Simplify and Accelerate Drug Discovery with TR-FRET
eBook
Last Updated: July 1, 2024
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Published: April 30, 2024
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Time-resolved fluorescence resonance energy transfer (TR-FRET) is a powerful tool for drug discovery, enabling the analysis of molecular interactions in a wide variety of biochemical processes.
This combinatorial approach results in a highly flexible, reliable and sensitive technique for studying cellular signaling pathways, protein–protein interactions, DNA–protein interactions, and receptor-ligand binding.
This eBook explores the principles, methodologies and diverse applications of TR-FRET, enabling researchers to streamline their drug discovery processes.
Download this eBook to explore:
- The advantages of TR-FRET in drug discovery
- Case studies using TR-FRET to measure ubiquitination, monitor ligand binding and more
- How to optimize your drug-screening processes
TR-FRET Assay Kits Simplify and
Accelerate Drug Discovery
1 bpsbioscience.com
FRET
Time-Resolved Fluorescence Resonance Energy
Transfer (TR-FRET) is a powerful technique commonly used
to analyze the binding of two interacting molecules. Since
most biological responses involve an interaction between
at least two partners, TR-FRET is well suited to study a wide
range of events, including many that characterize cellular
signaling pathways. Possible applications are limitless:
interaction between two proteins, between a receptor and
its ligand or between an enzyme and its substrate; binding
of a drug to its target or binding of a nucleic acid to a
protein; measure of post-transcriptional modifications;
and more.
The technology is a combination of Time-Resolved
Fluorescence and Förster’s Resonance Energy Transfer,
a phenomenon in which a light-excited fluorophore
can transfer its absorbed energy to a nearby acceptor
fluorophore.
This eBook provides the technical bases of TR-FRET
technology, with a few examples of assay design and
validation data.
Fluorophores absorb high-energy light and emit light
of lower energy than the absorbed light. Fluorescence
Resonance Energy Transfer (FRET) is a phenomenon
in which two fluorophores emitting at different
wavelengths are coupled: the donor fluorophore excited
by a high energy source transfers energy (not light) to
an acceptor fluorophore. This results in excitation of the
acceptor and fluorescence emission at the wavelengths
inherent to the properties of the acceptor fluorophore.
TR-FRET technology takes advantage of the
fact that the transfer of energy between the donor
and the acceptor depends on physical proximity
(<10 nm) and decreases rapidly with distance.
Thus, partner molecules distributed in a solution
are sufficiently far apart that FRET does not occur.
Figure 1: Illustration of FRET principle. In practice, one of the binding partners in the interaction of interest is labeled with a donor fluorophore such
as a Europium chelate, whereas the other partner is labeled with an acceptor fluorophore. When direct labeling of the partners is not possible, the
molecule of interest is tagged or biotinylated and the pairing is completed using streptavidin-coated donor or acceptor, or anti-tag antibodies.
Introduction
Upon interaction, the partners complete the FRET
pairing as they are now in proximity of each other.
Thus, FRET is applicable to any two interacting
molecules, or to reactions in which a new molecular form
appears providing that the new molecular form can be
distinguished from the initial molecule. Examples include
post-transcriptional modifications such as ubiquitination,
methylation, or phosphorylation. The molecules under
study can be directly labeled with a donor or acceptor
fluorophore. Alternatively, a secondary reagent (for
example, an antibody) that binds to the molecule of
interest can be labeled with one of the fluorophores for
indirect detection. This provides great flexibility regarding
the design of an assay.
FRET has a lower background signal than classic
fluorescence methods because the acceptor emission may
not share much spectral overlap with the excitation pulse.
The donor and the acceptor, on the other hand, must have
good spectral overlap (the emission range of the first
must overlap with the excitation range of the second), as
well as good spectral resolution for a specific signal to be
measured. However, it is the Time Resolved Fluorescence
(TRF) technology element that allows for the ultra-low
background advantage of TR-FRET.
Classic fluorescence intensity uses short-lived
fluorophores such as fluorescein, with an emission speed
in the order of the nanosecond. Excitation and emission
occur at specific wavelengths that can be differentiated by
a fluorescence reader. However, excitation and emission
happen at the same time. If there is any amount of
spectral overlap between excitation and emission, as there
usually is, the reader will capture some of the excitation
fluorescence, resulting in background signal and low
signal-to-noise ratios.
TRF solves this by using long-lived inorganic
fluorophores as donors and adding a time delay between
excitation and measurement, which means that the
excitation signal is gone by the time of the measurement,
which considerably decreases background signals (Figure 2).
TRF also uses excitation pulses (not continuous
excitation), so that a series of measurements are repeated
over time. It eliminates the very transient background
fluorescence generated from sample components such
as buffers, proteins, and chemicals, which hinders classic
FRET methods.
Ideal fluorophores have high signal intensity,
are highly stable, and offer excellent signalto-noise ratios. The most commonly used are
“Lanthanide probes” which are metal ions referring
to elements Cerium to Lutetium in the periodic table,
Time-Resolved Fluorescence
Figure 2: Illustration of TRF principle.
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Figure 3, left panel: Illustration of the assay principle (BPS Bioscience #50222). Right panel: Example data. Increasing concentrations
of ABT199 were added to a terbium-labeled donor, a dye-labeled acceptor, purified His-tagged BCL-2 protein, peptide ligand, and
incubated for 3 hours before TR-FRET reading. Two sequential measurements were performed: Tb-donor emission was measured at
620 nm followed by dye-acceptor emission at 665 nm.
Monitoring BCL-2 binding to ligand
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more specifically Europium and Terbium, which
fluoresce over milliseconds instead of nanoseconds.
Experimentally, a comparison measurement of the two
emitted wavelengths over time needs to be calculated.
BCL-2 (B-cell lymphoma 2), a member of the BCL
family, is an integral protein of the outer mitochondrial
membrane. BCL-2 family members form hetero- or
homodimers that regulate apoptosis. The main function
of BCL-2 is to inhibit apoptosis and promote cell survival
through control of mitochondrial membrane permeability,
blocking the release of cytochrome c from mitochondria
by pro-apoptotic BH3-containing proteins, and inhibition
of caspase activity. However, BCL-2 may either promote or
suppress apoptosis depending on context and partners.
Constitutive expression of BCL-2 in B lymphocytes, caused
by translocation of the BCL2 gene to the immunoglobulin
heavy chain locus, promotes follicular lymphoma. The
protein contributes to cancer resistance to treatment in
leukemia, melanoma, and breast and prostate cancer,
owing to its pro-survival effects. Inhibitors used in the
clinic are BH3-mimetic molecules that prevent BCL-2
interaction with its BH3-type partners. Navitoclax (ABT263) inhibits BCL-2, BCL-xL, and BCL-w, while venetoclax
(ABT-199) is highly selective of BCL-2.
As shown in Figure 3, a TR-FRET assay was designed
to monitor BCL-2 binding to its peptide ligand in a
homogeneous 384-well format. In this assay, a terbiumlabeled anti-His-tag antibody is used as the donor
fluorophore. The antibody binds to His-tagged BCL-2,
while the peptide ligand is labeled with biotin which
allows binding to the dye-labeled streptavidin acceptor.
The TR-FRET signal is generated by proximity induced
upon interaction of the protein with the peptide ligand.
The assay kit can be used to screen for small molecules
that inhibit the interaction of BCL-2 with its partner peptide
or to determine the IC50 of candidate inhibitors.
Measuring ubiquitination
Covalent conjugation of ubiquitin (Ub) to a protein is
a common post-translational modification that regulates
protein stability, function, and localization. Ubiquitination
is the concerted action of three enzymes: a Ub-activating
enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub
ligase (E3). The specificity and efficiency of ubiquitination
are determined by the E3 enzyme, which directs the last
step of the conjugation cascade by binding to both an
E2-Ub conjugate and a substrate protein, leading to its
mono- or poly-ubiquitination.
The Intrachain TR-FRET Assay kits were designed to
measure the auto-ubiquitination of a specific E3 enzyme
in a homogeneous 384-well format. The E3 ligase, such as
NEDD4, MDM2, VHL (Von Hippel Lindau), is affinity purified.
The assays use a Europium-labeled Ub donor and a Cy5-
labeled Ub acceptor to complete the TR-FRET pairing. Since
both the donor and acceptor are incorporated into polyubiquitin chains forming on the E3 enzyme, the assays
measure poly-ubiquitination and not mono-ubiquitination.
They are used for high-throughput screening of E3
ligase inhibitors, to perform real-time kinetic analyses,
or to accurately determine the IC50 of a compound.
Figure 4, upper panel: Illustration of the assay principle. Lower panels: Increasing concentrations of methyl-ubiquitin were added
to a Europium-labeled donor, a Cy5-labeled acceptor, purified E1 and E2 proteins and a purified E3 ligase (NEDD4, MDM2 or VHL)
in the presence of ATP, and incubated for 2 hours prior to TR-FRET reading. Two sequential measurements were performed: donor
emission was measured at 620 nm followed by dye-acceptor emission at 665 nm.
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Measuring the activity of RdRp
The pandemic coronavirus disease 2019 (COVID-19)
is caused by Severe Acute Respiratory Syndrome
Coronavirus 2 (SARS-CoV-2). One of the most promising
druggable SARS-CoV-2 targets is the RNA-dependent
RNA Polymerase (RdRp), a crucial enzyme in the life cycle
of coronaviruses which operates as a complex of NSP12,
NSP7, and NSP8 viral proteins.
The RdRp TR-FRET Assay kit is designed to measure
RdRp-mediated incorporation of biotinylated ATP into a
double-stranded RNA substrate. The increase in TR-FRET
signal is proportional to the amount of ATP incorporated
in the RNA and therefore directly measures enzymatic
activity. This assay is ideal for high-throughput screening
of enzyme inhibitors, to perform kinetic studies, or to
accurately measure a drug IC50.
Figure 5, upper panel: Illustration of the assay principle (BPS Bioscience #78553). Lower panels: the assay was performed in two
steps. First, a test compound was incubated with the purified RdRp enzyme in a reaction mixture consisting of optimized buffers,
substrate, and biotinylated ATP. Next, a dye-labeled acceptor and a Europium-labeled anti-Digoxigenin antibody were added
for one hour before TR-FRET reading. Two sequential measurements were performed: donor emission was measured at 620 nm
followed by dye-acceptor emission at 665 nm. The experiment was performed using increasing amounts of enzyme (lower left
panel) or in the presence of increasing concentrations of an inhibitor (lower right panel).
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Conclusion
TR-FRET is an ultra-low background technique allowing the measurement of any reaction in which two labeled entities
come in proximity. The main drawback of the technique is that it requires two optimized labeled entities, in addition to a
low dynamic range. However, these drawbacks are offset by several advantages:
• Small volumes
• Homogeneous: no need for washing steps or for physical separation
from the unbound entities
• Robust, sensitive signal
• Ultra-low background with high signal-to-noise ratio
• Stable signal: use of lanthanide donor fluorophores minimizes
photobleaching
The growing commercial availability of ready-to-use TR-FRET
immunoassay kits has opened the technique to mainstream use. BPS
Bioscience offers over 80 assay kits for drug discovery in the TR-FRET format
with new products developed regularly. Optimized, validated, high quality
assay kits ensure reliable results, quickly.
Simple operation
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