Tuned-affinity bivalent ligands for the characterization of opioid receptor heteromers

Article abstract of DOI:10.1021/ml300083p

Opioid receptors, including the μ- and δ-opioid receptors (MOR and DOR), are important targets for the treatment of pain. Although there is mounting evidence that these receptors form heteromers, the functional role of the MOR/DOR heteromer remains unresolved. We have designed and synthesized bivalent ligands as tools to elucidate the functional role of the MOR/DOR heteromer. Our ligands (L2 and L4) are comprised of a compound with low affinity at the DOR tethered to a compound with high affinity at the MOR, with the goal of producing ligands with "tuned affinity" at MOR/DOR heteromers as compared to DOR homomers. Here, we show that both L2 and L4 demonstrate enhanced affinity at MOR/DOR heteromers as compared to DOR homomers, thereby providing unique pharmacological tools to dissect the role of the MOR/DOR heteromer in pain.

Full text of DOI:10.1021/ml300083p

Letter
pubs.acs.org/acsmedchemlett
Tuned-Affinity Bivalent Ligands for the Characterization of Opioid
Receptor Heteromers
Jessica H. Harvey,†,‡ Darcie H. Long,† Pamela M. England,‡ and Jennifer L. Whistler*,†,‡
†Ernest Gallo Clinic and Research Center, 5858 Horton Street, Suite 200, Emeryville, California 94608, United States
‡University of California, San Francisco, Genentech Hall, 600 16th Street, San Francisco, California 94158, United States
S
* Supporting Information
ABSTRACT: Opioid receptors, including the μ- and δ-opioid
receptors (MOR and DOR), are important targets for the
treatment of pain. Although there is mounting evidence that
these receptors form heteromers, the functional role of the
MOR/DOR heteromer remains unresolved. We have designed
and synthesized bivalent ligands as tools to elucidate the
functional role of the MOR/DOR heteromer. Our ligands (L2
and L4) are comprised of a compound with low affinity at the
DOR tethered to a compound with high affinity at the MOR,
with the goal of producing ligands with “tuned affinity” at MOR/
DOR heteromers as compared to DOR homomers. Here, we
show that both L2 and L4 demonstrate enhanced affinity at
MOR/DOR heteromers as compared to DOR homomers,
thereby providing unique pharmacological tools to dissect the
role of the MOR/DOR heteromer in pain.
KEYWORDS: bivalent ligand, opioid receptors, heteromers, chronic pain
pioid receptors are important targets for the treatment of
Moreover, characterizing the affinity of these bivalent ligands
Oacute and chronic pain, but side effects, including at heteromers has not been possible, since cells coexpressing
MOR and DOR contain not only MOR/DOR heteromers but
also DOR (and MOR) homomers, all of which have high
affinity for both pharmacophores in the previously reported
bivalent ligands.
respiratory suppression, constipation, analgesic tolerance, and
dependence, limit their utility. The three types of opioid
receptor, μ-opioid receptor (MOR), δ-opioid receptor (DOR),
and κ-opioid receptor (KOR), associate with each other both in
vitro and in vivo, producing heteromeric receptors with novel
properties.1−4 Additionally, the crystal structure of the MOR
indicates that it is a dimer.5 Opioid receptor heteromers may
represent novel therapeutic targets, but their functions have not
been fully elucidated, in part due to a lack of heteromer-specific
ligands. There has been particular interest in ligands for the
MOR/DOR dimer that show agonism at the MOR and
antagonism at the DOR and/or the MOR/DOR heteromer,
since antagonism, knock down, and knock out of the DOR
improve the side effect profiles of classic opioid analgesics.6−9
We have designed a series of “tuned-affinity” bivalent ligands
that specifically target the MOR/DOR heteromer to provide
tools to evaluate its functional role. The design, synthesis, and
binding affinity of these ligands are reported here. Although
many bivalent opioid receptor ligands have been characterized
previously,10−13 none were designed to show unique activity on
heteromeric (MOR/DOR) versus homomeric (DOR/DOR or
MOR/MOR) receptors. Specifically, previous bivalent opioid
receptor ligands were comprised of two pharmacophores, each
with high affinity for both homomeric and heteromeric receptor
complexes. Consequently, evaluating the role of the MOR/
DOR heteromer was hindered by a lack of specificity.
Our ligands (L2 and L4, Figure 1) were designed to have
novel or “tuned” affinities at MOR/DOR heteromers as
compared to DOR homomers. Discrimination between DOR
and MOR/DOR was accomplished by tethering a low affinity
DOR ligand to a high affinity MOR ligand. These ligands were
predicted to have a low affinity at DOR homomers and high
affinity at MOR/DOR heteromers where binding of the high
affinity MOR pharmacophore would increase the effective
molarity of the tethered, low affinity DOR ligandeffectively
“tuning” its affinity in favor of the heteromer.
The ligands presented here are comprised of a high affinity
MOR compound, either an agonist or an antagonist, tethered
to a low affinity DOR compound, again, either an agonist or an
antagonist. This approach was used to determine whether
binding of agonist or antagonist at the MOR in the MOR/
DOR heteromer is more effective for tuning the affinity of the
DOR ligand. One bivalent ligand, L2, is comprised of the high
affinity MOR agonist oxymorphone and the low affinity DOR
Received: April 24, 2012
Accepted: June 6, 2012
Published: July 18, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Synthesis of L2
Figure 1. Tuned-affinity bivalent ligands.
antagonist ENTI.14 The second, L4, features the high affinity
MOR antagonist, naltrexone, and the low affinity DOR agonist,
DM-SNC8015 (Figure 1). The linker used here has been used
extensively in previous bivalent ligands, where its composition
and length were optimized for the heteromer.10,16 Controls for
these ligands are shown in Figure 2. Linker-attached controls
were used when available.
L2 and L4 were prepared from their component fragments
(antagonist, linker, and agonist) as shown in Schemes 1 and 2.
For L2, Fisher indole synthesis of naltrexone (8) with 2-
nitrophenyl hydrazine10 followed by alkylation of the 3-phenol,
reduction of the 7′-nitro group, and acylation of the resulting
amine [ENTI (4)] with diglycolic anhydride produced the
antagonist portion of the molecule (13). Next, the linker (15)10
was synthesized via mono-Boc protection of cadaverine (14).
The agonist synthesis is a known sequence10 involving the
selective reduction of oxymorphone (3),17 attachment of
diglycolic anhydride, peptide coupling with monoboc cadaver-
ine, and deprotection to yield the agonist and linker portion of
the molecule (18). Finally, the agonist linker (18) and
antagonist (13) were combined in a peptide coupling to give
L2 (1).
a
(a) (2-Nitrophenyl)hydrazine, HCl, CH3CO2H, 90 °C, 2 h, 50%. (b)
(2-Bromoethyl)benzene, K2CO3, DMF, 90 °C, 24 h, 92%. (c) 5% Pd/
C, MeOH, H2, 40 psi, 2 h, 92%. (d) Diglycolic anhydride, THF, 1 h,
95%. (e) tert-Butyl phenyl carbonate, EtOH, 65 °C, 16 h, 53%. (f) (i)
H2O, Na2CO3, 1 h; (ii) benzene, benzylamine, p-toluenesulfonic acid,
10 h; (iii) EtOH, NaBH4, 3 h, H2, 40 psi, 5% Pd/C, 72 h, 72%. (g)
Diglycolic anhydride, THF, 1 h, 33%. (h) Compound 15, DIC, HOBt,
DIEA, THF, 60 °C, 2 h, 30%. (i) 4 N HCl, dioxane, 2 h, 99%. (j)
Compound 13, DIC, HOBt, DIEA, THF, 60 °C, 2 h, 27%.
Figure 2. Monovalent control ligands for L2 and L4.
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ACS Medicinal Chemistry Letters
Letter
a
in cells expressing only DOR and in cells expressing both MOR
and DOR (which will contain a mixture of MOR homomers,
DOR homomers, and MOR/DOR heteromers). In these
conditions, both L2 and L4 show enhanced affinity at MOR/
DOR as compared to DOR. L4 shows a significant (10×) shift
in affinity (pKi = 8.60 at MOR/DOR as compared to pKi = 7.70
at DOR) (Table 1). Importantly, the L4 monovalent controls,
Scheme 2. Synthesis of L4
Table 1. Competition Binding
3H DPDPE (20 nM) pKi SEM
MOR/DOR
DOR
L2
7.34 0.06
6.29 0.10
7.46 0.09
8.26 0.15
8.60 0.07
5.78 0.14
8.11 0.13
7.79 0.11
6.69 0.18
6.14 0.09
7.04 0.21
8.58 0.23
7.70 0.11
5.54 0.20
7.78 0.23
7.51 0.10
Oxy
ENTI
NTI
L4
SNC-19
SNC80
NTX-19
3H DAMGO (10 nM) pKi SEM
MOR
MOR/DOR
L2
8.01 0.05
8.30 0.03
7.65 0.06
6.92 0.16
7.78 0.12
8.21 0.01
8.33 0.07
6.90 0.11
6.94 0.08
8.46 0.14
>9
Oxy
MA-19
ENTI
NTI
a
(a) TFAA, Br2, 0 °C, 1.5 h, 77%. (b) Bis(pinacolato)diboron, AcOK,
L4
>9
PdCl2(dppf), DMF, 80 °C, 16 h, 35%. (c) NBS, AIBN, CCl4, reflux, 7
h, 99%. (d) P(OMe)3, reflux, 5 h, 99%. (e) tert-Butyl 4-oxopiperidine-
1-carboxylate, LDA, THF, 12 h, −78 °C → rt, 32%. (f) Br2, K2CO3,
CH2Cl2, 0 °C → rt, 1.5 h, 78%. (g) NaOH, MeOH, 40 °C, 3 h, 87%.
(h) DMAP, SOCl2, CH2Cl2, Et2NH, −20 °C → rt, 12 h, 68%. (i)
Compound 21, K2CO3, THF/H2O, Pd(PPh3)2Cl2, 80 °C, 0.5 h, 41%.
(j) NaOH, H2O, MeOH, 12 h, 99%. (k) diglycolic anhydride, 0 °C →
rt, 12 h, 93%. (l) Compound 15, EDCI, HOBt, DMF, 12 h, 82%. (m)
HCl/EtOAc, 12 h, 99%. (n) Compound 27, EDCI, HOBt, DIEA,
DMF, 12 h, 44%. (o) HCl/EtOAC, iPrOH, 10 h, 99%.
SNC-19
NTX-19
naltrexone
6.26 0.15
9.49 0.11
9.19 0.23
5.92 0.07
8.38 0.04
9.06 0.32
NTX-19 and SNC-19, do not show this increase in affinity for
the heteromer. L2 also shows a change in affinity, but it did not
reach statistical significance (pKi = 7.34 at MOR/DOR and pKi
= 6.69 at DOR). Neither oxymorphone nor naltrindole
hydrochloride (NTI) show a change between MOR/DOR
and DOR. However, ENTI, the L2 low-affinity DOR control,
shows a nonsignificant shift in affinity similar to L2. These data
suggest that tuning may be achieved more productively by
binding of an antagonist.
The synthesis of L4 required dimethyl amino diary-
lmethylenepiperidine (26), which was made via Suzuki
coupling. The first coupling partner synthesis began with 3,5-
dimethylaniline (19), which was protected, brominated, and
transformed into the boronic ester (21). Next, methyl-4-
methylbenzoate (22) was subjected to radical bromination and
heating with triphenyl phosphite to give 23.15 Subsequent
Wittig−Horner olefination with N-tert-butyoxy-carbonyl-4-
piperidone gave 24, which was brominated and transformed
to the amide to yield the second coupling partner (25). Suzuki
coupling of 21 with 25 followed by deprotection and reaction
with diglycolic anhydride produced the agonist portion of L4
(27). Synthesis of the antagonist portion began with 6β-
naltrexamine (28),17 which was coupled to mono-Boc-
cadaverine (15), deprotected and coupled to 27.10 A final
Boc deprotection gave L4 (2).
We next examined whether these ligands showed tuned
affinity at MOR/DOR heteromers as compared to DOR
homomers. L2, L4, and their controls were analyzed in whole
cell radioligand competition binding assays to evaluate affinity
at DOR, MOR/DOR, and MOR. For both ligands, we expected
high affinity at MOR, low affinity at DOR, and increased affinity
at MOR/DOR as compared to DOR.
We also assessed the affinity of L2 and L4 at the MOR in
cells expressing MOR alone or both MOR and DOR using the
MOR radioligand H [DAla2,NMe-Phe4,Gly-ol5]-enkephalin
3
(DAMGO), where we do not expect tuned affinity. Indeed, L4
shows high affinity in both cell lines (pKi > 9 nM) (Table 1).
Naltrexone shows high affinity that is unchanged between the
cell lines, and naltrexone with the linker attached, NTX-19, has
a similar affinity to naltrexone on MOR but a decreased affinity
at MOR/DOR. SNC-19 shows low affinity at MOR, as
expected. L2 also shows a high “untuned” affinity in both cell
lines, similar to that of its high affinity MOR component,
oxymorphone (Table 1). Attachment of the linker to
oxymorphone decreased its affinity at MOR, as shown by
MA-19.10 However, while the affinity of MA-19 decreased on
MOR/DOR as compared to MOR cells, L2 shows the opposite
trend, indicating that tethering these two compounds has a
beneficial effect on affinity that overcomes adverse effects of the
linker. Finally, NTI, predominantly a DOR ligand, shows higher
affinity at MOR/DOR than at MOR.
We first evaluated affinity at DOR in competition with the
DOR radioligand H [D-Pen2,D-Pen5]-enkephalin (DPDPE)
Because the MOR/DOR cell line expresses a mixture of
MOR homomers, DOR homomers, and MOR/DOR hetero-
3
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mers, the observed affinity of L2 and L4 is a mixture of the
affinity at the three different receptor types. We evaluated the
relative amount of heteromers and homomers expressed on the
surface in our cell line by serial coimmunoprecipitation (Figure
3). The cells express substantially more DOR homomers than
We predicted that L2 and L4 would show low affinity at
DOR alone and enhanced affinity at MOR/DOR, and this is, in
fact, what we see. With L4, we see a significant change in
affinity that occurs only for the bivalent ligand and not for the
controls. Thus, we can postulate that L4 is bridging a
heteromer and that tethering is important for its enhanced
affinity at MOR/DOR. With L2, the change in affinity is not
significant, and ENTI also shows enhanced affinity at MOR/
DOR. Consequently, we cannot rule out that the shift in L2 is
due to a shift in ENTI affinity, rather than receptor bridging.
Intriguingly, ENTI shows equal affinity for both MOR and
DOR homomers while retaining a higher affinity for the
heteromer, suggesting that a nonbivalent ligand could itself
show a preference for a heteromer.
It is noteworthy that our results were obtained in a cell line
with a vastly greater population of DORs as compared to
MOR/DORs (Figure 3), reflecting the fact that DORs have a
higher affinity for one another than for the MOR.28
Consequently, we are likely greatly underestimating the change
in affinity of L2 and L4 on the heteromer.
Figure 3. Relative expression of MOR, DOR, and MOR/DOR.
HEK293 cells expressing both FLAG-MOR and HA-DOR were
biotinylated to label surface receptors, and the receptors were serially
immunoprecipitated to separate MOR, DOR, and MOR/DOR
heteromers.
MOR/DOR heteromers and few MOR homomers. Con-
sequently, we are likely significantly underestimating the shifts
in affinity for L2 and L4.
In conclusion, we have successfully designed and synthesized
compounds that specifically target the MOR/DOR heteromer.
L2 and L4 represent a novel class of bivalent ligand that
interacts differently with the heteromer, even in the presence of
its constituent monomers. These ligands, L4 in particular, can
be used as tools to characterize the heteromer as a novel
therapeutic target.
MOR/DOR heteromers may be important targets for pain
management, especially under chronic conditions such as stress,
alcoholism, or long-term opiate use.4,18 However, existing
bivalent ligands have equivalent affinity for the heteromer and
its constituent monomers, preventing validation of this target.
We developed our “tuned-affinity” ligands as tools to
distinguish between the MOR/DOR heteromer and the DOR
homomers.
ASSOCIATED CONTENT
* Supporting Information
■
S
We are specifically interested in the MOR/DOR heteromer
because of the untapped potential of DOR receptors in
controlling pain.19 The role of DORs is not fully understood, in
part because of conflicting evidence regarding their function.
Under some conditions, activation of DORs alleviates pain,
while in others, the DORs exacerbate pain or oppose analgesia.
For example, in acute conditions, DOR agonists are poor
analgesics for thermal pain20,21 and good analgesics in
mechanical nociception.22 However, in chronic conditions,
DOR agonists can become potent analgesics.23,24 Yet, in
morphine-tolerant animals, DOR antagonists enhance anti-
nociception.25 Additionally, disruption or knockout of the DOR
gene diminishes morphine tolerance.6,26,27 These findings have
led to the design of mixed and bivalent MOR agonist/DOR
antagonist ligands as potential analgesics with reduced side
effects. One such bivalent ligand, MDAN,10 designed to target a
MOR/DOR heteromer, showed analgesia with reduced
tolerance and dependence and did not support conditioned
place preference (CPP) in mice.16 MDAN is comprised of a
high affinity MOR agonist (oxymorphone) tethered to a high
affinity DOR antagonist (naltrindole) and, thus, has high
affinity at MOR homomers, DOR homomers, and the MOR/
DOR heteromer. Therefore, it is not possible to determine if it
is MDANs specific activity at the heteromer, nor what type of
activity it is (agonism or antagonism) that is responsible for its
biological action in vivo.
Synthetic procedures (for ENTI, L2, L4, SNC-19, and NTX-
19) and biological assay details. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 510.985.3127. E-mail: JenniferW@gallo.ucsf.edu.
■
Author Contributions
J.H.H. designed experiments, conducted experiments, and
wrote the paper; D.H.L. conducted experiments; P.E. designed
experiments; and J.L.W. designed experiments and wrote the
paper.
Funding
J.H.H. is supported by grants from the Program for
Breakthrough Biomedical Research (PBBR) and the A. P.
Giannini foundation. This research was supported by NIH R21
DA031574, R01 R01AA020401 and funds provided by the
State of California for substance abuse research through UCSF.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
P. S. Portoghese kindly provided MA-19. We thank Richard M.
van Rijn, Johan Enquist, and Laura Milan-Lobo for training,
technical assistance, and general support. Nanosyn (Menlo
Park, CA) carried out the synthesis of L2. Huaoxi Huang,
Chunchao Yue, and Xin Li (Chempartner, Shanghai, China)
assisted in the synthesis of L4.
Indeed, it remains unclear whether heteromer activation
increases or decreases pain perception, whether its actions are
specific for thermal or mechanical nociception, and if its activity
opposes that of the DOR homomer. Finally, when both an
agonist and an antagonist are bound to a heteromeric receptor,
it remains unclear which signal predominates (i.e., “who wins”).
We designed our tuned-affinity ligands to address these
questions.
ABBREVIATIONS
MOR, μ-opioid receptor; DOR, δ-opioid receptor; KOR, κ-
opioid receptor; HEK, human embryonic kidney; DPDPE, [D-
■
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(16) Lenard, N. R.; Daniels, D. J.; Portoghese, P. S.; Roerig, S. C.
Absence of conditioned place preference or reinstatement with
bivalent ligands containing mu-opioid receptor agonist and delta-
opiold receptor antagonist pharmacophores. Eur. J. Pharmacol. 2007,
566, 75−82.
Pen2,D-Pen5]-enkephalin; NTI, naltrindole hydrochloride;
DAMGO, [DAla2,NMe-Phe4,Gly-ol5]-enkephalin; HA, he-
magglutinin
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