Luminescent lanthanide cryptates: From the bench to the bedside

Article abstract of DOI:10.1021/ic402234k

The design and application of luminescent lanthanide cryptates for sensing biological interactions is highlighted through the review of the work performed in our laboratory and with academic collaborations. The path from the initial applications probing b

Full text of DOI:10.1021/ic402234k

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pubs.acs.org/IC
Luminescent Lanthanide Cryptates: from the Bench to the Bedside
Jurriaan M. Zwier, Herve Bazin, Laurent Lamarque, and Gerard Mathis*
́
́
Cisbio Bioassays, Parc Marcel Boiteux, BP 84175, Codolet, France
S
* Supporting Information
ABSTRACT: The design and application of luminescent lanthanide
cryptates for sensing biological interactions is highlighted through the
review of the work performed in our laboratory and with academic
collaborations. The path from the initial applications probing
biochemical interaction in vitro to “state-of-the-art” cellular assays
toward clinical applications using homogeneous time-resolved
fluorescence technology is described. An overview of the luminescent
lanthanide macrocyclic compounds developed at Cisbio in the recent
past is given with an emphasis on specific constraints required by
specific applications. Recent assays for drug-discovery and diagnostic
purposes using both antibody-based and suicide-enzyme-based
technology are illustrated. New perspectives in the field of molecular
medicine and time-resolved microscopy are discussed.
Therefore, because of their unique photophysical and
spectral properties, we identified the rare-earth complexes as
being the key elements that could contribute to the design of a
new homogeneous technology for simple and automated
immunoassays. At this time, rare-earth chelates were already
known as tracers in separation immunoassays. The luminescent
chelate is formed in an extra step, after separation of the
biological or blood sample, in controlled media containing
surfactants and organic ligands.4,5
Rare-Earth Cryptates. When rare-earth chelates are used
as tracers in homogeneous assays, incubation and measurement
occur in biological fluids that suppose a high stability of the
chelate in the presence of a high concentration of endogenous
proteins and ions; otherwise, a loss of the signal is expected.
The stability of the existing chelates in biological media did not
fit the more stringent constraints of homogeneous assays.
Instead of the well-accepted criteria of high thermodynamic
stability, which drove the synthesis of new rare-earth chelates at
that time, we stressed the key importance for a high kinetic
stability in biological media for these new applications.6
This new paradigm opened the way for a new class of rare-
earth chelates based on macrocyclic backbones and
chemistry: the cryptates. A fruitful collaboration with Lehn
starting in 1983 allowed us to illustrate the pertinence of our
analysis and to synthesize a series of new europium and terbium
rare-earth cryptates with tuned photophysical properties. Their
conjugation with a diversity of biomolecules such as proteins,
peptides, oligonucleotides, etc., was instrumental in the
development of homogeneous fluorescent technologies.7−12
INTRODUCTION
■
In the early 1980s, rare-earth chelates were used as long-lived
tracers in heterogeneous immunoassays.1 In the same era,
Ullman et al.2 pioneered homogeneous immunoassay methods
particularly by using fluorescence resonance energy transfer.
Shortly after, following evolution of the immunological and
biological analytical field toward automation and high-
throughput screening, we launched a long-term research
program for developing technologies and reagents suited for
the monitoring and quantification of biomolecular interactions
in biological fluids.
The following elements that we thought would be mandatory
for the development of such technologies were identified: (1) a
highly sensitive analytical detection technology using a tracer
stable in biological media, (2) a modulation process of the
tracer signal connected to the biomolecular interaction of
interest, and (3) a measurement process that should be
unaffected by the variations of the optical properties of the
biological media.
This original global approach led us to identify at an early
stage the interest of homogeneous technologies (no separation
between bound and free tracer in an immunoassay) and
particularly those using the modulation of the sensitive
fluorescence signal.
However, in a homogeneous assay, the measurement takes
place directly in the biological fluid where the intrinsic
fluorescence of compounds and proteins can lead to serious
limitations of sensitivity because of a high background
obscuring the signal from conventional fluorophores in a
steady-state fluorescent measurement.
We recognized the need of long-lived luminescent tracers,
which as pointed out by Wieder3 using time-resolved
fluorescence detection, should eliminate most of the short-
lived protein and diffusion background contributions.
Special Issue: Imaging and Sensing
Received: September 2, 2013
© XXXX American Chemical Society
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̈
Time-Resolved Forster Resonance Energy Transfer
(TR-FRET). At the same time, at Cisbio, we explored the
photophysical processes of rare-earth cryptates and cryptates
that could be used in the design of assaying a real biological
environment in a homogeneous way.
The rapidly growing knowledge in cellular biology opens up
new fields of investigation with additional constraints and
challenges such as working with living cells and native tissues.
Nowadays, the bridge is being made between the discovery of
these biological mechanisms and the clinic, requiring new
optimizations and developments.
We discovered that, despite a low global luminescence
quantum yield, rare-earth cryptates could be used as efficient
fluorescent donors in resonance energy transfer (the rare-earth
ion acting as the donor in this case), leading to long-lived
emission of the fluorescent acceptor. Moreover, when an
acceptor that emits between the characteristic emission lines of
the rare-earth ions is chosen, a simultaneous detection of donor
and acceptor emissions allows to us use the rare-earth emission
as an internal probe to correct the acceptor emission from
variations of the optical quality of the biological media.10
In the early 1990s, by combining these elements, we
developed homogeneous time-resolved fluorescence (HTRF)
and pioneered the TR-FRET technology. The power of the
technology was illustrated, using the specific fluorescent
properties of rare-earth cryptates, to probe molecular
interaction mechanisms in biology and showed the following
unique features: (1) the use of rare-earth cryptate emission with
their atomic-like spectra allowing simultaneous detection of
donor and acceptor signals; (2) rare-earth cryptates allowing
Besides a review of the basics of the chemistry of rare-earth
cryptates and HTRF technology, the purpose of this Forum
Article is also to illustrate recent research to further develop
and adapt the technology to these new constraints and
environments for answering new challenges in the field of
molecular medicine.
PRINCIPLES OF LANTHANIDE-BASED TR-FRET
AND HTRF
■
The interesting luminescent properties of lanthanides have
been used throughout the scientific world and discussed
extensively in reviews.18−24 Briefly, the long lifetime of the
excited state of the lanthanide ion, which is linked to its low
absorption cross section, is its main feature. Because of the low
absorption cross section, direct excitation of the lanthanide is
cumbersome. Because organic chromophores are quenched by
lanthanide ions (see ref 24 and references cited therein), it
became clear that, to achieve increased excitation and emission,
organic chromophores should be coordinated to the lanthanide
ions. Our strategy to develop new lanthanide complexes is
driven by the needs of the pharmacologic and diagnostic
industries and the availability of affordable instrumentation to
measure biological interactions. Detection systems in the near-
IR are relative expensive and for economic reasons not an
option to incorporate into plate readers. Therefore only
europium and terbium complexes with strong emission bands
in the visible are of direct interest. Furthermore, excitation
sources in plate readers are usually limited to 320−340 nm
excitation wavelengths; hence, complexes need sufficient
absorption cross sections in this spectral region to be of
interest for our technology. The europium cryptates developed
by Lehn, Mathis, and co-workers in the 1980s8,25 proved to be
excellently suited,10,16 and using these compounds led to
development of the HTRF technology described for the first
time in the early 1990s by Mathis.10,16 Basically, the lanthanide
cryptates are used as donors in a FRET system using
compatible acceptor fluorophores such as the modified
fluorescent protein allophycocyanine (XL665)10,26 or the d227
dye, a polysulfonated cyanine, emitting strongly in the 665 nm
region. When the donor and acceptor are in the proximity and
FRET occurs, because of the millisecond lifetime of the donor
excited state, a delayed acceptor emission is observed that can
be discriminated from the fluorescence of the medium and
direct excitation of the acceptor by using time gating, which is
typically set at 50 μs for plate-based assays (Figure 1).
high Forster distances to probe most of biological interactions;
̈
(3) homogeneous assays leading to a very simple mix and
measure format easy for automation; (4) homogeneous assays
allowing kinetic measurements; (5) ratiometric measurement,
which allows one to avoid measurement issues due to optical
quality variation between samples.
This allowed us to decipher biomolecular interactions in the
real world of biology with applications most notably in clinical
diagnostics and drug discovery.
Clinical Diagnostics. The set of unique properties offered
by this fluorescent homogeneous technology specific to the
usage of kinetically stable rare-earth cryptates allowed our
team to pioneer the field and develop original instruments and
immunoassays for the measurement of important blood
circulating haptens, hormones, proteins, and cancer bio-
markers.13 The Kryptor technology14 was introduced to the
market in the late 1990s, showing high robustness and
convenience, particularly for the measurement of tumor
markers. It is the only example of immunometric homogeneous
technology on the market14 in the field of clinical diagnostics.15
Drug Discovery. Starting in the mid-1990s, we turned our
interests toward the needs of the research community involved
in drug discovery.16
Using rare-earth cryptates and TR-FRET, we have
investigated directly or through collaborations most of the
biological mechanisms that can be of interest as models to
identify new chemical entities for important pathologies [DNA,
RNA hybridization and mutation, nuclear helicases, telo-
merases, caspases, kinases, phosphatases, proteases, inhibitors
or activators, P53 and other protein−protein interactions,
epidermal growth factor receptor (EGFR), and other receptor
ligand interactions, all mechanisms involved in important
pathologies such as cancer, metabolic disorders, and degener-
acy].17
A second important feature due to the large pseudo Stokes
shift and to the atomic-like emission spectra of the lanthanide
complexes is that the donor intensity in the acceptor channel
(at 665 nm) is very low compared to FRET with typical organic
fluorophores. The third major parameter in HTRF is the use of
an acceptor over the donor intensity ratio. The ratiometric
treatment of the 620 and 665 nm intensity measurement allows
real-time correction of the optical properties of the assay
medium. This solves the problem of performing unbiased
fluorescence measurement in the absorbing or turbid medium
often observed either in clinical analysis or in drug discovery.
The demonstrated effectiveness of the technology, to answer
specific needs of the researchers in this field, makes HTRF
nowadays a standard for drug-discovery processes in the
pharmaceutical industry.
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extinction coefficient, sensitization through organic chromo-
phores is mandatory to obtain a lanthanide complex displaying
high brightness (quantum yield × extinction coefficient).
Criteria to design a suitable lanthanide (europium and terbium)
complex have been extensively reviewed18,21,24 and can be
summarized as follows: a high absorption cross section above
330 nm and especially around 337 nm (nitrogen laser); the
highest quantum yield possible; no quenching in biological
media, strong kinetic stability, high solubility in an aqueous
buffer, a strong emission spectral band around 620 nm
5
corresponding either for europium to the transition D0 →
7F2 or for terbium to D4 → F3 and an excited-state lifetime
above 1 ms. Furthermore, an orthogonal functional group
allowing bioconjugation is necessary to set up robust
immunoassays. Cryptate 1 based on a bipyridine subunit
(Eu⊂tris-bipyridine) containing two amine functionalities for
bioconjugation was among the first lanthanide complexes that
fulfilled the requirements. In the Eu⊂tris-bipyridines 1, the
rigidity afforded by the bipyridine moiety tends to increase the
activation parameters, and a ΔG* of approximately 25−30 kcal·
mol−1 can be reached. On the contrary, the transition state of
europium chelates is easily reached with a ΔG* of 0−10 kcal·
mol−1, for instance, by protonation of the carboxylate groups.6
Thus, this cryptate has been used extensively until now for
HTRF assays. Although it displays both good photophysical
and chemical properties, this cryptate has several drawbacks. It
needs the addition of potassium fluoride to prevent quenching
(deactivation of the luminescent excited state) because of the
presence of two water molecules in the europium coordination
sphere.28 Second, the excited-state properties are not suitable
for the corresponding terbium cryptate, and the absorption
spectrum is not optimal at 337 nm. To circumvent these points,
we developed new lanthanide macrocyclic complexes. Because
of the very stringent set of physicochemical properties needed
for real-world applications, the main difficulty was to tune one
parameter without affecting the other factors, which contribute
also to the overall luminescent properties.
5
7
Figure 1. Schematic view of timing in HTRF assays. After excitation
by either a flash lamp or a laser source, the donor (black curve, 620
nm) and the delayed acceptor (red curve, 665 nm) emissions are long-
lived. Direct emission from the acceptor or compounds, proteins, and
medium (orange curve) present in the well and the background
emission/scattering from the matrix (purple curve) are suppressed by
applying a delay of about 50 μs. Typically, a measurement window
between 50 and 450 μs after excitation is applied in HTRF assays.
Taken together, this measurement strategy is of prime
importance in the development of highly efficient and robust
immunoassays.
LANTHANIDE COMPLEXES
■
Opposed to the main trends, we pointed out early on6 that the
key parameter to design an efficient fluorescent probe for
immunoassays was driven by kinetic considerations. Indeed, we
focus our research on lanthanide macrocycles that display high
kinetic stability in biological media, and the logical choice was
to investigate cryptates such as 18 and their derivatives. More
recently, we also started to investigate macrocycles bearing
pendant arms (see Charts 1 and 2) to stabilize the complex that
might be interesting enough to be incorporated into an
immunoassay. Because the lanthanide atoms possess a very low
To shift the maximum of the excitation wavelength
bathochromically, several antennas have been studied in the
cryptate framework.29−31 The best compromise was obtained
Chart 1. Europium Macrocycles Studied in This Paper
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Chart 2. Recently Developed Terbium and Europium Macrocycles Discussed in This Paper
with Eu⊂tris-bipyridine·4CO2H·cryptate 2 because the addi-
tional carboxylate function shifts the maximum of the excitation
wavelength through extended conjugation and does not affect
the solubility in the biological buffer.28 One of the europium
excited-state quenching processes is assigned to ligand-to-metal
charge transfer or photoinduced electron transfer enabling
reduction of Eu3+ (millisecond luminescent lifetime) to Eu2+
(nanosecond luminescent lifetime in solution32).28,33 Eu⊂pyr-
idinebipyridine cryptate 3 has been synthesized to prevent this
quenching by reducing the size of the cavity. Although the goal
to increase the luminescence quantum yield was achieved, the
environment around europium changed and therefore shifted
the 5D0 → 7F4 emission band within the acceptor measurement
channel, which dramatically affected the immunoassay
sensitivity.28
Complexes based on DOTA macrocycles have been designed
and assessed in collaboration with the Parker group.34,35 Several
complexes have been designed including 2-pyridylazaxanthone
4, 2-pyrazoylazaxanthone 5, or the less sterically hindered 3-
azaxanthone 6 antennae, allowing sensitization of either
europium or terbium. These complexes were not suitable for
our applications because they proved to be quenched or
partially quenched in the presence of divalent cations such as
Mn2+ possibly through an electron-transfer mechanism.35 In
lanthanide chelates, divalent cations present at high concen-
trations in biological fluids can displace the lanthanide ions,
destroying its luminescent properties.21
Some new complexes whose skeleton has been initially
developed by Picard and co-workers36 were synthesized with an
extra-cyclic functional group, allowing bioconjugation.37 The
new synthetic approach generated flexible framework useful to
screen chromophore efficiency. To validate the strategy,
Eu⊂tpy (2,2′:6′,2″-terpyridine) 7 and Eu⊂dpqc (dipyrido-
6,7,8,9-tetrahydrophenazine) 8 have been synthesized, and we
demonstrated that the conjugates maintain the photophysical
properties of the complex itself.37
Considering terbium complexes, spectacular improvements
have been reported by the group of Raymond with the so-called
Lumi4-Tb 9.38 In our hands, we confirmed the exceptional
brightness of the complex based on the 2-hydroxyisophthala-
mide moiety, and after bioconjugation, it is now broadly used in
HTRF applications. The benzylguanine derivative 9b (see the
Supporting Information for the syntheses; so far unpublished
data) proved to be excellently suited for studies on cell surface
receptors (vide infra).
Taking advantage of the results reported by Maury and co-
workers,39 where an electron-rich arylalkynylpyridyl chromo-
phore was used, we recently developed new europium
complexes involving an intramolecular charge-transfer mecha-
nism. Initially, we designed the new europium complex 10,
which displayed poor luminescent properties because of
unexpected nonradiative processes induced by an internal
isomerization equilibrium.40 We circumvented these issues by
incorporating the latest chromophore on a 1,4,9-triazacyclono-
nane macrocycle. Indeed, these complexes 11−13 (Chart 2)41
display exceptional brightness, optimal maxima of the excitation
wavelength (between 335 and 345 nm), high stability in the
presence of divalent cations, a very intense 620 nm emission
band (symmetry around europium is of the C3 type), the
absence of water molecules in the inner sphere, and excited-
state lifetimes around 1 ms. This new family of europium
macrocycles is probably the brightest europium complex we
have had in our hands, which allows us to envisage in the future
very broad applications in terms of immunoassay sensitivity but
also in the fluorescent microscopy field.41,42
BIOCHEMICAL APPLICATIONS
■
Luminescent lanthanide crypates, as illustrated in the previous
section, are developed and optimized in order to be applicable
for industrial applications. This translation is not evident and
needs continuous reevaluation depending on the demands. It is
interesting to observe the changes over a long period of time
not only in requirements but also of trends in the assay design.
The development of these applications was driven by the
naissance in the early 1990s of a new paradigm in the
pharmaceutical research based on the availability of large
chemical libraries and the belief that the possibility to screen as
many compounds as possible would increase the probability to
identify new active chemical entities. Therefore, technologies
like HTRF, allowing homogeneous (no separation steps)
testing of compounds on biochemical targets with high-
throughput rate, were introduced in the discovery process of
a major pharmaceutical company.
We have therefore developed a large panel of biochemical
assays for this purpose, exploring many different biological
interactions using TR-FRET technology. The first HTRF
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applications of europium cryptates such as 1−3 are exemplified
below. A sensor to detect prolactin in serum is one of the first
examples of an HTRF assay.10 Two antibodies, labeled with 1
as the FRET donor and a cross-linked allophycocyanine as the
FRET acceptor, were used to design a homogeneous sandwich
immunoassay (proximity assay).
The versatility of the HTRF technique was further
exemplified with various models representative of molecular
and cellular processes chosen from the signaling pathways
involved in cellular communication and expression. Assays such
as epidermal growth factor receptor (EGFR)−ligand inter-
action, EGFR kinase activity, and Jun/Fos protein−protein
interaction were developed as proof of concept.16
These early examples of HTRF assays43 paved the way for a
whole range of assays such as kinase,27,44 biomarker27 and
bioprocess assays,27 to name a few.
Time-Resolved Fluorescence Using Europium Tris-
bipyridine Cryptate Labeled DNA Probes. The earliest
application of 1 was performed using the corresponding
streptavidin conjugate (1−SA) for the indirect labeling of
either short oligodeoxynucleotide (ODN) or long DNA
probes.9 Such labeled probes were used for the detection of
DNA sequences by hybridization on membranes (dot blot); in
this assay, time-resolved detection could improve the signal/
noise ratio, decreasing dramatically the fluorescence back-
ground arising from the membrane. The sensitivity thus
obtained was of the same order as the colorimetric protocol
based on enzymatic signal amplification. The clear advantage is
that, as in radioisotopic labeling, one can perform a real-time
measurement in contrast with colorimetric methods, which are
typical end-point measurements.
A similar fluorescent-detection procedure was used to detect
amplified DNA combining the sensitivity and specificity of a
nested polymerase chain reaction (PCR)45 with the advantages
of time-resolved fluorescent detection.46 After a first PCR
amplification and dilution of these PCR products, a second
amplification was carried out, using a pair of labeled
oligonucleotide primers bearing biotin and dinitrophenyl
(DNP) groups, respectively. After affinity collection of the
double-labeled PCR amplicons on a SA-coated microtiter plate,
fluorescence detection was carried out using an anti-DNP
antibody labeled with 1 and time-resolved measurement using a
laser source. Interestingly, this protocol could be used with
either cryptate 1 or a 125I-labeled anti-DNP antibody to
compare the sensitivity of time-resolved fluorescence detection
versus radioisotopic detection.
Europium Tris-bipyridine Cryptate Labeled Nucleo-
side Triphosphates. The 5-(aminoallyl)deoxyuridine deriva-
tive was used as a thymidine triphosphate analogue and is
labeled with 1. This labeled triphosphate (K-11-dUTP) could
be enzymatically incorporated in the 3′ position of an ODN
using the terminal deoxynucleotide transferase and in DNA
fragments using DNA polymerases.47 K-11-dUTP was also used
to monitor the extension reaction of a biotinylated oligonucleo-
tide as the substrate for telomerase in a telomeric repeat
amplification protocol, as described in Figure 2. After the
addition of an allophycocyanine−SA conjugate, the extension
products give rise to FRET with the incorporated cryptate.48
1-labeled uridine triphosphate (K-11-UTP) was obtained
similarly by coupling the 1 moiety to a 5-aminoallyl-
functionalized UTP analogue. K-11-UTP can be directly
incorporated into RNA strands during enzymatic synthesis.49
This was demonstrated in an in vitro transcription reaction
Figure 2. HTRF extension assay by the incorporation of a cryptate-
labeled nucleotide into a biotinylated oligonuleotide primer. This
format is exemplified by the telomerase-mediated incorporation of K-
11-dUTP. The cryptate-labeled K-11-dUTP analogue is incorporated
during enzymatic elongation of the biotin−ODN primer. After the
addition of a SA−XL665 conjugate, a FRET signal is detected between
the incorporated cryptate donor and the XL665 acceptor. A similar
assay format can be used to monitor the incorporation of K-11-UTP in
RNA.
promoted by T(7) RNA polymerase. The reaction was
performed in the presence of K-11-UTP and biotin-labeled
cytidine triphosphate (biotin-16-CTP). After the addition of
the SA−XL665 conjugate, a FRET signal between the
europium cryptate and the SA−XL665 acceptor bound to the
biotin residues thus incorporated is observed. The study of this
FRET detection assay format showed that such doubly labeled
RNA can be easily detected even when a very low percentage of
K-11-UTP is used (less than 1% of the total UTP
concentration).
Detection of Single-Nucleotide Polymorphisms
(SNPs) Using an Oligonuleotide Extention Assay. Because
solid tumors contain a mixture of cancer cells representing
SNPs (or mutations) and normal cells, a mutation present in
the tumor sample may represent only a minor fraction of the
total DNA. Therefore, tumor sample analysis requires detection
strategies ensuring high sensitivity. Detection of known
mutations can be broadly classified as either hybridization- or
enzyme-based methods; the latter are inherently more specific
because they rely on discrimination by an enzyme.
This enzymatic reaction can be a ligation, as in
oligonuleotide ligation assay,50 or a polymerase, as in
minisequencing, also known as SNP extension (SNuPE) or
template-directed dye terminator incorporation.51
In SNuPE assays, a DNA polymerase is used to label a
specific primer hybridized to the target sequence by
incorporating a single-labeled dideoxynucleotide (ddNTP).
Such incorporation can be conveniently monitored using
HTRF detection, as described in Figure 3.52 The ODN primer
was labeled on the 5′ end by coupling with 1. In the presence of
polymerase (sequenase) and of the biotin-labeled dideoxynu-
cleoside triphosphate, complementary to the base next to the
primer 3′ end, this ddNTP was incorporated only if the primer
sequence was fully matching the sequence to be analyzed. This
was exemplified using a portion of the coding sequence of the
exon 7 p53 gene, CCG represented the wild-type codon 248
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because of the need to work with living cells. A selection of the
cell-based applications developed is discussed in this section.
In Vitro Assays To Detect Stimulation of Membrane
Receptors. In drug discovery, there is a need to use native and
more physiological models; hence, cell-based assays are
nowadays key technology to validate drug candidates. The
ultimate goal is to monitor interactions on unmodified cell lines
and even native tissues, but expression levels of the protein of
interest are often low. To circumvent this problem, the DNA of
the protein of interest is inserted into standard cell lines,
leading to its expression, and can be used to monitor cellular
responses of interest for drug discovery. Such responses can be
signaling events within the cell after agonist stimulation of the
receptor of interest. The changes of the concentrations of
molecules involved in these reactions can be measured with
HTRF-based biosensors.
Our main focus in recent years is the so-called G-protein-
coupled receptor (GPCR)54,55 or seven-transmembrane
receptor (7TM) family, which is one of the most important
drugable families56 with up to 600 different proteins present in
the human body. Basically, after stimulation of a GPCR with an
agonist, the G-protein(s) are activated, which involves the
displacement of guanosine diphosphate by guanosine triphos-
phate (GTP), and consequently signaling cascades within the
cells are triggered. In the past decade, it also became evident
that GPCR can also respond independently from G-
proteins;57,58 hence, the 7TM abbreviation has become more
popular. For pharmaceutical companies, it is of prime interest
to know the response of their libraries on the protein/receptor
of interest. Primary screening is done by monitoring
concentration changes of the second messengers involved in
the signaling cascades such as Ca2+ ions or 3′−5′ cyclic
adenosine monophosphate (cAMP). After analysis of the
results, counterscreens and/or secondary screening on a subset
of the initial library can be performed. This can involve ligand-
binding studies, receptor internalization, protein phosphor-
ylation, or β-arrestin recruitment as examples.
We have developed such functional assays or biosensors
based on HTRF to monitor these signaling cascades. Two
examples will be treated that form the basis of our GPCR
portfolio: cAMP27 and IP-One59 assays. cAMP has been
identified as a secondary messenger because its concentration is
dependent on the up or down regulation of adenylate cyclase,
which is involved in the signaling pathway of the G-proteins
Gαs- and Gαi-coupled GPCR, respectively. The assay principle is
shown in Figure 4. A monoclonal antibody labeled with 1
recognizes a cAMP molecule conjugated with a red-emissive
acceptor dye (d2). Upon formation of cAMP, the generated
HTRF signal will disappear because of competition of the
unlabeled cAMP with the d2-labeled cAMP. The Gq-coupled
receptors, which activate the phospholipase C pathway, have
traditionally been monitored by measuring the [Ca2+]
increase.60 However, this signal, triggered by D-myo-1,4,5-
inositol triphosphate, is transient, and thus for high-throughput
screening (HTS) is not an ideal indicator. However, D-myo-1-
inositol monophosphate (IP1), a further metabolite in the
cascade, can be accumulated by inhibiting the enzyme inositol
monophosphatase by the Li+ ion. By using this propensity, the
accumulation of IP1 can be measured59,61 using a biosensor
similar to the cAMP assay described above. The key to
developing these assays is not just choosing the right donor and
acceptors but also developing high-affinity antibodies. Both of
these assays are now routinely used in HTS.27,62 Current
Figure 3. Detection of SNPs using an oligonuleotide extension assay
based on HTRF detection. This figure describes the procedure used
for codon 248 minisequencing in the p53 tumor suppressor gene. It
exemplifies HTRF detection after the enzymatic incorporation of a
labeled nucleotide onto a cryptate-labeled oligonucleotide primer. The
DNA containing the SNP to analyze is first PCR-amplified and then
purified by enzymatic digestions. A cryptate-labeled sequence-specific
extension primer hybridizing to the antisense strand of the amplicon is
annealed immediately adjacent to the polymorphic site. Extension in
the liquid phase is performed in the presence of three nonlabeled
ddNTPs and one biotin-labeled ddNTP (here using biotin−ddTTP as
an example, leading to incorporation). Following the extension
reaction, SA−XL665 is added and a TR-FRET signal is observed.
and CCA the corresponding mutated sequence. Biotin−ddCTP
was incorporated by the enzyme on the primer 3′ end only in
the presence of the wild-type codon; conversely, biotin−
ddGTP was incorporated only in the presence of the CCG-
mutated codon. Neither the corresponding ddATP nor ddTTP
was incorporated. After the addition of SA−XL665, the
incorporation of biotin−ddNTP was monitored by the FRET
appearing between the europium cryptate donor and the SA−
XL665 acceptor interacting with the biotin residue incorpo-
rated.
Mutation Detection Using DNA−Protein Interaction.
The detection of mutation based on hybridization alone suffers
from high false-positive rates, the use of a protein as auxiliary
can improve the specificity, and this was exemplified using
mutS, which is a protein involved in DNA mismatch repair
processes. An indirect HTRF assay format was designed using
6-His-tagged mutS and XL665-labeled anti-(6-His) mAb in
association with 1−SA conjugate and biotinylated 21-mer DNA
duplexes.53 This assay was used to analyze the interactions
between mutS and mismatched DNA or DNA containing the
most common lesion of the anticancer drug cisplatin. A
semidirect DNA/mutS HTRF assay using a 1−mutS conjugate
was also tested. Interestingly, a mobility shift assays showed
that the 1 labeled mutS retains its DNA mismatch binding
affinity.
CELL-BASED ASSAYS
■
In the previous section, we have shown that HTRF assays can
address various biological interactions. The incredible progress
in the knowledge of cell function and the discovery of new
biological mechanisms were identified as possible important
targets for drug discovery. This opened up a new field for
development of the technology, however with new constraints
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dissociation, which will lead to artifacts in biological systems.
To develop assays at the outer surface of the cell membrane, it
would be necessary to tag a protein with a lanthanide cryptate.
One of the first requirements is that labeling would only occur
at the cell surface. If substrates would penetrate the cell
membranes easily, labeling of immature proteins and proteins
unavailable for response could increase nonspecific interactions
on the assay and reduce the efficacy and dynamic range of the
assay. Second, nonspecific labeling of the substrate to the cells
or plates should be minimal. Third, the reactivity of the
substrate should be elevated to reduce the costs and minimize
the chance for the first two requirements to occur. Although it
is often argued that protein tags might alter the functionality of
the protein, with the SNAP tag,67 we have encountered very
few examples where this was indeed the case.
Figure 4. cAMP HTRF assay principle. In this competitive assay,
cAMP is covalently labeled with the d2 dye, and an anti-cAMP
antibody is labeled with a cryptate donor. Upon stimulation of the
receptor of interest in living cells, cAMP is generated. After cell lyses
and the addition of two labeled compounds, unlabeled cAMP will
compete with cAMP−d2, and thus a decrease of FRET is observed.
The concentration of cAMP can be determined as well using this
cAMP sensor. The detection of inositol monophosphate (IP-One
assay59) operates through a similar strategy.
The first case study, in collaboration with the Pin laboratory
at the Institut de Genomique Fonctionnelle in Montpellier,
́
proved the power of the combination of the SNAP tag and
antibody labeling, and TR-FRET technology was done on
oligomerization studies of the GABAB and mGluR1 receptors,71
GPCRs, which are involved in brain signaling of the
neurotransmitters γ-aminobutyric acid and glutamate, respec-
tively. Tagging these receptors using the SNAP tag and labeling
with a combination of benzylguanine (BG)−europium cryptate
and a red fluorescent acceptor proved for the first time that
tetramers (or dimers of dimers of GABA-B1−GABA-B2)71 of the
GABA receptor exist in living cells. A further improvement of
the system was achieved by designing the BG derivative 9b
including the very bright Lumi4-Tb.38 This compound proves
to be very useful not only because of its brightness but also
because of its high reactivity toward SNAP-tag labeling and its
low nonspecific labeling of cells not expressing SNAP-tagged
proteins. Another advantage of the Tb3+ donor is that TR-
FRET can be measured at several spectral windows using
fluorophores emitting, for instance, in the green or red regions
of the spectrum. This opens the possibilities for multiplexing
(examples of terbium multiplexing are given by Geißler et al.72
in this Forum issue).
With this so-called Tag-lite technology in hand, we have
developed target-specific homogeneous ligand-binding assays
on living cells using in-house-developed pharmacologically
active fluorescent ligands.73,74 Traditionally, binding assays75
are done using radioactive compounds and have for a long time
been the only way to address receptor pharmacology. It is,
however, not practical for high-throughput screening purposes.
Fluorescent ligand-binding assays have appeared since,75,76 but
still washing steps are often required and nonspecific binding
involved often cannot be ruled out. In the Tag-lite binding
assay, TR-FRET only occurs when a true binding event is
measured because it is measured by a proximity interaction
between the two binding partners. In Figure 5, an example of
the fluorescent ligand binding and competition binding Tag-lite
assay on the cholecystokinin-2 (CCK2) receptor is shown. Tag-
lite assays for 18 GPCRs have been described by Zwier et al.74
Assays for more GPCRs such as the Bombesin, Serotonin,
Muscarinic, and Glucagon receptors have been addressed using
this technology.77 Currently (June 2013), there are 56 different
assays available.77
ongoing efforts are made to develop sensors for later signaling
events in these cascades and G-protein-independent signaling
such as the detection of mitogen-activated protein kinases,
tyrosine kinases, and phosphorylation. An example is an assay
to detect and quantify ERK1/2 phosphorylation.63
TR-FRET Assays on Living Cells To Determine the
Membrane Receptor Structure, Conformation, and
Ligand Binding. Performing TR-FRET assays on living cells
has supplementary requirements of the lanthanide complexes
used.
(1) Because high-ionic-strength buffers disturb the cell
structure, using concentrated KF solutions to enhance the
luminescence of 1 is cumbersome.
(2) Nonspecific interaction of the lanthanide with the cellular
environment needs to be negligible to reduce the signal from
events not of interest to the interaction under study.
(3) Studying interactions at the cell surface requires that the
lanthanide conjugate in question does not adhere to the cell
membrane and should not internalize.
In the last 2 decades, receptor pharmacology has been
revealed to be much more complex than previously thought.
One of these complexities arises from the notion that GPCRs
are not just occurring as entities on themselves but can form
complexes at the plasma membrane with other GPCRs, which
can modify the pharmacology of these receptors.64 Compounds
are discovered that can bind preferably to such homo- or
heterooligomers, and hence target-specific assays will become
more important. Antibody-based strategies are the most
straightforward way to proceed, and, indeed, upon using a
Eu⊂pyridinebipyridine cryptate (3)-labeled anti-HA antibody
and an Alexafluor-labeled antimyc antibody, it could be
validated that GABAB1−GABAB2 exists as heterooligomeric
species in living cells.65 Rondard et al.66 used the anti-HA- and
anti-FLAG-labeled antibodies to show that N-glycan at the
GABAB2−venus fly trap (VFT) domain interface prevents
dimerization with GABAB1. To address the need for target-
specific assays at the cell surface and to reduce the possible
steric effects of antibodies, we developed a strategy based on
the so-called “suicide enzymes”, such as the SNAP,67 CLIP,68 or
HALO tag.69 Because of their nature, these proteins can only
react once toward their substrate, forming a covalent bond.
Although many protein-tagging systems exist,70 a covalent
strategy is needed because we would like to monitor
interactions at low concentrations (picomolar to nanomolar
range) and noncovalent techniques are more prone to
Inhibition constants measured in these Tag-lite assays are in
the same range as radioactive assays, and signal-to-noise ratios
are elevated. Furthermore, these assays can be done on cell
suspensions and can be measured in a HTS setting using 384
and 1536 well plates. Furthermore, after cells are labeled with
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the ghrelin Tag-lite assay is more sensitive than the radioactive
assay and that they were equally well able to properly
characterize the ligands. A first opening toward HTS of the
pleiotropic cytokine hormone leptin receptor OB-R has also
been shown by labeling leptin with the d2 acceptor and
monitoring its binding and competition using the Tag-lite
format.78 Recently, by using three time-resolved ligand-binding
protocols using a d2 acceptor or 9-labeled substrates/ligands,
specific antibodies or both show that these techniques all are
capable of measuring similar binding constants with high signal-
to-noise ratios.79
Homo- and Heteromerization of GPCRs. Although
oligomerization (or the formation of higher order protein
complexes) of GPCRs has influenced the field for over a decade
now,80 assays to address this changed pharmacological
landscape have been sparse.81 The formation of homooligomers
can be addressed using the Tag-lite technology using the SNAP
tag. Basically, a standard concentration of 9b is set while
varying the concentration of the red or green acceptor BG
derivative. If there is oligomeric organization, one will see a
peak of the TR-FRET emission when there is an equivalent
concentration of donor- and acceptor-labeled receptors, as
shown for the GABAB and MgluR1 receptors.71 On the M3
receptor, this and other strategies have allowed to show that
this receptor is oligomeric and that its organization is regulated
by ligand binding.82,83 To address heterodimers, orthogonal
protein-labeling methods are necessary. In order to address the
dimeric landscape of mGluRs, such a strategy was developed by
Doumazane et al.84 All eight mGluRs were tagged with either
SNAP or CLIP tags and labeled with 9b and green fluorescent
SNAP or CLIP substrates. This strategy was used to
demonstrate that members of the mGluR family can form
heterodimers but that this is restricted to strict dimers and to
only certain groups within this receptor family. Using these
SNAP-tagged mgluRs, a very powerful TR-FRET sensor
Figure 5. Tag-lite binding assay for the CCK2 receptor.74 SNAP-
tagged CCK2 receptors expressed in HEK293 cells are covalently
labeled with 9b. Binding of the fluorescent CCK(26−33) ligand to the
receptor (green curve) can be followed by measuring the 665/620
HTRF ratio. Nonspecific binding (red dots) is measured by adding 10
μM CCK8S to these wells. A competition binding assay is done by
adding 10 nM red-CCK(26−33) to the SNAP-Lumi4-Tb-labeled cells,
which leads to a decrease in the HTRF ratio upon the addition of
increasing amounts of a competitor ligand. In this case, an assay
window (or dynamic range) of 67 is observed.
9b, the cells can be frozen and after shipment defrosted for use
in assays. For the ghrelin receptor GSH-R1a,73 14 different
ligands were tested in parallel with a radioactive assay on 384
well plates using prelabeled cells in suspension. It is shown that
Figure 6. Metabotropic glutamate receptor 2 (mGluR2) TR-FRET activation sensor on living cells.85 (a) Cartoon illustrating the assay principle.
The SNAP-tagged mGluR2 dimer is labeled with 9b and SNAP green, which leads to a green delayed emission signal at 520 nm upon excitation of
the donor. Upon addition of the natural agonist glutamate, it is bound to the VFT binding domain, activating the receptor. This increases the
distance between the chromophores, reducing the FRET intensity. Adding the antagonist LY341495 reverses the system. (b) TR-FRET output
monitored in time. After 30 s, 100 μM glutamate is added, decreasing the FRET ratio. At 60 s, LY341495 is added, restoring the original signal. See
Doumazane et al.85 for more details.
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Figure 7. TR-FRET assay format (left) to determine the expression levels of HER2 receptors in breast tumor samples. Trastuzumab labeled with 9
and d2-labeled FRP5 antibodies, which recognize different epitopes of the HER2 receptor, give rise to a TR-FRET signal.92 A comparison of the TR-
FRET assay with standard techniques for assessment of the HER2 status is depicted on the right (reproduced from Ho-Pun-Cheung et al.,92 an open
access source). The number of HER2/cells was evaluated by TR-FRET in breast tumors stratified according to their Herceptest score and the HER2
gene amplification status. A Herceptest score of 3+ is considered positive, and patients with this score are considered for Trastuzumap treatment.
technology was developed to study the conformational changes
of the extracellular ligand-binding domains of these receptors in
living cells.85 After stimulation of mGluR with agonist, the
labeled dimer changes conformation, resulting in an increased
distance between the fluorophores and reducing the TR-FRET
(see Figure 6). Adding antagonist reversibly increases the
FRET efficiency again, leading to a sensor with a dynamic range
of up to 10, which is exceptional for fluorescent protein-based
sensors. With this method, it is possible to study activation
processes of mGluR in a relatively straightforward way and to
identify the actions of partial agonists, positive- and negative-
allosteric modulators.
The dynamics of the formation and stability of GABAB1−
GABAB2 oligomers on living cells have equally been studied
using a TR-FRET multiplex detection strategy using SNAP/
CLIP-Lumi4-Tb and red- and green-emitting acceptors.86 On
the same receptor, Comps-Agrar et al.87 have used a TR-FRET
strategy with Lumi4-Tb- and d2-labeled antibodies to monitor
the existence of higher-ordered oligomers of GABAB1−GABAB2
in the brain and evaluated the cooperativity of these oligomers
using a mixed SNAP/antibody TR-FRET strategy.
al.90 using an approach based on labeling with 3 or 9 and
fluorescent acceptors of agonists and antagonists of the
vasopressin and oxytocin receptors. They succeeded in
demonstrating the presence of oxytocin receptor dimers or
oligomers in mammary glands of lactating rats.
Ex Vivo HER1 and HER2 TR-FRET Assays on Tumor
Tissue. The development of cancer immunotherapy using
monoclonal antibodies such as Cetuximab (Erbitux) targeting
EGFR (HER1) for colorectal cancer treatment or Trastuzumab
(Herceptin) targeting HER2 for treatment of breast cancer are
associated with a companion test predicting response to
therapy. For instance, treatment with Trastuzumab is indicated
in cases where HER2 expression has a score of 3+ determined
by immunohistochemistry (IHC) in association with either
chromogenic or fluorescent in situ hybridization (CISH/FISH)
for the ambiguous IHC scores. Efforts have been made to
standardize such IHC analysis and improve the accuracy and
reproducibility;91 however, the technique remains semiquanti-
tative.
In this respect, a quantitative HER2 assay on tumor tissue is
of interest.93 The development of new therapeutic monoclonal
antibody programs would also benefit from cellular or ex vivo
assay able to quantify receptor expressions and analyze receptor
associations (homo- and heterodimers). A TR-FRET protocol
has been developed to quantify EGFR/HER2 heterodimers on
the cell surface in order to predict the efficacy and explain the
mechanisms of action of therapeutic mAbs.94 Cancer cells
cultured in microtiter plates were incubated with various
anticancer agents, and after a short fixation step, 9-labeled anti-
HER2 and d2-labeled anti-EGFR antibodies were added. After
incubation and washing, the time-resolved fluorescence of the
Lumi4-Tb donor and d2 acceptor was measured respectively at
620 and 665 nm; the augmentation of a long-lived 665 nm
signal was correlated with the presence of EGFR/HER2 dimers.
The use of two anti-EGFR antibodies targeting distinct
epitopes and respectively labeled with 9 and d2 allowed
quantification of the EGFR expression. Similarly, the use of two
labeled anti-HER2 antibodies allowed quantification of the
HER2 expression in tumor cells, which is illustrated in Figure 7.
The use of a single antibody labeled with either Lumi4-Tb or
TR-FRET ON NATIVE TISSUES
■
Finally, we turn toward practical applications at the bedside. To
support the development of molecular medicine, there is a need
to bridge the gap between cellular models and native tissues
from a patient in order to stratify the responsive population
and/or provide biomarkers for treatment efficacy. One of the
new paradigms in this field is the importance of kinase receptor
expression and oligomerization. This is the new playground
where we focus our efforts in the development of the
technology. As a proof of concept, we are exploring EGF
receptor family receptors that are known as key proteins in the
development of cancer.88,89
GPCR Oligomerization in Native Tissue. Although the
evidence is growing that oligomers of GPCR play a significant
pharmacologic role,64 sensor technology to monitor the
protein−protein interactions on native tissue is still lacking. A
first example of TR-FRET assays between high-affinity
fluorescent ligands on native tissue is described by Albizu et
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Figure 8. TR-FRET assay principle to monitor HER2 dimerization on tumor samples. Using an antibody labeled with either 9 or d2, only a positive
signal will occur when there is a proximity interaction between the two units. The corrected TR-FRET intensity is proportional to the dimer
concentration. Using different labeled antibodies, EGFR−EGFR or EGFR−HER2 dimer concentrations can also be obtained. On the right, a
Kaplan−Meier curve for disease-free survival according to the presence or absence of HER2−HER2 homodimer assessed by TR-FRET in IHC−
HER2 negative/ER positive patients. Note: these data were previously published at a conference.97
d2 allowed quantification of receptor homodimers (EGFR/
EGFR or HER2/HER2). The assay principle is illustrated in
Figure 8. Similarly, this protocol was used to quantify EGFR/
HER2 dimers and study the efficiency of various anticancer
treatments on the EGFR/HER2 dimer disruption.95 Recently,
an adapted version was also used to quantify HER2/HER3
heterodimers and study the effect of an anti-HER3 and anti-
HER2 (trastuzumab) combination on HER2 low cancer cell
lines, for which trastuzumab alone shows no or moderate
efficiency.96 This protocol was extended to the ex vivo TR-
FRET detection of EGFR, HER2, and homo- and heterodimers
in breast tumors cryosections.92 In this ex vivo assay, with the
cell number present in the sample being unknown, the 665 nm
TR-FRET signal was normalized with the prompt fluorescence
of the Hoechst-stained DNA measured simultaneously; the
normalized signal is proportional to the receptor expression per
cell. The EGFR expression determined by TR-FRET was
significantly correlated with a EGFR mRNA copy number, and
HER2 overexpression was confirmed by the standard IHC,
FISH, and qPCR analyses.
suppressing background fluorescence, as has been demon-
strated to increase detection limits significantly.18,42 Ghose et
al.98 showed that using antibodies labeled with the
pyridinebipyridine cryptate 3 it was possible to monitor
GABAB1−GABAB2 heterodimers using a time-gated setup as a
basis for future TR-FRET imaging, which was indeed recently
described using derivatives of 9 by Rajapakse et al.99 Using the
SNAP-tag system, it proved also possible100 to monitor
lanthanide-based time-resolved imaging of GPCR, as is shown
in Figure 9. After labeling vasoactive intestinal peptide receptor
Analysis of the quantity of HER2 homodimers measured by
the normalized TR-FRET assay predicts a disease outcome in
IHC−HER2 negative/ER positive breast cancer patients for up
to 10 years of followup.97 Using Cox proportional hazard
analyses, the presence of a HER2 dimer was significantly
associated with reduced disease-free survival (p = 0.0001;
Figure 8).
Figure 9. VPAC1-ST internalization monitoring by time-resolved
imaging.100 (a) CHO cells expressing VPAC1-ST are labeled with 9b
and imaged in time-resolved mode. (b) Stimulation with 1 μM of the
natural agonist VIP leading to internalization of the VPAC1-ST−
Lumi4-Tb complex. Color bars represent time-resolved intensity
counts. Note: these data were previously published at a conference.100
LANTHANIDE-BASED TIME-RESOLVED IMAGING:
TOWARD TR-FRET IMAGING
■
1 (VPAC1)-ST receptors with SNAP−Lumi4-Tb, the labeled
receptors are mainly present at the cell surface. After
stimulation with the natural agonist vasoactive intestinal
peptide (VIP), internalization of the labeled receptors is
evident as vesicles are formed within the intercellular
compartments.
Time-resolved spectral imaging of the very bright europium
complex 13, which can be localized in mitochondria,41 was
recently also achieved. The development and time-resolved
imaging of pH101 or anion sensors102 derived from 13 prove
the versatility of these types of europium complexes, and its
possible future implication in time-resolved and TR-FRET
imaging in drug-discovery and clinical applications.
The HTRF and TR-FRET assays described above are all
performed in plate-based formats and have been proven to be
very successful, giving sensitive and quantitative data. However,
detailed information on the cellular structure such as expression
levels at different compartments of the cell cannot be retrieved
from such data. Afterall, they are “bulk” measurements, not
evaluating individual cells. Imaging of biological interactions or
expression of proteins at different locations is of particular
interest in the clinical field. Time-resolved (or time-gated)
microscopy using lanthanide complexes might be suited to
obtain better insight in cellular processes especially on native
tissue. Time-resolved microscopy has the big advantage of
J
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CONCLUDING REMARKS
ACKNOWLEDGMENTS
■
■
We thank the teams of Prof. Dr. David Parker (Durham
University) and Dr. Olivier Maury (ENS Lyon) for
collaboration on the development of novel europium
complexes. Eric Trinquet (Cisbio Bioaassays) and Dr. Jean-
On the bench side, biochemical assays are dealing mostly with
isolated biological elements taken out of their cellular
environment and are indeed far from being representative of
the reality of true molecular interactions in biology. Taking
advantage of the unique properties of rare-earth macrocycles
and particularly cryptates and HTRF to probe molecular
interactions in the nanometer range, we have opened up new
tools and paths to explore such interactions in living cells and
on the cell surface in collaboration with many academic
partners, and particularly with the IGF “Institut de Genomique
Fonctionnelle” in Montpellier in the field of GPCR.
From the early experiments using rare-earth cryptates and
fluorescent cyanine-labeled antibodies against tags fused to the
external parts of GABAB receptors to study the obligatory
heterodimer to more sophisticated experimental setups using a
suicide enzyme toolbox, we have investigated many different
biological processes including binding, oligomerization, inter-
nalization, modulation of receptor activity, and deciphered their
pharmacological behavior.
́
Philippe Pin and his laboratory of the Institut de Genomique
Fonctionnelle in Montpellier contributed in cell-based assay
development. Dr. Evelyne Lopez-Crapez from the CRLC Paul
Lamarque, Val d’Aurelle (Montpellier), and her team members
are acknowledged for their role in the development of HER
assays on tumor samples. For financial support, the following
organizations are acknowledged: Agence Nationale de la
Recherche, Fonds Unique Interminister
́
iel, Region Langue-
doc-Roussillon, Oseo, and the Eurobiomed cluster.
́
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NOTE ADDED AFTER ASAP PUBLICATION
■
Due to a production error, this paper was published on the
Web on January 6, 2014, before all of the corrections were
implemented. The corrected version was reposted on January
10, 2014.
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