Receptor-Ligand Interaction Measured by Inductively Coupled Plasma Mass Spectrometry and Selenium Labeling

Article abstract of DOI:10.1021/acs.jmedchem.8b01320

In the search for an alternative strategy to the radioactivity measurement conventionally performed to probe receptor-ligand interactions in pharmacological assays, we demonstrated that selenium labeling of the studied ligand combined with elemental mass spectrometry was as efficient and robust as the reference method but devoid of its environmental and health hazards. The proof-of-concept was illustrated on two GPCR receptors, vasopressin (V_1A) and cholecystokinin B (CCK-B), involving peptides as endogenous ligands. We proposed several methodologies to produce selenium-labeled ligands according to peptide sequences along with binding affinity constraints. A selection of selenopeptides that kept high affinities toward the targeted receptor were engaged in saturation and competitive binding experiments with subsequent sensitive RP-LC-ICP-MS measurements. Experimental values of affinity constant (K_i) were perfectly correlated to literature data, illustrating the general great potency of replacing radioactive iodine by selenium for ligand labeling to further undergo unaffected pharmacology experiments efficiently monitored by elemental mass spectrometry.

Full text of DOI:10.1021/acs.jmedchem.8b01320

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Article
Receptor-ligand interaction measured by Inductively
Coupled Plasma Mass Spectrometry and selenium labeling
Clémence Cheignon, Emmanuelle Cordeau, Nolween Prache, Sonia Cantel, Jean
Martinez, Gilles Subra, Carine Arnaudguilhem, Brice Bouyssiere, and Christine Enjalbal
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01320 • Publication Date (Web): 05 Nov 2018
Downloaded from http://pubs.acs.org on November 6, 2018
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Receptor-ligand interaction measured by Inductively
Coupled Plasma Mass Spectrometry and selenium
labeling
§
§
§
§
Clémence Cheignon, Emmanuelle Cordeau, Nolwenn Prache, Sonia Cantel, Jean
§
§


Martinez, Gilles Subra, Carine Arnaudguilhem Brice Bouyssiere and Christine
Enjalbal§*
§
IBMM, Univ Montpellier, CNRS, ENSCM, Montpellier, France.

CNRS/Univ Pau & Pays Adour / E2S UPPA, Institut des Sciences Analytiques et de Physico-
Chimie pour L’Environnement et les Matériaux, UMR 5254, 64000 Pau, France.
ABSTRACT
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In the search of an alternative strategy to radioactivity measurement conventionally
performed to probe receptor-ligand interactions in pharmacological assays, we
demonstrated that selenium labeling of the studied ligand combined with elemental mass
spectrometry was as efficient and robust as the reference method but devoid of its
environmental and health hazards. The proof-of-concept was illustrated on two GPCR
receptors, Vasopressin (V ) and Cholecystokinin B (CCK-B), involving peptides as
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1A
endogenous ligands. We proposed several methodologies to produce selenium labeled
ligands according to peptide sequences along with binding affinity constraints. A selection
of selenopeptides which kept high affinities towards the targeted receptor were engaged
in saturation and competitive binding experiments with subsequent sensitive RP-LC-ICP-
MS measurements. Experimental values of affinity constant (K) were perfectly correlated
i
to literature data illustrating the general great potency of replacing radioactive iodine by
selenium for ligand labeling to further undergo unaffected pharmacology experiments
efficiently monitored by elemental mass spectrometry.
INTRODUCTION
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The affinity of a drug candidate for a target is a parameter of upmost importance in the
search of new bioactive molecules. For this purpose, radioactive ligand labeling,
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developed in 1970, is considered as the reference method, providing robust and
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sensitive measurement through competitive binding experiments. However, in the light
of the obvious disadvantages of handling radioactive materials in research laboratories
(health hazardous, waste storage, …), other labeling strategies based on fluorescence
have emerged.3-5 Nevertheless, such approaches require the introduction of bulky
fluorescent tag which could potentially affect the pharmacological properties of the ligand,
leading to an affinity loss for the targeted receptor. Taking into account these drawbacks,
various label-free strategies have been developed, mainly based on mass spectrometry
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analysis. Höfner and Wanner introduced the concept of mass spectrometry (MS) binding
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assays where a target-bound marker is quantified after its displacement with the studied
compound by liquid chromatography hyphenated to tandem mass spectrometry (LC-
MS/MS). In contrast to such strategy that relies on small molecule detection with
electrospray ionization (ESI) technique and quantification following multiple reaction
monitoring (MRM) transitions on triple quadrupole mass analyzers, two other MS-based
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approaches (SUPREX and PLIMSTEX) dealing with entire protein analyses by either
MALDI-MS or ESI-MS techniques have also been developed.8-9 Both are based on H/D
exchange as indicator of protein-ligand complex formation. If such MS-based strategies
have been found very attractive to determine affinity constants in different model
systems,10-11 they are still limited by the ionization potency of the marker molecule
regarding the MS binding assay method and by the accuracy capacity of the analytical
technique to distinguish low mass changes for high molecular weight compounds for
SUPREX and PLIMSTEX methodologies. Besides such molecular mass spectrometry
approaches, elemental mass spectrometry also constitutes another very powerful tool for
quantification. Indeed, inductively coupled plasma mass spectrometry (ICP-MS) implies
sample atomization by means of a very hot plasma which brakes all covalent bonds and
release elements that are further ionized by removing an electron. Despite the fact that
the molecular information is lost, each of the produced atomic ions can be selected and
easily monitored with a cheap quadrupole mass analyzer (Q). Nevertheless, the
quantification sensitivity is directly dependent of the element ionization efficiency, the best
performances being obtained for metals allowing measurements of ultra-traces (ppb/ppt
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ranges). Thus, ICP-MS offers the advantages of very high sensitivity by targeting a stable
isotope of a known responsive element, few and simple sample preparation, wide
dynamic range, relative matrix robustness and finally the possibility to be hyphenated to
separation techniques such as liquid chromatography (LC) or electrophoresis.
Noteworthy, ICP-MS has found numerous applications in absolute quantification of
biomolecules provided that the targeted compound exhibits a hetero or metallic element
into its structure suitable for monitoring.12-14 Many labeling strategies have also been
described to introduce such ICP-MS mass reporter tag into biomolecules prior to
analyses.15-19 In particular, labeling antibodies with lanthanides instead of fluorescent
moieties was found a very valuable way to study the complexity of biology at the single-
cell level in ICP-MS based immunoassays (so called mass cytometry).20-21 Although
lanthanide isotopes have very low natural abundance in biological systems ensuring a
clean detection background, the fact that it requires the anchoring of a large chelating
moiety that may potentially affect the pharmacological properties of the investigated
ligand prompted us to pursue an alternative tagging approach for ICP-MS detection. Our
major requirements were to avoid bulky labels to preserve ligand pharmacological
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properties as well as chelating systems to fix the mass reporter onto the ligand since they
may be lost during the assays. Covalent binding of a hetero element was thus preferred
even if ionization efficacy linked to quantification limit and natural presence in biological
media governing detection specificity presented potent difficulties to overcome. Selenium
element was chosen for such purpose since many labeling strategies can be followed
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(insertion of selenium-containing proteinogenic or synthetic non-natural amino acid as
well as peptide substitution by a selenium containing moiety at the N- or C-termini or on
an amino acid side-chain). From a chemical point of view, selenium presents the
advantage to be inserted into the analyte through conventional sulfur organic chemistry,
since these both chalcogen atoms share similar physico-chemical properties. Moreover,
ICP-MS hyphenated to reversed phase chromatography (RP-LC-ICP-MS) was very
recently successfully investigated for quantification purpose of selenopeptides.22-23
Therefore, we chose such approach combining dedicated chemical labeling adapted to
elemental mass spectrometry analyses to achieve receptor-ligand interaction
measurement. We developed the methodology with the well characterized V subtype of
1A
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vasopressin receptor (V -R) implicated in a wide diversity of physiological processes
1A
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such as vasoconstriction and emotional behavior and involving Arginine Vasopressin
(
AVP), a cyclic nonapeptide, as native ligand. Then, we validated the new methodology
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with the type B cholecystokinin (CCK-B) receptor and two native peptidic ligands: CCK-4
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and non-sulfated CCK-8. This receptor is widely distributed in the brain and implicated
in numerous physiological functions such as mediation of anxiety, satiety and analgesia.27
Selenium tagged V and CCK-B ligands were designed according to different labeling
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strategies that can be applied to any peptide sequence as depicted in Scheme 1.
We have illustrated first amino acid substitution by the corresponding selenium
containing residue into peptide sequence such as cysteine (Cys) replaced by
selenocysteine (Sec) leading to either diselenide bridge or selenolanthionine formation,
methionine (Met) by selenomethionine (SeMet), proline (Pro) by selenazolidine (Sez) and
second N-terminal peptide derivatization with a selenium containing organic entity. The
affinities of these newly synthesized selenoligands towards V1A receptor were assessed
with conventional radiolabeling binding experiments and the compounds that have kept
their high affinity were further used as labeled ligands to establish the AVP affinity
constant with RP-LC-ICP-MS detection.
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Furthermore, the applicability of such methodology was confirmed through the affinity
measurement of a short panel of characteristic ligands exhibiting either high or poor
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affinity for V1A receptor. Finally, the possibility to evaluate K of selenium containing
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molecules through the displacement of a selenium tagged reference ligand was also
assessed. All obtained results were compared to the ones gathered with conventional
radiolabeling experiments.
In the second part, the established methodology was applied to the measurement of
non-sulfated CCK-8 interaction to CCK-B receptor with selenium tagged CCK-4 ligands.
RESULTS AND DISCUSSION
Peptide labeling: multimodal insertion of selenium tag
Nowadays, peptides benefit from a regain of interest as potential drug candidates and
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thus represent a promising target in drug discovery process. Thus, we concentrated our
effort on designing labeling strategies adapted to peptide ligands. Selenium introduction
into peptides is up to now predominantly achieved through the replacement of
proteinogenic sulfur containing amino acid residues (cysteine and methionine) by their
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selenium analogues that are selenocysteine (Sec) and selenomethionine (SeMet)29-31
according to standard Solid Phase Peptide Synthesis (SPPS) protocols. Moreover, many
bioactive peptides have been synthesized through disulfide bond substitution by
diselenide bridge for bioavailability improvement without pharmacological properties
loss.32-33 We followed the same approach for the peptide ligands that were exhibiting
sulfur-containing amino acids (Scheme 1, blue star). However, we went further by
proposing complementary tagging methods to be able to cope with any ligands not
possessing cysteine or methionine in their sequences. In order to enlarge the port-folio of
switchable amino acid residues, we prepared a non proteinogenic selenium-containing
amino acid, i.e. selenoproline (Sez) that was coupled in SPPS to the growing peptide
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sequence as a preformed dipeptide building block (Scheme 1, blue star). Because of its
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wide occurrence in human proteins, proline constitutes a valuable probe for the standard
insertion of a selenium atom through its replacement by selenazolidine (Sez). Formally,
in this proline surrogate, a methylene group of the cyclic side-chain was substituted by a
selenium atom which should guarantee minimal changes of pharmacological properties.
We finally investigated N-terminal substitution with various selenium-containing moieties
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introduced through the well-mastered acylation reaction (Scheme 1, red star). The
advantage of such approach relies in the fact that different positions can be targeted to
introduce the derivative according to the binding affinity constraints (e.g. lysine lateral
chain or N-terminal position). No difficulty was encountered to prepare and purify the
seven selenopeptides designed from three well-known ligands (AVP (1) and HO-Phpa-
LVA (4) for V -R, CCK-4 (7) for CCK-B-R) as shown in Figures 1 and 2.
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In the case of less straightforward syntheses, for instance for long sequences quite
difficult to produce upon SPPS elongation, native chemical ligation (NCL)36-38 approaches
and recombinant biosynthesis,39-41 that have already been reported to prepare long
selenopeptides and selenoproteins, would represent additional ways to produce such
seleno-labeled molecules.
Proof of concept: the V model
1A
Affinities of designed selenium labeled ligands
Among the diversity of ligands exhibiting a high affinity toward V1A receptor,42-45 we
selected the native AVP (1), a nonapeptide cyclized between two cysteine residues in
first and sixth positions, and the potent linear antagonist HO-Phpa-LVA (4) as two model
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ligands to introduce selenium atoms (Figure 1, dashed rectangles). Since sulfur
substitution by selenium has been demonstrated to preserve structural and
pharmacological properties of biomolecules,29-33 such approach represented the first
obvious way to perform selenium labeling (Figure 1a). In a previous work, we have
synthesized the AVP selenium analogue [Se-Se]-AVP (2) (Figure 1a, left) where both Cys
residues in positions 1 and 6 were substituted by Sec residues, which has shown to retain
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its affinity toward V1A receptor (K of 0.7 ± 0.2 nM). In order to confirm that selenium
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labeling through sulfur replacement does not impact the initial binding behavior of affine
or not affine ligands, we produced a second selenium-containing peptide which was
intended to represent a negative control. According to same SPPS Boc/Bzl protocol, [Se]-
AVP (3) cyclized via the formation of a selenolanthanine bridge between 1 and 6 positions
(Figure 1a, right) was designed since such ring shortening have been demonstrated by
Muttenthaler and co-workers to result in a drastic affinity loss with the oxytocin hormone,
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a very similar peptide to AVP (1). The K of 1280 nM observed for [Se]-AVP compound
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(Table 1) confirmed such affinity loss tendency indicating that the chalcogen atom was
not perturbing the receptor-ligand interaction.
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Moreover, to enlarge the possibilities of selenium tagging out of sulfur containing
peptides, we also investigated alternative ways to introduce selenium into peptide
sequences either in a residue side-chain or at the N-backbone terminus (Figure 1b). The
linear HO-Phpa-LVA (4) antagonist peptide was prone to such modifications, already
bearing an aromatic acetyl group onto the N-terminal amine function that was directly
replaced by the 2-phenylselenyl acetyl group (Ph-Se-acetyl) (Figure 1b, right). Based on
the tolerance for structural modifications in position 1 of linear vasopressin antagonist
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47
(LVA) family as evidenced by Manning et al., we chose to replace the phenylpropionyl
(Phpa) group in position 1 of HO-Phpa-LVA (4) ligand, to afford a selenium labeled
peptide, taking also advantage of the structure analogy between Ph-Se-acetyl and Phpa
(Figure1b, right). Regarding the latter strategy, proline constitutes a valuable probe for
the standard insertion of a selenium atom through its replacement by selenazolidine (Sez)
(
Figure 1b, left). In both cases, a methylene group (CH ) was just changed by a Se atom
2
which should guarantee minimal changes of pharmacological properties.
As expected, pharmacological properties were preserved by substitution of proline by
6
selenazolidine as illustrated by K values found at 0.9  0.5 nM and 0.8  0.1 nM for [Sez ]-
i
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HO-Phpa-LVA (5) and HO-Phpa-LVA (4), respectively (Table 1). Similarly, [Ph-Se-acetyl]-
LVA (6) was found to preserve affinity toward V receptor with a K determined at 0.9 nM
1A
i
1
1
1
1
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
0.5 nM (Table 1). According to these different approaches, [Se-Se]-AVP (2), [Ph-Se]-
6
HO-LVA (6) and [Sez ]-HO-Phpa-LVA (5) have been selected to be further used as new
tagged ligands for competitive binding experiments since their affinities for the V1A
receptor is now available. The negative control, [Se]-AVP (3) peptide which presented a
poor affinity for V receptor (Table 1), was not further investigated.
1A
RP-LC-ICP-MS analytical methodology
The analytical methodology was previously carefully developed and validated for
22
diselenide peptide quantification in pharmacological environment. Therefore we used
the same chromatographic conditions. All peptides were eluted within the isocratic step
78
at 50% of organic solvent. Quantification was performed on Se isotope, since it was less
interfered than 80Se as demonstrated previously. External calibration curves with 7
-1
concentrations from 50 to 2500 ng Se L (corresponding to either 0.3 nM to 15.8 nM or
0.6 nM to 31.7 nM for the peptides bearing either two or one selenium atoms, respectively)
were injected for each analytical batch in order to properly correct ICP-MS signal
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detection variations. Blank samples constituted of water supplemented with 1% TFA were
injected every 4 samples to evaluate possible cross contamination during analyses and
were found stable during all analyses.
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Furthermore, blank samples from experimental protocol (buffer incubation on cells
without ligands) were also injected, [Se-Se]-AVP (2) was interfered by a co-eluted
impurity found in the blank, therefore mean of three blank signals areas was
systematically subtracted to sample area. Because of their different retention times, no
major interfering peak was observed for the other compounds. Linearity was validated for
each compound with a regression coefficient superior to 0.999 (Figure 3). Accuracy was
6
also assessed for each curve with bias lower than 10%, except [Sez ]-HO-Phpa-LVA (5)
for which the concentration recalculation according to the linear regression equation of
one point showed a bias of 16%.
Establishment of the binding assays workflow
The protocol was performed in analogy to conventional radioligand binding assays. We
used adherent CHO cells that were previously transfected to stably over express the V1A
receptor. Cells were sowed into plates the days before the experiment. We selected a 6
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multi-wells plate format to reach about 100 000 cells per well, corresponding to an
increase of roughly a factor 10 in comparison to 96 multi-wells plates usually used in
radiolabeling experiments. Incubation of ligands was performed at 4°C for 4h, this
incubation temperature was chosen to avoid potent receptor internalization induced by
ligand binding as it has been observed for the growth hormone secretagogue receptor.48
After this incubation time, cells were washed to remove all unbound material. In contrast
to radiolabeling or fluorescent-labeling binding assays where measurement can be
directly performed from the plate, LC-ICP-MS analysis requires to recover receptor-
bounded ligands for their further injection into the analytical device. This was achieved
with the use of a dissociation buffer constituted of water supplemented with 1 % of
trifluoroacetic acid (TFA) which was previously assessed to perform efficient receptor
1
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22
denaturation as well as solubilization of whole dissociated ligands. Moreover, to ensure
total analyte recovery, we scratched the inner of the wells with a spatula. Samples were
then centrifuged to remove residual insoluble material and the supernatants were directly
analyzed with the optimized RP-LC-ICP-MS procedure. For saturation experiments,
increasing concentrations of selenium labeled peptides were incubated on cells to
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determine the total binding while unspecific binding was established under the same
conditions with 2000-fold excess of native AVP (1). In saturation assays, specific binding
curve was thus deduced from these data by subtracting unspecific to total values, allowing
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the determination of the maximum binding capacity (Bmax) and K values (Figure 4).
d
Competitive binding experiments were carried out by displacing 1 nM of selenium
labeled ligand with increasing concentrations of competitor (from 10-12 to 10-5 nM).
Recorded results were compared with the ones determined with the radiolabeling
reference method. No unspecific binding was observed for [Se-Se]-AVP (2) while non
6
specific binding for [Ph-Se-acetyl]-LVA (6) and [Sez ]-HO-Phpa-LVA (5) ligands represent
respectively 81% and 53% of the total binding at saturation doses. This feature was also
125
observed in [ I]-HO-Phpa-LVA (11) saturation experiment performed in conventional
radiolabeling conditions, which showed 74% of unspecific binding at saturation (Figure
S3, panel d). Introduction of phenylselanylacetyl group in position 1 presumably increases
the lipophilicity of the ligand in comparison to HO-Phpa-LVA (4) analogue bearing a
hydroxyl group. Nevertheless, K values established through selenium labeling/ICP-MS
d
signal measurement (Figure 4) were in good agreement with literature data. A K of 0.99
d
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
0.14 nM was obtained for [Se-Se]-AVP (2), a value correlated to the 0.6 - 3 nM range
3
42
gathered by Manning et al. for [ H]-AVP on V receptor. K values of 0.32  0.24 nM
1A
d
1
1
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and 0.45  0.21 nM for [Ph-Se-acetyl]-LVA (6) and [Sez ]-HO-Phpa-LVA (5), respectively,
125
were also in good adequacy with K obtained for [ I]-HO-Phpa-LVA (11) ligand
d
determined at 0.46  0.08 nM (value calculated from curve shown in Figure S3, panel d).
Regarding Bmax measurements, the differences observed for the recorded experimental
values may be due to varying expression rates of V1A receptor in CHO cells between
different batches.
AVP (1) affinity toward V1A receptor (Figure 5) evaluated through the displacement of
6
[Se-Se]-AVP (2) (K of 1.7  0.7 nM) as well as with [Sez ]-HO-Phpa-LVA (5) (K of 3.8 
i
i
1
.6 nM) were in good correlation with the ones evaluated through conventional
radiolabeling assay performed by us (K of 3.8  1.3 nM) as well as with the value reported
i
by Cotte et al. estimated at 3.4  1.1 nM.49
However, the use of [Ph-Se-acetyl]-LVA ligand (6) was found less suitable since the
inhibition constant determined for AVP (1) was about 4 times greater than the reference
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value. This may be explained by the very high unspecific binding observed for this
compound, which resulted in a flattened range for displacement measurement.
From these promising results, we selected the diselenide AVP (2) to validate the
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selenium tagging/ICP-MS quantification methodology by determining the K values of a
i
set of V -R ligands in comparison with radioactive assays. The V -R antagonist peptide
1A
1A
HO-Phpa-LVA (4) together with two organic molecules (the V1A receptor selective
antagonist SR 49059 (12) and V2 receptor selective antagonist SR 121463 (13)) were
50
chosen for this purpose, exhibiting respectively high and poor affinity toward V -R.
1A
Moreover, the capability of the developed methodology to evaluate selenium compound
affinity was also investigated through the displacement of a selenium-containing peptide,
i.e. [Se-Se]-AVP (2) by another selenopeptide, the selenazolidine analogue of HO-Phpa-
6
LVA ligand ([Sez ]-HO-Phpa-LVA) (5).
Since ICP-MS measurement does not provide molecular information, the success of
such challenge mainly relies on an efficient chromatographic separation before ICP-MS
detection. Methanol percentage decrease from 50% to 45% during the isocratic elution
step was found suitable to achieve sufficient separation between both compounds
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whatever their respective concentrations (chromatograms are displayed in Figure S4).
Dose-response curves of the different binding assays and related K values of different
i
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compounds are displayed in Figure 6. Good correlations were also obtained with
reference data, the three V selective ligands were found to retain affinity while the non
1A
affine behavior of SR 121463A (13) was confirmed.
Method validation: the CCK-B model
Design of selenium labeled ligands
51
Among the numerous native ligands of CCK-B receptor, the minimal active sequence
toward CCK-B receptor (Trp-Met-Asp-Phe-NH named CCK-4 (7), see Figure 2, dashed
2
rectangle) was selected to design selenium analogues, despite its lower affinity toward
CCK-B receptor. This choice was governed by the commitment to perform the simplest
labeling strategy as possible along with a tagged ligand presenting less affinity toward the
CCK-B receptor than the other natural ligands (sulfated or non-sulfated CCK-8, CCK-33,
gastrin).27
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In first instance, following the sulfur amino acid substitution with selenium analogue
approach, methionine residue in position 2 was substituted by a selenomethionine leading
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to the compound [SeMet ]-CCK-4 (9) (Figure 2, bottom). Secondly, N-terminal
derivatization with a seleno-organic compound was also applied for the labeling of CCK-
4
(7) ligand. Indeed, this position could tolerate modifications, as demonstrated by other
active sequences such as CCK-8 or CCK-33 ligands.
However, the direct introduction of Ph-Se-acetyl moiety led to a water insoluble
compound which was found deleterious to achieve pharmacological experiments in
aqueous media. Therefore, an arginine residue was added to the sequence to increase
polarity and thus hydrophilicity. Furthermore, the linker 6-amino-hexanoic acid (Ahx) was
additionally used for spacing the positively charged arginine residue from bounded
sequence, and thus to preserve affinity (Figure 2, left and right).
Saturation and competition assays
The three synthesized selenium labeled peptides were used for competition and
saturation assays with HEK cells over expressing CCK-B receptors. A similar protocol
was applied to these experiments (see experimental section) except for [Ph-Se-acetyl]-
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Arg-Ahx-CCK-4 (10) and Ac-SeMet-Arg-Ahx-CCK-4 (8), for which the LC elution gradient
has been adapted in order to more efficiently separate the selenium labeled peptides from
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selenium containing interferences. External calibration curves with 6 concentrations from
-1
7
5 to 2500 ng.Se.L (corresponding to 0.9 nM to 31.7 nM of selenium labeled peptide)
were injected for each analytical batch in order to properly correct ICP-MS signal
detection variations (Calibration curves are displayed in Figure S5).
Saturation experiments have been carried out by incubation of increasing
concentrations of selenium labeled peptide on HEK cells in order to determine the total
binding. Unspecific binding was established under the same conditions with 300-fold
excess of non-sulfated CCK-8 (14), and the subtraction of the non-specific binding from
the total binding values led to the specific binding curve (Figure 7).
2
Very little unspecific binding was observed for [SeMet ]-CCK-4 (9) and Ac-SeMet-Arg-
Ahx-CCK-4 (8) (5.5 % and 1.7 % of the total binding at saturation doses, respectively)
while Ph-Se-Acetyl-Arg-Ahx-CCK-4 (10) exhibits a higher unspecific binding value (14.6
%
the total binding at saturation doses), as shown in Figure S6.
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The K and B
values obtained from these experiments (Table in Figure 7) showed a
d
max
medium affinity of these selenium labeled ligands for the CCK-B receptor, in line with the
one of CCK-4 (7). Thus, the modifications undergone by the labeled peptides did not
induce a drastic impact on their affinity. It is worth noting that the differences observed for
the recorded experimental Bmax values probably resulted from varying expression rates
of CCK-B receptor in HEK cells between different batches.
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These promising results highlighted the specificity of the selenium tagged ligands for
the CCK-B receptor and their potential suitability as labeled ligands for affinity
measurements of new compounds with CCK-B receptor. In order to demonstrate it,
competition assays were carried out by using the three selenium labeled peptides with
non-sulfated CCK-8 (14) as competitor, in order to determine its affinity constant for CCK-
B receptor by using these new ligands.
Competitive binding experiments were carried out by displacing 34 nM of selenium
labeled ligand with increasing concentrations of the non-sulfated CCK-8 (14) competitor
-5
(
from 10-12 to 10 nM). The competition binding curves as well as the K values of non-
i
sulfated CCK-8 (14) for CCK-B receptor obtained for each selenium labeled ligand are
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shown in Figure 8. Recorded experimental results led to non-sulfated CCK-8 (14) Ki
values between 3.9 and 10.4 nM (Table in Figure 8). These values are in the same order
1
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of magnitude of the one found in the literature (non-sulfated CCK-8 (14) at 5.9 nM).27
CONCLUSION
We described here an alternative methodology to quantify receptor-ligand interaction
relying on selenium labeling combined to RP-LC-ICP-MS analyses, applied to both the
V1A and CCK-B receptors interaction measurement. Several approaches for selenium
labeling have been proposed according to the structural features of targeted ligands.
Provided that the overall structure was not perturbed, Met, Cys and Pro substitutions with
their respective selenium analogues SeMet, Sec and Sez as well as the introduction of a
selenium organic moiety at the N-terminal position afforded selenopeptide ligands
displayed similar binding properties to the native ones. New affine ligands for V1A and
CCK-B receptors were thus prepared. The proof-of-concept was demonstrated with the
V receptor where the developed nonradioactive binding assays were found as efficient
1A
and reliable as the conventional radiolabeling methodology. The versatility of our method
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was illustrated here with another receptor-ligand system, i.e. the CCK-B receptor and its
native CCK-4 ligand. This novel labeling strategy associated with bench elemental mass
spectrometer represents a highly valuable alternative to costly and hazardous
radiolabeling and radioactive measurements that must only be conducted in dedicated
laboratories with authorized trained scientists and thus opens up new opportunities in the
research of novel potential drugs in pharmacology.
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EXPERIMENTAL SECTION
Chemicals
SR 49059 (12) and SR 121463 (13) were purchased from Sigma-Aldrich. N,N-
dimethylformamide (DMF), anhydrous DMF, amine-free DMF and trifluoroacetic acid
(TFA) were obtained from Sigma–Aldrich. Protected amino acids, L-selenocystine, resins
and coupling reagents were purchased from Iris Biotech GmbH. Boc--Cl-Alanine was
purchased from Bachem and other reagents were purchased from Aldrich. All chemicals
and solvents used for analyses were of analytical grade. HF is a hazardous acid, which
can result in serious tissue damage if burns were not appropriately treated. HF treatment
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used for peptide synthesis should be performed in a well-ventilated fume hood with
appropriate face shield and double layered nitrile gloves.
Peptide synthesis
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The four selenium tagged V ligands ([Se-Se]-AVP (2), [Se]-AVP (3), [Sez ]-HO-Phpa-
1A
LVA (5) and [Ph-Se-acetyl]-LVA (6)) and three selenium tagged CCK-B ligands (Ac-
2
SeMet-Arg-Ahx-CK-4 (8), [SeMet ]-CCK-4 (9) and [Ph-Se-acetyl]-Arg-Ahx-CCK-4 (10))
were synthetized via Solid Phase Peptide Synthesis (SPPS) chemistry. The compounds
were characterized by MALDI-MS/MS and LC-ESI-MS experiments. Purities were
determined by integrating the UV chromatogram acquired in LC-ESI-MS as well as by
determining the Net Peptide Content (NPC) by elemental analysis. The synthesis as well
as the characterization of the peptides are described in the sections S1 and S2 of the
125
supporting information. The iodinated linear peptidic V1A antagonist [ I]OH-Phpa-LVA
(
11) 52 was available in the laboratory, its radioiodination was previously described.53
Native AVP (1), CCK-4 (7) and non-sulfated CCK-8 (14), ligands were available in the
laboratory and characterized prior to their use.53
Peptide purity:
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Purities were determined by NMR ( H spectra recorded at 298 K on a Bruker Avance
III 600 MHz NMR spectrometer, using TCI Cryoprobe Prodigy®), by LC-MS analyses
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(
signal corresponding to the peptide was integrated from the UV chromatogram recorded
at 214 nm) as well as by elemental analysis (by determining the NPC (Net Peptide
Content)). For the NMR spectra, chemical shift data are given in δ ppm calibrated with
1
residual protic solvent (e.g. DMSO : 2.50 ppm - H). All the peptides have been obtained
with an NMR purity percentage higher than 95%.
RP-LC-ICP-MS analyses
Standard solution of inorganic selenium was purchased from SCP Science
(Courtaboeuf, France). Water was distilled and purified by a Milli-Q system (Millipore,
Saint-Quentin en Yvelines, France). The quadrupole ICP-MS system was an Agilent 7900
Inductively Coupled Plasma Mass Spectrometer equipped with a Micromist nebulizer at
-1
4
00 μL min , X lenses, a Scott chamber cooled to -5°C and a collision reaction cell with
-1
hydrogen flow at 5 mL.min . The sampler and skimmer cones were made of platinum. 8
-1
%
of oxygen was added to the carrier gas (0.66 L.min argon) to avoid carbon deposition
on the cones. Two isotopes, 78Se and 80Se, were monitored with a dwell time of 960
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msec/isotope. LC-ICP-MS analyses were performed on a Dionex Ultimate 3000 HPLC
system, equipped with a degasser, a quaternary pump and an autosampler. The column
was a Zorbax Eclipse XDB-C8 2.1 x 100 mm, 3.5 µm (Agilent) thermostated at 20°C.
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-1
Analyses were carried out with a flow rate of 400 μL.min and an injection volume of 100
μL. Solvent A and solvent B were respectively constituted of water with 1 % of formic acid
(v/v) and methanol with 1 % of formic acid (v/v). An isocratic step in pure water (100 %
solvent A) was first applied for 1 minute in order to elute all polar contaminants (mainly
salts and very polar organic compounds). A second isocratic stage at 50 % of methanol
was reached in 0.5 minute. Then, the elution profile was slightly adapted to the peptide
2
sequences. For selenium tagged V ligands and [SeMet ]-CCK-4 (9), the best separation
1A
conditions were found to be an isocratic elution stage lasting 2 minutes at 50% of solvent
B, a gradient in 1 minute to reach 100% of solvent B with further 2 minutes of isocratic
elution (100 % Solvent B) whereas only 1 minute of isocratic elution at 50% of solvent B
and 4 minutes to reach 100% of solvent B suited better to [Ph-Se-acetyl]-Arg-Ahx-CCK-
4
(10) and Ac-SeMet-Arg-Ahx-CCK-4 (8) peptides, respectively. Finally, 100 % of solvent
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A (starting elution conditions) was reached within 1.5 minutes, an extra 3 minutes of
column conditioning gave a total run time of 11 minutes for all injected compounds.
Instrumental parameters were daily tuned with a 50% water/methanol solution of the
studied selenium analyte, corresponding to the conditions on which compounds are
eluted.
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Binding experiments
For the V1A receptor, binding experiments assays were performed using CHO cells
stably expressing the human V1A receptor cultured with pressure selection at 1 mg.mL-1
of Genetacin (G 418). Four selenium labeled ligands displayed in Figure 1 were used as
reference ligands. For the CCK-B receptor, binding experiments assays were performed
using HEK cells stably expressing the CCK-B receptor cultured with pressure selection
-1
at 1 mg.mL of hygromicin B. Three selenium labeled ligands displayed in Figure 2 were
used as reference ligands. For both V and CCK-B receptors, binding experiments were
1A
carried out on cells previously sowed on 6 multi-wells plate in 2 mL of binding buffer at
pH 7.4 (PBS with 1 mM of aqueous solution of CaCl ; 5 mM of aqueous solution of MgCl ;
2
2
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.1 % of bovine serum albumin (BSA); 40 µg.mL of Bacitracine and 1 mM of PMSF) with
1
or 34 nM of labeled ligand for V1A and CCK-B receptors respectively, and increasing
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concentrations of compound to be tested. Cells were counted the day of the experiment
using Malassez cells (1 000 000 CHO cells/well and 3 950 000 HEK cells/well, for V1A
and CCK-B receptors respectively). For saturation experiments, total binding was
determined with increasing concentrations of selenium labeled ligands (0.1 nM to 10 nM
or 0 to 75 nM for V and CCK-B receptors respectively) while for non-specific binding the
1A
same experiment was performed in the presence of a 2000-fold excess of AVP or a 300-
fold excess of non-sulfated CCK-8 for V and CCK-B receptors respectively.
1A
For both V1A and CCK-B receptors, incubation was performed for 4 h at 4°C. Assays
were performed in triplicate. After incubation, cells were washed two times with binding
buffer and then 200µL of dissociation buffer (1% TFA aqueous solution (v/v)) was added.
Mixture was transferred in Eppendorf tube and centrifuged 5 min at 12500 rpm.
Supernatant was directly analyzed by RP-LC-ICP-MS with the above described method.
Data were treated with non-linear model fitting programs (GraphPad PRISM 4). Bmax were
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