Multiparametric Homogeneous Method for Identification of Ligand Binding to G Protein-Coupled Receptors: Receptor–Ligand Binding and β‑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 β2-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 β2-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

Peptic Fluorescent “Signal-On” and “Signal-Off” Sensors Utilized for the Detection Protein Post-Translational Modifications

Protein post-translational modifications (PTMs) are typically enzyme-catalyzed events generating functional diversification of proteome; thus, multiple PTM enzymes have been validated as potential drug targets. We have previously introduced energy-transfer-based signal-modulation method called quenching resonance energy transfer (QRET), and utilize it to monitor PTM addition or removal using the developed peptide-break technology. Now we have reinvented the QRET technology, and as a model, we introduced the tunable fluorescent “signal-on” and “signal-off” detection scheme in the peptide-break PTM detection. Taking the advantage of time-resolved fluorescence-based single-label detection technology, we were able to select the signal direction upon PTM addition or removal by simply introducing different soluble Eu3+-signal-modulating molecule. This enables the selection of positive signal change upon measurable event, without any additional labeling steps, changes in assay condition or Eu3+-reporter. The concept functionality was demonstrated with four Eu3+-signal modulators in a high-throughput compatible kinase and phosphatase assays using signal-on and signal-off readout at 615 nm or time-resolved Förster resonance energy transfer at 665 nm. Our data suggest that the introduced signal modulation methodology provides a transitional fluorescence-based single-label detection concept not limited only to PTM detection

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

Nanomolar Protein Thermal Profiling with Modified Cyanine Dyes

Protein properties and interactions have been widely investigated by using external labels. However, the micromolar sensitivity of the current dyes limits their applicability due to the high material consumption and assay cost. In response to this challenge, we synthesized a series of cyanine5 (Cy5) dye-based quencher molecules to develop an external dye technique to probe proteins at the nanomolar protein level in a high-throughput one-step assay format. Several families of Cy5 dye-based quenchers with ring and/or side-chain modifications were designed and synthesized by introducing organic small molecules or peptides. Our results showed that steric hindrance and electrostatic interactions are more important than hydrophobicity in the interaction between the luminescent negatively charged europium-chelate-labeled peptide (Eu-probe) and the quencher molecules. The presence of substituents on the quencher indolenine rings reduces their quenching property, whereas the increased positive charge on the indolenine side chain improved the interaction between the quenchers and the luminescent compound. The designed quencher structures entirely altered the dynamics of the Eu-probe (protein-probe) for studying protein stability and interactions, as we were able to reduce the quencher concentration 100-fold. Moreover, the new quencher molecules allowed us to conduct the experiments using neutral buffer conditions, known as the peptide-probe assay. These improvements enabled us to apply the method in a one-step format for nanomolar protein–ligand interaction and protein profiling studies instead of the previously developed two-step protocol. These improvements provide a faster and simpler method with lower material consumption

MOESM2 of eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5′UTR

Additional file 2. Supplementary Tables S1-S2

MOESM1 of eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5′UTR

Additional file 1. Supplementary Figure S1-S11 and Supplemental References

MOESM3 of eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5′UTR

Additional file 3. Review history