HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications

Abstract

HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions.Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays.Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.

Keywords: Biomarker; Bioprocess.; GPCR; HTRF; Kinase; TR-FRET.

Fig. (1)

The general principle of fluorescence resonance energy transfer technology (FRET). When the donor and acceptor apart, there is no FRET signal. Once they are brought in proximity, the FRET signals are generated (for example, the emission spectrum at 665 nm).

Fig. (2)

The energy pulse from the excitation source (flash lamp, laser) is followed by a time delay, allowing interfering short-lived fluorescence (compounds, proteins, medium etc.) to decay. The red line: FRET signal intensity generated at 665 nm; black line, emission of donor cryptate at 620 nm; orange line, fluorescent signal generated from acceptor fluorophores.

Fig. (3)

Features of donor cryptate. a. Structure of cryptate trisbipyridine (TBP). Lambda max absorption: 305nm, molar extinction coefficient at 305nm: 30000 M-1 cm-1, molar extinction coefficient 337nm: 4500 M-1 cm-1. b. Detection wavelengths for Europium and Terbium cryptate. Both Europium and Terbium have emission spectra at 620 nm so that these signals can be used as reference for data analysis.

Fig. (4). Ratiometric reduction of the signal output data.

The specific signal at 665 nm may be affected by light transmission. The ratio (i.e. 665nm/620 nm) will normalize the signal measured and generate a variable that is independent of the optical properties of the medium in which the interaction is studied. In the case shown above, the 665 nm fluorescence decreases proportionally to the transmission, whereas the ratio corrects this interference and is equivalent in both situations.

Fig. (5)

cAMP assay principle. In this competitive assay, an anti-cAMP antibody is labeled with a cryptate donor and cAMP is labeled with acceptor d2. Once both of them are place together, the binding of antibody to cAMP brings donor and acceptor into proximity range. Upon the excitation of donor at 337 nm, the energy is transferred from donor to acceptor. Donor and acceptor generate emission at 620 nm and 665 nm, respectively. In the cAMP assay, the level of free cAMP generated by the signal cascade can be measured by competing with the cAMP d2 for antibody binding. IP-One assay from HTRF has the similar principle.

Fig. (6)

The comparison of “sandwich” assay and competition assay. a. “Sandwich” assay. HTRF signals are generated through the energy transfer from donor to acceptor that are labeled on the antibodies recognizing the target at different regions. b. Competition assay. Targets generated through biological events compete binding activity to the donor labeled antibody with the target labeled with acceptor so that the decrease of the HTRF signals are measured.

Fig. (7)

Kinase assay principle. The substrates used in HTRF kinase assays are tagged by fusion proteins, peptides, or biotin. An antibody to these proteins or peptides, or streptavidin (for biotin in this case) is labeled with acceptor. An antibody against phosphorylated substrate is labeled by donor cryptate. In the enzymatic reaction, once substrate is phosphorylated, donor and acceptor can be brought into proximity by affinity interactions of antibodies/substrate or streptavidin/biotin.

Fig. (8)

Principle of protein-protein interaction. Antibodies against the desired proteins (CD28 and CD 86 as examples) for interaction or the fused tags on these proteins are labeled with donor and acceptor, respectively. The FRET signals are generated once these proteins interact with each other to bring the labeled antibodies in into proximity.