast, modifications of the N-terminal part had little
effect on binding affinities. Acetylation of Nα-amino group in
ligand 16 (IC50 = 120 nM), 20 (IC50 = 140 nM) and 23 (IC50 =
140 nM) retained good binding affinities in the same range as
nonacetylated analogues. Even the inversion of chirality of the
Phe residue from L to D did not reduce their binding affinities
at the BR in 18 (IC50 = 360 nM), 21 (IC50 = 150 nM), and 24
(IC50 = 200 nM). On the other hand, the same modification at
the C-terminus by D-Lys decreased its binding more than 10-
fold in 17 (IC50 = 630 nM). However, thanks to the same basic
property, the substitution of a Lys residue at the C-terminus
with an Arg in ligands 19−24 was tolerated well to maintain the
same high binding affinities as 14, even with various
modifications of the N-terminal position.
NMR Structures. As discussed earlier, the most important
SAR result was that removal of the Arg residue at position 7 did
not affect binding affinities. Therefore, for the comparison of
topographical structures of [des-Arg7]-Dyn A and Dyn A,
ligands 7, Dyn A-(4−11), and 14, [des-Arg7]-Dyn A-(4−11),
were selected and tested for their conformations in membrane-
1
like sodium dodecyl sulfate (SDS) micelles by H 2D-NMR
spectroscopy.17 As G-protein coupled receptors (GPCRs) such
as BRs have their binding sites close to the lipophilic
transmembrane (TM) domains, their ligand−membrane
interactions play an important role in biological activities.18
The membrane can also promote ligand−receptor docking by
stabilizing their secondary structural elements.19,20 Therefore,
for further insight into biological profiles it is crucial to identify
the membrane-bound structures of the ligands in these
circumstances. In nuclear Overhauser effect (NOE) summary
(Figure 2), both ligands showed strong consecutive dαN(i, i +
In order to identify the role of hydrophobic amino acids
neighboring Arg residues, the Leu and Ile residues were
replaced by an Ala residue, and the resulting ligand 25 had
decreased binding affinity (IC50 = 450 nM). Even with the
slight loss of affinity, this result suggests that hydrophobicity
and size of the hydrophobic amino acid residues play a role in
isolating basic amino acid residues. When the two Ala residues
in 25 were replaced by two Nle, respectively, the modification
recovered binding affinity (22, IC50 = 140 nM) to the same
range as 19. Further acetylation did not change the binding
affinity in ligands 23 (IC50 = 140 nM) and 24 (IC50 = 200 nM).
These results clarify SAR at the N-terminal part, where neither
acetylation nor D-amino acid replacement affects binding
affinities of 14, 16, and 18.
Figure 2. NOE summary and temperature coefficient values for 7 and
14. The thickness of the line corresponds approximately to the
intensity of NOE cross peaks.
Heterogeneity of Bradykinin 2 Receptors (B2Rs).
Interestingly, the binding affinity of BK in the rat brain
membrane using [3H]BK (IC50 = 91 nM; log [IC50] = −7.04
0.11) or [3H]DALKD (IC50 = 120 nM; log [IC50] = −6.93
0.08) is lower than that (nanomolar range) reported previously
for the B2R, which is the predominant BR type constitutively
expressed in all tissues.13−16 On the other hand, DALKD,
which has been defined as a bradykinin type 1 receptor (B1R)
selective antagonist, binds to the BRs (IC50 = 130 nM; log
[IC50] = −6.90 0.07 and IC50 = 76 nM; log [IC50] = −7.12
0.11 vs, [3H]BK and [3H]DALKD, respectively) in rat brain
membranes in the same range as BK.11,14 The B2R selective
antagonist, HOE140, had very low affinity (IC50 > 10 000 nM)
against [3H]BK binding in rat brain membranes. These results
1) with a break at the Pro residue due to the imide nature of
the residue; i.e., the Pro residue lacks an amide proton.
Similarly they also displayed sequential dβN(i, i + 1) NOEs
throughout the sequence. Apart from trivial NOEs, consecutive
d
NN(i, i + 1) NOEs and a number of medium range dαN(i, i +
2) NOEs in the middle segment were observed. It is
noteworthy to observe consecutive medium range NOEs for
shorter linear peptides such as ligands 7 and 14. These
observations along with no α-helical signature NOEs such as
dαN(i, i + 3), dαN(i, i + 4) strongly suggest both ligands fold into
consecutive turns or a 310-type helical fold. The observation of
similar NOE patterns in both ligands indicates that the removal
of an Arg residue does not significantly affect the overall
conformation of these ligands in a membrane environment.
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CαH chemical shifts are very sensitive to conformational
structures, and their consecutive negative and positive
deviations from random coil values indicate a helical fold and
β-sheet structures, respectively.21 Chemical shift index (CSI)
plots (Figure 3) for both ligands 7 and 14 displayed a similar
Figure 4. Lowest energy structure and overlay of 10 low energy
structures of 7 (lower) and 14 (upper) from the simulated annealing
molecular dynamics calculations. The hydrogen atoms are not shown
for clarity. The ribbon diagram shows the secondary structure of the
peptide. RMSD between structures is 2.013 Å (1.694 Å for 14) when
all the atoms are considered but is reduced significantly to 0.502 Å
(0.155 Å for 7) when only backbone atoms are considered.
Figure 3. Chemical shift deviation of observed CαH values from the
random coil values (CSI plot). It should be noted here that peptide 14
does not contain the Arg4 residue.
trend. Although it is difficult to conclude simply on the basis of
the CSI plot since 7 and 14 are very short peptides, the
negative deviations from the random coil suggest a conforma-
tional space in the helical domain for both ligands. It could be
either a β-turn structure or a 310-type helical structure.
Simulated annealing molecular dynamics calculations were
carried out in order to obtain three-dimensional structures.
NMR-derived distance and dihedral angle constraints were
applied in the calculations as described in the Supporting
Information. The stereochemical quality for the final minimized
structures was verified by the distribution of per-residue
backbone dihedral angles (Phi and Psi) in the Ramachandran
plot. The distribution of Phi and Psi angles for 14 is
concentrated in only one region for all residues except the C-
terminal Lys residue, suggesting that the conformational
ensemble contains a single family of structures with conforma-
tional flexibility at the C-terminus around the Lys residue
(Figure 4). A closer examination of dihedral values and their
distribution in the Ramachandran plot identifies two consec-
utive type III β-turns (or a short 310-helix: type III β-turns and
310-helix have the same dihedral angle values) at the N-
terminus, and a distorted type 1 β-turn at C-terminal region
centered at Pro7-Lys8 in 7. However, the removal of an Arg
residue in 14 resulted in a single type III β-turn at the N-
terminus and the same distorted type I β-turn at the C-
terminus. The consecutive turn structure at the N-terminus in 7
may not be necessary for the B2R interactions considering the
same range of binding affinities of 7 and 14. Ramachandran
plots of each amino acid for all the final hundred structures of 7
revealed semi rigid conformations for all the residues except for
the Arg, or Arg and Lys residues where the Psi angle deviation
is greater than 30 degrees from the average value. We suggest
that the flexibility of the three basic amino acids in 14 could
contribute to the receptor recognition by adapting the same
conformation as 7.
important role in the receptor interactions and thus does not
affect ligand binding. Also one Arg residue at position 3 of
ligand 14 seems to be sufficient for receptor recognition similar
to the two Arg residues in ligand 7. In Figure 3, the Arg residue
at positions 5 or 6 showed high positive deviations, which
indicate the formation of β-sheet structures. When one more
Arg residue was inserted between positions 6 and 7 in ligand 7,
the resulting ligand 26 retained nearly the same binding affinity
(IC50 = 110 nM) at the receptor as 7 and 14.
Binding Affinities and pH Sensitivity. It has been
previously shown that the optimal binding conditions for the
BR to its endogenous ligands, including BK, is at pH 6.8.22
Thus, we also tested the effect of pH 6.8 on the ability of Dyn A
analogues to bind to the BR in rat brain membranes (Table 2,
Table 2. Binding Affinities of Dyn A Analogues at BRs in Rat
a
Brain at pH 6.8 or 7.4
[3H]DALKD, pH 7.4
[3H]DALKD, pH 6.8
ligand
log [IC50]
IC50 (nM)
log [IC50]
IC50 (nM)
7
−6.86 0.06
−6.78 0.09
−6.71 0.11
−6.96 0.20
−6.93 0.08
−7.12 0.11
140
170
190
110
120
76
−7.16 0.16
−7.67 0.05
−7.16 0.09
−7.13 0.04
−7.01 0.05
−6.98 0.10
69
21
10
14
69
26
74
BK
98
DALKD
100
a
Details described in Table 1.
Figure 5). These initial analyses found that the affinity for all 4
analogues selected was enhanced by 2 to 8 fold. Additional
analyses shown in Table 3 demonstrate that other analogues
also interact with the rat brain BR with affinities in the
nanomolar range. These data validate the optimal binding
conditions for the Dyn A analogues to be at pH 6.8, which is
consistent with the binding conditions previously established
for BRs.
The NMR study showed that the two ligands, 7 and 14, have
the same distorted type 1 β-turn structure at the C-terminus,
which is considered a key region for binding. This explains how
the two ligands bind to the receptor with the same affinity even
with a dissimilarity of structure at the N-terminus. As
mentioned above, the N-terminal part does not play an
Identification of Dyn A Pharmacophore for BRs. On
the basis of the binding affinities of the three ligands 7, 14, and
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considered as a minimum pharmacophore to fulfill the
structural requirement (l = 0, m = 2, one Pro). Together, it
suggests that the BR recognition depends on the electrostatic
interactions between positive charges of the ligand and negative
charges of the BR, and thus to amplify the electrostatic
interactions, the positive charges of the ligand should be
allocated by making a proper topographical structure. This may
be the role of hydrophobic amino acid residues including the
Pro.
Off-Target Screening and Functional Assay. As shown
in Figure 6, the pharmacophore of Dyn A for BRs represents a
simple amphipathic structure. In order to ensure the specificity
of the Dyn A analogues for the BR, lead ligands 14 and 32 were
screened at 43 off-target receptors. While the screen was
limited, the blinded analysis supports the notion that the
ligands’ interaction with BR is specific (see Supporting
Information). We also tested selected Dyn A analogues for
their binding to the three cloned opioid receptors ([3H]-
DAMGO, ([3H]deltorphin, and [3H]U69,593 for the rat mu
opioid receptor (rMOR), human delta opioid receptor
(hDOR), and human kappa opioid receptor (hKOR),
respectively). No ligand showed affinities for the μ and δ
opioid receptors but ligands 10−13, which include a longer N-
terminus part, exhibited low affinities (10, Ki = 560 nM; 11, Ki
= 2400 nM; 13, Ki = 2200 nM) at the κ opioid receptor (n = 2).
However, our lead ligands, 14 and 32, did not exhibit affinity at
the κ opioid receptor (Ki > 10 000 nM, n = 2). These results
show that our strategies have successfully differentiated opioid
and nonopioid functions.
Figure 5. Inhibition of [3H]DALKD binding to rat brain membrane at
pH 6.8. Rat brain membrane was incubated with [3H]DALKD and
increasing concentrations of ligands. Data for each nonlinear
regression analysis were collected from at least two independent
experiments.
Table 3. Amphipathic Dyn A Analogues and Their Binding
a
Affinities for BR in Rat Brain at pH 6.8
BR, [3H]DALKD
IC50
no.
structure
log [IC50]
(nM)
27 H-Leu-Arg-Ile-Arg-Pro-Lys-Leu-Lys
−7.52 0.09
30
-OH
28 H-Nle-Lys-Nle-Lys-Pro-Lys-Nle-Lys-
−7.04 0.10
91
OH
29 H- Lys-Nle-Lys-Pro-Lys-Nle-Lys-OH
30 H-Nle-Lys-Pro-Lys-Nle-Lys-OH
31 H- Lys-Pro-Lys-Nle-Lys-OH
32 H- Arg-Pro-Lys-Leu-Lys-OH
33 H-Pro-Lys-Leu-Lys-OH
−7.07 0.16
−7.11 0.14
−6.64 0.13
−7.24 0.12
−6.68 0.10
85
78
230
58
210
To determine the functional activity of ligand 14, we tested
its ability to stimulate the hydrolysis of [3H]inositol phosphates
in transfected HEK 293 cells expressing the human B2R. This
bioassay measures the production of the intracellular second
messenger, inositol triphosphate, which has been defined as the
primary signal produced upon the activation of B2R by BK.23 In
the HEKB2R cells (Figure 7), BK stimulated the production of
a
Details described in Table 1.
26, it was considered that two neighboring basic amino acid
residues are not necessary for BR recognition, and furthermore
it was observed that in relatively longer length analogues such
as ligand 27, truncation of the Arg residue increased their
binding affinities (log [IC50] = −7.17 0.10, IC50 = 68 nM at
pH 7.4, cf. 13) (Table 3). From the structure of ligand 27, it is
clear that the ligand consists of basic amino acids and
hydrophobic amino acids possessing amphipathic properties
and a Pro residue making a turn structure. This is similar to
other ligands that show good binding affinities to the BR.
Successive truncations of N-terminus and substitutions of a
basic amino acid residue and hydrophobic amino acid residue
with a Lys and Nle did not change their affinities. Analyzing the
structure of all the ligands that showed good binding affinities
at the BRs provides the following insight regarding the
pharmacophore of Dyn A analogues for the rat BR in the
CNS: It requires a basic amino acid residue such as a Lys and
Arg at the C-terminus, combinations of basic amino acid
residues and hydrophobic amino acid residues, and a Pro
residue as a hydrophobic residue to make a turn structure. On
the basis of the SAR results, the pharmacophore for the BR can
be simplified as an amphipathic structure as shown below in
Figure 6. Ligand 32, which retained high binding affinity (IC50
= 58 nM) after successive truncation at the N-terminus, is
Figure 7. Phosphatidylinositol (PI) assay. The effect of test drug on
production of [3H]inositol phosphates was expressed as a ratio of
stimulated over basal activity defined as the amount detected in the
absence of test drug. EC50: concentration at 50% of maximal
stimulation. Data are representative of 3 independent experiments.
[3H]inositol phosphates with an EC50 of 3.9 2.4 nM. Ligand
14, in contrast, showed no stimulation up to a dose of 10 μM.
Also, there was no overt cell death observed in ligand 14 up to
10 μM.
In Vivo Assay: Toxic and Hyperalgesic Effects. The
nontoxic effect of ligand 14 was also demonstrated by in vivo
̈
rotarod and hindlimb tests using naive rats (Figure 8). In the
rotarod test, intrathecal (i.th.) administrations (3 nmol) of 14
retained the similar latencies as vehicle and 10. In the hindlimb
test, i.th. administration of 14 did not show any motor
Figure 6. Pharmacophore of Dyn A for BRs.
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Figure 8. Rotarod (left) and Hindlimb (right) tests by i.th.
̈
administration of 10 or/and 14 in naive rats.
Figure 10. Dose-dependent reversal of thermal hyperalgesia (left,
radiant heat test) and tactile hypersensitivity (right, von Frey test)
using varying doses of 14 (i.th.) in L5/L6 SNL-operated male SD rats.
Statistical significance was determined by 95% confidence interval (*P
< 0.05, **P < 0.01 vs vehicle; n ≥ 6).
impairments at a high dose (30 nmol), but 10 (25 nmol, i.th)
induced paralysis that occurred within a few minutes post-
injection and lasted for 30 to 40 min. In addition, pretreatment
of 14 (30 nmol, i.th.) completely blocked ligand 10 (25 nmol,
i.th.)-induced paralysis, suggesting that ligand 14 may inhibit
Dyn A’s excitatory effects in vivo.
BRs as a therapeutic target for neuropathic pain. 14 is a
structure derived from Dyn A’s interaction at the BRs. Together
with our previous studies characterizing the role of spinal Dyn
A in neuropathic pain, the results of the present study not only
support a structural basis for Dyn A’s actions at the spinal BRs
to promote pain, but also show that it is possible to develop
antagonists like 14 that target CNS BRs for therapy.
To evaluate the hyperalgesic effects of 10 and 14, radiant
̈
heat and von Frey filaments tests were performed in naive rats
(Figure 9). Paw withdrawal latencies and thresholds of all
Peripheral Effects: Paw Edema and Plasma Extrava-
sation. Local administration of 14 had no effect on BK-
induced paw edema and plasma extravasation (Figure 11).
Figure 9. Effects of ligand 10 or/and 14 on thermal hyperalgesia (left,
radiant heat test) and tactile hypersensitivity (right, von Frey test) 2 h
̈
after i.th. administration in naive rats. Ligand 10 decreased thermal
latency and tactile thresholds after i.th. administration. Ligand 14
blocked ligand 10-induced thermal hyperalgesia and tactile hyper-
sensitivity after coadministration. Statistical significance was deter-
mined by 95% confidence interval (*P < 0.05, **P < 0.01, ***P <
0.001 vs vehicle; n ≥ 6).
Figure 11. Effect of i.pl. injection of BK, given alone or in combination
with HOE140 or 14 on paw edema (left) and plasma extravasation
(right). Values shown represent the differences between volumes
(mL) of vehicle and drug combination (BK/vehicle, BK/HOE140,
and BK/14) paws (left, *P < 0.05 vs vehicle) and the difference
absorbance (percent) of two hind paws (one vehicle only, the other
BK/vehicle, BK/HOE140, and BK/14) at 620 nm by the content of
Evans Blue dye (right).
animals were 18.7
0.6 s and 15
0 g, respectively. As
expected, i.th. administration (3 nmol/5 μL) of ligand 10
reduced these values to 11.4 1.0 s and 7.3 1.0 g in 1 h,
demonstrating thermal hyperalgesia (paw withdrawal latency:
area under curve (AUC) SEM = 2067 137 in 2 h) and
mechanical hypersensitivity (paw withdrawal threshold: AUC
SEM = 1074 68 in 2 h). In contrast, administration of 14
increased latency of paw withdrawal from a heat source when
compared with vehicle control, an effect that is defined as
analgesic (Figure 9, left panel). 14 did not alter mechanical
sensitivity (Figure 9, right panel). Coadministration of 10 and
14 prevented the hyperlalgegia induced by 10 when given
alone. These data suggest that ligand 14 can effectively block
ligand 10, to induce abnormal pain states in vivo, presumably
mediated by spinal BRs as an antagonist.
Thermal and tactile hypersensitivities were also assessed in
rats that had received unilateral L5/L6 spinal nerve ligation
(SNL) injury (Figure 10). Before injury, paw withdrawal
latencies and thresholds of all animals were between 19.3−20.9
s and 15 g, respectively. SNL injury reduced these values to
8.6−11.3 s and 2.2−2.9 g, respectively, indicating abnormal
sensitivities to thermal and tactile stimuli in the injured hind
paw. Injured animals treated with ligand 14 showed
antihyperalgesic effects in both tests in a dose dependent
manner in 2 h. The greatest antihyperalgesic effects occurred at
the highest dose of 3 nmol/10 μL in both tests.
Intraplantar (i.pl.) injection of BK (10 nmol) induced robust
paw swelling and edema that peaked at 30 min postdose. The
paw volume difference between the ipsi- and contralateral hind
paws increased from 0.03 0.09 mL (baseline) to 0.43 0.08,
0.35
0.08 and 0.23
0.07 mL at 30, 60, and 90 min
postdose, respectively. As expected, coadministration of a B2R
antagonist, HOE140 (10 nmol, i.pl.), reduced the BK-induced
paw volume increase to 0.22 0.03 mL at 30 min postdose by
50%. In contrast, 14 (10 nmol, i.pl.) had no effect on paw
edema with the same degree of paw volume increase (0.42
0.07 mL at 30 min postdose) as that of BK alone. The same
trend of effects was also observed in a plasma extravasation test.
Coadministration of BK with 14 did not affect the BK-induced
plasma extravasation, although HOE140 at this concentration
was effective in reducing the extravasation on its own. This
result suggests that our lead ligand 14 does not inhibit BK’s
action at the peripheral BRs, and therefore there will be little
impact on the BK’s cardiovascular function at the region. Paw
edema and plasma extravasation tests show the possibility of
ligand 14 as a drug candidate to block hyperalgesia in chronic
pain states without serious cardiovascular side effects.
The ability of 14 to reverse abnormal pain states in this
model of neuropathy underscores the potential role of spinal
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XWINNMR (Bruker, Inc.) and FELIX2000 (Accelrys, Inc., San Diego,
CA). Mixing times for TOCSY and NOESY spectrum were 60 and
300 ms, respectively. All experiments were 750 increments in t1, 16, or
32 scans each, 1.5 s relaxation delay, size 2 or 4K, and the spectral
processing was with shifted sine bell window multiplications in both
dimensions. The water suppression was achieved by using WATER-
GATE pulse sequence. Coupling constants (3JαH‑NH) were measured
from double quantum filtered correlation spectroscopy (DQF-COSY)
experiments.
Structure Calculation Methods. Distance constraints for the
structure calculation were obtained from integral volumes of the
NOESY peaks. The NOE integral volumes were classified into strong,
medium, and weak with 3.0, 4.0, and 5.0 Å as upper bound distance.
Molecular dynamics simulation was done with the INSIGHT/
DISCOVER package (Accelrys, Inc., San Diego, CA) with consistent
valency force field (CVFF). All calculations were done in vacuo. A
distance dependent dielectric constant (2.5r where r is the distance in
Å) was used. All peptide bonds were constrained to trans
conformation by a 100 kcal/mol energetic penalty. Distance restraints
with a force constant of 25 kcal/mol were applied in the form of a flat-
bottom potential well with a common lower bound of 1.8 Å and an
upper bound of 3.0, 4.0, and 5.0 Å, respectively, in accordance with
observed weak, moderate, or strong NOE intensities. Only the
distance restraints from inter-residue NOEs were included in the
calculation. Dihedral angle restraints based on CαH CSI were imposed
on the residues displaying negative deviation. Thus, for a CSI > −0.10
ppm, the ϕ and ψ restraints were in the range −90° to −30° and −60°
to 0°, respectively, while for a CSI ≤ −0.10 ppm, the corresponding
ranges were −150° to −30° for ϕ and −90° to 150° for ψ.
Radioligand Competition Binding Assays. Binding affinities of
Dyn A analogues at the BRs were determined by radioligand
competition analysis using [3H]DALKD or [3H]BK in rat brain
membranes or in transfected HEK 293 cells expressing the human
B2R. Crude rat brain membranes were pelleted and resuspended in 50
mM Tris buffer containing 50 μg/mL bacitracin, 10 μM captopril, 100
μM PMSF, and 5 mg/mL bovine serum albumin (BSA). Ten
concentrations of a test compound were each incubated with 50 μg of
membranes and [3H]DALKD (1 nM, 76.0 Ci/mmol) or [3H]BK (1
nM, 85.4 Ci/mmol) at 25 °C for 2 h. Nonspecific binding was defined
by that in the presence of 10 μM KD in all assays. Reactions were
terminated by rapid filtration through Whatman GF/B filters
presoaked in 1% polyethylenimine, followed with four washes of 2
mL each of cold saline. Radioactivity was determined by liquid
scintillation counting in a Beckman LS5000 TD. Data were analyzed
by nonlinear least-squares analysis using GraphPad Prism 4.
Logarithmic values were determined from nonlinear regression
analysis of data collected from at least two independent experiments.
Functional Assays. Phosphatidylinositol (PI) assays were
performed using [3H]inositol phosphates tracer growth media (myo
tritium inositol) in poly-D-Lys-coated cell culture plates, and the
method used to measure the accumulation of [3H]inositol phosphates
was according to that described earlier with additional wash with
water, 5 mM sodium tetraborate/60 mM sodium formate before
elution with 0.2 mM ammonium formate/0.1 M formic acid through
Biorad AG 1-X8 Resin.25
In Vivo Assays. The experiments were carried out using nonfasted
male Sprague−Dawley rats (250−300g; Harlan; Indianapolis, IN) kept
in a room controlled for temperature (22 2 °C) and illumination
(12 h on and 12 h off). All experiments were performed under a
protocol approved by Institutional Animal Care and Use Committee
(IACUC) of the University of Arizona, and in accordance with policies
and guidelines for the care and use of laboratory animals as adopted by
International Association for the Study of Pain (IASP) and the
National Institutes of Health (NIH). Intrathecal (i.th.) catheterization
was performed under ketamine/xylazine (80/12 mg/kg, i.p.)
anesthesia. Some groups of rats were implanted with i.th. catheters
(polyethylene 10, 7.8 cm) through atlanto-occipital membrane
extended to the level of the lumbar spinal cord for drug administration.
Animals were allowed to recover for 7 days. L5/L6 spinal nerve ligation
(SNL) injury was induced as described by Chung and colleagues.26
CONCLUSIONS
■
We have discovered a lead ligand 14 for CNS BRs and the
human B2R through a systematic SAR study on Dyn A. Further
modification of the lead ligand asserted the key structural
features of the BR pharmacophore: amphipathicity. NMR study
of two ligands 7 (Dyn A-(4−11)) and 14 ([des-Arg7]-Dyn A-
(4−11)) showed the same distorted type 1 β-turn structure at
the C-terminus, a key region for the binding. Considering the
pH dependence of the ligand’s binding affinities, the BR
recognitions seem to correlate with the electrostatic inter-
actions between Dyn and BRs and thus for the optimization of
their interactions, allocation of positive charges in ligands may
be critical.
Ligand 14 inhibited ligand 10-induced thermal and
mechanical hyperalgesia in naive animals. In nerve injured
̈
animals, ligand 14 blocked thermal and mechanical hyper-
sensitivity in a dose-dependent manner. At high dose, ligand 10
showed serious motor impairment in vivo. In contrast, 14 did
not show any toxicity, and motor impairment and furthermore
blocked ligand 10-induced paralysis in vivo. These in vivo
activities of ligand 14 may be localized in the CNS, since there
is no peripheral activity of ligand 14 shown in the paw edema
and plasma extravasation tests. As we predicted, ligand 14
inhibits the actions of endogenous Dyn A’s fragment, Dyn A-
(2−13), resulting in antihyperalgesic effects in the CNS. This
work further supports our hypothesis that the actions of spinal
Dyn A at BRs underlie pathological pain states. These results
also demonstrate that ligand 14 has the therapeutic potential
for chronic pain states via a novel mechanism of BRs in the
CNS.
EXPERIMENTAL SECTION
■
Synthesis. Dyn A analogues were synthesized by standard solid
phase peptide synthesis using Nα-Fmoc-chemistry (Fmoc = 9-
fluorenylcarboxy) on amino acid preloaded Wang resin (100−200
mesh, Novabiochem) in high yields (overall yield >40%), except for
the analogues with a Pro residue at the C-terminus. Because of serious
side reactions of Pro on the resin, Chlorotrityl resin (200−400 mesh,
1% DVB, Novabiochem) was used as an alternative.24 Coupling was
performed using 3 equiv 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethy-
laminium hexafluorophosphate (HBTU)/3 equiv N-hydroxybenzo-
triazole (HOBt)/6 equiv diisopropylethylamine (DIPEA) in N,N-
dimethylformamide (DMF) for 1 h at rt, and Nα-Fmoc-group was
deprotected by 20% piperidine in DMF for 20 min at rt. In most cases,
crude peptides were obtained by cleavage using a 95% trifluoroacetic
acid (TFA) solution containing 2.5% triisopropylsilane (TIS) and
2.5% water for 3 h in high purity (70−90%) and could be isolated with
more than 97% purity by preparative reverse phase high performance
liquid chromatography (RP-HPLC) using gradient (10−40% acetoni-
trile in water containing 0.1% TFA in 15 min) in a short time (<15
min) because of their hydrophilic characters (refer to aLogPs in
Supporting Information). The purified Dyn A analogues were
validated by analytical RP-HPLC and high resolution mass spectros-
copy in positive ion mode.
NMR Spectroscopy Methods. NMR studies of ligands 7 and 14
in SDS micelles were performed on a Bruker DRX600 (600 MHz) at
25 °C and at pH 5.5. Peptide concentrations for the NMR experiments
were 5.8 mM and 6.1 mM for 7 and 14, respectively. The micelle
samples were prepared by dissolving the peptides and 50 equiv of
perdeuterated SDS in 0.6 mL of acetate buffer (10 mM) containing
10% D2O. The pH of each sample was adjusted to 5.5 by using DCl or
NaOD as necessary. Deuterated 3-(trimethylsilyl)propionic acid
(TSP) was added as an internal standard for referencing. Two-
dimensional nuclear Overhauser enhancement spectroscopy (NOESY)
and total correlated spectroscopy (TOCSY) (Supporting Information)
were acquired using standard pulse sequences and processed using
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Rotarod latencies, paw-withdrawal latencies, and paw-withdrawal
thresholds were calculated and expressed as the mean AUC SEM
in Graph Pad Prism 6 (GraphPad Software, La Jolla, CA). One-way
analysis of variance (ANOVA) was performed in FlashCalc (University
of Arizona, Tucson), and statistical significance achieved when p ≤
0.05.
Rotarod Test. Rats were trained to walk on an automated rotating
rod (8 rev/min, Rotamex 4/8, Columbus Instrument, Columbus, OH)
for maximal cutoff time of 180s.27 Baseline values were recorded for
each animal. Compounds were administered (i.th.) and assessment
occurred every 20 min for the first 120 min. The rotarod latencies
were recorded at each time point.
Motor Function and Paralysis. Paralysis was evaluated as
flaccidity of the hind limbs following i.th. injection as previously
reported by Spampinato and colleagues.28 Drugs were injected in a
volume of 5 μL, followed by a 1 μL air bubble and a 9 μL saline flush.
Flaccidity of the hind limbs was measured for 2 h after drug
administration.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Jose Juan Ortiz Navarro, Ann Ngyuene, Alyssa Peake,
Alice Cai, and Robert Kupp for their help in synthesis and
bioassay of ligands. Off-target receptor screening was
generously provided by the National Institute of Mental
Health’s Psychoactive Drug Screening Program, Contract #
HHSN-271-2008-025C (NIMH PDSP). The NIMH PDSP is
directed by Bryan L. Roth at the University of North Carolina
at Chapel Hill and Project Officer Jamie Driscol at NIMH,
Bethesda MD, USA. This work was supported by the U.S.
Public Health Services, NIH, and NIDA (P01DA006284).
Thermal Hypersensitivity Test (Radiant Heat). Thermal
hypersensitivity was assessed using the rat plantar test (Ugo Basile,
Italy) as described earlier.27 Rats were allowed to acclimate within
Plexiglas enclosures on a clear glass plate. A mobile radiant heat source
(halogen bulb coupled to an infrared filter) was located under the glass
plate and focused onto the hind paw. Paw withdrawal latencies were
recorded in seconds. An automatic cut off point of 33 s was set to
prevent tissue damage. The apparatus was calibrated to give a paw
withdrawal latency of approximately 20 s on the uninjured paw. The
radiant heat source was activated with a timer and focused onto the
plantar surface of the hind paw.
Tactile Hypersensitivity Test (von Frey, Innocuous). The
assessment of mechanical hypersensitivity consisted of measuring the
withdrawal threshold of the paw ipsilateral to the site of nerve injury in
response to probing with a series of calibrated von Frey filaments.27
Each filament was applied perpendicularly to the plantar surface of the
ligated paw of rats kept in suspended wire-mesh cages. Measurements
were taken both before and after administration of drug or vehicle.
The paw withdrawal threshold was determined by sequentially
increasing and decreasing the stimulus strength (“up−down” method)
analyzed using a Dixon nonparametric test16.
Measurement of Rat Paw Edema and Plasma Extravasation.
Under ketamine/xylazine (80/12 mg/kg, i.p.) anesthesia animals
received an injection of Evans Blue (30 mg/mL/kg, i.v.) via tail vein,
and baseline paw volume for both hind paws was measured by use of a
plethysmometer (Ugo Basile). Animals then received 0.1 mL i.pl.
injections in one hind paw of normal saline (0.9% NaCl) containing
BK either alone or mixed with HOE140 or 14 (10 nmol/paw each).
The contralateral paw received 0.1 mL of saline and was used as a
control. Edema was measured at several 30, 60, and 90 min after i.pl.
injections and expressed in mL as the difference between the test and
control paws. Three hours after BK injections, animals were sacrificed,
and patches (10 × 5 mm) of the dorsal skin from both hind paws were
collected. The skin patches were then incubated separately in
Eppendorf tubes containing 1.8 mL of formamide at 60 °C water
bath for 24 h to extract the dye. The tissue extraction was then
centrifuged at 15 000 rpm for 15 min, and the supernatant was
pipetted to a 96 well plate as triplicates and the absorbance was
determined at 620 nm. The difference of the mean absorbance
between the two hind paws of each rat was used to compare the
degree of plasma extravasation in different treatment groups.
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ASSOCIATED CONTENT
* Supporting Information
Analytical data, binding affinity data, NMR spectroscopy. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
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AUTHOR INFORMATION
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hruby@email.arizona.edu
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