Developing Cyclic Opioid Analogues: Fluorescently Labeled Bioconjugates of Biphalin

Article abstract of DOI:10.1021/acsmedchemlett.9b00569

The development of bioconjugates is of pivotal importance in medicinal chemistry due to their potential applications as therapeutic agents to improve the targeting of specific diseases, decrease toxicity, or control drug release. In this work we achieved

Full text of DOI:10.1021/acsmedchemlett.9b00569

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Letter
DEVELOPING CYCLIC OPIOID ANALOGUES:
FLUORESCENTLY LABELED BIOCONJUGATES OF BIPHALIN
azzurra stefanucci, marilisa pia dimmito, gabriela molnar, John M.
Streicher, Ettore Novellino, Gokhan Zengin, and Adriano Mollica
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00569 • Publication Date (Web): 08 Jan 2020
Downloaded from pubs.acs.org on January 9, 2020
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DEVELOPING CYCLIC OPIOID ANALOGUES:
FLUORESCENTLY LABELED BIOCONJUGATES OF
BIPHALIN
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Azzurra Stefanucci,1 Marilisa Pia Dimmito,1 Gabriela Molnar,2 John M. Streicher,2 Ettore Novellino,3
Gokhan Zengin,4 Adriano Mollica1,*
1 Dipartimento di Farmacia, Università di Chieti-Pescara “G. d’Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy.
2 Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ USA.
3 Dipartimento di Farmacia, Università di Napoli “Federico II”, Via D. Montesano 49, 80131 Naples, Italy.
4 Department of Biology, Science Faculty, Selcuk University, Konya, Turkey.
KEYWORDS: biphalin, opioids, fluorescent probes, GPCRs, bioconjugates.
ABSTRACT: The development of bioconjugates is of pivotal importance in medicinal chemistry due to their potential applications
as therapeutic agents to improve the targeting of specific diseases, decrease toxicity, or control drug release. In this work we achieved
the synthesis and characterization of three novel opioid peptides fluorescently labeled, analogues of cyclic biphalin derivatives,
namely 1D, 1C and 2C. Among them, compound 1D, containing a dansyl-maleimide motif, exhibited an excellent binding affinity
and functional potency for the -opioid receptor (DOR). 1D also demonstrated a strong fluorescence emission spectrum ranging from
300 to 700 nm. These features could be highly desirable for medical and biological applications needed for targeting the DOR,
including in vivo imaging, and as a lead for the design of fluorescent probes.
INTRODUCTION
with peptidic or non-peptidic structures may help to localize
receptors at low density, allowing analysis of the receptor
ligand-interaction of OR-containing cell populations. Simple
labeling methods involve the anchorage of fluorescent dyes
through succinimidyl ester or maleimide functional groups with
primary amines or thiol groups.7,8 Maleimides may quench
fluorescence in the conjugated form because the C=C bond
undergoes to selective addition reaction for thiols, but when
they are linked to chromophores such as pyrene, phtalimide and
naphthopyranone, the fluorescence emission results increased.
The application of maleimide in peptides and protein
bioconjugation is highly desirable due to the prompt N-
functionalization of maleimide and the possibility to insert the
fluorescent molecule by a selective reaction with two cysteine
residues present in the peptides. It also allows to establish a
precise distance between the fluorescent core and the peptide by
the addiction of specific linkers.9 Biphalin has been used as
model scaffold in the design of bioconjugated opioid peptides.10
Diverse hydrophilic/hydrophobic components and biochemical
markers have been attached to biphalin in order to study the
biological properties as opioid ligand and to explore its possible
application as fluorescent probe.11-16 In 2002 Lipkowski et al.
demonstrated that one biphalin pharmacophore could be
replaced by hydrophobic groups without loss of receptor
Bioconjugate peptides occupy an important position in
fluorescent probes field, due to their potential application in
supramolecular structure design, targeting of biological tissues
and diagnostic medicine.1 Peptides containing a fluorescent dye
could be valuable tools for monitoring intracellular reactions
and molecular signaling, also fluorescent ligands could be used
for pharmacokinetic studies, among other potential
applications. Peptide bioconjugates are advantageous, due to
their high receptors specificity correlated to the amino acid
sequence, low toxicity and low disposal procedures compared
to radiolabeled compounds.2 Peptide conjugates also could be
used in fluorescent microscopy. Studies in living cells require
the management of selective probes to investigate the
mechanism of internalization of membrane receptors, including
the opioid receptors.3 In recent studies opioid peptides such as
Dermorphin, deltorphin, TIPP, and endomorphin have been
conjugated to the fluorescent dyes BODIPY TR and Alexa
Fluor 488.4 Most of these peptides were biologically active,
showing mixed -opioid receptor (MOR/DOR) binding
affinity and selectivity. They have been used in internalization
study for the MOR and DOR, showing real-time visual tracking
of receptor-ligand complexes in living cells, antagonist
(naloxone)-sensitive and temperature-dependent.5 Leong et al.
conjugated Endomorphin-1 (EM-1) to tetramethylrhodamine
(TAMRA) to analyze the cell biology of the MOR in
keratinocytes, revealing a rapid and complete internalization in
the endoplasmic reticulum (ER) under resting conditions.6
Furthermore, fluorophore-conjugated opioid receptor ligands
affinity, with the aim to develop
a useful tool for
pharmacokinetic and pharmacodynamic studies in vivo;17 the
first biphalin analogue so designed contained a dansyl moiety,
which expressed high receptor binding affinity for MOR and
DOR and antinociceptive activity in vivo.17
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Cl
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Scheme 1. Functionalization of 7-amino-4-methylcoumarin and dansylchloride with Boc--Ala-OH and tert-Butyl N-(2-
aminoethyl)carbamate respectively. Reagents and Conditions: (i) tert-Butyl N-(2-aminoethyl)carbamate, EDC.HCl, HOBt, DIPEA in DMF,
r.t., overnight; (ii) TFA:DCM =1:1, r.t. 1h; (iii) 3,4-dibromofuran-2,5-dione, AcOH at r.t. for 6h then reflux for 3h; (iv) Boc--Ala-OH,
POCl3, pyridine at -15°C, 1h.
The same fluorescent moiety has been applied to the
derivatization of DALDA sequence to give three potent MOR
agonists.18 Further replacement of the dansyl group of one
biphalin branch with amino-coumarin resulted in an increased
affinity for the MOR and high antinociceptive activity
comparable to that of biphalin.19 These studies establish proof-
of-concept for the use of biphalin as a bioconjugate scaffold for
targeting the MOR and DOR.
These N-Boc protected intermediates were deprotected with
a mixture of TFA:DCM = 1:1 at r.t. and promptly reacted with
3,4-dibromofuran-2,5-dione. Maleic anhydride was converted
in the organic scaffold 3,4-dibromofuran-2,5-dione, following
the procedure reported by Dubernet et al.23 The fluorescent
scaffolds were conjugated to 3,4-dibromofuran-2,5-dione
following the procedure reported by Stefanucci et al.20 so as to
give three fluorescent-maleimide moieties in good overall
yields (98% yield for 1, 40% yield for 2, 71% yield for 3) (see
SI). The cyclic biphalin fluorescent analogs 1C, 2C, 1D were
prepared following the well-established procedure reported by
us in solution (Scheme 2).20
Recently our research group published the first cyclic
biphalin analogue incorporating a fluorescein-maleimide
moiety with the aim to identify a good fluorescent ligand for in
vivo and in vitro assays, concerning receptor binding and
penetration of biological barriers e.g. the blood brain barrier
(BBB).20 This novel compound exhibited modest affinity for
MOR and DOR and was able to partially stimulate their G
protein activation. In this study we sought to improve on this
initial work. We designed and synthesized three novel cyclic
biphalin analogues incorporating different fluorescent-labeled
moieties in order to find a good candidate for probe
development on opioid receptors for both in vitro and in vivo
studies.
Compound 4 was prepared following the procedure
previously described by our research group,24 and was
submitted to in situ reduction with the tris(2-
carboxyethyl)phosphine hydrochloride solution (TCEP)
reagent in presence of the desired fluorescent probe to afford
the N-Boc protected cyclic compounds.20 They were then
treated with a mixture of TFA/DCM in solution at r.t. to give
the final products 1D, 1C and 2C as TFA salts in good overall
yields (75% yield for 1D, 61% yield for 1C and 58% yield for
2C) and excellent purity after purification by RP-HPLC (see
SI). The three novel cyclic peptides were then tested for their
ability to bind the MOR, DOR and Kappa OR (KOR) in vitro
RESULTS AND DISCUSSION
Commercially available dansylchloride and 7-amino-4-
methylcoumarin were functionalized with tert-Butyl N-(2-
aminoethyl)carbamate and Boc--Ala-OH respectively,
following the reactions depicted in Scheme 1. Dansyl chloride
was functionalized with tert-Butyl N-(2-aminoethyl)carbamate
following the procedure described by Youziel et al. so as to give
the N-Boc protected compound in 93% yield after reaction
work-up.21 7-Amino-4-methylcoumarin was also functionalized
with Boc--Ala-OH following the procedure reported by
Heltweg et al. to obtain a crude intermediate compound pure on
silica gel TLC plate, in 63% yield after reaction work-up.22
using radioligand displacement binding assays. All
3
compounds showed varying modest to strong binding affinity
for MOR and DOR, while all 3 showed very poor binding to the
KOR (Figure 1). All compounds also showed a higher affinity
for DOR over MOR, with 1D achieving a strong ~15 nM Ki
with all 3 compounds showing ~5-8 fold selectivity for
DOR>MOR. Notably, the compounds show incomplete
competition at the MOR, which is consistent with an allosteric
binding mode.
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Scheme 2. Synthesis of novel cyclic peptides 1C, 2C and 1D. Reagents and conditions. (ii) TFA:DCM =1:1, r.t. 1h; (v) TCEP, DMF, 1
for compound 1D, 2 for compound 2C and 3 for 1C, r.t. overnight.
3
Figure 1. Opioid Receptor Binding Affinity of Novel Compounds. All compounds were competed against H-diprenorphine using
membranes from CHO cells expressing the human opioid receptors (DOR, MOR, or KOR). Data reported as the mean ± SEM of N = 3
independent experiments. A) Concentration-response curves shown for all 3 compounds and positive control (naloxone for MOR and DOR;
U50,488 for KOR) at each receptor. All compounds show competition at each receptor, with affinity DOR>MOR>KOR. The compounds
show incomplete competition at the MOR, which is consistent with an allosteric binding mode. B) Affinity values (Ki) shown for each
compound at each receptor derived from the curves in A.
which we’ve used extensively to characterize the functional
activity of our compounds.20,25
These profiles of MOR/DOR binding are somewhat similar
to the biphalin parent scaffold, and also suggest that these
ligands could be effective labeling agents for the DOR and/or
MOR.We next measured the functional activity of all
compounds at the opioid receptors using the same CHO cell
lines used above for the binding. We used 35S-GTPγS coupling,
We first found that all 3 compounds were partial agonists at
the DOR (EMAX 47-62%), with modest potencies (EC50 40-250
nM, Figure 2). These potencies were all about 2 fold worse than
the binding affinities measured in Figure 1, suggesting that
these ligands have somewhat poor intrinsic efficacy at the DOR.
The reverse was true for the MOR, where all 3 compounds had
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improved potency vs. binding affinity, suggesting good
intrinsic efficacy, while all 3 were near-full to full agonists
(EMAX 79-120%). Lastly, all 3 compounds were weak potency
partial agonists at KOR, as suggested by the poor affinities
above, however they did show complete curves with improved
potency vs. affinity, suggesting good intrinsic efficacy at KOR.
These results altered the binding selectivity profile at the
different opioid receptors found in Figure 1, with all
compounds now nearly equi-potent at MOR/DOR with little to
Page 4 of 7
no selectivity between them, at least at the functional level.
These results should be kept in mind for evaluating these
compounds in vivo. For the purposes of binding and labeling,
for which the fluorescent moieties would be most useful, these
compounds will likely be DOR-preferring. However, for any
functional studies, they will likely be non-selective between
MOR and DOR, similar to the parent scaffold biphalin. Taken
together, these results show that the fluorescent moieties do not
prevent effective binding and activation of the opioid receptors.
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Figure 2. Opioid Receptor Functional Activity of Novel Compounds. All compounds were used to stimulate 35S-GTPγS accumulation
using membranes from CHO cells expressing the human opioid receptors (DOR, MOR, or KOR). Data reported as the mean ± SEM of N =
3 independent experiments. A) Concentration-response curves shown for all 3 compounds and positive control (DAMGO for MOR, SNC80
for DOR, U50,488 for KOR) at each receptor. All compounds show partial to full agonist activity at each receptor, with potency
DOR=MOR>KOR. B) Potency (EC50) and Efficacy (EMAX) values shown for each compound at each receptor derived from the curves in A.
After establishing the molecular pharmacology of our
compounds, we finally sought to evaluate their potential as
fluorescent labeling tools. We thus measured the excitation and
emission spectra of our highest affinity cyclic peptide 1D, using
an Edinburg Instruments FLS-920 fluorimeter in 100%
methanol matrix at r.t., with a sample concentration of 10-2 M
(Figure 3). The fluorescence of the flurescent-conjugated
molecule is strongly dependent on the polarity of the solvent
and chemical surroundings. The fluorescence emission
spectrum of our novel compound 1D shows an emission band
ranging from 300 to 700 nm, with a maximum at 450 nm in
methanol. In agreement with the findings of Guy et al. a
maximum at 605 nm is present in the spectra, which represents
the fluorescence emission of N-substituted dansylamide.26 Two
strong emission bands are present around 300 and 450 nm
associable to tyrosine residues and conjugated hydrazine into
the cyclic peptide.27,28 These results establish that 1D is indeed
a fluorescent ligand, with potential for use as a labeling and
imaging agent. The good separation between excitation and
emission spectra means that the compound would show little
bleed through or crosstalk if the excitation and emission gates
were correctly chosen. In conclusion three novel chemical
entities have been synthesized and characterized with the aim
to find a useful fluorescent bioconjugate of cyclic biphalin able
to bind opioid receptors. Among them, compound 1D exhibits
the best affinity profile, being able to bind MOR/DOR and to
stimulate the G protein coupled receptors with high affinity and
potency. This compound represents an ideal candidate for
further future work to define the pharmacokinetic properties of
cyclic biphalin compounds, and a valuable tool for binding
studies, cellular uptake, and tissue distribution measurement of
opioid receptors.
EXPERIMENTAL PROCEDURES
General information
Amino acids and reagents were acquired from Sigma-Aldrich
(Milano, Italy), and HPLC grade solvents from VWR (Milano,
Italy). The three novel cyclic peptides as TFA salts were
purified by RP-HPLC on a Waters XBridgeTM Prep BEH130
C18, 5.0 μm, 250 mm × 10 mm column; flow rate of 7 mL/min;
Waters Binary pump 1525 (Waters, MA, USA); eluent: linear
gradient of H2O/ACN 0.1% TFA from 5 to 90% ACN in 35
min. Nα-Boc-protected products were identified by NMR
analysis on a Varian Mercury 300 MHz instrument and mass
spectrometry ESI-LRMS (Thermo Finnigan, NJ, USA). The
purity of the final compounds as TFA salts was calculated using
analytical reverse phase high performance liquid
chromatography (RP-HPLC; C18-bonded 4.6 × 150 mm) at a
flow rate of 1 mL/min; gradient eluent of H2O/ACN 0.1% TFA
from 5 to 95% ACN in 26 min, and was found to be ≥90% pure.
The identity of all final compounds was determined by 1H NMR
and ESI-LRMS. Fluorescence measurement was performed
using an Edinburg Instruments FLS-920 fluorometer. The
fluorescence measure was done in 100% methanol matrix at r.t.,
sample concentration was 10-2 M; wavelength ranging from
200 to 900 nm in 10 minutes.
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Figure 3. Adsorption (blue)/emission (red) spectrum of compound 1D in 100% methanol. Y axis: Absorbance in A.U.; X axis: wavelength
in nm unit.
Chemistry
from ThermoFisher, Gibco brand). The cells were grown in a
37°C humidified incubator with 5% CO2. Cells were grown and
pellets harvested for both binding and G protein coupling assays
as reported.20 The competition binding assay was also
performed as reported.20 Briefly, 20-25 μg of cell membrane
protein was combined with concentration curves of
experimental compound or positive control and a fixed
concentration (1.2-4.6 nM) of 3H-diprenorphine (PerkinElmer)
in a 200 μL reaction volume. The reactions were incubated for
1 hr at r.t., and terminated by rapid filtration onto GF/B filter
plates (PerkinElmer) using a Brandel cell harvester. The plates
were dried, 40 μL of Microscint PS (PerkinElmer) was added,
and the data was collected using a MicroBeta2 96 well format
scintillation counter (PerkinElmer). The data was analyzed
using GraphPad Prism 8.2 with a 1-site competition binding
model using the previously measured KD of the 3H-
diprenorphine in each cell line. The data was further normalized
to radioligand alone (100%) or in the presence of 10 μM
naloxone (non-specific binding, 0%). The resulting affinity (Ki)
values were reported as the mean ± SEM of N=3 independent
experiments.
The organic scaffold 3,4-dibromofuran-2,5-dione was
prepared starting from the commercially available maleic
anhydride following the procedure reported by Dubernet et al.23
The fluorescent intermediates reported in Scheme 1 were
synthesized in solution as described by Youziel et al.,21 and
Heltweg et al.22 respectively. Compounds 1-3 were prepared
starting from the treatment of dibromomaleic anhydride with
the appropriate fluorescent intermediates as TFA salts or 7-
amino-4-methylcoumarin at room temperature (r.t.), followed
by reflux in AcOH to give the desired fluorescent maleimides
in good yields. All linear intermediate peptides were obtained
by solution phase peptide synthesis using the EDC·HCl/HOBt
hydrate/N,N-Diisopropylethylamine
(DIPEA)
in
the
dimethylformamide (DMF) coupling method. Deprotection of
the Nα terminus of peptides from Boc-protecting group was
performed with a mixture of TFA in DCM 1:1 at r.t. The
intermediate TFA salts were used for subsequent reactions
without further purification. NBoc-protected intermediates
were isolated by silica gel column chromatography when
required. The cyclic biphalin intermediate 4 was synthesized in
solution, following the well-established procedure reported by
us.24 Then this intermediate was treated with tris(2-
carboxyethyl)phosphine hydrochloride solution (TCEP)
reagent in DMF at r.t. and the fluorescent-probes 1-3 were
added in situ to promptly react with the thiol groups, so as to
form the cyclic compounds 1C, 1D and 2C after N-Boc
deprotection.20 The fluorescent cyclic intermediates were
obtained in good yields after an easy work-up, and used as such
for the following reaction without further purification. All final
biphalin fluorescent analogs were deprotected with a mixture of
TFA:DCM =1:1, purified by RP-HPLC and characterized as
TFA salts. 1H NMR, ESI-LRMS and analytical RP-HPLC were
performed for all final compounds, details of the experimental
procedures are reported in the SI.
35S-GTPγS coupling assay
The cells were grown and harvested as above; the assay
protocol used was also reported previously in Stefanucci et
al.20,25 15 μg of cell membrane protein was combined with
concentration curves of experimental or positive control agonist
and 0.1 nM 35S-GTPγS (PerkinElmer) in a 200 μL volume.
Reactions were incubated at 30°C for 1 hr, then harvested and
data collected as above. The data was normalized to the
stimulation caused by positive control agonist (100%) or
vehicle treatment (0%). GraphPad Prism 8.2 was used to
analyze the data using a 3-variable non-linear regression model
(Hill Slope defined as 1). The resulting potency (EC50) and
efficacy (EMAX) values were reported as the mean ± SEM of
N=3 independent experiments.
Competition-binding assays
ASSOCIATED CONTENT
Supporting Information
Opioid receptor-expressing CHO cells were used as
previously reported.19 The cells were grown using 50:50
DMEM/F12 culture media with 10% heat-inactivated fetal
bovine serum, 1X penicillin/streptomycin, with propagation
cultures maintained with 500 μg/mL G418 (all culture reagents
The Supporting Information is available free of charge on the
ACS Publications website. Details of compound synthesis and
characterization included (PDF).
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5. Dado, R. J.; Law, P. Y.; Loh, H. H.; Elde, R. Immunofluorescent
AUTHOR INFORMATION
identification of a -opioid receptor on primary afferent nerve
terminals. Neuroreport 1993, 5, 341-344.
1
Corresponding Author
2
3
4
5
6
7
8
9
6. Leong, C.; Neumann, C.; Ramasamy, S.; Rout, B.; Yi, W. L.;
Bigliardi-Qi, M. Investigating endogenousµ-opioidreceptors in
human keratinocytes as pharmacological targets using novel
* Corresponding author email: a.mollica@unich.it.
Author Contributions
fluorescent
ligand.
Plos
One
2017,
12(12):
AS designed and he novel compounds synthesized the novel
compounds, co-wrote and organized the manuscript. MPD
helped in the purification and characterization of the novel
compounds. GL and EN revised the entire manuscript for
English language; GZ collaborated in the interpretation and
rationalization of the data. JMS participated with and
supervised GM in the performance of the in vitro experiments
and co-wrote the manuscript. AM, co-wrote and organized the
manuscript, and coordinated all the research units.
e0188607.https://doi.org/10.1371/journal. pone.0188607
7. Smith, M. E. B.; Felix, F.; Schumacher, C. P.; Lauren, R.; Tedaldi,
M.; Papaioannou, D.; Waksman, G.; Caddick, S.; Baker, J. R.
Protein modification, bioconjugation, and disulfide bridging
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Funding Sources
This study was funded in part by institutional funds from the
University of Arizona to JMS. The authors have no relevant
conflicts of interest to declare.
ABBREVIATIONS
DOR, δ-opioid receptor; MOR, μ-opioid receptor; KOR, k-
opioid receptor; EDC, 1-ethyl-(3-(dimethylamino)propyl)-
carbodiimide; DAMGO, [DAla(2), N-Me-Phe-(4), Gly-ol(5)]
enkephalin; SNC80, (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-
dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-
diethylbenzamide; TAMRA, tetramethylrhodamine; ER,
endoplasmic reticulum; SEM, Standard error of measurement;
HOBt,
1-hydroxybenzotriazole;
DIPEA,
N,N-
Diisopropylethylamine; DMF, dimethylformamide; EtOAc,
ethyl acetate; RP-HPLC, reversed phase high performance
liquid chromatography; ACN, acetonitrile; NMR, nuclear
magnetic resonance; ESI-LRMS, electrospray ionization low
resolution mass spectrometry; HRMS, high resolution mass
spectrometry;
TFA,
trifluoroacetic
acid;
DCM,
dichloromethane, TLC, thin layer chromatography; BBB, blood
brain barrier; CNS, central nervous system; GTP, guanosine
triphosphate; DMSO, dimethylsulfoxide; CHO, chinese
hamster ovary; DMEM/F12, Dulbecco’s modified eagle
medium/nutrient
mixture
F-12;
EDTA,
ethylenediaminetetraacetic acid; PBS, phosphate buffered
saline.
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H H
N N
HN
NH
HN
O
O
O
O
HN
O
O
O
O
H
H
S
Boc
N
S
N Boc
HO
OH
H H
N N
H H
N N
H H
N N
HN
NH
HN
NH
HN
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
HN
O
S
HN
S
O
HN
O
S
HN
S
O
HN
O
S
HN
S
O
O
O
O
O
O
O
TFA . H2N
NH2 . TFA
TFA . H2N
NH2 . TFA
TFA . H2N
.
NH2 TFA
N
N
H
N
N
H
N
N
H
H
H
H
O
O
O
O
O
O
O
N
N
N
O
HO
OH
HO
OH HO
OH
O
S
O
HN
NH
O
N
O
2C
1D
Ki = 15 nM
1C
Ki = 98 nM
Ki = 120 nM
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