Eija Martikkala

A New Simple Cell-Based Homogeneous Time-Resolved Fluorescence QRET Technique for Receptor-Ligand Interaction Screening

In this article, a single-label separation-free fluorescence technique is presented as a potential screening method for cell-based receptor antagonists and agonists.The time-resolved fluorescence technique, quenching resonance energy transfer (QRET), relies on a single-labeled binding partner in combination with a soluble quencher. The quencher efficiently suppresses the luminescence of the unbound labeled ligand, whereas the luminescence of the bound fraction is not affected. This approach allows the development of cell-based screening assays in a simple and cost-effective manner. The authors have applied the technique to the screening of β2-adrenoreceptor (β2AR) antagonists and agonists in intact human embryonic kidney HEK293i cells overexpressing human β2-adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for β2AR were investigated in a displacement assay using europium(III)-labeled pindolol ligand. The assay Z′ values ranged from 0.68 to 0.78, the coefficient of variation was less than 10%, and the Ki values were 19 nM for propranolol and alprenolol and 14 and 5.9 µM for metaproterenol and terbutaline, respectively. The QRET technique with β2AR was also applied to LOPAC compound library screening, yielding nearly error-free recognition of known binders. This simple and cost-effective technique can be readily adapted to laboratory and industrial-scale screening. (Journal of Biomolecular Screening 2009:936-943)

Sensitive Fluorometric Nanoparticle Assays for Cell Counting and Viability

We have developed easy-to-use homogeneous methods utilizing time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence quenching for quantification of eukaryotic cells. The methods rely on a competitive adsorption of cells and fluorescently labeled protein onto citrate-stabilized colloidal gold nanoparticles or carboxylate-modified polystyrene nanoparticles doped with an Eu(III) chelate. In the gold nanoparticle sensor, the adsorption of the labeled protein to the gold nanoparticles leads to quenching of the fluorochrome. Eukaryotic cells reduce the adsorption of labeled protein to the gold particles increasing the fluorescence signal. In the Eu(III) nanoparticle sensor, the time-resolved fluorescence resonance energy transfer between the nanoparticles and an acceptor-labeled protein is detected; a decrease in the magnitude of the time-resolved energy transfer signal (sensitized time-resolved fluorescence) is proportional to the cell-nanoparticle interaction and subsequent reduced adsorption of the labeled protein. Less than five cells were detected and quantified with the nanoparticle sensors in the homogeneous microtiter assay format with a coefficient of variation of 6% for the gold and 12% for the Eu(III) nanoparticle sensor. The Eu(III) nanoparticle sensor was also combined with a cell impermeable nucleic acid dye assay to measure cell viability in a single tube test with cell counts below 1000 cells/tube. This sensitive and easy-to-use nanoparticle sensor combined with a viability test for a low concentration of cells could potentially replace existing microscopic methods in biochemical laboratories.

A Homogeneous Single-Label Time-Resolved Fluorescence cAMP Assay

G-protein–coupled receptors (GPCRs) are an important class of pharmaceutical drug targets. Functional high-throughput GPCR assays are needed to test an increasing number of synthesized novel drug compounds and their function in signal transduction processes. Measurement of changes in the cyclic adenosine monophosphate (cAMP) concentration is a widely used method to verify GPCR activation in the adenylyl cyclase pathway. Here, a single-label time-resolved fluorescence and high-throughput screening (HTS)–feasible method was developed to measure changes in cAMP levels in HEK293i cells overexpressing either β2-adrenergic or δ-opioid receptors. In the quenching resonance energy transfer (QRET) technique, soluble quenchers reduce the signal of unbound europium(III)-labeled cAMP in solution, whereas the antibody-bound fraction is fluorescent. The feasibility of this homogeneous competitive assay was proven by agonist-mediated stimulation of receptors coupled to either the stimulatory Gs or inhibitory Gi proteins. The reproducibility of the assays was excellent, and Z′ values exceeded 0.7. The dynamic range, signal-to-background ratio, and detection limit were compared with a commercial time-resolved fluorescence resonance energy transfer (TR-FRET) assay. In both homogeneous assays, similar assay parameters were obtained when adenylyl cyclase was stimulated directly by forskolin or via agonist-mediated activation of the Gs-coupled β2AR. The advantage of using the single-label approach relates to the cost-effectiveness of the QRET system compared with the two-label TR-FRET assay as there is no need for labeling of two binding partners leading to reduced requirements for assay optimization.

Homogeneous GTP binding assay employing QRET technology.

Functional cell signaling assays have become important tools for measuring ligand-induced receptor activation in cell-based biomolecular screening. Guanosine-5′-triphosphate (GTP) is a generic signaling marker responsible for the first intracellular signaling event of the G-protein-coupled receptors (GPCRs). [35S]GTPγS binding assay is the classical well-established method for measuring agonist-induced G-protein activation requiring a separation of free and bound fractions prior to measurement. Here a novel, separation-free, time-resolved fluorescence GTP binding assay has been developed based on a non–fluorescence resonance energy transfer (FRET) single-label approach and quenching of a nonbound europium-labeled, nonhydrolyzable GTP analog (Eu-GTP). The quenching resonance energy transfer (QRET) method relies on the use of Eu-GTP, providing a time-resolved fluorescent detection as an alternative to the radiolabel [35S]GTPγS assay. Upon activation of recombinant human α2A-adrenoceptors (α2A-AR) expressed in Chinese hamster ovary cells, guanosine-5′-diphosphate is released from the α-subunit of Gi-proteins, enabling the subsequent binding of Eu-GTP. Activation of α2A-AR with 5 different α2-AR agonists was measured quantitatively using the developed QRET GTP assay and compared to [35S]GTPγS and heterogeneous Eu-GTP filtration assays. Equal potencies and efficacy rank orders were observed in all 3 assays but with a lower signal-to-background ratio and increased assay variation in the QRET assay compared to the Eu-GTP filtration and the nonhomogeneous [35S]GTPγS binding assays.

Homogeneous Single-Label Biochemical Ras Activation Assay Using Time-Resolved Luminescence

Mutations of the small GTP-binding protein Ras have been commonly found in tumors, and Ras oncogenes have been established to be involved in the early steps of cancerogenesis. The detection of Ras activity is critical in the determination of the cell signaling events controlling cell growth and differentiation. Therefore, development of improved methods for primary screening of novel potential drugs that target small GTPase or their regulators and their signaling pathways is important. Several assays have been developed for small GTPases studies, but all these methods have limitations for a high-throughput screening (HTS) use. Multiple steps including separation, use of radioactive labels or time-consuming immunoblotting, and a need of large quantities of purified proteins are decreasing the user-friendliness of these methods. Here, we have developed a homogeneous H-Ras activity assay based on a single-label utilizing the homogeneous quenching resonance energy transfer technique (QRET). In the QRET method, the binding of a terbium-labeled GTP (Tb-GTP) to small GTPase protein H-Ras protects the signal of the label from quenching, whereas the signal of the nonbound fraction of Tb-GTP is quenched by a soluble quencher. This enables a rapid determination of the changes in the activity status of Ras. The assay optimization showed that only 60 nM concentration of purified H-Ras protein was needed. The functionality of the assay was proved by detecting the effect of H-Ras guanine nucleotide exchange factor, Son of Sevenless. The signal-to-background ratio up to 7.7 was achieved with an average assay coefficient of variation of 9.1%. The use of a low concentration of purified protein is desirable and the signal-to-background ratio of 3.4 was achieved in the assay at a concentration of 60 nM for H-Ras and SOS proteins. The need of only one labeled molecule and the ability to decrease the quantities of purified proteins used in the experiments are valuable qualities in HTS showing the potential of the QRET method.

A homogeneous single-label quenching resonance energy transfer assay for a δ-opioid receptor–ligand using intact cells

This study, a homogeneous assay system for delta opioid receptor binding ligands has been developed using Quenching Resonance Energy Transfer (QRET). The QRET system allows receptor-ligand binding assays on intact cells using a single-label approach and a nonspecific quenching mechanism. Binding of antagonists or agonists to the receptor can be defined using a europium(III) labeled ligand. In the presence of the unlabeled ligand the labeled ligand is displaced and remains in solution. The non-bound labeled ligand is not protected by the target receptor, and the luminescence signal is quenched. For this objective, a Eu(III) labeled peptide molecule with three different linkers (AX0, AX1 and AX2) was designed. Peptides were evaluated using the homogeneous QRET technique, radioligand binding assays and the heterogeneous time-resolved luminescence (TRL) technique. Using the Eu-AX0 peptide and the QRET method, a panel of opioid compounds (naltrexone, naltrindole, SCN-80, DPDPE and DAMGO) was tested to prove the assay performance. The signal-to-background ratio for the tested opioid ligand ranged from 3.3 to 12.0. The QRET method showed prominent performance also in high DMSO concentrations. QRET is a homogenous and a non-radioactive detection system for screening and this is the first attempt to utilize peptide ligands in the QRET concept.

Single-label time-resolved luminescence assay for estrogen receptor--ligand binding.

Homogeneous luminescence-based microplate assays are desirable in high-throughput screening of new nuclear receptor regulators. Time-resolved fluorescence resonance energy transfer (TR-FRET) assays provide high sensitivity due to low background signal. The TR-FRET concept requires labeling of both ligand and receptor, making the assay format and its development relatively expensive and complex compared with single-label methods. To overcome the limitations of the multilabel methods, we have developed a single-label method for estrogen receptor (ER)-ligand binding based on quenching resonance energy transfer (QRET), where estradiol labeled with luminescent europium(III) chelate (Eu-E(2)) is quenched using soluble quencher molecules. The luminescence signal of Eu-E(2) on binding to full-length ER is protected from quenching while increasing competitor concentrations displace Eu-E(2) from the receptor, reducing the signal. The QRET method was paralleled with a commercial fluorescence polarization (FP) assay. The measured signal-to-background (S/B) values for estradiol, estrone, fulvestrant, and tamoxifen obtained for the QRET assay (5.8-9.2) were clearly higher than the S/B values for the FP assay (1.3-1.5). A K(d) value of 30nM was calculated for binding of Eu-E(2) to ER from a saturation binding isotherm. The QRET method provides an attractive new single-label assay format for nuclear receptor ligand screening.

Nonspecific particle-based method with two-photon excitation detection for sensitive protein quantification and cell counting.

A novel easy-to-use homogeneous method utilizing two-photon excitation (TPX) for quantification of proteins or counting of eukaryotic cells in solution has been developed. This highly sensitive technique is based on the adsorption competition between the sample and fluorescently labeled protein to micrometer-sized carboxylate modified polystyrene particles and detection of two-photon excited fluorescence. The adsorption of the labeled protein to the particles was detected as a distinct fluorescence on individual microparticles. Analyte protein or eukaryotic cells interacted with particle surface and reduced the adsorption of labeled protein to the particles resulting in a decrease of the fluorescence. The optimizations of assay conditions were performed separately for protein quantification and cell counting, and the principle of the method was confirmed with the fluorescence microscopy imaging. The protein quantification assay allowed the determination of picogram quantities (1.2 μg/L) of protein, and the cell counting assay allowed three cells in the sample with an average variation of approximately 10% in the signal. The protein assay sensitivity was more than 500-fold improved from the common most sensitive commercial methods. Moreover, the dynamic range of the assay was broad, approximately 4 orders of magnitude. The cell assay has sensitivity comparable to the most sensitive commercial method. The developed method tolerates interfering agents such as neutral detergents found in cell lysate samples even at high concentrations. The method is experimentally fairly simple and allows the expansion for the use of the TPX technology.