GTP-specific fab fragment-based GTPase activity assay.

GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2'/3'-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4(GTP)) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations.

Multiparametric homogeneous method for identification of ligand binding to G protein-coupled receptors: receptor-ligand binding and ß-arrestin assay.

Two homogeneous assay systems have been combined to provide a new cell-based functional assay. The assay can be used to identify ligand binding to ß(2)-adrenergic receptors, but also the downstream response can be determined in the same assay. Both the quenching resonance energy transfer (QRET) and the DiscoveRx PathHunter assay formats allow the use of intact cells. The homogeneous QRET technique is a single-label approach based on nonspecific quenching of the time-resolved luminescence, enabling agonist and antagonist receptor binding measurements. The commercial PathHunter assay is in turn based on enzyme fragment complementation, which can be detected on the basis of chemiluminescence signal. In the PathHunter technology the enzyme complementation is recorded immediately downstream of agonist-induced receptor activation. The new multiparametric detection technology combines these two assay methods enabling the identification of agonist, and antagonist binding to the receptor, and the agonist-induced response. Using the QRET and the PathHunter methods a panel of ß(2)-adrenergic receptor ligands (epinephrine, terbutaline, metaproterenol, salmeterol, propranolol, alprenolol, bisoprolol, ICI 118,551, and bucindolol) was tested to prove the assay performance. The signal-to-background ratio for tested ligands ranged from 5 to 11 and from 6 to 18 with QRET and PathHunter, respectively. Combined homogeneous assay technique can provide an informative method for screening purposes and an efficient way to monitor receptor-ligand interaction, thus separating agonist from antagonist.

Sensitive luminometric method for protein quantification in bacterial cell lysate based on particle adsorption and dissociation of chelated europium.

A sensitive and rapid assay for the quantification of proteins, based on sample protein adsorption to Eu(3+)-chelate-labeled nanoparticles, was developed. The lanthanide ion of the surface-conjugated Eu(3+) chelate is dissociated at a low pH, decreasing the luminescence signal. The increased concentration of the sample protein prevents dissociation of the chelate, leading to a high luminescence signal due to the nanoparticle-bound protein. The assay sensitivity for the quantification of proteins was 130 pg for bovine serum albumin (BSA), which is an improvement of nearly 100-fold from the most sensitive commercial methods. The average coefficient of variation for the assay of BSA was 8%. The protein-to-protein variability was sufficiently low; the signal values varied within a 28% coefficient of variation for nine different proteins. The developed method is relatively insensitive to the presence of contaminants, such as nonionic detergents commonly found in biological samples. The existing methods tested for the total protein quantification failed to measure protein concentration in the presence of bacterial cell lysate. The developed method quantified protein also in samples containing insoluble cell components reducing the need for additional centrifugal assay steps and making the concept highly attractive for routine laboratory work.

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.

Cell-based beta2-adrenergic receptor-ligand binding assay using synthesized europium-labeled ligands and time-resolved fluorescence.

High-sensitivity, high-throughput, and user-friendly lanthanide-based assays for receptor-ligand interactions provide an attractive alternative to the traditional radioligand displacement assays. In this study, three small-molecule pindolol ligand derivatives were synthesized and their binding properties were tested in a radioligand displacement assay. The ligand derivatives were further labeled with fluorescent europium(III) chelate for beta(2)-adrenergic receptor-ligand binding assay. The europium-labeled pindolol ligands having no spacer (C0) or a 12-carbon spacer (C12) arm bound to the human beta(2)-adrenergic receptors overexpressed in human embryonic kidney HEK293(i) cells. Europium ligand with a 6-carbon spacer arm (C6) showed no binding. Competitive binding assays were developed with the functional labeled ligands. The IC(50) values for beta(2)-adrenergic antagonist propranolol were 60 and 37 nM, the Z' values were 0.51 and 0.77, and the signal-to-background ratios were 5.5 and 16.0 for C0 and C12, respectively. This study shows that functional time-resolved fluorescent assays can be constructed using fluorescent lanthanide chelates conjugated to small-molecule ligands.

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 HEK293(i) cells overexpressing either ß(2)-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 G(s) or inhibitory G(i) 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 G(s)-coupled ß(2)AR. 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). [(35)S]GTPgammaS 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 [(35)S]GTPgammaS assay. Upon activation of recombinant human alpha(2A)-adrenoceptors (alpha(2A)-AR) expressed in Chinese hamster ovary cells, guanosine-5'-diphosphate is released from the alpha-subunit of Gi-proteins, enabling the subsequent binding of Eu-GTP. Activation of alpha(2A)-AR with 5 different alpha(2)-AR agonists was measured quantitatively using the developed QRET GTP assay and compared to [(35)S]GTPgammaS 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 [(35)S]GTPgammaS binding assays.

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 beta(2)-adrenoreceptor (beta(2)AR) antagonists and agonists in intact human embryonic kidney HEK293(i) cells overexpressing human beta(2)-adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for beta(2)AR 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 K(i) values were 19 nM for propranolol and alprenolol and 14 and 5.9 microM for metaproterenol and terbutaline, respectively. The QRET technique with beta(2)AR 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.