New Biotechnology Volume 00, Number 00 June 2015
REVIEW
Review
Quenching resonance energy transfer (QRET): a single-label technique for inhibitor screening and interaction studies Kari Kopra and Harri Ha¨rma¨ Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Kiinamyllynkatu 10, 3rd Floor, FI-20520 Turku, Finland
The increased number of therapeutic targets has led to a growing need for screening methods enabling possible inhibitor compound selection. Information for new therapeutic targets has been found mostly from sequencing of the human genome but this knowledge cannot be directly converted into clinically relevant drug molecules. After target identification, the multistep drug development process takes many years and hundreds of millions of dollars are spent without certainty of the outcome. The first and the most critical step in the drug development process is hit selection. The optimal high throughput screening method should provide the highest possible number of true positive hits for further studies and lead discovery. The result should be achieved with low material consumption in a rapid and automated process. Radioactive label based methods are sensitive, but due to the problems arising from the radioactivity, luminescence-based methods have become increasingly popular in screening. In this review, the time-resolved luminescence based quenching resonance energy transfer (QRET) technique is discussed for primary screening. Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The primary screening process . . . . . . . . . . . . . . . . . . . . . . The quenching resonance energy transfer (QRET) technique Mix-and-read assays for screening . . . . . . . . . . . . . . . . . . . . QRET based screening techniques . . . . . . . . . . . . . . . . . . . . . . GPCR inhibitor screening . . . . . . . . . . . . . . . . . . . . . . . . . . Small GTPase inhibitor screening . . . . . . . . . . . . . . . . . . . . Protein–protein interaction inhibitor screening . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . .
Introduction The primary screening process There is a major gap between medical therapy and drug development, and thus new drugs are being approved at a decreasing rate [1]. The cycle from idea to marketed medicine takes usually 12–15 Corresponding author: Kopra, K. ([email protected]) http://dx.doi.org/10.1016/j.nbt.2015.02.007 1871-6784/ß 2015 Elsevier B.V. All rights reserved.
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
000 000 000 000 000 000 000 000 000 000 000
years and the costs are astronomical [2,3]. Before molecular screening, target validation needs to be carried out to select a ‘druggable’ target meeting clinical and commercial needs [3]. Bioinformatics and phenotypic screening have increased the number of potential targets, but not all targets are ‘druggable’ [3]. Upon target selection, primary screening is performed to provide candidates for further studies. High throughput screening (HTS) assays enable the www.elsevier.com/locate/nbt
Please cite this article in press as: Kopra, K., and Ha¨rma¨, H., Quenching resonance energy transfer (QRET): a single-label technique for inhibitor screening and interaction studies, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.02.007
1
NBT-762; No of Pages 6 REVIEW
New Biotechnology Volume 00, Number 00 June 2015
Review
study of large compound libraries to identify molecules with desired activity [4,5]. Primary screening aims to find a set of potentially active compounds. This means that not all compounds found are active or suitable as drug leads. When primary screening methods are developed, the aim is to find all those compounds which might be active and discard all others. However, all methods also find wrong hits, which can arise, for example, aggregation, autofluorescence or quenching, depending on the chosen HTS method [6,7]. Thus all HTS hits need to be confirmed in a secondary lower throughput screen to remove artifacts and to ensure the correct binding. In the following sections, different biophysical assay methods enabling inhibitor and/or interaction screening are introduced.
The quenching resonance energy transfer (QRET) technique Quenching resonance energy transfer (QRET) is based on a separation between target bound and non-bound lanthanide (Ln3+) chelate conjugated to a low molecular weight ligand [8]. When the labeled ligand is free in solution, the Ln3+-chelate (Eu3+ or Tb3+) is exposed to soluble quencher and a low time-resolved luminescence (TRL) signal is monitored. The interaction between Ln3+ligand and the target molecule interferes with the quencher/Ln3+chelate interaction, increasing the distance between quencher and Ln3+-chelate. The increased distance decreases energy transfer between the Ln3+-ligand and the quencher, thus increasing the luminescence lifetime of the Ln3+-chelate and the monitored overall TRL-signal in the measurement window from 400 to 800 ms [9]. The labeled ligand could be, for example, a DNAfragment, peptide, nucleotide or small hormone incapable of protecting the label as such [8–11]. The QRET based screening is easy to convert to new targets if a small molecule ligand of the target can be labeled. On the basis of the selected Ln3+-chelate and assay buffer, the quencher molecule is selected to enable the highest possible assay performance. The quencher can be basically any luminophore having a spectral overlap with the Ln3+-chelate [8,9]. QRET has an exceptional property of enabling detection in assays with purified proteins, membranes or intact cells. We have introduced screening assays for the most important drug targets, G protein-coupled receptors (GPCRs), as well as other receptor-ligand pairs and a variety of other targets, for example, small GTPases. In following sections the wide suitability of the QRET technique for screening will be addressed and compared to most important commercial methods for HTS purposes.
Mix-and-read assays for screening Traditional heterogeneous methods for screening have been mostly replaced by homogeneous mix-and-read assays. These can utilize radioactive labels as in heterogeneous methods but increasingly luminescence-based detection has gained ground in both assay types [12–21]. Comparing luminescence-based homogeneous methods with heterogeneous methods such as enzymelinked immunosorbant assay (ELISA) and dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), detection sensitivity is decreased but multiple separation and incubation steps can be avoided [6,15]. Luminescence-based homogeneous mix-and-read assays provide a platform with reasonable sensitivity, easy 2
FIGURE 1
Principles of the commercial screening assays for cAMP. (a) AlphaScreen and AlphaLISA techniques employ singlet oxygen mediated energy transfer between donor and acceptor beads. Competitive, distance-dependent energy transfer between beads enables a longer distance between interacting molecules than Fo¨rster resonance energy transfer (FRET). (b) TRFRET is an energy transfer assay where energy is transferred from lanthanide donor to acceptor fluorophore. In competitive TR-FRET assays, two chromophore-conjugated molecules are needed and the TRL-signal is distance dependent. Both homogeneous time-resolved fluorescence assay (HTRF) and lanthanide fluorescence energy transfer assay (LANCE) are based on TR-FRET. (c) Fluorescence polarization (FP) provides a single-label detection platform to monitor cAMP. FP assays rely on competitive binding between cAMP and labeled cAMP. In FP, binding induced effects on molecular rotation and polarization are monitored, due to increased complex size.
automation and miniaturization, and fast native-like detection in solution. Because of these properties, luminescence-based homogeneous methods are the most suitable for HTS purposes today. The most important luminescence-based HTS methods are presented in Fig. 1.
www.elsevier.com/locate/nbt Please cite this article in press as: Kopra, K., and Ha¨rma¨, H., Quenching resonance energy transfer (QRET): a single-label technique for inhibitor screening and interaction studies, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.02.007
NBT-762; No of Pages 6 New Biotechnology Volume 00, Number 00 June 2015
REVIEW
QRET based screening techniques GPCR inhibitor screening GPCRs are the most significant pharmacological drug targets, with 30–40% share of marketed drugs [34,35]. This is due to their critical role in transduction of extracellular into intracellular signals [35]. Most GPCR studies are performed on cell membranes or with intact cells. GPCR signaling can be studied either by monitoring ligand binding or receptor activation markers, for example, increasing concentration of GTP binding and cAMP. Ligand, either agonist or antagonist, binding to the cell surface receptor is the first step in GPCR activation. The QRET assay enables sensitive ligand binding and inhibitor/ligand screening in a whole cell format (Fig. 2a) [8,36]. The signal-to-background (S/B) ratio in
Review
AlphaScreen and AlphaLISA are widely used homogeneous screening methods (Fig. 1a) [14,15]. Methods utilizing non-radioactive latex-bead particles and energy transfer from acceptor to donor are mediated through singlet oxygen [22]. AlphaScreen and ¨ rster resonance enAlphaLISA are proximity-based assays, like Fo ergy transfer (FRET) assays, but unlike FRET the acceptor emission is monitored at a lower wavelength than that at which the donor is excited [22]. Conventional FRET between two rapidly decaying fluorophores is rarely used in screening due to problems arising from autofluorescence, acceptor direct excitation, and emission signal leakage to the detection window [6]. These issues can be avoided using TRL detection [23]. This decreases the luminescence background from the assay matrix, due to time delay between the excitation signal and emission detection [6,8,23–25]. Commercial TR-FRET based assays, homogeneous time-resolved fluorescence assay (HTRF) and lanthanide fluorescence energy transfer assay (LANCE) both utilize lanthanides and TRL-signal detection (Fig. 1b) [15–19]. When comparing conventional FRET and TRFRET, not only is the background signal decreased but the allowed distance between acceptor and donor is also increased [6]. However, when commercial proximity based assays are compared, only AlphaScreen enables the study of large protein complexes. In AlphaScreen the distance between donor and acceptor can be increased up to 200 nm due to singlet oxygen approach. This is a 10-fold to 100-fold higher distance than in FRET and TR-FRET [6,15]. Fluorescence polarization (FP) enables single-label detection which simplifies assay principle (Fig. 1c) [8,20,21,26–28]. In FP, the polarized excitation light is emitted by the fluorophore with a degree of polarization which reflects the molecular mass of the complex [27,28]. The use of a single fluorophore reduces the costs of the assay and thus FP has been applied to a wide variety of targets [29,30]. Unfortunately, FP detection is limited by autofluorescence and scattering from the conventional fluorophores used in the assay matrix [6,8,26,31]. As in the QRET assay, the labeled compound in FP needs to be small to enable sufficient polarization change when bound to the target. However, the QRET technique utilizes Ln3+-chelates and TRL detection, and thus does not suffer from background problems like FP. Unlike HTRF or LANCE based on the TRL detection and energy transfer, only a single label conjugation is needed in QRET assays. Thus greater simplicity is achieved with QRET compared to HTRF or LANCE, without loss of assay dynamics and sensitivity [32,33]. In addition, QRET is not limited to the use of purified molecules and nanomolar limit-of-detection (LOD) as is FP [26,27].
FIGURE 2
The principle of the quenching resonance energy transfer (QRET) technique for inhibitor screening. (a) Receptor–ligand interactions can be monitored using intact cells and secondary messengers such as cAMP can be measured in a competitive fashion, using lysed cells. (b) Small GTPase cycle monitoring using purified proteins and Ln3+-chelate conjugated guanosine triphosphate enables inhibitor screening and reaction kinetic monitoring. (c) Indirect protein–protein interactions monitoring has been shown using small molecule ligand binding in the interaction surface of the two binding partners, for example, growth factor and heparin.
www.elsevier.com/locate/nbt 3 Please cite this article in press as: Kopra, K., and Ha¨rma¨, H., Quenching resonance energy transfer (QRET): a single-label technique for inhibitor screening and interaction studies, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.02.007
NBT-762; No of Pages 6 REVIEW
Review
these competitive binding assays using unmodified cells and Ln3+chelate conjugated ligand are usually from 5 to 10, which is in the same range as with commercial methods [8,36]. The QRET binding assay provides knowledge on ligand binding without distinguishing agonists and antagonists. QRET is not so far applicable to studying signaling cascades or binding events within cells, and thus the technique was combined with the DiscoveRx PathHunter assay to enable agonist/antagonist separation [37]. The PathHunter assay utilizes enzyme fragment complementation (EFC) to monitor agonist-induced receptor activation [38,39]. Performing the QRET assay with the PathHunter cell line, both receptor– ligand binding and receptor activation can be monitored [37]. In this multiparametric assay, the performance of either assay is not compromised and activation signal inhibiting antagonists can be separated from activating agonists. If GPCR activation is the reaction of interest, either GTP binding to the Ga subunit or cAMP secondary messenger can be monitored [10,32]. GTP binding to Ga in the membrane is usually performed in a heterogeneous manner [12,40]. QRET is the first method enabling homogeneous GTP binding monitoring in cell membranes [10]. When QRET is compared to heterogeneous radioactivity or TRL assays, no membrane modification is needed, reducing risks of lowered assay performance associated with such modifications. In the downstream reaction pathway, Ga-mediated activation of adenylyl cyclase increases cAMP concentration. There are several homogeneous methods to monitor cAMP mostly based on proximity based signaling, for example, HTRF, LANCE, and Alphascreen (Fig. 1). The major advantage of the QRET cAMP assay over the commercial methods is the need for only a single labeled molecule as in FP (Figs 1c and 2a) [32]. Compared to the LANCE assay for cAMP, the Z-factor is slightly reduced in the QRET assay but no significant effect on sensitivity or S/B ratio was monitored [32].
Small GTPase inhibitor screening Small GTPases are a family of cytosolic G proteins acting in many ways equally to Ga proteins in the case of GPCR. Small GTPases are involved in regulation of key cellular processes, including cell division, signal transduction, and apoptosis. Thus misregulated small GTPase are highly linked to cancer and other diseases [41– 44]. Small GTPases are difficult drug targets due to their similarities between families and high affinity binding to guanosine nucleotides. Also there are no obvious small molecule binding sites in the small GTPase surface [45]. Current screening methods are partially ineffective in finding all possible inhibitors, since they measure decrease in interaction between GTPase and regulator/effector. Interfacial inhibitors act oppositely by increasing the interaction between small GTPase and regulator/effector, and thus cannot be found with present methods [46]. NMR-based fragment screening is currently the most widely used method to find inhibitors affecting the small GTPase cycle [45]. Luminescence based methods, which are used to study nucleotide exchange and GTP hydrolysis kinetics, are normally too consuming of materials for efficient screening [47,48]. Methods based on environmentally sensitive labels for example, Nmethylanthraniloyl and BODIPY, also suffer from low S/B ratio which predispose to interference from the assay matrix [48]. The commercial Transcreener method based on FP or TR-FRET 4
New Biotechnology Volume 00, Number 00 June 2015
detection is one of the only luminescence base methods validated for small GTPase inhibitor screening [49,50]. Like the Transcreener FP assay, QRET enables small GTPase inhibitor screening in singlelabel fashion (Fig. 2b) [51,52]. The QRET technique is based on Eu3+-GTP monitoring, unlike the Transcreener method which monitors GDP produced after GTP hydrolysis. GTP monitoring in QRET is advantageous over GDP, because it can enable an easy detection of both GTP association (small GTPase activation) and GTP hydrolysis (small GTPase inactivation) [53]. In addition, QRET for GTP association detection has functional elements, such as the association reaction which can be monitored kinetically in a microtiter plate format [52]. Both the QRET assay for GTP association and GTP hydrolysis detection have been shown to be feasible in 1280 small compound pilot screens (Kopra et al., unpublished data).
Protein–protein interaction inhibitor screening In most of the cellular processes, protein–protein interactions (PPIs) play crucial roles. However, PPI inhibitors are not easy to discover due to problems with conventional inhibitor libraries [6,54]. The latter are normally selected by adherence to the Rule of 5, which is poorly suitable for PPI inhibitors [54]. Also the PPI contact area is usually a flat, large surface, which complicates finding the effective inhibitors [6,54]. Despite these challenges, a multitude of PPI inhibitors have been introduced [55,56]. PPI inhibitor screening in non-cellular assay formats is often performed using the same techniques as introduced in the mixand-read assays for screening section [6]. For PPI and PPI inhibitor screening, we have developed a competitive assay based on DNAaptamers [9]. This utilizes Eu3+-chelate conjugated aptamers which have a binding pocket in the heparin–growth factor interaction interface and can be replaced with competing molecules (Fig. 2c) [9,57,58]. The QRET method is sensitive and provides very high S/B ratios. Wider PPI studies utilizing QRET are yet to be performed. However, QRET should enable similar assays with similar limitations as FP. In both QRET and FP assays, the PPI interaction is monitored indirectly using label small molecular ligand [6,9].
Concluding remarks There are a wide variety of different techniques for inhibitor and interaction screening. Properties of the most widely used homogeneous screening methods are presented in Table 1. The final selection of a screening method is case dependent, but for any efficient screening the assay should have low material and time consumption, easy optimization and automation, and should tolerate detergents and DMSO [6]. The method should enable not only single-concentration screening, but also dose–response assays. Despite the selection technique for primary screening, no single method can be used alone and re-evaluation and confirmation of the results should always be performed with an alternative method. The QRET method provides a fast-growing set of assays for different targets. The single-label QRET technique can be converted to new targets by a relative easy procedure, because only a single Ln3+-chelate conjugated small molecule ligand is needed. QRET combines the benefits of TRL detection, an integral part of the well-accepted HTRF and LANCE techniques, with the single-label detection used in FP. Because of TRL detection, the
www.elsevier.com/locate/nbt Please cite this article in press as: Kopra, K., and Ha¨rma¨, H., Quenching resonance energy transfer (QRET): a single-label technique for inhibitor screening and interaction studies, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.02.007
NBT-762; No of Pages 6 New Biotechnology Volume 00, Number 00 June 2015
REVIEW
TABLE 1
Method
Dye
Sample
Time
Cost
Sensitivity
Pros
Cons
Alphascreen
Polystyrene beads
Complex samples
1–3 hours
High
nM
Simple single-label method
Assay background, need for small ligand
HTRF
Ln3+-cryptate and organic fluorophore
Complex samples
1–2 hours
Moderate
pM to nM
Wide protein size range, multiplexing
Dye orientation and distance
LANCE
Ln3+-chelate and organic fluorophore
Complex samples
1–2 hours
Moderate
pM to nM
Wide protein size range, multiplexing
Dye orientation and distance
EFC
P-galactosidase
Complex samples
1–3 hours
Moderate to high
pM to nM
Validated to wide variety of targets
Special cell lines needed
QRET
Ln3+-chelate
Complex samples
REVIEW
Review
Quenching resonance energy transfer (QRET): a single-label technique for inhibitor screening and interaction studies Kari Kopra and Harri Ha¨rma¨ Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Kiinamyllynkatu 10, 3rd Floor, FI-20520 Turku, Finland
The increased number of therapeutic targets has led to a growing need for screening methods enabling possible inhibitor compound selection. Information for new therapeutic targets has been found mostly from sequencing of the human genome but this knowledge cannot be directly converted into clinically relevant drug molecules. After target identification, the multistep drug development process takes many years and hundreds of millions of dollars are spent without certainty of the outcome. The first and the most critical step in the drug development process is hit selection. The optimal high throughput screening method should provide the highest possible number of true positive hits for further studies and lead discovery. The result should be achieved with low material consumption in a rapid and automated process. Radioactive label based methods are sensitive, but due to the problems arising from the radioactivity, luminescence-based methods have become increasingly popular in screening. In this review, the time-resolved luminescence based quenching resonance energy transfer (QRET) technique is discussed for primary screening. Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The primary screening process . . . . . . . . . . . . . . . . . . . . . . The quenching resonance energy transfer (QRET) technique Mix-and-read assays for screening . . . . . . . . . . . . . . . . . . . . QRET based screening techniques . . . . . . . . . . . . . . . . . . . . . . GPCR inhibitor screening . . . . . . . . . . . . . . . . . . . . . . . . . . Small GTPase inhibitor screening . . . . . . . . . . . . . . . . . . . . Protein–protein interaction inhibitor screening . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . .
Introduction The primary screening process There is a major gap between medical therapy and drug development, and thus new drugs are being approved at a decreasing rate [1]. The cycle from idea to marketed medicine takes usually 12–15 Corresponding author: Kopra, K. ([email protected]) http://dx.doi.org/10.1016/j.nbt.2015.02.007 1871-6784/ß 2015 Elsevier B.V. All rights reserved.
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
000 000 000 000 000 000 000 000 000 000 000
years and the costs are astronomical [2,3]. Before molecular screening, target validation needs to be carried out to select a ‘druggable’ target meeting clinical and commercial needs [3]. Bioinformatics and phenotypic screening have increased the number of potential targets, but not all targets are ‘druggable’ [3]. Upon target selection, primary screening is performed to provide candidates for further studies. High throughput screening (HTS) assays enable the www.elsevier.com/locate/nbt
Please cite this article in press as: Kopra, K., and Ha¨rma¨, H., Quenching resonance energy transfer (QRET): a single-label technique for inhibitor screening and interaction studies, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.02.007
1
NBT-762; No of Pages 6 REVIEW
New Biotechnology Volume 00, Number 00 June 2015
Review
study of large compound libraries to identify molecules with desired activity [4,5]. Primary screening aims to find a set of potentially active compounds. This means that not all compounds found are active or suitable as drug leads. When primary screening methods are developed, the aim is to find all those compounds which might be active and discard all others. However, all methods also find wrong hits, which can arise, for example, aggregation, autofluorescence or quenching, depending on the chosen HTS method [6,7]. Thus all HTS hits need to be confirmed in a secondary lower throughput screen to remove artifacts and to ensure the correct binding. In the following sections, different biophysical assay methods enabling inhibitor and/or interaction screening are introduced.
The quenching resonance energy transfer (QRET) technique Quenching resonance energy transfer (QRET) is based on a separation between target bound and non-bound lanthanide (Ln3+) chelate conjugated to a low molecular weight ligand [8]. When the labeled ligand is free in solution, the Ln3+-chelate (Eu3+ or Tb3+) is exposed to soluble quencher and a low time-resolved luminescence (TRL) signal is monitored. The interaction between Ln3+ligand and the target molecule interferes with the quencher/Ln3+chelate interaction, increasing the distance between quencher and Ln3+-chelate. The increased distance decreases energy transfer between the Ln3+-ligand and the quencher, thus increasing the luminescence lifetime of the Ln3+-chelate and the monitored overall TRL-signal in the measurement window from 400 to 800 ms [9]. The labeled ligand could be, for example, a DNAfragment, peptide, nucleotide or small hormone incapable of protecting the label as such [8–11]. The QRET based screening is easy to convert to new targets if a small molecule ligand of the target can be labeled. On the basis of the selected Ln3+-chelate and assay buffer, the quencher molecule is selected to enable the highest possible assay performance. The quencher can be basically any luminophore having a spectral overlap with the Ln3+-chelate [8,9]. QRET has an exceptional property of enabling detection in assays with purified proteins, membranes or intact cells. We have introduced screening assays for the most important drug targets, G protein-coupled receptors (GPCRs), as well as other receptor-ligand pairs and a variety of other targets, for example, small GTPases. In following sections the wide suitability of the QRET technique for screening will be addressed and compared to most important commercial methods for HTS purposes.
Mix-and-read assays for screening Traditional heterogeneous methods for screening have been mostly replaced by homogeneous mix-and-read assays. These can utilize radioactive labels as in heterogeneous methods but increasingly luminescence-based detection has gained ground in both assay types [12–21]. Comparing luminescence-based homogeneous methods with heterogeneous methods such as enzymelinked immunosorbant assay (ELISA) and dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), detection sensitivity is decreased but multiple separation and incubation steps can be avoided [6,15]. Luminescence-based homogeneous mix-and-read assays provide a platform with reasonable sensitivity, easy 2
FIGURE 1
Principles of the commercial screening assays for cAMP. (a) AlphaScreen and AlphaLISA techniques employ singlet oxygen mediated energy transfer between donor and acceptor beads. Competitive, distance-dependent energy transfer between beads enables a longer distance between interacting molecules than Fo¨rster resonance energy transfer (FRET). (b) TRFRET is an energy transfer assay where energy is transferred from lanthanide donor to acceptor fluorophore. In competitive TR-FRET assays, two chromophore-conjugated molecules are needed and the TRL-signal is distance dependent. Both homogeneous time-resolved fluorescence assay (HTRF) and lanthanide fluorescence energy transfer assay (LANCE) are based on TR-FRET. (c) Fluorescence polarization (FP) provides a single-label detection platform to monitor cAMP. FP assays rely on competitive binding between cAMP and labeled cAMP. In FP, binding induced effects on molecular rotation and polarization are monitored, due to increased complex size.
automation and miniaturization, and fast native-like detection in solution. Because of these properties, luminescence-based homogeneous methods are the most suitable for HTS purposes today. The most important luminescence-based HTS methods are presented in Fig. 1.
www.elsevier.com/locate/nbt Please cite this article in press as: Kopra, K., and Ha¨rma¨, H., Quenching resonance energy transfer (QRET): a single-label technique for inhibitor screening and interaction studies, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.02.007
NBT-762; No of Pages 6 New Biotechnology Volume 00, Number 00 June 2015
REVIEW
QRET based screening techniques GPCR inhibitor screening GPCRs are the most significant pharmacological drug targets, with 30–40% share of marketed drugs [34,35]. This is due to their critical role in transduction of extracellular into intracellular signals [35]. Most GPCR studies are performed on cell membranes or with intact cells. GPCR signaling can be studied either by monitoring ligand binding or receptor activation markers, for example, increasing concentration of GTP binding and cAMP. Ligand, either agonist or antagonist, binding to the cell surface receptor is the first step in GPCR activation. The QRET assay enables sensitive ligand binding and inhibitor/ligand screening in a whole cell format (Fig. 2a) [8,36]. The signal-to-background (S/B) ratio in
Review
AlphaScreen and AlphaLISA are widely used homogeneous screening methods (Fig. 1a) [14,15]. Methods utilizing non-radioactive latex-bead particles and energy transfer from acceptor to donor are mediated through singlet oxygen [22]. AlphaScreen and ¨ rster resonance enAlphaLISA are proximity-based assays, like Fo ergy transfer (FRET) assays, but unlike FRET the acceptor emission is monitored at a lower wavelength than that at which the donor is excited [22]. Conventional FRET between two rapidly decaying fluorophores is rarely used in screening due to problems arising from autofluorescence, acceptor direct excitation, and emission signal leakage to the detection window [6]. These issues can be avoided using TRL detection [23]. This decreases the luminescence background from the assay matrix, due to time delay between the excitation signal and emission detection [6,8,23–25]. Commercial TR-FRET based assays, homogeneous time-resolved fluorescence assay (HTRF) and lanthanide fluorescence energy transfer assay (LANCE) both utilize lanthanides and TRL-signal detection (Fig. 1b) [15–19]. When comparing conventional FRET and TRFRET, not only is the background signal decreased but the allowed distance between acceptor and donor is also increased [6]. However, when commercial proximity based assays are compared, only AlphaScreen enables the study of large protein complexes. In AlphaScreen the distance between donor and acceptor can be increased up to 200 nm due to singlet oxygen approach. This is a 10-fold to 100-fold higher distance than in FRET and TR-FRET [6,15]. Fluorescence polarization (FP) enables single-label detection which simplifies assay principle (Fig. 1c) [8,20,21,26–28]. In FP, the polarized excitation light is emitted by the fluorophore with a degree of polarization which reflects the molecular mass of the complex [27,28]. The use of a single fluorophore reduces the costs of the assay and thus FP has been applied to a wide variety of targets [29,30]. Unfortunately, FP detection is limited by autofluorescence and scattering from the conventional fluorophores used in the assay matrix [6,8,26,31]. As in the QRET assay, the labeled compound in FP needs to be small to enable sufficient polarization change when bound to the target. However, the QRET technique utilizes Ln3+-chelates and TRL detection, and thus does not suffer from background problems like FP. Unlike HTRF or LANCE based on the TRL detection and energy transfer, only a single label conjugation is needed in QRET assays. Thus greater simplicity is achieved with QRET compared to HTRF or LANCE, without loss of assay dynamics and sensitivity [32,33]. In addition, QRET is not limited to the use of purified molecules and nanomolar limit-of-detection (LOD) as is FP [26,27].
FIGURE 2
The principle of the quenching resonance energy transfer (QRET) technique for inhibitor screening. (a) Receptor–ligand interactions can be monitored using intact cells and secondary messengers such as cAMP can be measured in a competitive fashion, using lysed cells. (b) Small GTPase cycle monitoring using purified proteins and Ln3+-chelate conjugated guanosine triphosphate enables inhibitor screening and reaction kinetic monitoring. (c) Indirect protein–protein interactions monitoring has been shown using small molecule ligand binding in the interaction surface of the two binding partners, for example, growth factor and heparin.
www.elsevier.com/locate/nbt 3 Please cite this article in press as: Kopra, K., and Ha¨rma¨, H., Quenching resonance energy transfer (QRET): a single-label technique for inhibitor screening and interaction studies, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.02.007
NBT-762; No of Pages 6 REVIEW
Review
these competitive binding assays using unmodified cells and Ln3+chelate conjugated ligand are usually from 5 to 10, which is in the same range as with commercial methods [8,36]. The QRET binding assay provides knowledge on ligand binding without distinguishing agonists and antagonists. QRET is not so far applicable to studying signaling cascades or binding events within cells, and thus the technique was combined with the DiscoveRx PathHunter assay to enable agonist/antagonist separation [37]. The PathHunter assay utilizes enzyme fragment complementation (EFC) to monitor agonist-induced receptor activation [38,39]. Performing the QRET assay with the PathHunter cell line, both receptor– ligand binding and receptor activation can be monitored [37]. In this multiparametric assay, the performance of either assay is not compromised and activation signal inhibiting antagonists can be separated from activating agonists. If GPCR activation is the reaction of interest, either GTP binding to the Ga subunit or cAMP secondary messenger can be monitored [10,32]. GTP binding to Ga in the membrane is usually performed in a heterogeneous manner [12,40]. QRET is the first method enabling homogeneous GTP binding monitoring in cell membranes [10]. When QRET is compared to heterogeneous radioactivity or TRL assays, no membrane modification is needed, reducing risks of lowered assay performance associated with such modifications. In the downstream reaction pathway, Ga-mediated activation of adenylyl cyclase increases cAMP concentration. There are several homogeneous methods to monitor cAMP mostly based on proximity based signaling, for example, HTRF, LANCE, and Alphascreen (Fig. 1). The major advantage of the QRET cAMP assay over the commercial methods is the need for only a single labeled molecule as in FP (Figs 1c and 2a) [32]. Compared to the LANCE assay for cAMP, the Z-factor is slightly reduced in the QRET assay but no significant effect on sensitivity or S/B ratio was monitored [32].
Small GTPase inhibitor screening Small GTPases are a family of cytosolic G proteins acting in many ways equally to Ga proteins in the case of GPCR. Small GTPases are involved in regulation of key cellular processes, including cell division, signal transduction, and apoptosis. Thus misregulated small GTPase are highly linked to cancer and other diseases [41– 44]. Small GTPases are difficult drug targets due to their similarities between families and high affinity binding to guanosine nucleotides. Also there are no obvious small molecule binding sites in the small GTPase surface [45]. Current screening methods are partially ineffective in finding all possible inhibitors, since they measure decrease in interaction between GTPase and regulator/effector. Interfacial inhibitors act oppositely by increasing the interaction between small GTPase and regulator/effector, and thus cannot be found with present methods [46]. NMR-based fragment screening is currently the most widely used method to find inhibitors affecting the small GTPase cycle [45]. Luminescence based methods, which are used to study nucleotide exchange and GTP hydrolysis kinetics, are normally too consuming of materials for efficient screening [47,48]. Methods based on environmentally sensitive labels for example, Nmethylanthraniloyl and BODIPY, also suffer from low S/B ratio which predispose to interference from the assay matrix [48]. The commercial Transcreener method based on FP or TR-FRET 4
New Biotechnology Volume 00, Number 00 June 2015
detection is one of the only luminescence base methods validated for small GTPase inhibitor screening [49,50]. Like the Transcreener FP assay, QRET enables small GTPase inhibitor screening in singlelabel fashion (Fig. 2b) [51,52]. The QRET technique is based on Eu3+-GTP monitoring, unlike the Transcreener method which monitors GDP produced after GTP hydrolysis. GTP monitoring in QRET is advantageous over GDP, because it can enable an easy detection of both GTP association (small GTPase activation) and GTP hydrolysis (small GTPase inactivation) [53]. In addition, QRET for GTP association detection has functional elements, such as the association reaction which can be monitored kinetically in a microtiter plate format [52]. Both the QRET assay for GTP association and GTP hydrolysis detection have been shown to be feasible in 1280 small compound pilot screens (Kopra et al., unpublished data).
Protein–protein interaction inhibitor screening In most of the cellular processes, protein–protein interactions (PPIs) play crucial roles. However, PPI inhibitors are not easy to discover due to problems with conventional inhibitor libraries [6,54]. The latter are normally selected by adherence to the Rule of 5, which is poorly suitable for PPI inhibitors [54]. Also the PPI contact area is usually a flat, large surface, which complicates finding the effective inhibitors [6,54]. Despite these challenges, a multitude of PPI inhibitors have been introduced [55,56]. PPI inhibitor screening in non-cellular assay formats is often performed using the same techniques as introduced in the mixand-read assays for screening section [6]. For PPI and PPI inhibitor screening, we have developed a competitive assay based on DNAaptamers [9]. This utilizes Eu3+-chelate conjugated aptamers which have a binding pocket in the heparin–growth factor interaction interface and can be replaced with competing molecules (Fig. 2c) [9,57,58]. The QRET method is sensitive and provides very high S/B ratios. Wider PPI studies utilizing QRET are yet to be performed. However, QRET should enable similar assays with similar limitations as FP. In both QRET and FP assays, the PPI interaction is monitored indirectly using label small molecular ligand [6,9].
Concluding remarks There are a wide variety of different techniques for inhibitor and interaction screening. Properties of the most widely used homogeneous screening methods are presented in Table 1. The final selection of a screening method is case dependent, but for any efficient screening the assay should have low material and time consumption, easy optimization and automation, and should tolerate detergents and DMSO [6]. The method should enable not only single-concentration screening, but also dose–response assays. Despite the selection technique for primary screening, no single method can be used alone and re-evaluation and confirmation of the results should always be performed with an alternative method. The QRET method provides a fast-growing set of assays for different targets. The single-label QRET technique can be converted to new targets by a relative easy procedure, because only a single Ln3+-chelate conjugated small molecule ligand is needed. QRET combines the benefits of TRL detection, an integral part of the well-accepted HTRF and LANCE techniques, with the single-label detection used in FP. Because of TRL detection, the
www.elsevier.com/locate/nbt Please cite this article in press as: Kopra, K., and Ha¨rma¨, H., Quenching resonance energy transfer (QRET): a single-label technique for inhibitor screening and interaction studies, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.02.007
NBT-762; No of Pages 6 New Biotechnology Volume 00, Number 00 June 2015
REVIEW
TABLE 1
Method
Dye
Sample
Time
Cost
Sensitivity
Pros
Cons
Alphascreen
Polystyrene beads
Complex samples
1–3 hours
High
nM
Simple single-label method
Assay background, need for small ligand
HTRF
Ln3+-cryptate and organic fluorophore
Complex samples
1–2 hours
Moderate
pM to nM
Wide protein size range, multiplexing
Dye orientation and distance
LANCE
Ln3+-chelate and organic fluorophore
Complex samples
1–2 hours
Moderate
pM to nM
Wide protein size range, multiplexing
Dye orientation and distance
EFC
P-galactosidase
Complex samples
1–3 hours
Moderate to high
pM to nM
Validated to wide variety of targets
Special cell lines needed
QRET
Ln3+-chelate
Complex samples
